No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Influence of fatty acids on energy metabolism. 1. Stimulation of oxygen consumption, ketogenesis and CO
Abstract
Changes in metabolic rates (oxygen consumption, ketogenesis, 14CO2 production from labelled fatty acids, glycolysis) following the addition of octanoate or oleate were studied in isolated livers from fed and starved rats perfused with Krebs-Henseleit bicarbonate buffer in a non-recirculating system. The following results were obtained.
In conclusion, the increase in hepatic oxygen consumption due to addition of fatty acids reflects a mitochondrial process; it is, in part, independent of the ATP demand of the cell. An uncoupling-like effect of fatty acids on the respiratory chain and its possible physiological significance in ketogenesis are discussed.
... Like other MCFAs, and in contrast to LCFAs, octanoate is rapidly degraded and is stored as triglyceride in the adipose or other tissues only in a very low extent. Octanoate as a fuel for the energy metabolism in mammals has been studied in highenergy requiring tissues such as skeletal muscle, heart, liver and brain (89)(90)(91)(92)(93)(94). Concerning the latter organ, it is important to remember that SCFAs and MCFAs are able to permeate the blood-brain barrier (95). ...
... The effects of SCFAs and MCFAs on the hepatic energy metabolism were studied mostly either by the perfusion technique of isolated rat liver (89,(91)(92)(93)(96)(97)(98) indicate that these fatty acids effectively supply reducing equivalents (NADH, FADH 2 ) to the mitochondrial respiratory chain. In addition, octanoate raised the mitochondrial energization, an observation based on the in situ measurement of the mitochondrial membrane potential (∆Ψ m ) (101). ...
Short- and medium-chain fatty acids (SCFAs and MCFAs), independently of their cellular signalling functions, are important substrates of the energy metabolism and anabolic processes in mammals. SCFAs are mostly generated by colonic bacteria and are predominantly metabolized by enterocytes and liver, whereas MCFAs arise mostly from dietary triglycerides, among them milk and dairy products. A common feature of SCFAs and MCFAs is their carnitine-independent uptake and intramitochondrial activation to acyl-thioesters. Contrary to long-chain fatty acids, the cellular metabolism of SCFAs and MCFAs depends in a lesser extent on fatty acid-binding proteins. SCFAs and MCFAs modulate tissue metabolism of carbohydrates and lipids as manifested by mostly inhibitory effect on glycolysis and stimulation of lipogenesis or gluconeogenesis. SCFAs and MCFAs exert in mitochondria no or only weak protonophoric and lytic activities and do not significantly impair the electron transport in the respiratory chain. SCFAs and MCFAs modulate mitochondrial energy production by two mechanism: they provide reducing equivalents to the respiratory chain and partly decrease efficacy of the oxidative ATP synthesis.
... It has often been suggested that high rates of ketogenesis provide more ATP than is conventionally thought to be required by hepatocytes, and that there is therefore some uncoupling or that an ATP-consuming 'futile cycle' may be operating [31,32]. Indeed, it is well known that high concentrations of long-chain fatty acids uncouple isolated mitochondria [33]. ...
... This supports conclusions that the addition of long-chain fatty acids does not uncouple hepatocyte mitochondria [28]. It is difficult to assess reports that anabolic reactions in hepatocytes do not account for all of the extra 0, uptakes recorded [31,32], particularly in view of modern ideas about the lack of exact stoichiometries of ATP synthesis. ...
... Unfortunately, misguided observations have been used to promote the assumedly 'healthy' intake of low-carbohydrate ketogenic diets (especially those with reduced energy content) [588,589]. Ketogenesis (in adults, since maternal milk constitutes an example of growth-promoting obesogenic ketogenic diet [590] until weaning) is an emergency mechanism to distribute pre-metabolized 2C providers (i.e., 3hydroxy-butyrate or acetoacetate) between most tissues (including muscle) to provide rapidly usable energy [591,592] and prevent/limit the forced storage of 2C as TAG, given the low-temporal capacity of the liver mitochondria to oxidize any massive flow of 2C consequence as of excessive dietary carbohydrate (6C hexoses glycolyzed to 3C and, eventually, yielding in 2C as main final energy staple) [593,594]. The so-called 'refined diets' and the excessive use of sugars and TAG in our daily food intake enhance the need to oxidize the massive availability of 2C in order to remove the excess energy already in the system [595,596]. ...
This review focuses on the question of metabolic syndrome (MS) being a complex, but essentially monophyletic, galaxy of associated diseases/disorders, or just a syndrome of related but rather independent pathologies. The human nature of MS (its exceptionality in Nature and its close interdependence with human action and evolution) is presented and discussed. The text also describes the close interdependence of its components, with special emphasis on the description of their interrelations (including their syndromic development and recruitment), as well as their consequences upon energy handling and partition. The main theories on MS’s origin and development are presented in relation to hepatic steatosis, type 2 diabetes, and obesity, but encompass most of the MS components described so far. The differential effects of sex and its biological consequences are considered under the light of human social needs and evolution, which are also directly related to MS epidemiology, severity, and relations with senescence. The triggering and maintenance factors of MS are discussed, with especial emphasis on inflammation, a complex process affecting different levels of organization and which is a critical element for MS development. Inflammation is also related to the operation of connective tissue (including the adipose organ) and the widely studied and acknowledged influence of diet. The role of diet composition, including the transcendence of the anaplerotic maintenance of the Krebs cycle from dietary amino acid supply (and its timing), is developed in the context of testosterone and β-estradiol control of the insulin-glycaemia hepatic core system of carbohydrate-triacylglycerol energy handling. The high probability of MS acting as a unique complex biological control system (essentially monophyletic) is presented, together with additional perspectives/considerations on the treatment of this ‘very’ human disease.
... With CII considered as the involved dehydrogenase, the major criticisms about Mitchell's Q cycle were as follows: (1) no CII-CIII binding was observed with the technology of the time; (2) CIII activity does not require CII; (3) Ant A does not inhibit CII; and (4) the isolated Cyt bc 1 complex (prokaryotic CIII analogue) works autonomously with a self-contained mechanism 5,6 . In this study, we used different approaches to demonstrate that (1) Skm CIII interacts with ETFDH; (2) CIII activity is dependent on ETFDH; and (3) both CIII inhibitors, Ant A and myxothiazol, inhibit ETFDH activity 50 and FAO 51,52 . The observation that CIII retains ~40-50% of its activity following ETFDH deletion means that it can still function autonomously d,e, Body weight over time (d) and weight loss after fasting (150-day-old mice) (e) in wt and Etfdh −/− mice. ...
Coenzyme Q (Q) is a key lipid electron transporter, but several aspects of its biosynthesis and redox homeostasis remain undefined. Various flavoproteins reduce ubiquinone (oxidized form of Q) to ubiquinol (QH2); however, in eukaryotes, only oxidative phosphorylation (OXPHOS) complex III (CIII) oxidizes QH2 to Q. The mechanism of action of CIII is still debated. Herein, we show that the Q reductase electron-transfer flavoprotein dehydrogenase (ETFDH) is essential for CIII activity in skeletal muscle. We identify a complex (comprising ETFDH, CIII and the Q-biosynthesis regulator COQ2) that directs electrons from lipid substrates to the respiratory chain, thereby reducing electron leaks and reactive oxygen species production. This metabolon maintains total Q levels, minimizes QH2-reductive stress and improves OXPHOS efficiency. Muscle-specific Etfdh−/− mice develop myopathy due to CIII dysfunction, indicating that ETFDH is a required OXPHOS component and a potential therapeutic target for mitochondrial redox medicine.
... Collectively, above inconsistent findings from multiple studies including ours in terms of KD effects on exercise efficiency would be stemmed from differences in EE. The increase in EE when consuming a KD, compared to a MD, may be due to the thermic effect of food [34], uncoupling protein [35], and increased hepatic oxygen consumption proportional rate to the rate of ketone production [36]. However, the mechanism regarding KD and increased EE remains unclear [37]. ...
Objective
We examined the effects of short-term KD on exercise efficiency and hormonal response during and after the graded exercise testing.
Methods
Fourteen untrained healthy adults (8 males, 6 females, age 26.4 ± 3.1 [SD] years; BMI 24.8 ± 4.6 kg/m²; peak VO2max 54.0 ± 5.8 ml/kg FFM/min) completed 3-days of a mixed diet (MD) followed by another 3-days of KD after 3-days of washout period. Upon completion of each diet arm, participants underwent graded exercise testing with low- (LIE; 40% of VO2max), moderate- (MIE; 55%), and high-intensity exercise (HIE; 70%). Exercise efficiency was calculated as work done (kcal/min)/energy expenditure (kcal/min).
Results
Fat oxidation during the recovery period was higher in KD vs. MD. Despite identical workload during HIE, participants after having KD vs. MD showed higher energy expenditure and lower exercise efficiency (10.1 ± 0.7 vs. 12.5 ± 0.3%, p < .01). After KD, free fatty acid (FFA) concentrations were higher during MIE and recovery vs. resting, and beta-hydroxybutylate (BOHB) was lower at HIE vs. resting. Cortisol concentrations after KD was higher during recovery vs. resting, with no significant changes during graded exercise testing after MD.
Conclusions
Our data suggest that short-term KD is favorable to fat metabolism leading increased circulating FFA and BOHB during LIE to MIE. However, it is notable that KD may cause 1) exercise inefficiency manifested by increased energy expenditure and 2) elevated exercise stress during HIE and recovery. Trial registration: KCT0005172, International Clinical Trials Registry Platform.
... Overall, it has been hypothesized [77] that during the first phase of KDs, when glucose utilization is still prevalent, an increase in EE may occur possibly due to increased hepatic oxygen consumption, proportional to the rate of ketogenesis and consequent to a raise in the energy request for gluconeogenic pathways and for triglyceride-fatty acid recycling [81][82][83]. Later on, a reduction of gluconeogenesis caused by the shifting from glucose to ketone bodies oxidation by the brain, a decrease in the respiratory quotient, and the possible direct action of additional hormonal signals (i.e., thyroid hormone, adipokines and catecholamines) might lead to a reduction in EE [84][85][86][87] (Figure 1). ...
A dysregulation between energy intake (EI) and energy expenditure (EE), the two components of the energy balance equation, is one of the mechanisms responsible for the development of obesity. Conservation of energy equilibrium is deemed a dynamic process and alterations of one component (energy intake or energy expenditure) lead to biological and/or behavioral compensatory changes in the counterpart. The interplay between energy demand and caloric intake appears designed to guarantee an adequate fuel supply in variable life contexts. In the past decades, researchers focused their attention on finding efficient strategies to fight the obesity pandemic. The ketogenic or “keto” diet (KD) gained substantial consideration as a potential weight-loss strategy, whereby the concentration of blood ketones (acetoacetate, 3-β-hydroxybutyrate, and acetone) increases as a result of increased fatty acid breakdown and the activity of ketogenic enzymes. It has been hypothesized that during the first phase of KDs when glucose utilization is still prevalent, an increase in EE may occur, due to increased hepatic oxygen consumption for gluconeogenesis and for triglyceride-fatty acid recycling. Later, a decrease in 24-h EE may ensue due to the slowing of gluconeogenesis and increase in fatty acid oxidation, with a reduction of the respiratory quotient and possibly the direct action of additional hormonal signals.
... As is evident in Fig. 1A (light blue trace), the addition of oleate in the range 10-40 μM increased practically immediately the oxygen consumption rate (thermogenesis); the response reached saturation at 50 μM (here and in the following, these values refer to nominal concentrations; due to binding of fatty acids to many different components, free concentrations cannot be given). This was expected since fatty acid addition to cells and tissues has long been known to induce an uncoupling effect [18][19][20]. After the steady state level had been reached, addition of the artificial uncoupler FCCP only modestly further enhanced the rate (Fig. 1A), indicating either that maximal uncoupling had been achieved or that oleate had inhibited further oxidative stimulation (see below). ...
The possibility that N-acyl amino acids could function as brown or brite/beige adipose tissue-derived lipokines that could induce UCP1-independent thermogenesis by uncoupling mitochondrial respiration in several peripheral tissues is of significant physiological interest. To quantify the potency of N-acyl amino acids versus conventional fatty acids as thermogenic inducers, we have examined the affinity and efficacy of two pairs of such compounds: oleate versus N-oleoyl-leu and arachidonate versus N-arachidonoyl-gly in cells and mitochondria from different tissues. We found that in cultures of the muscle-derived L6 cell line, as well as in primary cultures of murine white, brite/beige and brown adipocytes, the N-acyl amino acids were proficient uncouplers but that they did not systematically display higher affinity or potency than the conventional fatty acids, and they were not as efficient uncouplers as classical protonophores (FCCP). Higher concentrations of the N-acyl amino acids (as well as of conventional fatty acids) were associated with signs of deleterious effects on the cells. In liver mitochondria, we found that the N-acyl amino acids uncoupled similarly to conventional fatty acids, thus apparently via activation of the adenine nucleotide transporter-2. In brown adipose tissue mitochondria, the N-acyl amino acids were able to activate UCP1, again similarly to conventional fatty acids. We thus conclude that the formation of the acyl-amino acid derivatives does not confer upon the corresponding fatty acids an enhanced ability to induce thermogenesis in peripheral tissues, and it is therefore unlikely that the N-acyl amino acids are of physiological relevance as UCP1-independent thermogenic compounds.
... In addition, TG are neutral lipids that can provide energy for living organisms through oxidative decomposition (Ohshima, Li, & Koizumi, 1993). With sufficient oxygen supply, fatty acids can be decomposed into acetyl-CoA, which are completely oxidized to CO 2 and H 2 O and release considerable energy (Mao et al., 2006;Munday, 2002;Scholz, Schwabe, & Soboll, 2010). ...
To explore energy utilization during molting stage, matured and juvenile red claw crayfish were selected to determine changes in lipid composition and fatty acid contents of hepatopancreas. Moreover, the expression of the fatty acid binding protein (FABP) gene was determined to verify changes of fatty acids at the molecular level. Results showed that after molting, the crude lipid content was significantly lower than before molting (p <.05). Among them, the triglyceride content in the hepatopancreas showed no significant differences both before and after molting. The cholesterol content only increased significantly after the matured red claw crayfish had molted. Phospholipids of matured red claw crayfish decreased significantly after molting, but increased significantly after molting in juvenile (p <.05). Saturated fatty acid content did not differ significantly before and after molting in matured red claw crayfish, while it increased after molting in juveniles. The monounsaturated fatty acid content increased, while polyunsaturated fatty acid content decreased significantly after molting (p <.05). The expression of FABP was consistent with the phospholipids change trend of both matured and juvenile red claw crayfish, which may be the molecular indicator of phospholipids during the molting stage. The specific lipid metabolism pathway still needs further exploration.
... Substrate-free perfused livers from fed rats do not depend solely on the oxidation of endogenous FA. Actually, the glycolytic activity, that is enhanced in the arthritic state [19,20], leads by itself to the production of reducing equivalents and acetyl-CoA from pyruvate. Glycolysis is one of the reasons why substrate-free perfused livers from fed rats produce much less ketone bodies [53]. It is thus likely that the contribution of endogenous FA oxidation to the 23% increased citric acid cycle activity was relatively modest. ...
Severe rheumatoid cachexia is associated with pronounced loss of muscle and fat mass in patients with advanced rheumatoid arthritis. This condition is associated with dyslipidemia and predisposition to cardiovascular diseases. Circulating levels of triglycerides (TG) and free fatty acids (FFA) have not yet been consistently defined in severe arthritis. Similarly, the metabolism of these lipids in the arthritic liver has not yet been clarified. Aiming at filling these gaps this study presents a characterization of the circulating lipid profile and of the fatty acids uptake and metabolism in perfused livers of rats with adjuvant-induced arthritis. The levels of TG and total cholesterol were reduced in both serum (10–20%) and liver (20–35%) of arthritic rats. The levels of circulating FFA were 40% higher in arthritic rats, possibly in consequence of cytokine-induced adipose tissue lipolysis. Hepatic uptake and oxidation of palmitic and oleic acids was higher in arthritic livers. The phenomenon results possibly from a more oxidized state of the arthritic liver. Indeed, NADPH/NADP⁺ and NADH/NAD⁺ ratios were 30% lower in arthritic livers, which additionally presented higher activities of the citric acid cycle driven by both endogenous and exogenous FFA. The lower levels of circulating and hepatic TG possibly are caused by an increased oxidation associated to a reduced synthesis of fatty acids in arthritic livers. These results reveal that the lipid hepatic metabolism in arthritic rats presents a strong catabolic tendency, a condition that should contribute to the marked cachexia described for arthritic rats and possibly for the severe rheumatoid arthritis.
... The rapid increase in SEE and EE chamber within the first week of the KD may have been caused by increased hepatic oxygen consumption proportional to the rate of ketogenesis (39). For ketogenesis to fully explain the observed early w200-kcal/d increase in SEE requires w150 g/d of ketogenesis (16), which is commensurate with both the observed circulating ketone concentrations as well as the urinary excretion rate, and implies a rate of ketogenesis approximately half of that achieved within 1 wk of fasting when ketogenesis reaches a maximum (15). ...
Background:
The carbohydrate-insulin model of obesity posits that habitual consumption of a high-carbohydrate diet sequesters fat within adipose tissue because of hyperinsulinemia and results in adaptive suppression of energy expenditure (EE). Therefore, isocaloric exchange of dietary carbohydrate for fat is predicted to result in increased EE, increased fat oxidation, and loss of body fat. In contrast, a more conventional view that "a calorie is a calorie" predicts that isocaloric variations in dietary carbohydrate and fat will have no physiologically important effects on EE or body fat.
Objective:
We investigated whether an isocaloric low-carbohydrate ketogenic diet (KD) is associated with changes in EE, respiratory quotient (RQ), and body composition.
Design:
Seventeen overweight or obese men were admitted to metabolic wards, where they consumed a high-carbohydrate baseline diet (BD) for 4 wk followed by 4 wk of an isocaloric KD with clamped protein. Subjects spent 2 consecutive days each week residing in metabolic chambers to measure changes in EE (EEchamber), sleeping EE (SEE), and RQ. Body composition changes were measured by dual-energy X-ray absorptiometry. Average EE during the final 2 wk of the BD and KD periods was measured by doubly labeled water (EEDLW).
Results:
Subjects lost weight and body fat throughout the study corresponding to an overall negative energy balance of ∼300 kcal/d. Compared with BD, the KD coincided with increased EEchamber (57 ± 13 kcal/d, P = 0.0004) and SEE (89 ± 14 kcal/d, P < 0.0001) and decreased RQ (-0.111 ± 0.003, P < 0.0001). EEDLW increased by 151 ± 63 kcal/d (P = 0.03). Body fat loss slowed during the KD and coincided with increased protein utilization and loss of fat-free mass.
Conclusion:
The isocaloric KD was not accompanied by increased body fat loss but was associated with relatively small increases in EE that were near the limits of detection with the use of state-of-the-art technology. This trial was registered at clinicaltrials.gov as NCT01967563.
... Ketogenesis was increased $30% in livers from FGF21 transgenic mice ( Figure 2D). By comparison, fasting alone induces ketone production by $60% in isolated rodent livers perfused with 0.5 mM oleate (Scholz et al., 1984). These data suggest that FGF21 contributes substantially to fasting-induced ketogenesis. ...
... However, practically nothing has been reported on the effect of these major fatty acids accumulating in MCADD on the liver and skeletal muscle, tissues that have been shown to be altered in patients affected by this disorder [7,25]. To our knowledge, the only observations available in the literature indicate that OA increases oxygen consumption and decreases cytosolic ATP concentrations and the glycolytic rate in perfused rat liver [26][27][28]. The present study extended these investigations by initially studying the effects of OA and DA on important energy metabolism parameters in liver and skeletal muscle of young rats, namely the respiratory chain complexes and creatine kinase activities. ...
The accumulation of octanoic (OA) and decanoic (DA) acids in tissue is the common finding in medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), the most frequent defect of fatty acid oxidation. Affected patients present hypoketotic hypoglycemia, rhabdomyolysis, hepatomegaly, seizures and lethargy, which may progress to coma and death. At present, the pathophysiological mechanisms underlying hepatic and skeletal muscle alterations in affected patients are poorly known. Therefore, in the present work, we investigated the in vitro effects of OA and DA, the accumulating metabolites in MCADD, on various bioenergetics and oxidative stress parameters. It was verified that OA and DA decreased complexes I-III, II-III and IV activities in liver and also inhibit complex IV activity in skeletal muscle. In addition, DA decreased complexes II-III activity in skeletal muscle. We also verified that OA and DA increased TBA-RS levels and carbonyl content in both tissues. Finally, DA, but not OA, significantly decreased GSH levels in rat skeletal muscle. Our present data show that the medium-chain fatty acids that accumulate in MCADD impair electron transfer through respiratory chain and elicit oxidative damage in rat liver and skeletal muscle. It may be therefore presumed that these mechanisms are involved in the pathophysiology of the hepatopathy and rhabdomyolysis presented by MCADD-affected patients.
... Increased hepatic fatty acid oxidation during infusion of fatty acids generates more reducing equivalents (NADH) and greater oxidative stress, as evidenced by higher 8 OHdG in NASH in this study [48,49]. Plasma ceramides increased in patients with NASH compared with controls during intralipid infusion and these may also contribute to the oxidative injury [50]. ...
Data from studies in patients with nonalcoholic steatohepatitis (NASH) suggest an increased hepatic fatty acid oxidation. We have previously shown higher fasting plasma bile acid concentrations in patients with NASH. In-vivo and in-vitro studies suggest that bile acids by binding to peroxisome proliferator-activated receptor α activate fibroblast growth factor 21 (FGF21) and increase hepatic fatty acid oxidation.
Plasma bile acid levels were quantified in healthy controls (n=38) and patients with biopsy-proven NASH (n=36). Plasma concentration of fatty acids, β-hydroxybutyrate, insulin, glucose, leptin, alanine aminotransferase, FGF21, and 8-hydroxydeoxyguanosine, a measure of oxidative stress, were measured in 16 healthy controls and 10 patients with NASH in the fasted state and in response to 3 h of infusion of intralipid. In a subgroup of these patients (n=6 each), plasma ceramide subspecies were quantified.
Fasting plasma bile acids, FGF21, and leptin concentrations were significantly higher in patients with NASH. In response to intralipid infusion there was an increase in plasma β-hydroxybutyrate and free fatty acid levels in both controls and NASH; however, the ratio of β-hydroxybutyrate/free fatty acid was higher in NASH (P=0.02). Plasma FGF21 concentration increased in response to intralipid in patients with NASH only (P<0.01). Plasma leptin, insulin, glucose, and alanine transferase concentrations did not change in either group after infusion of intralipid. Increase in total ceramides in response to intralipid was greater in NASH.
Elevated bile acids and FGF21 may be responsible for the higher hepatic fatty acid oxidation in NASH.
... However, it remains disputed whether stimulation of respiration by fatty acids in vivo is accounted for by their intrinsic mitochondrial uncoupling activity, or due to their availability as substrates for oxidation, combined with stimulation of extramitochondrial ATP-consuming reactions. Indeed, increased oxygen consumption induced by fatty acids in the perfused liver or in isolated liver cells has been reported to be essentially or partly eliminated upon blocking oxidative phosphorylation by added oligomycin or atractyloside, thus refuting classic mitochondrial uncoupling (12)(13)(14)(15). Furthermore, inner mitochondrial membrane potential measured in isolated hepatocytes, or phosphorylation potential measured in situ in the perfused heart were found to be unaffected or rather increased by added fatty acids. ...
The role played by long chain fatty acids (LCFA) in promoting energy expenditure is confounded by their dual function as substrates
for oxidation and as putative classic uncouplers of mitochondrial oxidative phosphorylation. LCFA analogs of the MEDICA (MEthyl-substituted
DICarboxylic Acids) series are neither esterified into lipids nor β-oxidized and may thus simulate the uncoupling activity
of natural LCFA in vivo, independently of their substrate role. Treatment of rats or cell lines with MEDICA analogs results in low conductance gating
of the mitochondrial permeability transition pore (PTP), with 10–40% decrease in the inner mitochondrial membrane potential.
PTP gating by MEDICA analogs is accounted for by inhibition of Raf1 expression and kinase activity, resulting in suppression
of the MAPK/RSK1 and the adenylate cyclase/PKA transduction pathways. Suppression of RSK1 and PKA results in a decrease in
phosphorylation of their respective downstream targets, Bad(Ser-112) and Bad(Ser-155). Decrease in Bad(Ser-112, Ser-155) phosphorylation
results in increased binding of Bad to mitochondrial Bcl2 with concomitant displacement of Bax, followed by PTP gating induced
by free mitochondrial Bax. Low conductance PTP gating by LCFA/MEDICA may account for their thyromimetic calorigenic activity
in vivo.
... It is of interest that the transient change in NAD(P)H fluorescence seen in the present studies and by others (Sugano et al., 1980;Kimura et al., 1984;Balaban & Blum, 1982) was not reflected in a transient change in the ,-hydroxybutyrate/acetoacetate ratio (P. T. Quinlan & A. P. Halestrap, unpublished work;Williamson et al., 1969b;Berry et al., 1983a,b;Soboll et al., 1984;Scholz et al., 1984). This could indicate that the fluorescence changes observed are not reflecting the mitochondrial NADH/NAD+ ratio as has been predicted (Williamson et al., 1969b;Sugano et al., 1980;Balaban & Blum, 1982;Kimura et al., 1984). ...
The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)
... Several mechanisms have been proposed by which fibrates decrease hepatic production of triacylglycerol, including: (i) decreased fatty acid availability [1]; (ii) increased intrahepatic diversion of fatty acids towards peroxisomal and mitochondrial fl-oxidation, as opposed to esterification to glycerolipid [13]; (iii) inhibition of glycerolipid formation [14]; and (iv) inhibition of very-low-densitylipoprotein secretion [15]. Studies on perfused liver or hepatocytes from rats treated with fibrates in vivo have shown increased rates of fatty acid f-oxidation [5,10,[16][17][18][19], associated with either decreased [16,19] or unchanged or increased [5,10] rates of fatty acid esterification. Clofibrate has been shown to lower plasma insulin in the rat [20]. ...
The direct effects of clofibrate analogues on carnitine acyltransferase activities and fatty acid metabolism were studied in cultured hepatocytes. Rat hepatocytes cultured with bezafibrate or ciprofibrate (0.1-10 micrograms/ml) for 48 h had increased activities of carnitine acetyltransferase (CAT; 4-6-fold) and carnitine palmitoyltransferase (CPT; 12-34%). The increase in CAT was higher in hepatocytes from the periportal zone (440%) of rat liver compared with cells from the perivenous zone (266%). In human hepatocytes, in contrast with rat, the fibrates did not cause a marked increase in CAT activity. The effects of fibrates on palmitate metabolism were dependent on the carnitine status. In the presence of exogenous carnitine (1 mM), rat hepatocytes cultured with bezafibrate had higher rates of total palmitate metabolism (29-34%) without increased partitioning of palmitate towards beta-oxidation, relative to control cultures. At low endogenous carnitine concentrations, cells cultured with bezafibrate had a greater increase in palmitate metabolism, esterification and cellular accumulation of triacylglycerol compared with the corresponding increases in the presence of carnitine. The changes in palmitate metabolism at either high or low carnitine concentrations were small in comparison with the changes in CAT activity. It is concluded that the increase in hepatic carnitine that occurs in vivo after fibrate feeding probably plays the major role in the changes in partitioning of fatty acid between beta-oxidation and esterification.
... However the stimulation decreases with increasing [pyruvate]. The inhibition of [1-14C]pyruvate decarboxylation by fatty acids that has been observed in perfused liver (Dennis et al., 1978;Scholz et al., 1978;Patel et al., 1984), but not in isolated hepatocyte suspensions (Demaugre et al., 1984), may be due to 02 limitation in perfused liver, because of the increased 02 consumption caused by the fatty acids (Scholz et al., , 1984 and the increased respiration required to support gluconeogenesis (Tutwiler & Brentzel, 1982). 02 limitation would result in a more reduced NADH/NAD+ couple, which may cause a decreased flux through the enzyme as well as inactivation of the enzyme by increased phosphorylation because of stimulation of the kinase. ...
The contribution of pyruvate to ketogenesis was determined in rat hepatocyte suspensions by using [14C]pyruvate. The rates of conversion of pyruvate into ketone bodies in hepatocytes from fed and 24 h-starved rats were 10 and 17 mumol/h per g wet wt. respectively, and accounted for 50 and 29% of the total ketone bodies formed. In hepatocytes from fed rats, the addition of palmitate (0.25-1 mM) increased the rate of conversion of pyruvate into ketone bodies (80-140%), but decreased the relative contribution of pyruvate to total ketogenesis. In hepatocytes from starved rats, palmitate did not increase pyruvate conversion into ketone bodies.
... Normally the liver operates under highly aerobic conditions. Values for hepatic 0 2 consumption vary between 2 and 10 kmol/g liver wet weight per min (Berry et al. 1973; Krebs et al. 1974; Scholz et al. 1984;Rabkin & Blum, 1985;Seifter & Englard, 1988). Based on a P:O ratio of 3 (i.e. ...
Background:
Acetaminophen (APAP)-associated transaminase elevation, induced by N-acetyl-p-benzoquinone imine (NAPQI) protein adduction, remains an area of research interest. Distinct from known genetic, physiologic, and dosage associations dictating severity of hepatic injury, no known factors predict an absence of protein adduct formation at therapeutic APAP dosing.
Hypothesis:
Sex-based physiology is predictive of APAP-induced protein adduct formation and differential metabolite expression at therapeutic doses.
Methods:
This retrospective study interrogated serum samples collected for a prior study investigating fluctuations of alanine aminotransferase (ALT) over time with 4G daily APAP dosing for ≥ 16 days in subjects from Denver, Colorado. Subjects were grouped by adduct formation (n = 184) vs no adducts (n = 20). Samples were run on ultra-high-performance liquid chromatography mass spectrometry from study days 0, 7, 16, and 31. Significant metabolite expressions were identified using t-tests with false discovery rate correction (FDR), partial least squares discriminant, and ANOVA simultaneous comparison analyses. Demographic and clinical data were explored using t-tests with FDR (age, weight, BMI, ALT) and Chi-square (sex, ethnicity, race) analyses.
Results:
In pre-treatment samples, relative quantitation caprylic acid was expressed ninefold higher and 6-carboxyhexanoate was expressed threefold lower in subjects who did not develop adducts. Lactate had greater expression in the no adducts group (p = 0.001). Using absolute quantitation, glutathione was expressed 2.6-fold greater among no adduct subjects. Odds of males developing NAPQI protein adducts at therapeutic APAP dosing were 5.91 times lower than females (95% CI = 2.3-14.9; p = 0.0001).
Conclusion:
Multiple metabolites were differentially expressed based on adduct group and sex. Metabolites were identified unique to adduct development independent of sex. At therapeutic APAP dosing, males were less likely to develop APAP protein adducts. Further research into lipid biosynthesis and metabolism may provide further insight into physiology associated with adduct production.
Since the discovery of the process of oxidative phosphorylation, biochemical textbooks have tacitly assumed that most, if not all, of mammalian mitochondrial respiration is tightly coupled to ATP turnover, so that any increase in cellular oxygen uptake (JO) must necessarily indicate an augmented demand for ATP. On the other hand, much evidence has now accrued that the increase in JO observed when hepatocytes are presented with substrates, particularly fatty acids (Berry, 1974a; Williamson et al., 1969; Debeer et al., 1974; Berry et al., 1983a), cannot be wholly or even mainly accounted for by increased utilization of ATP in biosynthetic processes such as gluconeogenesis and urea formation (Berry, 1974a; Berry et al., 1983a; Hems et al., 1966; Krebs et al., 1964). The mechanism by which oxygen consumption is stimulated to a greater extent than predicted from any increased metabolic activity of the cells has not been established unequivocally. Possibilities include uncoupling of the mitochondria (Scholz et al., 1984; Soboll and Stucki, 1985), changes in eficiency of coupling, perhaps by alterations in the H+/e- ratio of mitochondrial proton pumping (Nicholls, 1974; Pietrobon et al., 1981), induction of futile cycles of ATP synthesis and hydrolysis (Debeer et al., 1974; Newsholme and Crabtree, 1976; Katz and Rognstad, 1976; Plomp et al., 1985), or stimulation of some other pathway such as reversed electron flow (Berry et al., 1983a). None of these explanations seems entirely satisfactory.
The phosphorylation state of cytosolic ATP ([ATP]/([ADP][Pi)) can be calculated from the measured reactants of creatine kinase, including H+. Measured total tissue ATP/(ADP x Pi) ratio underestimates [ATP]/([ADP][Pi]) by up to two orders of magnitude, mainly because total tissue ADP grossly overestimates the thermodynamic concentration of ADP ([ADP]) in the soluble cytoplasm (cytosol). Both ADP concentration estimates using the creatine kinase equilibrium and published estimates of cytoplasmic binding sites for ADP indicate that, in muscle and heart in particular, ADP is compartmented between cytosol and mitochondria and that most of cytoplasmic ADP is bound to actin. Current data on [ADP] place its concentration between 16 to 60 μM as a function of cardiac energy output which determines oxygen usage (MVO2). Similarly, employing the myokinase equilibrium to estimate the thermodynamic concentration of AMP ([AMP]), the calculated free cytosolic AMP concentrations exhibited highly significant square dependencies on MVO2, [ADP], and the [ADP]/[ATP] ratio, respectively. Calculated [AMP] ranged from approximately 100 to 800 nM as a function of the [ADP]/[ATP] ratio in normal and ischemic hearts. Considering that measured total AMP was in the submillimolar range, the data suggested that most of cardiac AMP was not in catalytic contact with myokinase, i. e., probably located in the mitochondrial compartment. Net release of adenosine plus inosine (V(AR+INO)) was directly related to [AMP] and cellular lactate/pyruvate ratio, respectively. These results suggested that the availability of free AMP may be a determinant of 5′-nucleotidase plus adenylate deaminase activities and that V(AR+INO) was reciprocally linked to moycardial energy state as quantitated by the [ATP]/([ADP][Pi]) ratio. Such energy-linked V(AR+INO) allows for adenosine formation in normoxic heart without assuming a microhypoxia stimulus. According to this model the key trigger for net ATP degradation and V(AR+INO) during both normoxia and ischemia is the actually incurred myocardial energy deficit or the cytosolic ATP potential, not a change in the oxygen supply-demand ratio per se.
The effects of fatty acids (FA)-carrier, egg-lecithin liposomes (LIPO) as alternative to BSA, on ATP, glycogen and glucose contents in isolated perfused liver of fed rats were non-invasively studied using 31P/13C nuclear magnetic resonance (NMR). Oxidative phosphorylation was studied in isolated mitochondria from the same liver consecutively to the NMR experiments. ATP content decreased slowly and ATP turnover was similar during the perfusion with saline solution (KHB) or LIPO. However, LIPO induced an enhancement of respiratory control ratio in isolated mitochondria. Tissue glycogen and glucose content decreased when FA (linoleate or linolenate) were perfused with defatted BSA (3%) or LIPO (600 mg/l) whereas glucose excretion level was unchanged and lactate excretion tended to increase, reflecting changes in the cytosolic redox state and/or an enhancement of glycolysis. Addition of FA (0.5 or 1.5 mM) to LIPO caused a dramatic fall in liver ATP, a mitochondrial uncoupling and an impairment of the phosphorylation activity. Perfusion with FA (1.5 mM) carried by BSA significantly increased the ATP degradation without change of mitochondrial function. Owing to the higher affinity of BSA than LIPO for FA, these latter could be more easily released from complex LIPO–FA, increasing their uncoupling effect. Hence, the FA concentrations have to be largely decreased from the above currently used concentrations to avoid this effect. It will then be possible to minimize the effector action of FA and to study their more specific metabolic function as fuel. It was concluded that LIPO were appropriate carriers to study the different metabolic effects of FA.
In liver, 13CO2 can be generated from [1-13C]pyruvate via pyruvate dehydrogenase or anaplerotic entry of pyruvate into the TCA cycle followed by decarboxylation at phosphoenolpyruvate carboxykinase (PEPCK), the malic enzyme, isocitrate dehydrogenase, or α-ketoglutarate dehydrogenase. The purpose of this study was to determine the relative importance of these pathways in production of hyperpolarized (HP) 13CO2 after administration of hyperpolarized pyruvate in livers supplied with a fatty acid plus substrates for gluconeogenesis. Isolated mouse livers were perfused with a mixture of thermally-polarized 13C-enriched pyruvate, lactate and octanoate in various combinations prior to exposure to HP pyruvate. Under all perfusion conditions, HP malate, aspartate and fumarate were detected within ~3 s showing that HP [1-13C]pyruvate is rapidly converted to [1-13C]oxaloacetate which can subsequently produce HP 13CO2 via decarboxylation at PEPCK. Measurements using HP [2-13C]pyruvate allowed the exclusion of reactions related to TCA cycle turnover as sources of HP 13CO2. Direct measures of O2 consumption, ketone production, and glucose production by the intact liver combined with 13C isotopomer analyses of tissue extracts yielded a comprehensive profile of metabolic flux in perfused liver. Together, these data show that, even though the majority of HP 13CO2 derived from HP [1-13C]pyruvate in livers exposed to fatty acids reflects decarboxylation of [4-13C]oxaloacetate (PEPCK) or [4-13C]malate (malic enzyme), the intensity of the HP 13CO2 signal is not proportional to glucose production because the amount of pyruvate returned to the TCA cycle via PEPCK and pyruvate kinase is variable, depending upon available substrates.
In order to elucidate the relation between the hepatotoxicity of salicylate (SA) and the pathogenesis of Reye's syndrome (RS), urea production, gluconeogenesis and ketogenesis were investigated in isolated perfused rat livers in the presence of salicylate (SA) and oleate. Although urea formation from 0.5 mM NH4Cl, 2 mM ornithine and 0.3 mM oleate was not inhibited by infusion of SA, 3 mM SA caused a 26% decrease of ketogenesis, 85% decrease of 3-hydroxybutyrate/acetoacetate ratio (30HB/AcAc) and 45% increase of oxygen consumption. Glucose production from 2 mM pyruvate in the presence of 0.3 mM oleate decreased by 33% after administration of 3 mM SA, and 30HB/AcAc ratio also decreased by 33%. The decrement of gluconeogenesis and that of the 30HB/AcAc ratio were very close.
These results suggested that ATP production was maintained but that the intra-mitochondrial redox state was changed to a more oxidized state after SA administration in perfused rat livers. This change in redox state could be responsible for the decrease of gluconeogenesis.
Metabolic characteristics found in RS were not obtained by infusion of 3mM SA and 0.3 mM oleate in rat livers. Therefore, some other factors in addition to SA seem necessary to establish an animal model of RS.
An animal model experiment was conducted with nine adult sows to study the effects of long-chain fatty acids on thermogenesis when different fatty acids were replaced for 30% of the energy of a basal diet based on cereals and soybean meal. The acids were fed as commercial products containing as main constituent either palmitic acid, oleic acid, or linoleic acid, according to a latin square design in experimental periods 2 to 4. In periods 1 and 5 the sows were submitted to basal diet alone.
Digestibility of palmitic acid was only 36%, whereas the unsaturated fatty acids were highly absorbed (90%). Interaction effects of the undigested proportion of the long-chain fatty acids with the basal diet in hindgut fermentation could be ruled out since a supplementary experiment on three sows showed no influence of infusion of oleic or linoleic acid into the caecum on the energy utilization of the basal diet. There was no significant differences in thermogenesis among the fatty acids. Heat production in the treatment periods averaged −1.2% as compared to the basal diet periods. This result was in accordance with the value −1.0% calculated theoretically for the reduction in heat production in the treatment periods. Thus, the data did not indicate any stimulating effect of long-chain fatty acids on heat production, and utilization of energy of fatty acids occurred within the obligatory thermogenesis.
It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-(14)C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-(14)C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD(+) ratio, but stimulated (14)CO(2) production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.
The association of TGF beta1 polymorphisms and atrial fibrillation (AF) in essential hypertensive (EH) subjects remains unknown. Methods EH subjects with AF (EH+AF+) and sinus rhythm (EH+AF-) were enrolled. The polymorphisms of +869 T --> C at codon 10 and + 915 G --> C at codon 25, were genotyped. The clinical characteristics including serum TGF beta1 levels were detected.
The GG genotypes of TGF beta1 +915 G --> C at codon 25 were more prevalent in subjects from EH+AF+ group than those from EH+AF- group (P = 0.009). The subjects with GG genotype from EH+AF+ group had the highest mean serum TGF beta1 level, which was significantly higher than that of GG genotype subjects from EH+AF- group (3.18 +/- 0.24 ng/dl vs.2.29 +/- 0.14 ng/dl, P < 0.05). Multiple analyses revealed that the TGF beta1 GG genotype of +915 G --> C at codon 25 presented a 3.09 times higher risk in developing AF in the multivariate model after adjusting for age and gender.
The polymorphisms of TGF beta1 +915 G --> C at codon 25 were associated with occurrence of AF and serum TGF beta1 level in EH subjects.
Glutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable.
Zonation of glutamine metabolism was investigated in the bivascularly perfused rat liver with [U-14C]glutamine infusion (0.6 mM) into the portal vein (antegrade perfusion) or into the hepatic vein (retrograde perfusion).
Ammonia infusion into the hepatic artery in retrograde and antegrade perfusion allowed to promote glutamine metabolism in the periportal region and in the whole liver parenchyma, respectively. The results revealed that the space-normalized glutamine uptake, indicated by (14)CO(2) production, gluconeogenesis, lactate production and the associated oxygen uptake, predominates in the periportal region. Periportal predominance was especially pronounced for gluconeogenesis. Ureogenesis, however, tended to be uniformly distributed over the whole liver parenchyma at low ammonia concentrations (up to 1.0 mM); periportal predominance was found only at ammonia concentrations above 1 mM. The proportions between the carbon and nitrogen fluxes in periportal cells are not the same along the liver acinus.
In conclusion, the results of the present work indicate that the glutaminase activity in periportal hepatocytes is not the rate-controlling step of the glutamine-derived nitrogen flow through the urea cycle. The findings corroborate recent work indicating that ureogenesis is also an important ammonia-detoxifying mechanism in cells situated downstream to the periportal region.
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an inherited metabolic disorder of fatty acid oxidation in which the affected patients predominantly present high levels of octanoic (OA) and decanoic (DA) acids and their glycine and carnitine by-products in tissues and body fluids. It is clinically characterized by episodic encephalopathic crises with coma and seizures, as well as by progressive neurological involvement, whose pathophysiology is poorly known. In the present work, we investigated the in vitro effects of OA and DA on various parameters of energy homeostasis in mitochondrial preparations from brain of young rats. We found that OA and DA markedly increased state 4 respiration and diminished state 3 respiration as well as the respiratory control ratio, the mitochondrial membrane potential and the matrix NAD(P)H levels. In addition, DA-elicited increase in oxygen consumption in state 4 respiration was partially prevented by atractyloside, indicating the involvement of the adenine nucleotide translocator. OA and DA also reduced ADP/O ratio, CCCP-stimulated respiration and the activities of respiratory chain complexes. The data indicate that the major accumulating fatty acids in MCADD act as uncouplers of oxidative phosphorylation and as metabolic inhibitors. Furthermore, DA, but not OA, provoked a marked mitochondrial swelling and cytochrome c release from mitochondria, reflecting a permeabilization of the inner mitochondrial membrane. Taken together, these data suggest that OA and DA impair brain mitochondrial energy homeostasis that could underlie at least in part the neuropathology of MCADD.
The unidirectional fluxes of palmitate across the liver cell membrane and metabolic uptake rates were measured employing the multiple-indicator dilution technique. The following results were obtained: (1) Influx and net uptake rates do not vary proportionally to each other when albumin and palmitate concentrations are varied. (2) Efflux is significant for albumin concentrations in the range between 1.5 and 500 microM. (3) At 150 microM albumin net uptake rates are proportional to the total (bound plus free) extracellular palmitate concentration in the range from 10 to 600 microM; the dependence of influx rates on the palmitate concentration is rather concave up. (4) When albumin and palmitate are both varied at an equimolar ratio, pseudo-saturation appears in the net uptake rates; the influx rates also show pseudo-saturation, but with a declining tendency at the higher concentrations. (5) The intracellular palmitate concentration is strongly influenced by albumin. At very low concentrations of the protein (1.5 microM) the intracellular concentration is practically equal to the extracellular one; at physiological albumin concentrations, however, the intracellular palmitate concentration is less than 2% of the extracellular one. (6) Saturation of net uptake with respect to the intracellular palmitate concentration was not observed with concentrations up to 46 microM.
1. Previous studies have shown that an arterial-to-portal glucose concentration gradient may be an important signal for insulin-dependent net hepatic glucose uptake. It is not known whether intestinal factors also contribute to the regulation of hepatic glucose utilization. This problem was studied in a newly developed model which allows luminal perfusion of the small intestine via the pyloric sphincter and a combined vascular perfusion of the small intestine via the gastroduodenal artery and superior mesenteric artery, and of the liver via the hepatic artery and portal vein. 2. In both the presence and the absence of 1 mM-glutamine in the vascular perfusate, only about 7% of a luminal bolus of 5500 mumol (1 g) of glucose was absorbed by the small intestine, and nothing was taken up by the liver. 3. With small doses of 75-380 mumol (11-55 mg) of luminal glutamine, but not with 300 mumol of alanine, the intestinal absorption of the luminal glucose bolus was increased almost linearly from 7% to a maximum of 40% and the hepatic uptake from 0% to a maximum of 22%. 4. The increase of hepatic glucose uptake caused by luminal glutamine was only observed when the glucose load was applied into the intestinal lumen, rather than into the superior mesenteric artery. 5. The relative hepatic glucose uptake (uptake/portal supply) was enhanced from 0% to 55% with an increase in portal supply by luminal glutamine, whereas with a similar range of portal glucose supply the relative hepatic uptake by the isolated liver, perfused simultaneously via the hepatic artery and portal vein, was slightly decreased, from 20% to 15%. 6. Addition of various amounts of portal glutamine and/or alterations in the Na+ content of the portal perfusate failed to mimic the luminal glutamine-dependent activation of hepatic glucose uptake. Therefore the luminal-glutamine-elicited activation of hepatic glucose uptake was apparently not caused by a simple increase in the portal-arterial glucose gradient, by glutamine itself or by Na(+)-dependent alterations in hepatic cell volume. The results suggest that luminal glutamine caused not only an increase in intestinal glucose absorption by unknown mechanisms but also the generation of one or more humoral or nervous 'hepatotropic' signals in the small intestine which enhanced the hepatic uptake of absorbed glucose.
The relative effectiveness of British Anti-Lewisite (BAL), dimercaptopropanesulfonic acid (DMPS), dimercaptosuccinic acid (DMSA), and a new metal binding agent 2,3-bis(acetylthio)propanesulfonamide (BAPSA) was compared by determining their effect on pyruvate metabolism in perfused livers of guinea pigs after repeated treatment with As2O3. Guinea pigs received As2O3, 2.5 mg/kg s.c. twice daily on 5 consecutive days (total dose 25 mg/kg). Sixteen hours after the last dose the livers were perfused (2.5 ml/min/g liver) with Krebs-Henseleit buffer and glucose (10 mmol/l) as substrate for 80 min. After 50 min of perfusion 0.1 or 0.7 mmol/l BAL, DMPS, DMSA, or BAPSA were added to the perfusate for 30 min. Samples of the effluent were collected every 10 min; lactate and pyruvate were determined enzymatically. As compared to controls, a significant decrease in the pyruvate and lactate efflux was observed in perfused livers of guinea pigs treated with As2O3. After influx of BAL (0.1 mmol/l), DMSA (0.7 mmol/l), and BAPSA (0.1 and 0.7 mmol/l) respectively, the pyruvate and lactate efflux and the oxygen consumption (exception BAL 0.1 mmol/l) increased and reached control values without arsenic treatment. On the other hand, the pyruvate and lactate efflux and the oxygen consumption was further significantly decreased after influx of 0.7 mmol/l BAL.
P/O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30-180 min. P/O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P/O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P/O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting/exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.
Addition of fatty acids to isolated hepatocytes raised respiration rate by 92% and raised mitochondrial membrane potential (delta psi m) in situ from 155 to 162 mV suggesting that the increased fuel supply had a greater effect on respiration rate than any increases in processes that consumed mitochondrial protonmotive force (delta p). The relationship between delta psi m and respiration rate was changed by addition of fatty acids or lactate, showing that there was also stimulation of delta p-consuming reactions. In the presence of oligomycin the relationship between delta psi m and respiration rate was unaffected by substrate addition, showing that the kinetics of delta p consumption by the H+ leak across the mitochondrial inner membrane were unchanged. The stimulation of delta p consumers by fatty acids therefore must be in the pathways of ATP synthesis and turnover. Inhibition of several candidate ATP-consuming reactions had little effect on basal or fatty acid-stimulated respiration, and the nature of the ATP turnover reactions in hepatocytes remains speculative. We conclude that fatty acids (and other substrates) stimulate respiration in hepatocytes in two distinct ways. They provide substrate for the electron transport chain, raising delta p and increasing the non-ohmic proton leak across the mitochondrial inner membrane and the rate of oxygen consumption. They also directly stimulate an unidentified delta p-consuming reaction in the cytoplasm. They do not work by uncoupling or by stimulation of intramitochondrial ATP-turnover reactions.
During oxidative phosphorylation by mammalian mitochondria part of the free energy stored in reduced substrates is dissipated and energy is released as heat. Here I review the mechanisms and the physiological significance of this phenomenon.
The effects of acetaminophen on the metabolism of the isolated perfused rat liver were investigated. The following results were obtained: Acetaminophen increased glucose release and glycolysis from endogenous glycogen (glycogenolysis).
Oxygen uptake, gluconeogenesis from either pyruvate or fructose and glycogen synthesis were inhibited.
In isolated rat liver mitochondria acetaminophen decreased state III and state IV respiration; it also decreased the ADP/O ratio and the respiratory control ratio.
The action of acetaminophen on glycogenolysis was not affected by N ‐acetylcysteine; this compound, however, increased glycogen synthesis.
The effects of acetaminophen are reversible.
It was concluded that glycogen depletion by acetaminophen can be produced by two mechanisms. The first, as previously demonstrated by several workers, depends on irreversible binding of a reactive metabolite. The second, however, is reversible and depends primarily on an inhibition of mitochondrial energy metabolism.
The action of mefenamic acid, a nonsteroidal anti-inflammatory drug, on energy metabolism in the isolated perfused rat liver was investigated. Mefenamic acid in the range between 0.1 and 1.0 mM was infused to livers from well-fed rats and from 24-hr fasted rats. The former were perfused with substrate-free Krebs/Henseleit-bicarbonate buffer, allowing the measurement of glycogenolysis and glycolysis from endogenous glycogen. The livers from 24-hr fasted rats, on the other hand, were perfused with Krebs/Henseleit-bicarbonate buffer containing fructose, thus allowing the measurement of fructolysis and glucose synthesis. Oxygen consumption was measured in both cases. When present in the range between 0.1 and 0.5 mM, mefenamic acid increased glycolysis, oxygen uptake, glycogenolysis and fructolysis. Higher concentrations, depending on the perfusion conditions, were inhibitory. Glucose production from exogenous fructose, on the other hand, was inhibited at low mefenamic acid concentrations. In general terms, the effects of mefenamic acid on energy metabolism seemed to be the primary consequence of its uncoupling action on the respiratory chain. This conclusion is supported mainly by the opposite effects on glucose synthesis (inhibition) and oxygen consumption (activation). The intracellular concentration of mefenamic acid is much higher than the extracellular one, a phenomenon which may represent binding to intracellular membrane or proteins.
Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the 'efficiency' of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a 'safety valve' preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.
To answer the question ‘What controls the rate of respiration?’ requires a clear definition of control and an explicit description of the limits of the system to be considered. In this review we use a neutral definition of control in which A controls B if changes in A cause changes in B. A useful system to define when discussing the control of respiration consists of the electron transport chain, the H+‐ATPase, the adenine nucleotide carrier, the intramitochondrial adenine nucleotide and phosphate pools, δ p and the proton leak across the mitochondrial inner membrane.
Controls operating within this system are designated internal controls and many of them are fairly well characterized. Several models have been advanced to describe the rates of these internal processes in isolated mitochondria, including control of respiration rate by cytochrome oxidase with all other steps near to equilibrium, control by the adenine nucleotide carrier or control by the extent of displacement of individual reactions from equilibrium. More recently, analysis using control theory has shown that in the resting state (state 4) most control over flux is exerted by the leak of protons through the inner membrane, whereas in more active, phosphorylating states (up to state 3) control is distributed between a number of steps, including the proton leak, the adenine nucleotide carrier and cytochrome oxidase. This approach seems a very useful framework within which to pose further questions.
This system may be treated as a ‘black box’ interacting with its environment (the rest of the mitochondrion and the experimental cuvette or living cell) through the redox states of NAD, Q and O 2 and through the phosphorylation state of the extramitochondrial adenine nucleotides. Very few external effectors cross the system boundary; the only well‐characterized ones are long‐chain fatty acyl‐CoA, which inhibits the adenine nucleotide carrier, and fatty acids, which activate a specific uncoupling protein found only in the inner membrane of mitochondria from brown adipose tissue. At this level respiration rate is determined only by the internal properties of the ‘black box’, by the redox states of NAD, Q and O 2 , and by the phosphorylation status of the extramitochondrial adenine nucleotide pool.
Within a cell the rate of respiration is controlled primarily by the rates of reactions feeding electrons to the electron transport chain (through their effects on NADH/NAD and QH 2 /Q ratios) and by the rates of reactions consuming or producing ATP (through the cytosolic phosphorylation potential or ATP/ADP ratio). Control of reducing equivalent supply occurs through availability of oxidizable substrates (determined by diet and hormonal status), through regulation of pathways such as glycolysis or fatty acid catabolism and, importantly, through Ca ²⁺ activation of intramitochondrial dehydrogenases.
Hormonal control over respiration can occur at all the levels mentioned. Hormones may alter the kinetic properties of the oxidative phosphorylation system by altering the concentrations of individual proteins or by altering their kinetic properties either by affecting the lipid environment or, possibly, more directly. Important controls by hormones occur through changes in ATP demand altering the cytoplasmic adenine nucleotide pool and by changes in free Ca ²⁺ concentration in the mitochondrial matrix, altering the activity of dehydrogenases and the supply of electrons to NAD and Q. Hormones also affect the supply of reducing equivalents to the mitochondria by their catabolic or anabolic effects on other pathways.
Brief incubation of isolated rat hepatocytes in the presence of the oleate-bovine serum albumin complex resulted in a release to the cytosol of a portion of hexokinase (EC 2.7.1.1) normally bound to intracellular membranes. This was correlated with an increase of the negative surface potential of the outer mitochondrial membrane, as measured in situ by determining changes of Km of monoamine oxidase (EC 1.4.3.4). It is suggested that non-esterified fatty acids produce a partial release of bound hexokinase in the liver cell by changing the surface charge of intracellular membranes.
Brown adipocytes from cold-adapted guinea-pigs (C-cells) are more sensitive to uncoupling by exogenous palmitate than are cells from warm-adapted animals (W-cells) with much less uncoupling protein. Half-maximal respiratory stimulation of C-cells requires 80 nM free palmitate. Noradrenaline-stimulated lipolysis is not rate-limiting for the respiration of either C-cells or W-cells. Half-maximal stimulation of fatty acid oxidation by mitochondria from warm-adapted guinea-pigs (W-mitochondria) and cold-adapted guinea-pigs (C-mitochondria) both require 12 nM free palmitate. Palmitate uncouples C-mitochondria much more readily than M-mitochondria, paralleling its action on the adipocytes. The uncoupling is partially saturable, about 100 nM free palmitate being required for half-maximal response of C-mitochondria. W- and C-mitochondria show identical binding characteristics for palmitate. The respiratory increase of mitochondria is calculated as a function of bound palmitate. After correcting for the residual uncoupling protein present in W-mitochondria, palmitate is estimated to be almost ineffective as an uncoupler of brown fat mitochondria in the absence of the protein. It is concluded that fatty acids display characteristics required of a necessary and sufficient physiological activator of the uncoupling protein.
The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ , in intact hepatocyte suspensions.
The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1‐ ¹⁴ C]pyruvate into ¹⁴ CO 2 plus [ ¹⁴ C]glucose. The flux through PDH was determined from the rate of formation of ¹⁴ CO 2 from [1‐ ¹⁴ C]pyruvate correted for other decarboxylation reactions (citrate cycle, phospho enol pyruvate carboxykinase and malic enzyme), and the flux through PC was determined by substracting the flux through PDH from the total pyruvate metabolized.
With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h‐starved rats.
In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59–76%) and PC (80–83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats.
In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC.
2[5(4‐Chlorophenyl)pentyl]oxirane‐2‐carboxylate (100 μM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate‐stimulated but not the octanoate‐stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the β‐oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation.
It is concluded that the effect of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.
The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.
The uncoupling-like effect of fatty acids [Scholz, R., Schwabe, U., and Soboll, S. (1984) Eur. J. Biochem. 141, 223–230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze-fixed and dried tissues in non-aqueous solvents. The following results were obtained.
The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.
The effect of palmitate and metabolizable and nonmetabolizable monosacharides (D-glucose, D-fructose and 2-deoxy-D-glucose = 2-DG) on the membrane potential (Vm) of mouse hepatocytes was investigated employing a superfused mouse liver slice technique. Palmitate hyperpolarized the liver cell membrane in a concentration dependent manner whereas the monosaccharides tested did not. When mice were fed a fat-rich diet, the hyperpolarisation was greater in comparison to mice fed a low fat diet. The hyperpolarization was reversed by ouabain, an inhibitor of the Na+/K(+)-ATPase, by the K(+)-channel blockers tetra-ethyl-ammonium (TEA) and cetiedil and by three inhibitors of fatty acid oxidation (2-bromopalmitate, 2-bromooctanoate and 4-pentenoate). The results suggest that hyperpolarization of the liver cell membrane is due to fatty acid oxidation and that both activation of Na+/K(+)-ATPase and opening of K(+)-channels are involved. The implications of these findings with regard to control of food intake by fatty acid oxidation are discussed. The results are consistent with a role of the hepatic membrane potential in control of food intake by fatty acid oxidation.
We have investigated the effects of imposing an ATP demand, generated by the addition of lactate, on hepatocytes isolated from fasted normal and streptozocin-induced diabetic rats. The stimulation of O2 consumption upon lactate addition was much greater in hepatocytes from diabetic rats, as a result of a lactate-induced stimulation of beta-oxidation that was not observed in control cells. This lactate-induced increment in beta-oxidation was extremely sensitive to inhibition by low levels of a number of inhibitors of energy transduction, implying that the increment was tightly coupled to ATP synthesis. Such sensitivity of the beta-oxidative pathway to the addition of similar low concentrations of these inhibitors was not seen in control cells. Inhibitors of the gluconeogenic pathway were also more effective in decreasing beta-oxidation in cells from diabetic animals than in cells from normal rats. The increment in beta-oxidation was not accompanied by increased rates of glucose synthesis, fatty acid esterification or ureogenesis. We propose that it may be associated with higher rates of glucose cycling in cells from diabetic rats.
An animal model experiment was conducted with nine adult sows to study the effects of long-chain fatty acids on thermogenesis when different fatty acids were replaced for 30% of the energy of a basal diet based on cereals and soybean meal. The acids were fed as commercial products containing as main constituent either palmitic acid, oleic acid, or linoleic acid, according to a latin square design in experimental periods 2 to 4. In periods 1 and 5 the sows were submitted to basal diet alone. Digestibility of palmitic acid was only 36%, whereas the unsaturated fatty acids were highly absorbed (90%). Interaction effects of the undigested proportion of the long-chain fatty acids with the basal diet in hindgut fermentation could be ruled out since a supplementary experiment on three sows showed no influence of infusion of oleic or linoleic acid into the caecum on the energy utilization of the basal diet. There was no significant differences in thermogenesis among the fatty acids. Heat production in the treatment periods averaged -1.2% as compared to the basal diet periods. This result was in accordance with the value -1.0% calculated theoretically for the reduction in heat production in the treatment periods. Thus, the data did not indicate any stimulating effect of long-chain fatty acids on heat production, and utilization of energy of fatty acids occurred within the obligatory thermogenesis.
Long-chain fatty acids are natural uncouplers of oxidative phosphorylation in mitochondria. The protonophoric mechanism of this action is due to transbilayer movement of undissociated fatty acid in one direction and the passage of its anion in the opposite direction. The transfer of the dissociated form of fatty acid can be, at least in some kinds of mitochondrion, facilitated by adenine nucleotide translocase. Apart from dissipating the electrochemical proton gradient, long-chain fatty acids decrease the activity of the respiratory chain by mechanism(s) not fully understood. In intact cells and tissues fatty acids operate mostly as excellent respiratory substrates, providing electrons to the respiratory chain. This function masks their potential uncoupling effect which becomes apparent only under special physiological or pathological conditions characterized by unusual fatty acid accumulation. Short- and medium-chain fatty acids do not have protonophoric properties. Nevertheless, they contribute to energy dissipation because of slow intramitochondrial hydrolysis of their activation products, acyl-AMP and acyl-CoA. Long-chain fatty acids increase permeability of mitochondrial membranes to alkali metal cations. This is due to their ionophoric mechanism of action. Regulatory function of fatty acids with respect to specific cation channels has been postulated for the plasma membrane of muscle cells, but not demonstrated in mitochondria. Under cold stress, cold acclimation and arousal from hibernation the uncoupling effect of fatty acids may contribute to increased thermogenesis, especially in the muscle tissue. In brown adipose tissue, the special thermogenic organ of mammals, long-chain fatty acids promote operation of the unique natural uncoupling protein, thermogenin. As anionic amphiphiles, long-chain fatty acids increase the negative surface charge of biomembranes, thus interfering in their enzymic and transporting functions.
The relative contributions of beta-oxidation and citric acid cycle activity to total O2 consumption during fatty acid oxidation were examined in isolated hepatocytes. When hepatocytes were incubated with palmitate alone, a rise in fatty acid concentration induced an increase in O2 uptake that reflected a large stimulation of beta-oxidation and an accompanying smaller inhibition of citric acid cycle oxidation. In the presence of lactate, successive increments in palmitate concentration over the range from 0 to 1.0 mM stimulated glucose synthesis and brought about a concomitant incremental stimulation of both beta-oxidation and citric acid cycle flux. However, above 1.5 mM palmitate, additional increments in fatty acid concentration depressed gluconeogenesis and citric acid cycle activity but induced a further stimulation of beta-oxidation. These findings demonstrate that, during fatty acid oxidation, the rate of citric acid cycle turnover is more closely linked to the rate of glucose synthesis than is the rate of beta-oxidation. This may be relevant to observations that the stimulation of hepatic O2 consumption, induced by fatty acid oxidation, is much greater than can be explained in terms of the ATP-demand arising from exposure of hepatocytes to fatty acid.
In the present study, ethanol oxidation by the perfused rat liver has been used to investigate the interrelationships between the pathways of glucose metabolism, fatty acid oxidation, and the citric acid cycle. In the absence of exogenous fatty acids, the production of glucose from alanine was stimulated 2-fold by 10 mM ethanol, whereas, in the presence of 1 mM oleate, ethanol caused an inhibition of net glucose production. Measurements of the rates of ethanol utilization and acetate formation showed that over 80% of the ethanol metabolized was converted to acetate. The increased rate of generation of reducing equivalents in the cytosol during ethanol oxidation increased the oxidation-reduction state of pyridine nucleotides in both the intra- and extramitochondrial compartments. This fact was established by analyses of the tissue content of pyridine nucleotides and substrate couple ratios, and directly by surface fluorometry. Changes of flavin and pyridine nucleotide fluorescence intensity from the surface of the liver showed that the transfer of reducing equivalents from cytosol to mitochondria during ethanol oxidation was very rapid.
Analyses of intermediates in the gluconeogenic pathway of livers perfused in the absence of fatty acids indicated an activational site at the glyceraldehyde-3-P dehydrogenase step upon ethanol addition. The stimulatory effect of ethanol on gluconeogenesis from alanine, therefore, results from the increased availability of the NADH in the cytosol. On the other hand, when ethanol was added to livers perfused in the presence of oleate, an inhibitory site was observed between fructose-1, 6-di-P and fructose-6-P. Measurement of the tissue levels of the known modifiers of phosphofructokinase and fructose diphosphate phosphatase indicated that this effect was caused primarily by deinhibition of phosphofructokinase resulting from a fall of the citrate content.
Oxidation of NADH produced during ethanol metabolism inhibited the activity of the citric acid cycle. Sites of inhibition were identified at the citrate synthase and iso-citrate dehydrogenase steps. The relative strengths of the inhibitory interactions at these sites were dependent on the rate of β oxidation. It is proposed that a coordinated inhibition of citrate synthase and isocitrate dehydrogenase is mediated primarily by the increased state of reduction of intramitochondrial pyridine nucleotides.
The time course of changes in a variety of physiological parameters concerned with carbohydrate and lipid metabolism has been
studied both in vivo and in the isolated perfused liver during induction and reversal of starvation ketosis in the rat. The data obtained demonstrate
that surprisingly brief periods of starvation and refeeding exert dramatic effects on glucose and fatty acid metabolism in
the intact animal and that generally synchronous changes occur in the ketogenic and gluconeogenic capacities of the perfused
liver.
In agreement with previous findings it was shown that the enhanced conversion of labeled oleate into ketone bodies by livers
from fasted rats was associated with a concomitant depression in its incorporation into triglycerides, and that the antiketogenic
effect of lactate was accompanied by a diversion of the fatty acid from the β oxidation sequence into the esterification pathway.
The key observation, however, was that blockade of long chain fatty acid oxidation by (+)-decanoylcarnitine, an inhibitor
of long chain acylcarnitinetransferase, stopped ketone body formation and acutely changed the pattern of metabolism of oleic
acid in livers from fasted rats to that exhibited by livers from normal animals, i.e. the fatty acid was now virtually completely esterified.
The data are consistent with the view that hepatic fatty acid oxidation and ketogenesis are under strict dietary and hormonal
control exerted primarily by regulation of an early step in the oxidative sequence, probably the acylcarnitinetransferase
reaction. The possibility is also raised that the effects of lactate and other antiketogenic agents are related to interactions
at this site.
Purified rat liver peroxisomes contain a cyanide-insensitive fatty acyl-CoA oxidizing system that uses O2 and NAD as electron acceptors. The system was detected by the ability of added palmitoyl-CoA to elicit O2 consumption, H2O2 production, and O2-dependent NAD reduction. The activity of this system is increased approximately one order of magnitude in rats treated with clofibrate, a hypolipidemic drug known to cause peroxisomal proliferation.
Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.
Two hepatic cytoplasmic protein fractions, designated Y and Z, which bind sulfobromophthalein (BSP), bilirubin, and other organic anions, have been separated by G75 Sephadex gel filtration. The physiologic role of these protein fractions has been investigated. They are present in the 110,000 g supernatant fraction from the livers of all the species tested (rats, mice, guinea pigs, Rhesus monkeys, sheep, and man). Tissues which do not preferentially extract BSP or bilirubin from plasma do not contain these fractions, with the exception of small intestinal mucosa which contains Z. Anion binding by Y and Z fractions is not due to contamination with albumin. These fractions are responsible for the cytoplasmic localization of bilirubin in Gunn rats, and the fractions bind bilirubin, BSP, or indocyanine green (ICG), whether given in vivo or added in vitro to liver supernate from normal rats. Flavaspidic acid-N-methylglucaminate, bunamiodyl, and iodipamide, drugs known to interfere with the hepatic uptake mechanism, compete with bilirubin and BSP for binding to Z. These proteins appear to be important in the transfer of organic anions from plasma into the liver and provide a tool for the investigation of hepatic uptake mechanisms.
The calorigenic action of norepinephrine in isolated brown adipocytes was selectively mimicked by theophylline, dibutyryl cyclic AMP, and the principal fatty acids known to be present in the acyl moieties of brown adipose tissue triglycerides (palmitic, oleic, and linoleic acids). The stimulatory effects of fatty acids were entirely reversible, occurred at physiological concentrations, and were critically dependent upon the molar ratio of extracellular fatty acids to albumin. The calorigenic potency of fatty acids increased with their chain length. The apparent synchrony between the switching "on and off" of lipolysis and respiration by norepinephrine and propranolol indicated that the two phenomena are functionally inter-related and that they are both mediated by beta-adrenoreceptors. Respiratory stimulation by palmitic acid was accompanied by an inhibition of glycerol release suggesting that fatty acids retroinhibit lipolysis while simultaneously activating respiration. Studies with 2-tetradecylglycidic acid, oligomycin, and uncouplers of oxidative phosphorylation support the view that fatty acids exert their calorigenic effects by increasing mitochondrial proton permeability and by simultaneously serving as substrates for beta-oxidation via carnitine-dependent pathways. Since fatty acids mimicked the calorigenic action of norepinephrine even when beta-adrenoreceptors were blocked by propranolol, it is concluded that cyclic AMP controls respiration indirectly, most probably by modulating lipolysis. It is suggested that endogenous long chain fatty acids released in consequence of cyclic AMP activation of lipolysis play a fundamental role in the control of brown adipose tissue metabolism by self-regulating lipolysis and by serving as physiological modulators of mitochondrial oxygen consumption.
Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH-cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and beta-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun.89, 580-584].
Fatty acid-binding protein (FABP) was identified and isolated from rat liver cytosol by gel filtration, thin layer isoelectric focusing, and affinity chromatography. FABP (Mr 12,080 +/- 80) exists in several immunochemically identical forms differing in isoelectric pH, which may in part reflect differences in their respective complements of bound endogenous ligand. FABP-bound fatty acids accounted for 60% of total cytosolic long chain fatty acids but contained no detectable phospholipid; the substantial enrichment of FABP in 18:2 and 20:4 as compared with whole liver homogenate was not influenced by homogenization of tissue in EDTA. The amino acid composition of FABP suggests that it is closely related or identical with certain similar neutral and acidic cytosolic proteins reported from other laboratories. By quantitative radial immunodiffusion, FABP concentration in cytosol from livers of sexually mature female rats exceeded that from mature males (51.7 +/- 3.0 versus 39.8 +/- 4.0 micrograms/mg of protein, p less than 0.05), confirming earlier studies in which sex steroid effects on rates of fatty acid utilization were correlated with FABP concentration as determined by means of a binding assay. The abundance of FABP, its importance in the cytosolic binding of endogenous as well as exogenous fatty acids, and its demonstrated correlation with rates of hepatocyte fatty acid utilization provide additional evidence for its relationship to the cellular metabolism of long chain fatty acids.
The rates of O 2 consumption during fatty acid oxidation to acetyl‐CoA and to CO 2 were determined for isolated liver cells from starved rats on the basis of measured rates of respiration and ketogenesis. About 60% of the endogenous O 2 uptake was associated with acetyl‐CoA formation. The remainder was assumed to represent total combustion of fatty acid to CO 2 and H 2 O through the Krebs cycle. In the absence of added fatty acid, 1.90 μmol · min ‐1 · g ‐1 of acetyl‐CoA was generated, of which 1.40 μmol · min ‐1 · g ⁻¹ gave rise to ketone bodies. O 2 consumption was stimulated about 30% by the addition of 2 mM palmitate or 4 mM hexanoate. This increase was entirely due to stimulation of O 2 consumption related to oxidation of fatty acid to acetyl‐CoA. The extra acetyl‐CoA produced was channelled into ketone body formation.
The gluconeogenic substrate lactate, which increases hepatic demand for ATP, stimulated O 2 consumption almost 40%, but in this case the increase was due to an enhancement of Krebs cycle activity, and acetyl‐CoA generation was depressed 25%. In the absence of added fatty acid, 83% of the total acetyl‐CoA produced by cells incubated with lactate was oxidized in the Krebs cycle. Addition of fatty acid caused a further stimulation of O 2 uptake which reflected a promotion of acetyl‐CoA production with an associated increase in ketone body formation.
The uncoupling agent, 2,4‐dinitrophenol, stimulated total O 2 consumption of cells incubated without exogenous substrate or with added fatty acid. In each circumstance this stimulation of O 2 uptake was related entirely to enhancement of Krebs cycle activity and depression of ketogeneis. The large increment in O 2 consumption induced by the combination of lactate and 2,4‐dinitrophenol in the presence of added fatty acid was likewise attributable solely to a marked stimulation of Krebs cycle flux. Added ethanol caused only a small inhibition of total acetyl‐CoA production and had little effect on respiration, but considerably depressed Krebs cycle activity. This depression was relieved by 2,4‐dinitrophenol.
Oligomycin and antimycin had markedly different effects on fatty acid catabolism although both inhibitors depressed O 2 consumption to the same extent. Oligomycin caused only a small diminution of rates of acetyl‐CoA formation but induced a strong depression of cellular ATP concentrations and of acetyl‐CoA oxidation to CO 2 . In contrast antimycin had no effect on acetyl‐CoA oxidation and brought about only a small decline in cellular ATP levels, but considerably depressed formation of acetyl‐CoA.
These results are interpreted as indicating that addition of fatty acid to liver cells induces various energy‐dependent processes, including ATP synthesis, reversed electron transfer and metabolite translocation, all of which contribute to the observed stimulation of O 2 uptake. It is inferred that oxidation of acetyl‐CoA to CO 2 is preferentially coupled to ATP synthesis whereas oxidation of fatty acid to acetyl‐CoA is obligatorily associated with reversed electron transfer. Thus, ketogenesis is not constrained by the hepatic phosphorylation potential and provides a mechanism for the rapid disposal of fatty acids, independent of the ATP demands of the liver.
The levels of serum insulin, glucagon, and free fatty acids (FFA) and the tissue concentrations of hepatic cyclic AMP, long-chain acyl-CoA (LCA), adenine nucleotides, inorganic phosphate, the intermediates of the Embden-Meyerhof pathway, the citric acid cycle (including acetyl-CoA and free CoA), and the cytoplasmic and mitochondrial redox couples were determined in the rat 12, 24, and 48 h after food withdrawal and 5, 10, 20, 40, 60, and 120 min after the refeeding of glucose. Using the measured metabolite contents in the liver, the alterations in the concentration of malate, oxaloacetate, citrate, and α-ketoglutarate and the changes in the energy state of the adenine nucleotide system and the redox state of the NAD system were attributed to the cytoplasmic and mitochondrial compartments by applying established calculation methods. Glucose refeeding provoked significant alterations in all parameters investigated. These changes occurred within minutes, reversing the hormone and metabolite pattern which had developed within 24 h in response to food withdrawal. Particularly, glucose refeeding resulted in a drastic increase in the insulin/glucagon ratio. Simultaneously, the level of serum FFA and the concentration of LCA in the liver declined. The latter alteration was accompanied by an increase in the cytoplasmic and a decrease in the mitochondrial ratios. Moreover, the redox state of the cytoplasmic NAD system was shifted toward the oxidized state. When the appropriate data were plotted against each other, highly significant correlations were obtained (i) between the insulin/glucagon ratio and the serum FFA concentration, (ii) between the serum FFA concentration and the concentration of hepatic LCA, (iii) between the hepatic LCA concentration and the cytoplasmic energy state, and (iv) between the cytoplasmic energy state and the redox state of the cytoplasmic NAD system. These findings are interpreted to support the hypothesis derived from experiments carried out in vitro that the insulin/glucagon ratio via the FFA-dependent concentration of hepatic LCA might affect the translocation of adenine nucleotides between the cytoplasmic and the mitochondrial compartment, thereby regulating the cytoplasmic energy state and the redox state of the cytoplasmic NAD system, consequently. Glucose refeeding provoked rapid coordinate changes in the concentration of the intermediates of both the citric acid cycle and the Embden-Meyerhof chain, indicating the altered substrate flow through these pathways. Those metabolites, known to modulate the activity of key regulatory enzymes in vitro, were analyzed with respect to their suggested regulatory function. As to the established shift from pyruvate carboxylation to pyruvate decarboxylation after glucose refeeding, the data revealed that the decrease in pyruvate carboxylase activity can be attributed to the decrease in the intramitochondrial ratio and the simultaneous fall in acetyl-CoA concentration, while the coordinate increase in pyruvate dehydrogenase activity can be ascribed to the decline in the concentration of LCA and, consequently, in the ratios of , , and acetyl- within the mitochondria. As for the citric acid cycle, increased citrate synthesis from acetyl-CoA and oxaloacetate was supported by the rapid drop in the concentration of the established inhibitor of citrate synthesis, LCA. In contrast, the concentration of succinyl-CoA, an inhibitor of the enzyme in vitro, remained practically constant, questioning its regulatory function under the present experimental conditions. In addition to the activation of citrate synthase, the coordinate activation of isocitrate dehydrogenase was indicated by the LCA-mediated decline in both the mitochondrial and the ratios. Glucose refeeding immediately reduced urea excretion to basal values. This alteration was preceded by a drastic fall in the tissue concentration of cyclic AMP, supporting the physiological role of the nucleotide in the control of hepatic gluconeogenesis. In contrast, the observed changes in the concentration of the effectory acting metabolites (ATP, AMP, fructose 1,6-diphosphate, citrate, and alanine) were incompatible with the suggested function of these intermediates in switching over the substrate flow through the Embden-Meyerhof pathway from gluconeogenesis to glycolysis. The results are discussed in reference to the known rapid stimulation of fatty acid biosynthesis in the liver and to the transfer of reducing equivalents by the different shuttles of the inner mitochondrial membrane. In summary, it can be concluded that the insulin/glucagon ratio in a moment-to-moment fashion controls the glucose balance across the liver by regulating hepatic intermediary metabolism via the concentration of both LCA and cyclic AMP.
1The relationship between the rate of oxygen uptake in isolated rat-liver cells and the cytosolic phosphorylation state was studied under steady-state conditions. The cells were perifused with different concentrations of the gluconeogenic substrates lactate + pyruvate in order to obtain different steady-state rates of oxygen consumption and glucose production.2The cytosolic phosphorylation state was calculated by the metabolite method. For this calculation, the apparent equilibrium constant of the combined glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase reactions was redetermined taking into account the strong Mg2+ dependence of the overall reactions. At the free Mg2+ concentration in the cytosol (0.4 mM) the value of Kapp is 912 M−1. Using this value, a cytosolic phosphorylation state of 2700 M−1 can be calculated for cells incubated in the presence of 10 mM alanine. This value agrees very closely with that obtained by direct measurement after fractionation of the cells by the digitonin procedure.3It is possible to describe the regulation of the rate of O2 uptake in perifused liver cells in terms of the principles of irreversible thermodynamics. It is concluded that the transition from one steadystate to another is accompanied by a thermodynamics regulation of the rate of O2 consumption by the cytosolic phosphorylation state, the mitochondrial redox level and the partial pressure of oxygen.
The effect of fatty acids on the rate of pyruvate decarboxylation was studied in perfused livers from fed rats. The production of 14CO2 from infused [1-14C]pyruvate was employed as a monitor of the flux through the pyruvate dehydrogenase reaction. A correction for other decarboxylation reactions was made using kinetic analyses. Fatty acid (octanoate or oleate) infusion caused a stimulation of pyruvate decarboxylation at pyruvate concentrations in the perfusate below 1 mM (up to 3-fold at 0.05 mM pyruvate) but decreased the rate to one-third of control rates at pyruvate concentrations near 5 mM. These effects were half-maximal at fatty acid concentrations below 0.1 mM. Infusion of 3-hydroxybutyrate also caused a marked stimulation of pyruvate decarboxylation at low pyruvate concentrations.
The data suggest that the mechanism by which fatty acids stimulate the flux through the pyruvate dehydrogenase reaction in perfused liver at low (limiting) pyruvate concentrations involves an acceleration of pyruvate transport into the mitochondrial compartment due to an exchange with acetoacetate. Furthermore, it is proposed that a relationship exists between ketogenesis and the regulation of pyruvate oxidation at pyruvate concentrations approximating conditions in vivo.
The effect of ethanol on hepatic respiration and glycolysis was studied in perfused rat livers.
These data indicate that the ethanol-stimulated oxygen uptake is due to an increased flux through the mitochondrial respiratory chain and that it involves the NAD+-dependent oxidation of ethanol by alcohol dehydrogenase. The data are consistent with the hypothesis that the ethanol-stimulated respiration results from an increased demand for mitochondrial oxidative phosphorylation as a consequence of the decreased extramitochondrial ATP generation following inhibition of glycolysis by ethanol.
Gluconeogenesis from lactate was studied in perfused livers from phenobarbital pretreated rats fasted for 24 hr. Maximal rates of glucose production obtained with lactate concentrations of more than 2mM were suppressed to about 50% following the addition of aminopyrine, a substrate for NADPH utilizing mixed function oxidations. Submaximal rates (i.e. with 0.5-2 mM lactate) or gluconeogenesis from dihydroxyacetone were only slightly affected by aminopyrine, whereas no inhibition was observed at low gluconeogenic rates. The data are consistent with the following hypothesis. In the presence of active NADPH utilizing processes (such as mixed function oxidation of aminopyrine) a futile cycle involving malic enzyme occurs in the pathway of gluconeogenesis which is compensated for by an increased flux through the pyruvate carboxylase reaction. Thus, gluconeogenesis is suppressed when maximal activity of pyruvate carboxylase is reached. It is assumed that the observed inhibition of gluconeogenesis is not specific for mixed function oxidation, but may be an example for a more general mechanism of metabolic interdependences involving the extramitochondrial NADPH pool.
The notion is presented that recycling of carbon between the steps pyruvate to P-enolpyruvate, and fructose diphosphate to fructose-6-P, is involved in the overall control of hepatic gluconeogenesis in both normal and pathological states. This results in a higher than theoretical energy cost for gluconeogenesis, which may be revealed by low apparent P:O ratios when calculated on the basis of the theoretical increment of 6 moles of ATP used per mole of glucose formed, compared with the observed increment of respiration when the rate of gluconeogenesis is altered. The concept is developed that the rate of gluconeogenesis is controlled either directly by changes in the concentration of factors modifying pyruvate carboxylase or indirectly by alterations of the activity of pyruvate kinase. If the activity of the latter enzyme is increased, flux through the loop Pyr → OAA → PEP → Pyr will be increased and net gluconeogenesis decreased. Since the inhibitory effects of alanine and ATP on pyruvate kinase can be overcome by low concentrations of fructose diphosphate, interactions in the segment of the gluconeogenic pathway from P-enolpyruvate to glucose, although not able to affect gluconeogenesis flux directly, can control it indirectly by alterations of the fructose diphosphate concentration. Interactions discussed are variations of the citrate concentration, which will affect the activity of phosphofructokinase, and altered metabolic states caused by changes of the state of reduction of the cytosolic pyridine nucleotides. By taking into account the possibility that high rates of carbon recycling between pyruvate and P-enol-pyruvate occur relative to the net rate of gluconeogenesis, the relationship between changes of the cytosolic pyridine nucleotide oxidation-reduction state and the overall control of gluconeogenesis is clarified.
The effects of octanoate and oleate were studied in the isolated fasted rat liver perfused without substrate and were compared with the effects of lactate. Measurements were made of hepatic adenine nucleotide content, formation of ketone bodies, urea and glucose and uptake of fatty acids, lactate and oxygen. Calculations of the Krebs cycle activity and the flux through the oxidation at C-3 were carried out. Comparing the extra oxygen consumption with extra ATP needs after addition of the fatty acids or lactate, ADP: O ratios were estimated for fatty acid oxidation and lactate gluconeogenesis.
1. Octanoate as well as oleate induced a net decrease in ATP and an increase in AMP content of the liver. Total adenine nucleotides were unaltered. ATP:ADP ratios were lowered while the adenylate kinase mass-action ratio increased.
2. The Krebs cycle activity was suppressed by both fatty acids and enhanced by lactate. Oxidation at C-3 was strongly stimulated by the fatty acids and was unaffected by lactate.
3. An apparent ADP:O ratio of 2.7 was obtained after lactate addition, indicating a tightly coupled oxidative phosphorylation for the liver preparations used. Octanoate and oleate gave extremely low ratios of near one and near zero respectively.
4. Perfusion with oligomycin caused a severe drop in oxygen uptake by the liver, which was unaltered after addition of fatty acids. Oligomycin added after the fatty acids caused an immediate fall in oxygen uptake to the level observed with oligomycin alone. 2,4-Dinitrophenol was able to stimulate the oligomycin-depressed respiration, both in the absence and in the presence of fatty acids. These results indicate that under our experimental conditions the fatty acids had no uncoupling effect and that microsomal fatty acid oxidation had to be minimal.
5. Incorporation of [3H]leucine into proteins in liver and medium remained unchanged after addition of fatty acids, indicating that the low apparent ADP:O ratios are not the result of an enhanced synthesis of proteins.
6. The possible relationship between fatty acid transport, energy-wasting futile cycles and the low apparent ADP:O ratios observed during fatty acid oxidation is discussed.
The fate of labelled free fatty acids in isolated perfused livers shows that on entering the liver they are esterified or oxidized. The more acid which enters the oxidation pathway, the more goes into ketogenesis and the less into the citric acid cycle, so that the total production of energy remains constant.
Phenylalkyloxirane carboxylic acids and esters are a new class of potent hypoglycaemic substances. Sodium 2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (B 807-27) produces a dose-dependent hypoglycaemic effect when administered orally or intravenously to several fasted laboratory animals, i.e. rats (with and without adrenalectomy), guinea pigs, dogs, streptozotocin-treated diabetic pigs and db/db-mice. In addition, the substances have a more pronounced lowering effect on ketone bodies in the blood than any other known substance. The minimal dose for lowering blood glucose significantly in rats is 15 mumol/kg. The corresponding dose for a significant lowering of ketone bodies in the blood is less than 2.4 mumol/kg. Hence, the substance B 807-27 is approximately five times more potent than tolbutamide and 30 times more potent than the biguanide buformin with respect to lowering blood glucose levels. B 807-27 differs from the sulphonylureas in that it fails to stimulate insulin secretion. In contrast to the biguanides, the substance decreases rather than increases blood lactate concentration and blocks both fatty acid oxidation and gluconeogenesis. The therapeutic usefulness of these compounds in diabetic man remains to be elucidated.
Kinetic analysis of the uptake of carbon-14-labeled oleate in a single-pass perfusion of rat liver and saturable and specific binding of iodine-125-labeled albumin to hepatocytes in suspension suggest the existence of a receptor for albumin on the liver cell surface. The putative receptor appears to mediate uptake of albumin-bound fatty acids by the cell and may account for the efficient hepatic extraction of many other substances tightly bound to albumin.
Changes in the rates of glucose, lactate and pyruvate production following infusion of glucagon were studied in isolated livers from fed or fasted rats perfused with non‐recirculating Krebs‐Henseleit bicarbonate buffer.
Evidence was presented that under these experimental conditions, the release of lactate plus pyruvate into the perfusate represents 80–90% of the total glycolytic flux; oxidation of pyruvate derived from glucose and/or glycogen accounts for the remaining 10–20%, whereas recycling of pyruvate to glucose is negligible.
In the presence of glucagon, rates of lactate plus pyruvate production were diminished to less than 30% in the fed state (glycogen as substrate) and to less than 10% in the fasted state (glucose as substrate). Rates of pyruvate oxidation were unchanged. Although recycling of pyruvate to glucose was enhanced, it could account for not more than 20% of the decrease in lactate plus pyruvate production. The data indicate a strong inhibition of the substrate flux through glycolysis.
The glucagon concentration for half‐maximal inhibition of glycolysis was 0.2 nM, independent of the substrate (glucose or glycogen) and the nutritional state. The effective concentrations were within the physiological range of glucagon concentrations reported for portal venous blood.
Transient state analyses indicated that the inhibition of glycolysis precedes the stimulation of glycogenolysis. When after a delay glycogenolysis was accelerated, it was followed by a transient stimulation of glycolysis. The stimulatory component in the glucagon effect on glycolysis was diminished in glycogen‐depleted livers and when glucose was the main substrate. The coordinated control of glycolysis and glycogenolysis by glucagon and the interaction of the two processes in the transient state are discussed.
The uncoupling-like effect of fatty acids [Scholz, R., Schwabe, U., and Soboll, S. (1984) Eur. J. Biochem. 141, 223–230] was further substantiated in experiments with perfused rat livers by two ways: firstly the kinetics of changes in metabolic rates (oxygen consumption, ketogenesis, fatty acid oxidation) were analysed; secondly subcellular contents of adenine nucleotides and pH gradients across the mitochondrial membrane were determined following fractionation of freeze-fixed and dried tissues in non-aqueous solvents. The following results were obtained.
The data support the hypothesis that the increase in hepatic oxygen consumption due to octanoate or oleate is, in part, caused by a mechanism similar to uncoupling of oxidative phosphorylation. This mechanism seems not to be an artifact of isolated systems; it may be of physiological importance for processes in which reducing equivalents are removed independently of the ATP demand of the hepatocyte.
1.1. At all pH values between 6.0 and 9.5 unsaturated long-chain fatty acids are more effective than the saturated acids in stimulating the latent ATPase activity of fresh rat-liver mitochondria, provided Mg2+ is added. Oleic, linoleic, linolenic, arachidonic and elaidic acids are about equally effective.2.2. Whereas the saturated fatty acids scarcely stimulate above pH 8 the unsaturated acids are maximally active at PH 9.3.3. Mg2+ was required for maximal stimulation by oleic acid. In the absence of added Mg2+ oleic acid completely inhibited the ATPase stimulated by 2,4-dinitro-phenol.4.4. Oleic acid concentration that gave complete loss of ADP respiratory control or phosphate respiratory control lowered the P:O ratio with glutamate as substrate below I. With palmitic acid loss of respiratory control was only partial.5.5. Uncoupling by oleic could be reserved by adding serum albumin.
- R G Thurman
- R Scholz
Thurman, R. G. & Scholz, R. (1977) Eur. J. Biochem. 75,13 -21.
- B Riistow
- S Riser
- D Kunze
Riistow, B., Riser, S. & Kunze, D. (1982) Acta Biol. Med. Germ.
107 -110.
Institut fur Physiologische Chemie Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universitat Miinchen
- R Scholz
- U Schwabe
R. Scholz and U. Schwabe,
Institut fur Physiologische Chemie, Physikalische Biochemie und Zellbiologie, Ludwig-Maximilians-Universitat Miinchen,
GoethestraRe 33, D-8000 Munchen 2, Federal Republic of Germany
S. Soboll, Institnt fur Physiologische Chemie 1, Universitat Diisseldorf,
Moorenstralje 5, D-4000 Diisseldorf 1, Federal Republic of Germany
- R K Ockner
- J A Manning
Ockner, R. K. & Manning, J. A. (1982) J. Biol. Chem. 257,7872-
485.
- M J Reddy
- P F Hollenberg
- J F Reddy
Reddy, M. J., Hollenberg, P. F. & Reddy, J. F. (1980) Biochem. J.
- S Soboll
- S Griindel
- U Schwabe
- R Scholz
Soboll, S., Griindel, S., Schwabe, U. & Scholz, R. (1984) Eur. J.
- W Braun
Braun, W. (1978) Eur. J. Biochem. 86, 519-530.
- Mc Garry
- J D Meier
- J M Forster
Mc Garry, J. D., Meier, J. M. & Forster, D. W. (1973) J. Biol.
- R G Langdon
Langdon, R. G. (1960) in Lipid Metabolism (Block, K., ed.) pp.
Biol. Chem. 256, 12840-12848.
- P D Lazarow
- C De Duve
- G P Mannaerts
- L J Debeer
- J T Thomas
- P De Schepper
Lazarow, P. D. & de Duve, C. (1976) Proc. NutlAcad. Sci. USA 73,
Mannaerts, G. P., Debeer, L. J., Thomas, J. T. & De Schepper, P.
- J R Williamson
- A Jakob
- R Scholz
- L J Debeer
- G Mannaerts
- P J De Schepper
- J Eur
- R Van Der Meer
- T P M Akerboom
- A K Groen
- J Tager
Williamson, J. R., Jakob, A. & Scholz, R. (1971) Metabolism 20,
Debeer, L. J., Mannaerts, G. & De Schepper, P. J. (1974) Eur. J.
Van der Meer, R., Akerboom, T. P. M., Groen, A. K. & Tager, J.
- H U Bergmeyer
Bergmeyer, H. U. ed. (1974) Methods ofEnzymatic Analyses, 2nd
- A Lehninger
- L F Remmer
- P Borst
- J A Loos
- E J Christ
- E C Slater
Lehninger, A. & Remmer, L. F. (1959) J. Biol. Chem. 234,2459-Borst, P., Loos, J. A., Christ, E. J. & Slater, E. C. (1962) Biochem.
- Ch Bode
- M Klingenberg
Bode, Ch. & Klingenberg, M. (1965) Biochem. Z. 341,271 -299.
- L J Bukowiecki
- N Follea
- J Lupien
- A Paradis
Bukowiecki, L. J., Follea, N., Lupien, J. & Paradis, A. (1981) J.
248, John Wiley, New York.