[show abstract][hide abstract] ABSTRACT: Net hepatic glucose uptake (NHGU) is an important contributor to postprandial glycemic control. We hypothesized that NHGU is reduced during normal pregnancy and in a pregnant diet-induced model of impaired glucose intolerance/gestational diabetes mellitus (IGT/GDM). Dogs (n = 7 per group) that were nonpregnant (N), normal pregnant (P), or pregnant with IGT/GDM (pregnant dogs fed a high-fat and -fructose diet [P-HFF]) underwent a hyperinsulinemic-hyperglycemic clamp with intraportal glucose infusion. Clamp period insulin, glucagon, and glucose concentrations and hepatic glucose loads did not differ among groups. The N dogs reached near-maximal NHGU rates within 30 min; mean ± SEM NHGU was 105 ± 9 µmol⋅100 g liver(-1)⋅min(-1). The P and P-HFF dogs reached maximal NHGU in 90-120 min; their NHGU was blunted (68 ± 9 and 16 ± 17 µmol⋅100 g liver(-1)⋅min(-1), respectively). Hepatic glycogen synthesis was reduced 20% in P versus N and 40% in P-HFF versus P dogs. This was associated with a reduction (>70%) in glycogen synthase activity in P-HFF versus P and increased glycogen phosphorylase (GP) activity in both P (1.7-fold greater than N) and P-HFF (1.8-fold greater than P) dogs. Thus, NHGU under conditions mimicking the postprandial state is delayed and suppressed in normal pregnancy, with concomitant reduction in glycogen storage. NHGU is further blunted in IGT/GDM. This likely contributes to postprandial hyperglycemia during pregnancy, with potential adverse outcomes for the fetus and mother.
[show abstract][hide abstract] ABSTRACT: The cellular events mediating the pleiotropic actions of portal vein glucose (PoG) delivery on hepatic glucose disposition have not been clearly defined. Likewise, the molecular defects associated with postprandial hyperglycemia and impaired hepatic glucose uptake (HGU) following consumption of a high-fat, high-fructose diet (HFFD) are unknown. Our goal was to identify hepatocellular changes elicited by hyperinsulinemia, hyperglycemia, and PoG signaling in normal chow-fed (CTR) and HFFD-fed dogs. In CTR dogs, we demonstrated that PoG infusion in the presence of hyperinsulinemia and hyperglycemia triggered an increase in the activity of hepatic glucokinase (GK) and glycogen synthase (GS), which occurred in association with further augmentation in HGU and glycogen synthesis (GSYN) in vivo. In contrast, 4 weeks of HFFD feeding markedly reduced GK protein content and impaired the activation of GS in association with diminished HGU and GSYN in vivo. Furthermore, the enzymatic changes associated with PoG sensing in chow-fed animals were abolished in HFFD-fed animals, consistent with loss of the stimulatory effects of PoG delivery. These data reveal new insight into the molecular physiology of the portal glucose signaling mechanism under normal conditions and to the pathophysiology of aberrant postprandial hepatic glucose disposition evident under a diet-induced glucose-intolerant condition.
[show abstract][hide abstract] ABSTRACT: The purpose of this study was to determine the effect of liver glycogen loading on net hepatic glycogen synthesis during hyperinsulinemia or hepatic portal vein glucose infusion in vivo. Liver glycogen levels were supercompensated (SCGly) in two groups (using intraportal fructose infusion) but not in two others (Gly) during hyperglycemic-normoinsulinemia. Following a 2-h control period during which fructose infusion was stopped, there was a 2-h experimental period in which the response to hyperglycemia plus either 4× basal insulin (INS) or portal vein glucose infusion (PoG) was measured. Increased hepatic glycogen reduced the percent of glucose taken up by the liver that was deposited in glycogen (74 ± 3 vs. 53 ± 5% in Gly+INS and SCGly+INS, respectively, and 72 ± 3 vs. 50 ± 6% in Gly+PoG and SCGly+PoG, respectively). The reduction in liver glycogen synthesis in SCGly+INS was accompanied by a decrease in both insulin signaling and an increase in AMPK activation, whereas only the latter was observed in SCGly+PoG. These data indicate that liver glycogen loading impairs glycogen synthesis regardless of the signal used to stimulate it.
[show abstract][hide abstract] ABSTRACT: We previously showed that hepatic nitric oxide regulates net hepatic glucose uptake (NHGU), an effect that can be eliminated by inhibiting hepatic soluble guanylate cyclase (sGC), suggesting that the sGC pathway is involved in the regulation of NHGU. The aim of the current study was to determine whether hepatic cyclic guanosine monophosphate (cGMP) reduces NHGU. Studies were performed on conscious dogs with transhepatic catheters. A hyperglycemic-hyperinsulinemic clamp was established in the presence of portal vein glucose infusion. 8-Br-cGMP (50 µg/kg/min) was delivered intraportally, and either the glucose load to the liver (CGMP/GLC; n = 5) or the glucose concentration entering the liver (CGMP/GCC; n = 5) was clamped at 2× basal. In the control group, saline was given intraportally (SAL; n = 10), and the hepatic glucose concentration and load were doubled. 8-Br-cGMP increased portal blood flow, necessitating the two approaches to glucose clamping in the cGMP groups. NHGU (mg/kg/min) was 5.8 ± 0.5, 2.7 ± 0.5, and 4.8 ± 0.3, whereas the fractional extraction of glucose was 11.0 ± 1, 5.5 ± 1, and 8.5 ± 1% during the last hour of the study in SAL, CGMP/GLC, and CGMP/GCC, respectively. The reduction of NHGU in response to 8-Br-cGMP was associated with increased AMP-activated protein kinase phosphorylation. These data indicate that changes in liver cGMP can regulate NHGU under postprandial conditions.
[show abstract][hide abstract] ABSTRACT: Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is the most potent allosteric activator of liver 6-phosphofructo-1-kinase enzyme, which is crucial for glycolysis. It is present in skeletal muscle but its importance is controversial as a regulator of muscle glycolysis. This study aims to determine the role of Fru-2,6-P(2) in the control of muscle glycolysis during contraction. Muscle contraction was produced by chronic low-frequency stimulation of rabbit tibialis anterior for 24 h, followed by a rest period of 48 h. To determine muscle glycolysis adaptation, we applied a short functional electrostimulation test using the same system of low-frequency stimulation for 1, 3, and 10 s. The variation in concentration of lactate and pyruvate was used to calculate the flux along the glycolysis pathway and the Fru-1,6-P(2)/Fru-6-P ratio permitted to analyze the 6-phosphofructo-1-kinase activation. Fru-2,6-P(2) levels increased over the 24 h of stimulation and remained elevated after the rest period, this being the only metabolite that kept the changes produced by chronic low-frequency stimulation during the rest. During the short functional electrostimulation test, the glycolytic pathway in stimulated and rested muscle was more active than in control muscle, which coincided with higher kinase activity of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) enzyme. Furthermore, we found a decrease in muscle, liver, and ubiquitous PFK-2/FBPase-2 isoform expression and an increase in heart isoform expression. For the first time, we demonstrate that a persistent increase in Fru-2,6-P(2) produced by a change in PFK-2/FBPase-2 isoform expression may play an important role in the regulation of muscle glycolysis during the first moments of exercise.
Pflügers Archiv - European Journal of Physiology 04/2012; 463(4):603-13. · 4.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood.
Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest.
In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 5'-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels.
Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches.
PLoS ONE 01/2012; 7(7):e42453. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: The objective of this study was to determine how increasing the hepatic glycogen content would affect the liver's ability to take up and metabolize glucose.
During the first 4 h of the study, liver glycogen deposition was stimulated by intraportal fructose infusion in the presence of hyperglycemic-normoinsulinemia. This was followed by a 2-h hyperglycemic-normoinsulinemic control period, during which the fructose infusion was stopped, and a 2-h experimental period in which net hepatic glucose uptake (NHGU) and disposition (glycogen, lactate, and CO(2)) were measured in the absence of fructose but in the presence of a hyperglycemic-hyperinsulinemic challenge including portal vein glucose infusion.
Fructose infusion increased net hepatic glycogen synthesis (0.7 ± 0.5 vs. 6.4 ± 0.4 mg/kg/min; P < 0.001), causing a large difference in hepatic glycogen content (62 ± 9 vs. 100 ± 3 mg/g; P < 0.001). Hepatic glycogen supercompensation (fructose infusion group) did not alter NHGU, but it reduced the percent of NHGU directed to glycogen (79 ± 4 vs. 55 ± 6; P < 0.01) and increased the percent directed to lactate (12 ± 3 vs. 29 ± 5; P = 0.01) and oxidation (9 ± 3 vs. 16 ± 3; P = NS). This change was associated with increased AMP-activated protein kinase phosphorylation, diminished insulin signaling, and a shift in glycogenic enzyme activity toward a state discouraging glycogen accumulation.
These data indicate that increases in hepatic glycogen can generate a state of hepatic insulin resistance, which is characterized by impaired glycogen synthesis despite preserved NHGU.
[show abstract][hide abstract] ABSTRACT: We previously showed that elevating hepatic nitric oxide (NO) levels reduced net hepatic glucose uptake (NHGU) in the presence of portal glucose delivery, hyperglycemia, and hyperinsulinemia. The aim of the present study was to determine the role of a downstream signal, soluble guanylate cyclase (sGC), in the regulation of NHGU by NO.
Studies were performed on 42-h-fasted conscious dogs fitted with vascular catheters. At 0 min, somatostatin was given peripherally along with 4× basal insulin and basal glucagon intraportally. Glucose was delivered at a variable rate via a leg vein to double the blood glucose level and hepatic glucose load throughout the study. From 90 to 270 min, an intraportal infusion of the sGC inhibitor 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) was given in -sGC (n = 10) and -sGC/+NO (n = 6), whereas saline was given in saline infusion (SAL) (n = 10). The -sGC/+NO group also received intraportal SIN-1 (NO donor) to elevate hepatic NO from 180 to 270 min.
In the presence of 4× basal insulin, basal glucagon, and hyperglycemia (2× basal ), inhibition of sGC in the liver enhanced NHGU (mg/kg/min; 210-270 min) by ∼55% (2.9 ± 0.2 in SAL vs. 4.6 ± 0.5 in -sGC). Further elevating hepatic NO failed to reduce NHGU (4.5 ± 0.7 in -sGC/+NO). Net hepatic carbon retention (i.e., glycogen synthesis; mg glucose equivalents/kg/min) increased to 3.8 ± 0.2 in -sGC and 3.8 ± 0.4 in -sGC/+NO vs. 2.4 ± 0.2 in SAL (P < 0.05).
NO regulates liver glucose uptake through a sGC-dependent pathway. The latter could be a target for pharmacologic intervention to increase meal-associated hepatic glucose uptake in individuals with type 2 diabetes.
[show abstract][hide abstract] ABSTRACT: Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.
Journal of Biological Chemistry 11/2010; 285(45):34960-71. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: In individuals with type 1 diabetes, hypoglycemia is a common consequence of overinsulinization. Under conditions of insulin-induced hypoglycemia, glucagon is the most important stimulus for hepatic glucose production. In contrast, during euglycemia, insulin potently inhibits glucagon's effect on the liver. The first aim of the present study was to determine whether low blood sugar augments glucagon's ability to increase glucose production. Using a conscious catheterized dog model, we found that hypoglycemia increased glucagon's ability to overcome the inhibitory effect of insulin on hepatic glucose production by almost 3-fold, an effect exclusively attributable to marked enhancement of the effect of glucagon on net glycogen breakdown. To investigate the molecular mechanism by which this effect comes about, we analyzed hepatic biopsies from the same animals, and found that hypoglycemia resulted in a decrease in insulin signaling. Furthermore, hypoglycemia and glucagon had an additive effect on the activation of AMPK, which was associated with altered activity of the enzymes of glycogen metabolism.
The Journal of clinical investigation 11/2010; 120(12):4425-35. · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Approximately 90% of cases of Lafora disease, a fatal teenage-onset progressive myoclonus epilepsy, are caused by mutations in either the EPM2A or the EPM2B genes that encode, respectively, a glycogen phosphatase called laforin and an E3 ubiquitin ligase called malin. Lafora disease is characterized by the formation of Lafora bodies, insoluble deposits containing poorly branched glycogen or polyglucosan, in many tissues including skeletal muscle, liver, and brain. Disruption of the Epm2b gene in mice resulted in viable animals that, by 3 months of age, accumulated Lafora bodies in the brain and to a lesser extent in heart and skeletal muscle. Analysis of muscle and brain of the Epm2b(-/-) mice by Western blotting indicated no effect on the levels of glycogen synthase, PTG (type 1 phosphatase-targeting subunit), or debranching enzyme, making it unlikely that these proteins are targeted for destruction by malin, as has been proposed. Total laforin protein was increased in the brain of Epm2b(-/-) mice and, most notably, was redistributed from the soluble, low speed supernatant to the insoluble low speed pellet, which now contained 90% of the total laforin. This result correlated with elevated insolubility of glycogen and glycogen synthase. Because up-regulation of laforin cannot explain Lafora body formation, we conclude that malin functions to maintain laforin associated with soluble glycogen and that its absence causes sequestration of laforin to an insoluble polysaccharide fraction where it is functionally inert.
Journal of Biological Chemistry 08/2010; 285(33):25372-81. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen
is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption
of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop
liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but
dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise
compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice
reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO
mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression
of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates
with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model
mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in
whole body glucose homeostasis.
Journal of Biological Chemistry 04/2010; 285(17):12851-12861. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.
Journal of Biological Chemistry 02/2010; 285(17):12851-61. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Skeletal muscle glycogen is considered to be an important source of energy for contraction and increasing the level of the glucose polymer is generally thought to improve exercise performance in humans. A genetically modified mouse model (GSL30), which overaccumulates glycogen due to overexpression of a hyperactive form of glycogen synthase, was used to examine whether increasing the level of the polysaccharide enhances the ability of mice to run on a treadmill. The skeletal muscle of the GSL30 mice had large deposits of glycogen. There were no significant increases in the work performed by GSL30 mice as compared to their respective wild type littermates when exercised to exhaustion. The amount of muscle glycogen utilized by GSL30 mice, however, was greater, while the amount of liver glycogen consumed during exhaustive exercise was less than wild type animals. This result suggests that increased muscle glycogen stores do not necessarily improve exercise performance in mice.
Biochemical and Biophysical Research Communications 07/2005; 331(2):491-6. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.
Journal of Biological Chemistry 05/2005; 280(17):17260-5. · 4.65 Impact Factor