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Up-regulation of liver glucose-6-phosphatase in rats fed with a P-i-deficient diet
Abstract
Because P(i) deprivation markedly affects the Na/P(i) co-transporter in kidney and has been related to insulin resistance and glucose intolerance, the effect of a P(i)-deficient diet on the liver microsomal glucose-6-phosphatase (G6Pase) system was investigated. Rats were fed with a control diet (+P(i)) or a diet deficient in phosphate (-P(i)) for 2 days and killed on the morning of the third day, after an overnight fast (fasted) or not (fed). Kinetic parameters of P(i) transport (t((1/2)) and equilibration) into liver microsomes were not changed by the different nutritional conditions. In contrast, it was found that G6Pase activity was significantly increased in the (-P(i)) groups. This was due to an increase in the V(max) of the enzyme, without change in the K(m) for G6P. There was no correlation between liver microsomal glycogen content and G6Pase activity, but both protein abundance and mRNA of liver 36 kDa catalytic subunit of G6Pase (p36) were increased. The mRNA of the putative G6P translocase protein (p46) was changed in parallel with that of the catalytic subunit, but the p46 immunoreactive protein was unchanged. These findings indicate that dietary P(i) deficiency causes increased G6Pase activity by up-regulation of the expression of the 36 kDa-catalytic-subunit gene.
... We previously reported [Xie, Li, Me! chin and van de Werve (1999) Biochem. J. 343, 393-396] that dietary phosphate deprivation for 2 days up-regulated both the catalytic subunit and the putative glucose-6-phosphate translocase of the rat liver microsomal glucose-6-phosphatase system, suggesting that increased hepatic glucose production might be responsible for the frequent clinical association of hypophosphataemia and glucose intolerance. ...
... It has been reported that the overexpression of p36 in rat liver is sufficient to perturb glucose and lipid homeostasis in whole animals [4]. We have recently shown that a P i -deficient (kP i ) diet up-regulates rat liver microsomal G6Pase activity by the increased expression of both the p36 and p46 genes and the protein abundance of p36 only [5]. This finding suggests that the overproduction of glucose by the liver might contribute to glucose intolerance and insulin resistance, which have been documented in several diseases (hypophosphataemia rickets, adult-onset hypophosphataemia osteomalacia and renal P i leak) characterized with hypophosphataemia [6]. ...
... Our previous report demonstrated that a (kP i ) diet up-regulates liver G6Pase activity [5]. To verify whether hepatic glucose production might be enhanced in this condition, we measured plasma P i , insulin and glucose concentrations, insulin secretion, glucose disposal and endogenous glucose appearance after an IVGTT, and also liver glycogen and key regulatory steps of gluconeogenesis. ...
We previously reported [Xie, Li, Méchin and van de Werve (1999) Biochem. J. 343, 393-396] that dietary phosphate deprivation for 2 days up-regulated both the catalytic subunit and the putative glucose-6-phosphate translocase of the rat liver microsomal glucose-6-phosphatase system, suggesting that increased hepatic glucose production might be responsible for the frequent clinical association of hypophosphataemia and glucose intolerance. We now show that liver cAMP was increased in rats fed with a diet deficient in P(i) compared with rats fed with a control diet. Accordingly, in the P(i)-deficient group pyruvate kinase was inactivated, the concentration of phosphoenolpyruvate was increased and fructose 2, 6-bisphosphate concentration was decreased. Phosphoenolpyruvate carboxykinase activity was marginally increased and glucokinase activity was unchanged by P(i) deprivation. The liver glycogen concentration decreased in the P(i)-deficient group. In the fed state, plasma glucose concentration was increased and plasma P(i) and insulin concentrations were substantially decreased in the P(i)-deficient group. All of these changes, except decreased plasma P(i), were cancelled in the overnight fasted P(i)-deficient group. In the fasted P(i)-deficient group, immediately after a glucose bolus, the plasma glucose level was elevated and the inhibition of endogenous glucose production was decreased. However, this mild glucose intolerance was not sufficient to affect the rate of fall of the glucose level after the glucose bolus. Taken together, these changes are compatible with a stimulation of liver gluconeogenesis and glycogenolysis by the P(i)-deficient diet and further indicate that the liver might contribute to impaired glucose homeostasis in P(i)-deficient states.
... It is therefore important to establish whether such an association exists independently of the cause (diet or genetic). We previously reported that liver G6Pase is upregulated in rats fed with a Pi-deficient diet for two days [15]. These animals had their phosphatemia decreased almost by half, were hypoinsulinemic, and their glycemia increased by about 35 % [16]. ...
... Preparation of microsomes and G6Pase activity assay Mouse liver was homogenized using a glass homogenizer in an ice-cold buffer containing 50 mM Hepes-Tris, 250 mM sucrose, at pH 7.3. Microsomes were prepared as previously described [15]. G6Pase activity was assayed at 0.2 mM and 5 mM G6P at 30 8C before and after detergent (0.8 % Chapso) treatment [15]. ...
... Microsomes were prepared as previously described [15]. G6Pase activity was assayed at 0.2 mM and 5 mM G6P at 30 8C before and after detergent (0.8 % Chapso) treatment [15]. ...
We previously showed that a phosphate-deficient diet resulting in hypophosphatemia upregulated the catalytic subunit p36 of rat liver glucose-6-phosphatase, which is responsible for hepatic glucose production. A possible association between phosphate and glucose homeostasis was now further evaluated in the Hyp mouse, a murine homologue of human X-linked hypophosphatemia. We found that in the Hyp mouse as in the dietary Pi deficiency model, serum insulin was reduced while glycemia was increased, and that liver glucose-6-phosphatase activity was enhanced as a consequence of increased mRNA and protein levels of p36. In contrast, the Hyp model had decreased mRNA and protein levels of the putative glucose-6-phosphate translocase p46 and liver cyclic AMP was not increased as in the phosphate-deficient diet rats. It is concluded that in genetic as in dietary hypophosphatemia, elevated glucose-6-phosphatase activity could be partially responsible for the impaired glucose metabolism albeit through distinct mechanisms.
... Moreover, our data confirms this accompanies an increase in circulating ASARM peptides in mice 35,42 . Consistent with this, increased Glucose-6-Phosphatase activity (a gluconeogenic enzyme) occurs in rats fed with Pi-deficient diets 199,200 . These Pi diet-restricted animals are also hypophosphatemic, hypoinsulinemic, and hyperglycemic (35% increase) 199,200 . ...
... Consistent with this, increased Glucose-6-Phosphatase activity (a gluconeogenic enzyme) occurs in rats fed with Pi-deficient diets 199,200 . These Pi diet-restricted animals are also hypophosphatemic, hypoinsulinemic, and hyperglycemic (35% increase) 199,200 . Furthermore, clinical hypophosphatemia is associated with impaired glucose tolerance and insulin resistance 197,198 . ...
The eggshell is an ancient innovation that helped the vertebrates' transition from the oceans and gain dominion over the land. Coincident with this conquest, several new eggshell and noncollagenous bone-matrix proteins (NCPs) emerged. The protein ovocleidin-116 is one of these proteins with an ancestry stretching back to the Triassic. Ovocleidin-116 is an avian homolog of Matrix Extracellular Phosphoglycoprotein (MEPE) and belongs to a group of proteins called Small Integrin-Binding Ligand Interacting Glycoproteins (SIBLINGs). The genes for these NCPs are all clustered on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of the SIBLING proteins is an Acidic Serine Aspartate-Rich MEPE (ASARM)-associated motif. The ASARM motif and the released ASARM peptide play roles in mineralization, bone turnover, mechanotransduction, phosphate regulation and energy metabolism. ASARM peptides and motifs are physiological substrates for phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX), a Zn metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets. PHEX interacts with another ASARM motif containing SIBLING protein, Dentin Matrix Protein-1 (DMP1). DMP1 mutations cause bone-renal defects that are identical with the defects caused by loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both X-linked hypophosphatemic rickets and ARHR, increased fibroblast growth factor 23 (FGF23) expression occurs, and activating mutations in FGF23 cause autosomal dominant hypophosphatemic rickets (ADHR). ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. This review will discuss the evidence for a new integrative pathway involved in bone formation, bone-renal mineralization, renal phosphate homeostasis and energy metabolism in disease and health.
... 26,34 Consistent with this, increased glucose-6-phosphatase activity (a gluconeogenic enzyme) occurs in rats fed with PI-deficient diets. 187,188 These PI-diet-restricted animals are also hypophosphatemic, hypoinsulinemic, and hyperglycemic (35% increase). 187,188 Furthermore, clinical hypophosphatemia is associated with impaired glucose tolerance and insulin resistance. ...
... 187,188 These PI-diet-restricted animals are also hypophosphatemic, hypoinsulinemic, and hyperglycemic (35% increase). 187,188 Furthermore, clinical hypophosphatemia is associated with impaired glucose tolerance and insulin resistance. 185,186 Also, in type 2 diabetes patients subjected to a 4-h euglycemichyperinsulinemic clamp show major increases in FGF23 that correlate positively with insulin infusion. ...
More than 300 million years ago, vertebrates emerged from the vast oceans to conquer gravity and the dry land. With this transition, new adaptations occurred that included ingenious changes in reproduction, waste secretion, and bone physiology. One new innovation, the egg shell, contained an ancestral protein (ovocleidin-116) that likely first appeared with the dinosaurs and was preserved through the theropod lineage in modern birds and reptiles. Ovocleidin-116 is an avian homolog of matrix extracellular phosphoglycoprotein (MEPE) and belongs to a group of proteins called short integrin-binding ligand-interacting glycoproteins (SIBLINGs). These proteins are all localized to a defined region on chromosome 5q in mice and chromosome 4q in humans. A unifying feature of SIBLING proteins is an acidic serine aspartate-rich MEPE-associated motif (ASARM). Recent research has shown that the ASARM motif and the released ASARM peptide have regulatory roles in mineralization (bone and teeth), phosphate regulation, vascularization, soft-tissue calcification, osteoclastogenesis, mechanotransduction, and fat energy metabolism. The MEPE ASARM motif and peptide are physiological substrates for PHEX, a zinc metalloendopeptidase. Defects in PHEX are responsible for X-linked hypophosphatemic rickets (HYP). There is evidence that PHEX interacts with another ASARM motif containing SIBLING protein, dentin matrix protein-1 (DMP1). DMP1 mutations cause bone and renal defects that are identical with the defects caused by a loss of PHEX function. This results in autosomal recessive hypophosphatemic rickets (ARHR). In both HYP and ARHR, increased FGF23 expression plays a major role in the disease and in autosomal dominant hypophosphatemic rickets (ADHR), FGF23 half-life is increased by activating mutations. ASARM peptide administration in vitro and in vivo also induces increased FGF23 expression. FGF23 is a member of the fibroblast growth factor (FGF) family of cytokines, which surfaced 500 million years ago with the boney fish (i.e., teleosts) that do not contain SIBLING proteins. In terrestrial vertebrates, FGF23, like SIBLING proteins, is expressed in the osteocyte. The boney fish, however, are an-osteocytic, so a physiological bone-renal link with FGF23 and the SIBLINGs was cemented when life ventured from the oceans to the land during the Triassic period, approximately 300 million years ago. This link has been revealed by recent research that indicates a competitive displacement of a PHEX-DMP1 interaction by an ASARM peptide that leads to increased FGF23 expression. This review discusses the new discoveries that reveal a novel PHEX, DMP1, MEPE, ASARM peptide, and FGF23 bone-renal pathway. This pathway impacts not only bone formation, bone-renal mineralization, and renal phosphate homeostasis but also energy metabolism. The study of this new pathway is relevant for developing therapies for several diseases: bone-teeth mineral loss disorders, renal osteodystrophy, chronic kidney disease and bone mineralization disorders (CKD-MBD), end-stage renal diseases, ectopic arterial-calcification, cardiovascular disease renal calcification, diabetes, and obesity.
... Some mechanisms have been suggested in an attempt to explain this relationship: phosphate that is required for ATP production is an important component for energy metabolism (9) . Moreover, it is shown that phosphate-defi cient diet upregulates rat liver microsomal G6Pase activity (18) . This fi nding suggests that the overproduction of glucose by the liver might contribute to glucose intolerance and insulin resistance, which was documented in several diseases (hypophosphatemia rickets, adultonset hypophosphatemia osteomalacia and renal phosphate leak) characterized with hypophosphatemia (13,18) . ...
... Moreover, it is shown that phosphate-defi cient diet upregulates rat liver microsomal G6Pase activity (18) . This fi nding suggests that the overproduction of glucose by the liver might contribute to glucose intolerance and insulin resistance, which was documented in several diseases (hypophosphatemia rickets, adultonset hypophosphatemia osteomalacia and renal phosphate leak) characterized with hypophosphatemia (13,18) . ...
To investigate the relationship between serum phosphate levels with obesity and insulin resistance in childhood.
A total of 298 children and adolescents (190 obese subjects and 108 controls) were included in the study. Serum glucose, insulin, phosphate, calcium and alkaline phosphatase levels were measured after 12 h fasting at 08:00-08:30 h. We assessed insulin sensitivity by using the HOMA-IR (homeostasis model of insulin resistance) index as a surrogate marker of insulin resistance.
Serum levels of phosphate were significantly lower in the 6- to 12-year-old obese subjects than controls (p = 0.02, p < 0.05). At the same time, there was a moderate negative correlation between serum phosphate levels and the HOMA-IR index in the 6- to 12-year-old IR (-) obese children (r = -0.26, p = 0.02).
Low serum phosphate levels could contribute to the development of insulin resistance in 6- to 12-year-old obese children.
... Thus, inorganic phosphate plays multiple roles in the regulation of oxidative stress and mitochondrial function in health and disease (Kuro-o 2008(Kuro-o , 2010. Although a low phosphate diet does not reduce blood insulin levels, it alters expression of insulin-responsive genes in a way similar to that induced by diet restriction (Xie et al. 1999(Xie et al. , 2000, resulting in increased gluconeogenesis and decreased glycolysis. This may be owed to the fact that low phosphate diet induces moderate insulin resistance (Paula et al. 1998;Haap et al. 2006). ...
... Evidences suggest that a misbalance in phosphorus metabolism is related with the risk of development and severity of metabolic syndrome in humans [17][18][19], and phosphorus depletion is associated with fulminant hepatic failure, a consequence of severe liver injury [20]. Additionally, dietary phosphorus increased endogenous glucose production by stimulating gluconeogenic and glycogenolysis pathways and may be partially responsible for glucose intolerance and insulin resistance [21,22]. Hasi et al. [6] investigated the effects of butaphosphan solution on endurance capability and energy metabolism in mice and found that butaphosphan increased glycogen, adenosine triphosphate (ATP), and adenosine diphosphate (ADP) in liver and skeletal muscle, indicating that the solution can significantly enhance the energy metabolism in mice by improving liver and muscle glycogen storage and promoting the synthesis of ATP and ADP. ...
Butaphosphan is an organic phosphorus compound used in several species for the prevention of rapid catabolic states, however, the mechanism of action remains unclear. This study aimed at determining the effects of butaphosphan on energy metabolism of mice receiving a normal or hypercaloric diet (HCD) and submitted or not to food restriction. Two experiments were conducted: (1) during nine weeks, animals were fed with HCD (n = 28) ad libitum, and at the 10th week, were submitted to food restriction and received butaphosphan (n = 14) or saline injections (n = 14) (twice a day, for seven days) and; (2) during nine weeks, animals were fed with a control diet (n = 14) or HCD (n = 14) ad libitum, and at the 10th week, all animals were submitted to food restriction and received butaphosphan or saline injections (twice a day, for seven days). In food restriction, butaphosphan preserved epididymal white adipose tissue (WAT) mass, increased glucose, NEFA, and the HOMA index. In mice fed HCD and submitted to food restriction, the butaphosphan preserved epididymal WAT mass. Control diet influences on PI3K, GCK, and Irs1 mRNA expression. In conclusion, butaphosphan increased blood glucose and reduced fat mobilization in overweight mice submitted to caloric restriction, and these effects are influenced by diet.
... In mammals, a low-phosphate diet causes similar metabolic changes as a low-carbohydrate diet with increased gluconeogenesis and decreased glycolysis [159,160,161,162]. Although a lowphosphate diet does not affect blood insulin levels, it does alter the expression of insulindependent genes [163,164] with subsequent moderate insulin resistance [165,166]. ...
Without phosphate terrestrial life could not have come into existence because phosphate is an essential component of the genotype, an integral part of cellular and osteogenetic structures. Moreover, phosphate is an indispensable energy source for molecular, enzymatic, and metabolic processes in plants, animals, and humans. But phosphate on Earth is limited, which makes it necessary to deal with these resources responsibly and sustainably in order to maintain life on Earth. A phosphate deficit, but even more, an excess of phosphate is detrimental for the human organism and eventually leads to death. Phosphate homeostasis is warranted and maintained by different factors and hormonal mechanisms. Perturbation of these mechanisms leads to cardiovascular disease, tumors, accelerated aging, and increased mortality. In fact, phosphate can be considered central for human life from the beginning to its end. This review highlights the different aspects of phosphate, from its cosmo-geological origin, the efforts around a sustainable phosphate handling, the regulation of phosphate metabolism as well as its role in the human organism to the problems of hypo-and hyperphosphatemia for human health.
... Bei Säugetieren verursacht eine phosphatarme Diät ähnliche Veränderungen des Stoffwechsels wie eine kohlenhydratarme Ernährung mit vermehr-ter Gluconeogenese und verminderter Glykolyse [157,158,159,160]. Obwohl eine phosphatarme Ernährung die Blut-Insulin-Spiegel nicht beeinflusst, so verändert sie doch die Expression von insulinabhängigen Genen [161,162] mit nachfolgender moderater Insulinresistenz [163,164]. ...
Phosphorus is essential for terrestrial life but also a limited resource. A deficit but even more an excess of phosphate is detrimental for the human organism, leading tocardiovascular disease, Tumors, accelerated aging and finally to death. Phosphate can be considered central for human life from the beginning to its end. This review highlights the different aspects of phosphate.
... In humans, hypophosphatemia has been associated to glucose intolerance due to tissue insensitivity to insulin [19] and glucose disposal rate has been reported to increase after phosphate infusion [20]. Experiments in rats have shown that dietary P deprivation stimulates liver gluconeogenesis and glucogenolysis suggesting that the liver may be implicated in the modulation of glucose homeostasis induced by P deficiency [21,22]. In mice, dietary P restriction has been reported to induce lipid accumulation in the liver through dysregulation of genes involved in the hepatic metabolism of cholesterol [23]. ...
The effect of dietary phosphorus (P) on fibroblast growth factor 21 (FGF21)/β-klotho axis was investigated in rats that were fed diets with: Normal (NP) or high P (HP) and either normal (NC), high (HC) or low calories (LC). Sampling was performed at 1, 4 and 7 months. Plasma FGF21 concentrations were higher (p < 0.05) in NC and HC than in LC groups. Increasing P intake had differing effects on plasma FGF21 in rats fed NC and HC vs. rats fed LC at the three sampling times. When compared with the NP groups, FGF21 concentrations decreased at the three sampling points in rats fed NC-HP (80 vs. 194, 185 vs. 382, 145 vs. 403 pg/mL) and HC-HP (90 vs. 190, 173 vs. 353, 94 vs. 434 pg/mL). However, FGF21 did not decrease in rats fed LC-HP (34 vs. 20, 332 vs. 164 and 155 vs. 81 pg/mL). In addition, LC groups had a much lower liver FGF21 messenger ribonucleic acid/glyceraldehyde 3-phosphate dehydrogenase (mRNA/GAPDH) ratio (0.51 ± 0.08 and 0.56 ± 0.07) than the NC-NP (0.97 ± 0.14) and HC-NP (0.97 ± 0.22) groups. Increasing P intake reduced liver FGF21 mRNA/GAPDH in rats fed NC and HC to 0.42 ± 0.05 and 0.37 ± 0.04. Liver β-klotho mRNA/GAPDH ratio was lower (p < 0.05) in LC groups (0.66 ± 0.06 and 0.59 ± 0.10) than in NC (1.09 ± 0.17 and 1.03 ± 0.14) and HC (1.19 ± 0.12 and 1.34 ± 0.19) groups. A reduction (p < 0.05) in β-klotho protein/α-tubulin ratio was also observed in LC groups (0.65 ± 0.05 and 0.49 ± 0.08) when compared with NC (1.12 ± 0.11 and 0.91 ± 0.11) and HC (0.93 ± 0.17 and 0.87 ± 0.09) groups. In conclusion β-klotho is potently regulated by caloric restriction but not by increasing P intake while FGF21 is regulated by both caloric restriction and increased P intake. Moreover, increased P intake has a differential effect on FGF21 in calorie repleted and calorie depleted rats.
... Indeed, a deficiency of phosphate is found in rickets disease from children, osteomalacia in adults and Toni-Fanconi syndrome which leads to bone loss [3] [4]. In mammals, it is shown that a low phosphate diet alters glucose metabolism [5] [6] and the expression of insulin-sensitive genes [7] [8]. In addition, it is shown that phosphate increases oxidative stress such as Klotho deficiency in mice, causing phosphate retention and impairment of cognition due to increased oxidative damage and apoptosis in Hippocampus neurons [9]. ...
... low serum phosphate levels with glucose intolerance, insulin sensitivity and insulin secretion in non-diabetic healthy-subjects [95] and a phosphate deplete diet impairs rat insulin secretion (markedly reduced) by pancreatic islets ex vivo [109]. HYP mice also have increased hepatic glucose-6-phosphatase activity [53] and rats fed a phosphate deplete diet up-regulate expression and activity of this enzyme [97,110]. Also, overexpression of glucose-6phosphatase in rats induces glucose intolerance, hyperglycemia with changes in circulating free fatty acids and triglycerides [111]. ...
Context
PHEX or DMP1 mutations cause hypophosphatemic-rickets and altered energy metabolism. PHEX binds to DMP1-ASARM-motif to form a complex with α5β3 integrin that suppresses FGF23 expression. ASARM-peptides increase FGF23 by disrupting the PHEX-DMP1-Integrin complex. We used a 4.2 kDa peptide (SPR4) that binds to ASARM-peptide/motif to study the DMP1-PHEX interaction and to assess SPR4 for the treatment of energy metabolism defects in HYP and potentially other bone-mineral disorders.
Design
Subcutaneously transplanted osmotic pumps were used to infuse SPR4-peptide or vehicle (VE) into wild-type mice (WT) and HYP-mice (PHEX mutation) for 4 weeks.
Results
SPR4 partially corrected HYP mice hypophosphatemia and increased serum 1.25(OH)2D3. Serum FGF23 remained high and PTH was unaffected. WT-SPR4 mice developed hypophosphatemia and hypercalcemia with increased PTH, FGF23 and 1.25(OH)2D3. SPR4 increased GAPDH HYP-bone expression 60× and corrected HYP-mice hyperglycemia and hypoinsulinemia. HYP-VE serum uric-acid (UA) levels were reduced and SPR4 infusion suppressed UA levels in WT-mice but not HYP-mice. SPR4 altered leptin, adiponectin, and sympathetic-tone and increased the fat mass/weight ratio for HYP and WT mice. Expression of perlipin-2 a gene involved in obesity was reduced in HYP-VE and WT-SPR4 mice but increased in HYP-SPR4 mice. Also, increased expression of two genes that inhibit insulin-signaling, ENPP1 and ESP, occurred with HYP-VE mice. In contrast, SPR4 reduced expression of both ENPP1 and ESP in WT mice and suppressed ENPP1 in HYP mice. Increased expression of FAM20C and sclerostin occurred with HYP-VE mice. SPR4 suppressed expression of FAM20C and sclerostin in HYP and WT mice.
Conclusions
ASARM peptides and motifs are physiological substrates for PHEX and modulate osteocyte PHEX-DMP1-α5β3-integrin interactions and thereby FGF23 expression. These interactions also provide a nexus that regulates bone and energy metabolism. SPR4 suppression of sclerostin and/or sequestration of ASARM-peptides improves energy metabolism and may have utility for treating familial rickets, osteoporosis, obesity and diabetes.
... These data might suggest that dietary cholesterol can regulate renal and intestinal Pi homeostasis through LXR, and hypophosphatemia by Npt2a gene ablation induce imbalance of cholesterol and lipid metabolism through suppression of hepatic LXR alpha gene expression. Moreover, Xie et al. reported Pi restriction diet could change glucose metabolism in liver (25,26). It has been reported that the Npt2b known as intestinal Pi transporter is highly expressed in the intestine, lung and liver (27). ...
The type IIa sodium-dependent phosphate co-transporter (Npt2a) is important to maintain renal inorganic phosphate (Pi) homeostasis and the plasma Pi levels. It has reported that disorder of Pi metabolism in kidney can be risk factors for cardiovascular disease as well as hypercholesterolemia. However, the relationship between Pi and cholesterol metabolism has not been clarified. The current study investigated the effects of Npt2a gene ablation that is known as hypophosphatemia model on cholesterol metabolism in mice. Npt2a deficient (Npt2a(-/-)) mice and wild type mice were fed diets with or without 2% cholesterol for 12 days. Plasma lipid and lipoprotein profile analysis revealed that plasma lipid levels (total, LDL and HDL cholesterol) were significantly higher in Npt2a(-/-) mice than wild type (WT) mice. Interestingly, high cholesterol diet markedly increased plasma levels of total, LDL and HDL cholesterol in WT mice, but not Npt2a(-/-) mice. On the other hand, there were no differences in body and liver weight, intake and hepatic lipid accumulation between WT and Npt2a(-/-) mice. These results suggest that ablation of Npt2a gene induces hypercholesterolemia and affects the ability to respond normally to dietary cholesterol. J. Med. Invest. 60: 191-196, August, 2013.
... Previous reports showed that hyperphosphatemia is associated with left ventricular hypertrophy and progression of chronic kidney disease (CKD) [11,12]. Interestingly, Xie et al [13,14] reported that dietary Pi restriction affected hepatic glucose metabolism. Cholesterol homeostasis is maintained through 3 major pathways: biosynthesis, absorption from the intestine, and elimination as bile acids [15]. ...
... From the few studies conducted so far, it seems that the regulation of the expression of P46 is parallel to that of the catalytic subunit P36. Dietary P i -deficiency indeed increases both P36 and P46 mRNAs [173]. In the streptozocin-diabetic rat, the abundance of P46 mRNA (and protein) was enhanced in liver, kidney and intestine similarly to that of P36 [172]. ...
The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.
... Animals under diet restriction reduce blood insulin levels to adapt reduced carbohydrate availability and alter expression of insulin-responsive genes, which leads to changes in glucose metabolism including increased gluconeogenesis and decreased glycolysis among others (Cao et al., 2001; Kayo et al., 2001; Lee et al., 1999; Masoro, 2006; Wetter et al., 1999). Although low phosphate diet does not reduce blood insulin levels, it indeed alters expression of insulin-responsive genes in a way similar to that induced by diet restriction (Xie et al., 1999; Xie et al., 2000), resulting in increased gluconeogenesis and decreased glycolysis as well. This may be partly explained by the fact that low phosphate diet induces moderate insulin resistance by unknown mechanisms (Haap et al., 2006; Paula et al., 1998). ...
Phosphate homeostasis is maintained primarily by a bone-kidney endocrine axis. When phosphate is in excess, fibroblast growth factor-23 (FGF23) is secreted from bone and acts on kidney to promote phosphate excretion into urine. FGF23 also reduces serum vitamin D levels to suppress phosphate absorption from intestine. Thus, FGF23 functions as a hormone that induces negative phosphate balance. One critical feature of FGF23 is that it requires Klotho, a single-pass transmembrane protein expressed in renal tubules, as an obligate co-receptor to bind and activate cognate FGF receptors. Importantly, defects in either FGF23 or Klotho not only cause phosphate retention but also a premature-aging syndrome in mice, which can be rescued by resolving hyperphosphatemia. In addition, changes in extracellular and intracellular phosphate concentration affect glucose metabolism, insulin sensitivity, and oxidative stress in vivo and in vitro, which potentially affect aging processes. These findings suggest an unexpected link between inorganic phosphate and aging in mammals.
... We suggest that BBM Na/glucose cotransport increased in the hypophosphatemic mutants in response to the reduction in P i supply. Consistent with this hypothesis is the demonstration that glucose-6-phosphatase, an enzyme that increases glucose production and glycemia and is abundantly expressed in kidney, is also upregulated in the liver of Hyp mice (38) and of P i -deprived rats (37). ...
The present study was undertaken to define the mechanisms governing the regulation of the novel renal brush-border membrane (BBM) Na-phosphate (Pi) cotransporter designated type IIc (Npt2c). To address this issue, the renal expression of Npt2c was compared in two hypophosphatemic mouse models with impaired renal BBM Na-Pi cotransport. In mice homozygous for the disrupted Npt2a gene (Npt2-/-), BBM Npt2c protein abundance, relative to actin, was increased 2.8-fold compared with Npt2+/+ littermates, whereas a corresponding increase in renal Npt2c mRNA abundance, relative to beta-actin, was not evident. In contrast, in X-linked Hyp mice, which harbor a large deletion in the Phex gene, the renal abundance of both Npt2c protein and mRNA was significantly decreased by 80 and 50%, respectively, relative to normal littermates. Pi deprivation elicited a 2.5-fold increase in BBM Npt2c protein abundance in Npt2+/+ mice but failed to elicit a further increase in Npt2c protein in Npt2-/- mice. Pi restriction led to an increase in BBM Npt2c protein abundance in both normal and Hyp mice without correcting its renal expression in the mutants. In summary, we report that BBM Npt2c protein expression is differentially regulated in Npt2-/- mice and Hyp mice and that the Npt2c response to low-Pi challenge differs in both hypophosphatemic mouse strains. We demonstrate that Npt2c protein is maximally upregulated in Npt2-/- mice and suggest that Npt2c likely accounts for residual BBM Na-Pi cotransport in the knockout model. Finally, our data indicate that loss of Phex function abrogates renal Npt2c protein expression.
Objective
The liver releases glucose into the blood using the glucose-6-phosphatase (G6Pase) system, a multiprotein complex located in the endoplasmic reticulum (ER). Here, we show for the first time that the G6Pase system is also expressed in hypothalamic tanycytes, and it is required to regulate energy balance.
Methods
Using automatized qRT-PCR and immunohistochemical analyses, we evaluated the expression of the G6Pase system. Fluorescent glucose analogue (2-NBDG) uptake was evaluated by 4D live-cell microscopy. Glucose release was tested using a glucose detection kit and high-content live-cell analysis instrument, Incucyte s3. In vivo G6pt knockdown in tanycytes was performed by AAV1-shG6PT-mCherry intracerebroventricular injection. Body weight gain, adipose tissue weight, food intake, glucose metabolism, c-Fos, and neuropeptide expression were evaluated at 4 weeks post-transduction.
Results
Tanycytes sequester glucose-6-phosphate (G6P) into the ER through the G6Pase system and release glucose in hypoglycaemia via facilitative glucose transporters (GLUTs). Strikingly, in vivo tanycytic G6pt knockdown has a powerful peripheral anabolic effect observed through decreased body weight, white adipose tissue (WAT) tissue mass, and strong downregulation of lipogenesis genes. Selective deletion of G6pt in tanycytes also decreases food intake, c-Fos expression in the arcuate nucleus (ARC), and Npy mRNA expression in fasted mice.
Conclusions
The tanycyte-associated G6Pase system is a central mechanism involved in controlling metabolism and energy balance.
BACKGROUND & AIMS:
Hyperglycemia occurs in more than half of the extremely low birth weight (ELBW) neonates during the first weeks of life, and is correlated with an increased risk of morbi-mortality. Hypophosphatemia is another frequent metabolic disorder in this population. Data from animal, adult studies and clinical observation suggest that hypophosphatemia could induce glucose intolerance. Our aim was to determine whether a low phosphatemia is associated with hyperglycemia in ELBW neonates.
METHODS:
This observational study included ELBW infants admitted in a tertiary neonatal care center (2010-2011). According to the center's policy, they received parenteral nutrition from birth and human milk from day 1. Phosphatemia and glycemia were measured routinely during parenteral nutrition. Hyperglycemia was defined by two consecutives values >8.3 mmol/L (150 mg/dL). Statistical analysis used a joint model combining a mixed-effects and a survival submodels to measure the association between phosphate and hyperglycemia.
RESULTS:
The study included 148 patients. Mean gestational (Standard Deviation) age was 27.3 (1.6) weeks; mean birth weight was 803 (124) grams; 57% presented hyperglycemia. The multivariate joint model showed that the hazard of hyperglycemia at a given time was multiplied by 3 for each 0.41 mmol/L decrease of phosphate level at this time (p = 0.002) and by 3.85 for the same decreased of phosphate the day before (p = 0.0015).
CONCLUSION:
To our knowledge, this is the first study suggesting that low phosphatemia can be associated with hyperglycemia in ELBW neonates. Further studies will have to demonstrate whether better control of phosphatemia could help in preventing hyperglycemia.
Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
KEYWORDS:
Extremely low birth weight infant; Hyperglycemia; Hypophosphatemia; Insulin; Metabolic disorder; Preterm neonate
Hepatic microsomal glucose-6-phosphatase is a multicomponent system composed of substrate/product translocases and a catalytic subunit. Previously we (Foster et al. (1996) Biochim. Biophys. Acta 12, 244–254) demonstrated that N-bromoacetylethanolamine phosphate (BAEP) is a time-dependent, irreversible inhibitor of glucose-6-phosphate hydrolysis in intact but not disrupted microsomes. We proposed that BAEP manifests its inhibitory effect by binding with a glucose-6-phosphate translocase protein of the glucose-6-phosphatase system. Here we provide additional evidence that BAEP inhibits glucose-6-phosphate transport in microsomal vesicles and utilize [32P]BAEP as an affinity label in the identification of a glucose-6-phosphate transport protein. In this study, we identify 51-kDa rat and mouse liver microsomal proteins involved in glucose-6-phosphate transport into and out of microsomal vesicles by utilizing (1) an Ehrlich ascites tumor-bearing mouse model, which displays a decreased sensitivity to the time-dependent inhibitory effect of BAEP, and (2) another glucose-6-phosphate translocase inhibitor, tosyl-lysine chloromethyl ketone, in conjunction with [32P]BAEP as an affinity label.
The klotho gene was identified as an "aging-suppressor" gene in mice that accelerates aging when disrupted and extends life span when overexpressed. It encodes a single-pass transmembrane protein and is expressed primarily in renal tubules. The extracellular domain of Klotho protein is secreted into blood and urine by ectodomain shedding. The two forms of Klotho protein, membrane Klotho and secreted Klotho, exert distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors and functions as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging. Secreted Klotho functions as a humoral factor that regulates activity of multiple glycoproteins on the cell surface, including ion channels and growth factor receptors such as insulin/insulin-like growth factor-1 receptors. Potential contribution of these multiple activities of Klotho protein to aging processes is discussed.
The effect of streptozocin diabetes on the expression of the catalytic subunit (p36) and the putative glucose-6-phosphate translocase (p46) of the glucose-6-phosphatase system (G6Pase) was investigated in rats. In addition to the documented effect of diabetes to increase p36 mRNA and protein in the liver and kidney, a approximately 2-fold increase in the mRNA abundance of p46 was found in liver, kidney, and intestine, and a similar increase was found in the p46 protein level in liver. In HepG2 cells, glucose caused a dose-dependent (1-25 mM) increase (up to 5-fold) in p36 and p46 mRNA and a lesser increase in p46 protein, whereas insulin (1 microM) suppressed p36 mRNA, reduced p46 mRNA level by half, and decreased p46 protein by about 33%. Cyclic AMP (100 microM) increased p36 and p46 mRNA by >2- and 1.5-fold, respectively, but not p46 protein. These data suggest that insulin deficiency and hyperglycemia might each be responsible for up-regulation of G6Pase in diabetes. It is concluded that enhanced hepatic glucose output in insulin-dependent diabetes probably involves dysregulation of both the catalytic subunit and the putative glucose-6-phosphate translocase of the liver G6Pase system.
The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46). Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm. P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin. P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36. Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter. Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively. Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion. The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined. The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism.
Glucose-6-phosphatase (G6Pase), an enzyme found mainly in the liver and the kidneys, plays the important role of providing glucose during starvation. Unlike most phosphatases acting on water-soluble compounds, it is a membrane-bound enzyme, being associated with the endoplasmic reticulum. In 1975, W. Arion and co-workers proposed a model according to which G6Pase was thought to be a rather unspecific phosphatase, with its catalytic site oriented towards the lumen of the endoplasmic reticulum [Arion, Wallin, Lange and Ballas (1975) Mol. Cell. Biochem. 6, 75--83]. Substrate would be provided to this enzyme by a translocase that is specific for glucose 6-phosphate, thereby accounting for the specificity of the phosphatase for glucose 6-phosphate in intact microsomes. Distinct transporters would allow inorganic phosphate and glucose to leave the vesicles. At variance with this substrate-transport model, other models propose that conformational changes play an important role in the properties of G6Pase. The last 10 years have witnessed important progress in our knowledge of the glucose 6-phosphate hydrolysis system. The genes encoding G6Pase and the glucose 6-phosphate translocase have been cloned and shown to be mutated in glycogen storage disease type Ia and type Ib respectively. The gene encoding a G6Pase-related protein, expressed specifically in pancreatic islets, has also been cloned. Specific potent inhibitors of G6Pase and of the glucose 6-phosphate translocase have been synthesized or isolated from micro-organisms. These as well as other findings support the model initially proposed by Arion. Much progress has also been made with regard to the regulation of the expression of G6Pase by insulin, glucocorticoids, cAMP and glucose.
Glucose transport was investigated in rat liver microsomes in relation to glucose 6-phosphatase (Glu-6-Pase) activity using
a fast sampling, rapid filtration apparatus. 1) The rapid phase in tracer uptake and the burst phase in glucose 6-phosphate
(Glu-6-P) hydrolysis appear synchronous, while the slow phase of glucose accumulation occurs during the steady-state phase
of glucose production. 2) [14C]Glucose efflux from preloaded microsomes can be observed upon addition of either cold Glu-6-P or Glu-6-Pase inhibitors,
but not cold glucose. 3) Similar steady-state levels of intramicrosomal glucose are observed under symmetrical conditions
of Glu-6-P or vanadate concentrations during influx and efflux experiments, and those levels are directly proportional to
Glu-6-Pase activity. 4) The rates of both glucose influx and efflux are characterized by t values that are independent of Glu-6-P concentrations. 5) Glucose efflux in the presence of saturating concentrations of
vanadate was not blocked by 1 mM phloretin, and the initial rates of efflux appear directly proportional to intravesicular
glucose concentrations. 6) It is concluded that glucose influx into microsomes is tightly linked to Glu-6-Pase activity, while
glucose efflux may occur independent of hydrolysis, so that microsomal glucose transport appears unidirectional even though
it can be accounted for by diffusion only over the accessible range of sugar concentrations.
Phosphorus is the sixth most abundant element in the body after oxygen, hydrogen, carbon, nitrogen, and calcium. It comprises about 1% of the total body weight of humans. Eighty-five percent of it is stored in the bone in the form of hydroxyapatite crystal; 14% is in the soft tissues in the form of energy-storing bonds with nucleotides (ATP, GTP), nucleic acids in chromosomes and ribosomes, 2,3-DPG in the red blood cells, and phospholipids in the cells' membranes. Less than 1% is in the extracellular fluids. Phosphate balance is maintained by multiple systems. The gut is responsible for the absorption of two thirds of the 4-30 mg/kg/day of phosphate intake. Absorption sites are all along the gut; in humans the most active site is the jejunum. The kidney filters 90% of the plasma phosphate and reabsorbs it in the tubuli. In states of hypophosphatemia the kidney can reabsorb the filtered phosphates very efficiently, reducing the amount excreted in the urine virtually to zero. The healthy kidney can excrete high loads of phosphate and rid the body of phosphate overload. Through the vitamin D-PTH axis the endocrine system regulates the phosphate balance by influencing the kidney, gut, and bone. Other hormones, including thyroid, insulin, glucagon, glucocorticosteroid, and thyrocalcitonin, play a lesser role in regulation of phosphate metabolism. Because of the complex control of phosphate homeostasis, various clinical conditions may lead to hypophosphatemia. These include nutritional repletion, gastrointestinal malabsorption, use of phosphate binders, starvation, diabetes mellitus, and increased urinary losses due to tubular dysfunction. The clinical picture of phosphate depletion is manifested in different organs and is due mainly to the fall in intracellular levels of ATP and decreased availability of oxygen to the tissues, secondary to 2,3-DPG depletion. The various manifestations of phosphate depletion are listed in Table 2. The treatment of hypophosphatemia consists of administering enteral or parenteral phosphate salts. An important aspect of dealing with the potentially serious effects of phosphate depletion is to prevent the depletion from happening in the first place. Hyperphosphatemia can occur in renal failure, hemolysis, tumor lysis syndrome, and rhabdomyolysis. The treatment of hyperphosphatemia usually consists of fluid administration (in the absence of kidney failure). In chronic hyperphosphatemia, phosphate binders such as aluminum and magnesium salts can reduce the phosphate load. The use of these phosphate binders is limited by their potential side effects.
The mechanism of liver glycogen synthesis after refeeding has been investigated in diabetic rats, diabetic insulin-treated rats, and in control rats fasted for 48 h. The accumulation of liver glycogen was the same in diabetic rats and in control rats after 2 h of feeding, but did not proceed any further in the diabetic group during the next 2 h. Insulin-treated diabetic rats synthesized five times more hepatic glycogen than the control rats after 1 h of refeeding, but the amount accumulated at the end of the refeeding period was the same. Feeding resulted in a transient activation of glycogen synthase in untreated as well as in treated diabetic rats. In control rats, however, glycogen synthase was already partially in the active form before access to food, and the onset of glycogen synthesis occurred without further activation of the enzyme. A transient inactivation of phosphorylase was observed in all groups during the meal, but was very slight in the untreated diabetic rats in which phosphorylase a values were already reduced before the access to food.
Peripheral glycemia was markedly increased upon refeeding in treated and untreated diabetic rats, but remained normal in control rats. Peripheral insulinemia was increased by feeding in the control rats and remained low in the diabetic rats and high in the insulin-treated diabetic rats.
The results indicate that, in normal controls in contrast to diabetic rats, synthase activation is not a prerequisite for the initiation of glycogen synthesis after a meal; phosphorylase inactivation may be of major importance in normal controls, but also appears to play a role in the diabetic animals. The changes are triggered by glucose as well as insulin.
Although hypophosphatemia is commonly present in diabetics, little is known about its isolated effects on glucose and insulin metabolism. We therefore investigated glucose metabolism in six nondiabetic subjects with chronic hypophosphatemia. When glucose was infused to maintain a constant hyperglycemic level (125 mg per deciliter [6.9 mmol per liter] above basal levels), the glucose infusion rate was 36 per cent less in the hypophosphatemic group than in controls (4.90 +/- 0.34 mg per kilogram of body weight per minute vs. 7.64 +/- 0.37, P < 0.001), although responses to endogenous insulin were similar. When exogenous insulin was infused at a constant rate to maintain an insulin level about 100 microU per milliliter (718 pmol per liter) above basal levels and glucose was infused as necessary to maintain fasting glucose levels, the infusion rate of glucose was 43 per cent lower in the hypophosphatemic group than in controls (3.80 +/- 0.58 mg per kilogram per minute vs. 6.70 +/- 0.33, P < 0.001), although the clearance rate of insulin was similar in both groups. These results indicate that hypophosphatemia is associated with impaired glucose metabolism in both the hyperglycemic and euglycemic states, and that this associated primarily reflects decreased tissue sensitivity to insulin. (N Engl J Med. 1980; 303; 1259-63.).
Although renal Na(+)-P(i) cotransporter gene expression is decreased in X-linked Hyp mice, the mutants do respond to P(i) restriction with an adaptive increase in Na(+)-P(i) cotransport maximal velocity in renal brush-border membrane vesicles. In the present study, we examined the mechanism for the adaptive increase in Na(+)-P(i) cotransport in P(i)-deprived Hyp mice and normal littermates, using a cDNA probe encoding a rat, renal-specific Na(+)-P(i) cotransporter (NaPi-2) and a rabbit polyclonal antibody raised against a synthetic NaPi-2-derived peptide. The low-P(i) diet elicited an increase in Na(+)-P(i) cotransport in normal (141 +/- 13 to 714 +/- 158) and Hyp mice (59 +/- 6 to 300 +/- 62 pmol.mg protein-1.6 s-1; means +/- SE, n = 3, P < 0.01) that was accompanied by an increase in brush-border membrane NaPi-2 protein, relative to ecto-5'-nucleotidase, in normal (1.0 +/- 0.1 to 7.6 +/- 1.5) and Hyp mice (0.3 +/- 0.1 to 7.7 +/- 1.4) (means +/- SE, n = 4; P < 0.01). The low-P(i) diet also elicited an increase in the abundance of NaPi-2 mRNA, relative to the 18S RNA, in normal (157 +/- 9% of control diet, P < 0.05) and Hyp mice (194 +/- 10% of control diet, P < 0.01). Immunohistochemistry revealed that NaPi-2 protein was localized to the brush-border membrane of the proximal tubule and that both intensity of the signal and number of immunostained proximal tubules were increased in renal sections from normal and Hyp mice fed the low-P(i) diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Rapid kinetics of glucose-6-phosphate (G6P) uptake and hydrolysis as well as of orthophosphate uptake were investigated in microsomes prepared from normal and glycogen storage disease type 1a (GSD 1a) human livers using a fast sampling, rapid filtration apparatus and were compared to those of rat liver microsomes. As shown before with rat microsomes, the production of [U-14C]glucose from 0.2 mmol/L [U-14C]G6P by untreated normal human microsomes was characterized by a burst in activity during the first seconds of incubation, followed by a slower linear rate. The initial velocity of the burst was equal to the rate of glucose production in detergent-treated microsomes. In untreated and detergent-treated GSD 1a microsomes, no glucose-6-phosphatase activity was observed. When untreated normal human or rat microsomes were incubated in the presence of 0.2 mmol/L [U-14C]G6P, an accumulation of [U-14C]glucose was observed, whereas no radioactive compound (G6P and/or glucose) was taken up by GSD 1a microsomes. Orthophosphate uptake was, however, detectable in both GSD 1a and normal untreated vesicles. These results do not support a rate-limiting transport of G6P in untreated normal human microsomes and further show that in this case of GSD 1a, no distinct G6P transport activity is present.
Glucose-6-phosphatase catalyzes the final step of glucose production by liver and kidney. Though its strategic position has sparked interest in its regulation, difficulty with isolating a pure, stable enzyme has slowed progress. Virtually all previous work examining the physiologic regulation of this enzyme has relied on estimates of glucose-6-phosphatase activity in crude microsome preparations. The recent cloning of human and murine glucose-6-phosphatase cDNAs has now allowed study of its mRNA expression. We studied the effect of acute, streptozotocin-induced diabetes on hepatic microsomal glucose-6-phosphatase activity and mRNA expression in young (89 +/- 3 g), juvenile (304 +/- 4 g) and adult (512 +/- 10 g) rats. In control rats, mRNA expression and enzyme activity was similar among the three age groups. Streptozotocin-induced diabetes significantly increased the enzyme activities in both intact and triton-treated microsomes in all groups of rats (p < 0.001). Glucose-6-phosphatase mRNA expression was increased in the diabetic rats as well (p < 0.0001). Blood glucose concentrations correlated significantly with glucose-6-phosphatase mRNA level (p < 0.005) and both intact (p < 0.002) and triton-treated (p < 0.001) microsomal glucose-6-phosphatase activity. Both intact and triton-treated microsomal glucose-6-phosphatase activity correlated with mRNA level (p < 0.001, for each). We conclude that acute streptozotocin-diabetes increase expression of glucose-6-phosphatase mRNA and this contributes to the increased glucose-6-phosphatase activity seen with diabetes mellitus.
cDNA clones coding for the catalytic subunit of rat liver glucose-6-phosphatase (EC 3.1.3.9) were isolated from a rat liver cDNA library in lambda gt11 phage. The sequence of the cDNA and the amino acid sequence derived from it were greater than 90% identical to the corresponding sequences for the mouse and human forms of liver glucose-6-phosphatase. Northern blot analysis of RNA from FAO hepatoma cells revealed that dexamethasone induced the glucose-6-phosphatase mRNA while insulin suppressed its expression. When both hormones were added together insulin completely suppressed the effect of glucocorticoid. cAMP addition alone decreased the abundance of glucose-6-phosphatase mRNA. The results demonstrate multihormonal regulation of gene expression of hepatic glucose-6-phosphatase and support a dominant role for insulin.
Glycogen storage disease (GSD) type 1a is caused by the deficiency of D-glucose-6-phosphatase (G6Pase), the key enzyme in
glucose homeostasis. Despite both a high incidence and morbidity, the molecular mechanisms underlying this deficiency have
eluded characterization. In the present study, the molecular and biochemical characterization of the human G6Pase complementary
DNA, its gene, and the expressed protein, which is indistinguishable from human microsomal G6Pase, are reported. Several mutations
in the G6Pase gene of affected individuals that completely inactivate the enzyme have been identified. These results establish
the molecular basis of this disease and open the way for future gene therapy.
Polyclonal antibodies were raised in rabbits against the C-terminal portion of the rat renal brush-border membrane sodium/phosphate cotransporter NaPi-2. Antibody specificity and molecular sizes of proteins related to NaPi-2 were assayed by Western blot analysis. Proteins of 40 and 70-75 kDa (p40 and p70) were immunodetected in rat and mouse brush-border membranes and proteins of 72 and 82 kDa were detected in rabbit. The absence or presence of beta-EtSH in the samples before electrophoresis greatly influenced the immunodetection profile of the rat proteins. Since the 40 kDa protein (p40) can only be detected under reducing conditions, it probably originates from reduction of disulfide bonds in p70. Tryptic cleavage of p40 and p70 revealed identical protein fragments showing the close structural identity of those proteins. Both proteins were more abundant in the outer cortex portion of the rat kidney than in the juxtamedullary portion. Furthermore, rats fed a low-phosphate diet for 24 h showed a 20- and 14-fold increase in the amount of p40 and p70, respectively, compared to control rats, showing that the adaptation to P(i) deprivation by increasing renal phosphate reabsorption is not only the result of overproduction of p70, as previously shown, but is also due to the novel p40 which most probably derives from p70.
We have studied the effects of fatty-acyl-CoA esters on the activity of glucose-6-phosphatase (Glc6Pase) in untreated and detergent-treated liver microsomes. Fatty-acyl-CoA esters with chain lengths less than or equal to nine carbons do not inhibit Glc6Pase. Medium-chain fatty-acyl-CoA esters (10-14 carbons) inhibit Glc6Pase of untreated microsomes in a dose-dependent manner in the range 1-20 microM. The inhibitory effect is also dependent on the acyl-chain length. The higher the chain length, the stronger the inhibitory effect. It is also dependent on the microsomal protein concentration. The higher the protein concentration, the lower the inhibitory effect. Fatty-acyl-CoA esters with longer chain length (equal to or higher than 16 carbons) inhibit Glc6Pase of untreated microsomes within the range 1-2 microM. However, the inhibitory effect is either partially or totally cancelled, or even changed into an activation effect at higher concentrations. This is due to the release of mannose-6-phosphatase latency. The inhibition is fully reversible in the presence of bovine serum albumin. The mechanism of the Glc6Pase inhibition in untreated microsomes is uncompetitive (Ki for myristoyl-CoA = 1.2 +/- 0.3 microM, mean +/- SD, n = 3). Glc6Pase of detergent-treated microsomes is also inhibited by fatty-acyl-CoA esters, albeit less efficiently. In this case, the mechanism is non-competitive (Ki for myristoyl-CoA = 29 +/- 3 microM).
We have studied the role of Glc6Pase mRNA abundance in the time course of Glc6Pase activity in liver and kidney during long-term fasting in rat. Refered to the mRNA level in the fed state, Glc6Pase mRNA abundance was increased by 3.5 +/- 0.5 and 3.7 +/- 0.5 times (mean +/- S.E.M., n = 5) in the 24 h and 48 h-fasted liver, respectively. Then, the liver Glc6Pase mRNA was decreased to the level of the fed liver after 72 and 96 h of fasting (1.0 +/- 0.3 and 1.4 +/- 0.3). In the kidney, Glc6Pase mRNA abundance was increased by 2.7 +/- 1.0 and 5 +/- 1.2 times at 24 and 48 h of fasting, respectively. Then, it plateaued at the level of the 48 h fasted kidney after 72 h and 96 h of fasting (4.5 +/- 1.0 and 4.3 +/- 1.0). After 24 and 48 h-refeeding, the abundance of Glc6Pase mRNA in 48 h-fasted rats was decreased to the level found in the liver and kidney of fed rats. The time course of the activity of Glc6Pase catalytic subunit during fasting and refeeding was strikingly parallel to the time course of Glc6Pase mRNA level in respective tissues. These data strongly suggest that the differential expression of Glc6Pase activity in liver and kidney in the course of fasting may be accounted for by the respective time course of mRNA abundance in both organs.
Chronic renal adaptation to dietary deprivation of Pi is accompanied by increased Na+/Pi co-transport across the brush border membrane of the renal proximal tubule. The increased activity of this co-transport system depends on de novo protein synthesis and insulin. The present study used normal and diabetic rats to determine if the endosomal pool of Na+/Pi co-transporters was altered by Pi deprivation and the possible role of insulin. In response to 5 days of dietary Pi deprivation there was a significant increase in endosomal Na+/Pi co-transport in control rats but there was no change in diabetic rats. The increase in endosomal Pi uptake was restored in diabetic rats treated with exogenous insulin. Na(+)-independent Pi uptake and proline uptake remained unchanged in all groups. The changes in endosomal Na+/Pi co-transport correlated with the abundance of the specific Na+/Pi co-transporter protein, as determined by Western blots. The pattern of endosomal changes paralleled that observed in brush border membranes. One possibility consistent with these findings is that the endosomal fraction contains newly synthesized Na+/Pi co-transporters targeted for delivery to the apical brush border membrane. Increased synthesis and delivery is required to maintain the adaptation to chronic Pi deprivation.
Work on the glucose-6-phosphatase system has intensified and diversified extensively in the past 3 years. The gene for the catalytic unit of the liver enzyme has been cloned from three species, and regulation at the level of gene expression is being studied in several laboratories worldwide. More than 20 sites of mutation in the catalytic unit protein have been demonstrated to underlie glycogenesis type 1a. inhibition of glucose-6-P hydrolysis by several newly identified competitive and time-dependent, irreversible inhibitors has been demonstrated and in several instances the predicted effects on liver glycogen formation and/or breakdown and on blood glucose production have been shown. Refinements in and additions to the presently dominant "substrate transport-catalytic unit" topological model for the glucose-6-phosphatase system have been made. A new model alternative to this, based on the "combined conformational flexibility-substrate transport" concept, has emerged. Experimental evidence for the phosphorylation of glucose in liver by high-K(m),glucose enzyme(s) in addition to glucokinase has continued to emerge, and new in vitro evidence supportive of biosynthetic functions of the glucose-6-phosphatase system in this role has appeared. High levels of multifunctional glucose-6-phosphatase have been shown present in pancreatic islet beta cells. Glucose-6-P has been established as the likely insulin secretagog in beta cells. Interesting differences in the temporal responses of glucose-6-phosphatase in kidney and liver have been demonstrated. An initial attempt is made here to meld the hepatic and pancreatic islet beta-cell glucose-6-phosphatase systems, and to a lesser extent the kidney tubular and small intestinal mucosal glucose-6-phosphatase systems into an integrated, coordinated mechanism involved in whole-body glucose homeostasis in health and disease.
We report the sequence of a human cDNA that encodes a 46 kDa transmembrane protein homologous to bacterial transporters for phosphate esters. This protein presents at its carboxy terminus the consensus motif for retention in the endoplasmic reticulum. Northern blots of rat tissues indicate that the corresponding mRNA is mostly expressed in liver and kidney. In two patients with glycogen storage disease type Ib, mutations were observed that either replaced a conserved Gly to Cys or introduced a premature stop codon. The encoded protein is therefore most likely the glucose 6-phosphate translocase that is functionally associated with glucose-6-phosphatase.
Parathyroid hormone (PTH) regulates serum calcium and phosphate levels, which, in turn, regulate PTH secretion and mRNA levels. PTH mRNA levels are markedly increased in rats fed low calcium diets and decreased after low phosphate diets, and this effect is post-transcriptional. Protein-PTH mRNA binding studies, with parathyroid cytosolic proteins, showed three protein-RNA bands. This binding was to the 3'-untranslated region (UTR) of the PTH mRNA and was dependent upon the terminal 60 nucleotides. Parathyroid proteins from hypocalcemic rats showed increased binding, and proteins from hypophosphatemic rats decreased binding, correlating with PTH mRNA levels. There is no parathyroid cell line; however, a functional role was provided by an in vitro degradation assay. Parathyroid proteins from control rats incubated with a PTH mRNA probe led to an intact transcript for 40 min; the transcript was intact with hypocalcemic proteins for 180 min and with hypophosphatemic proteins only for 5 min. A PTH mRNA probe without the 3'-UTR, or just the terminal 60 nucleotides, incubated with hypophosphatemic proteins, showed no degradation at all, indicating that the sequences in the 3'-UTR determine PTH mRNA degradation. Hypocalcemia and hypophosphatemia regulate PTH gene expression post-transcriptionally. This correlates with binding of proteins to the PTH mRNA 3'-UTR, which determines its stability.
Phosphate is an active participant in energy metabolism, and its deficiency has been associated with changes in insulin sensitivity and glucose tolerance. In the present study, we have investigated insulin secretion and glucose tolerance in individuals with moderate and acute phosphate deprivation and in patients with chronic hypophosphatemia. The individuals with dietary phosphate deprivation, evidenced by a significant reduction in phosphaturia from 232.3 +/- 37.1 to 56.8 +/- 23.9 mmol/24 hours, but with normal serum levels of inorganic phosphorus, presented circulating glucose and insulin levels similar to those of the pre-dietary period during the oral and intravenous glucose tolerance tests. In contrast, patients with chronic hypophosphatemia (inorganic phosphorus < 0.65 mmol/l) presented in hyperinsulinemia during the postabsorptive state and during the early and late phases of insulin secretion after the oral and intravenous glucose stimulus. The physiological response of a fall in serum phosphate after glucose administration observed in individuals with chronic hypophosphatemia was similar to that of normal individuals. The presence of hyperinsulinemia both basally and after glucose stimulation, with normal glycemia, in phosphate-depleted individuals suggests that this condition is associated with reduced insulin sensitivity. However, severe phosphate deprivation is necessary for the manifestation of this undesirable association. The deviation of phosphate to the intracellular medium occurring after glucose administration in hypophosphatemic individuals is similar to that of normal individuals and explains the occurrence of severe hypophosphatemia in malnourished hypophosphatemic individuals when submitted to parenteral refeeding.
There are differences in the kinetic properties of the liver and brain microsomal glucose-6-phosphate transport systems suggesting the possibility of tissue specific isoforms. The availability of a human liver cDNA sequence which is mutated in patients with deficiencies of liver microsomal glucose-6-phosphate transport (glycogen storage disease 1b) made it possible to determine if a brain isoform exists. Northern blots of liver and brain RNA revealed that the mRNA of the brain form is slightly longer than the liver one. Isolation and sequencing of the respective human brain cDNA revealed that the brain protein has an additional 22 amino acid sequence.
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