Rate Of Hepatic Gluconeogenesis Research Articles

195-OR: A Novel 13C5 Glutamine Tracer Method (Q Flux) Reveals a Key Role of Succinyl CoA Anaplerosis in Promoting Increased Rates of Hepatic Gluconeogenesis during Hyperglucagonemia

Hyperglucagonemia is a hallmark of type 2 diabetes mellitus (T2DM) and contributes to increased rates of endogenous glucose production (EGP) , mostly through increased rates of hepatic gluconeogenesis (GNG) , but the anaplerotic pathways and respective substrates that support this process are unclear. To address this question, we developed a novel and extensively validated 13C5 glutamine-based in vivo metabolic flux analysis method (Q Flux) , which allows for quantitation of both the relative and absolute rates of hepatic gluconeogenesis from pyruvate (via pyruvate carboxylase [PC]) , succinyl CoA (via methylmalonyl CoA mutase [MUT]) , glutamine (via glutaminase [GLS2]) , and glycerol. Male Sprague-Dawley rats (∼300 g) were infused for 90 minutes with either low-dose (LD) glucagon (1.25 ng/[kg-min]) or high-dose (HD) glucagon (ng/[kg-min]) , plus insulin (0.5 mU/[kg-min]) , somatostatin (4 μg/[kg-min]) , and 13C5 glutamine (3 μmol/[kg-min]) after a 16-hour fast to deplete hepatic glycogen. HD animals displayed significantly increased plasma glucose concentrations and EGP relative to LD controls. Analyses of liver tissues collected at sacrifice revealed that in LD animals, 70% of mitochondrial GNG was from pyruvate, 15% from succinyl CoA, and 15% from glutamine. In HD animals, 59% of mitochondrial GNG was derived from pyruvate (p=0.vs. LD) , 26% from succinyl CoA (p=0.0vs. LD) , and 15% from glutamine (p=0.99 vs. LD) . Conclusion: We find an unexpected role for succinyl CoA anaplerosis and methylmalonyl CoA mutase flux in supporting increased rates of mitochondrial GNG during hyperglucagonemia. Furthermore, relative contributions of glutamine to hepatic gluconeogenesis were unchanged, contrary to previous reports. Taken together, these results identify novel targets to reduce hyperglucagonemia-induced increases in rates of gluconeogenesis and hyperglycemia in T2DM. Disclosure B.T.Hubbard: None. G.I.Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc. , Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. .

Silencing of FGF‐21 expression promotes hepatic gluconeogenesis and glycogenolysis by regulation of the STAT3–SOCS3 signal

Insulin resistance is a metabolic disorder associated with type2 diabetes. Recent reports have shown that fibroblast growth factor-21 (FGF-21) plays an important role in the progression of insulin resistance. However, the biochemical and molecular mechanisms by which changes in FGF-21 activation result in changes in the rates of hepatic gluconeogenesis and glycogenolysis remain to be elucidated. In this study, we developed adenovirus-mediated shRNA against FGF-21 to inhibit FGF-21 expression in ApoE knockout mice. Using this mouse model, we determined the effects of FGF-21 knockdown invivo on hepatic glucose production, gluconeogenesis and glycogenolysis, and their relationship with the signal transducer and activator of transcription3 (STAT3)/suppressor of cytokine signaling3 (SOCS3) signal pathways. We show that liver-specific knockdown of FGF-21 in high-fat diet-fed ApoE knockout mice resulted in a 39% increase in glycogenolysis and a 75% increase in gluconeogenesis, accompanied by increased hepatic expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Furthermore, FGF-21 knockdown decreased phosphorylation of STAT3 and SOCS3 expression in high-fat diet-fed mice. Our data suggest that hepatic FGF-21 knockdown increases gluconeogenesis and glycogenolysis by activation of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase via the STAT3/SOCS3 pathway, ultimately leading to exacerbation of hepatic insulin resistance.

A method for the determination of glucose synthesis in isolated bovine hepatocytes

A simple method for determining glucose synthesis from radiolabeled precursors in isolated bovine hepatocytes using ion exchange resins is presented. This method allows processing of multiple small volume samples using suspensions of anion and cation exchange resins rather than traditional stacked column separation methods. Hepatocytes were isolated from calf liver by collagenase perfusion of the caudate lobe and were incubated with 14C-labeled lactate or propionate as gluconeogenic substrates. Glucose synthesis was determined in an aliquot of cell suspension that was vortexed with a slurry of anion exchange (acetate form) resin, followed by a slurry of cation exchange resin. Newly synthesized, labeled glucose was recovered in the supernatant after centrifugation and quantitated by scintillation counting. Using this procedure, more than 98% of the unused labeled precursor was bound to the ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant. Pretreatment of hepatocyte suspensions with glucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for measurement of newly synthesized glucose. This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substrate concentration or the addition of glucagon or fatty acids to the hepatocyte incubations.

Effects of prolonged treatment of lactating goats with bovine somatotropin on aspects of adipose tissue and liver metabolism

The effects of prolonged (22 weeks) treatment of lactating goats with bovine somatotropin on the metabolism of adipose tissue and liver has been investigated. Somatotropin treatment resulted in smaller adipocytes, decreased rate of fatty acid synthesis and decreased total acetyl-CoA carboxylase activity of adipocytes, but with no change in the proportion of this enzyme in the active state. The rate of acylglycerol glycerol synthesis from glucose of adipocytes tended to decrease as did total glucose utilization by the tissue. Glucose conversion to lactate was unchanged by somatotropin treatment but glucose conversion to other products was decreased. Maximum response of adipose tissue to insulin was unchanged but the sensitivity to insulin decreased on somatotropin treatment. Treatment with somatotropin had no effect on basal lipolysis and decreased maximum response to the beta-agonist isoproterenol, but this probably reflects the rate of isoproterenol-stimulated lipolysis varying with cell volume in adipocytes. No apparent change in response either to alpha 2-adrenergic agonists or to adenosine was apparent. The number of beta-adrenergic receptors was unchanged in adipocyte membranes but the number of alpha 2-adrenergic receptors increased. The rate of hepatic gluconeogenesis in vitro, the activity of key gluconeogenic enzymes and the modulation of the rate of gluconeogenesis by butyrate were unchanged except for the effect of this latter agent on gluconeogenesis from propionate. Hepatic ketogenic activity, as indicated by the activity of carnitine palmitoyl-CoA-transferase-1 and the concentrations of carnitine and acyl carnitines, was unchanged by treatment. Thus at the end of a prolonged period of treatment with somatotropin in lactating goats, lipid synthesis in adipose tissue is still decreased but no effects on liver lipid and carbohydrate metabolism were apparent.

Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation.

Conceptus energy and nitrogen demands in late pregnancy are mostly met by placental uptake of maternal glucose and amino acids. The resulting 30 to 50% increase in maternal requirements for these nutrients is met partly by increased voluntary intake and partly by an array of maternal metabolic adaptations. The latter include increased hepatic gluconeogenesis from endogenous substrates, decreased peripheral tissue glucose utilization, increased fatty acid mobilization from adipose tissue, and, possibly, increased amino acid mobilization from muscle. Within 4 d of parturition, mammary demands for glucose, amino acids, and fatty acids are several-fold those of the pregnant uterus before term. Even unusual postparturient increases in voluntary intake cannot satisfy this increased nutrient demand. Therefore, rates of hepatic gluconeogenesis and adipose fat mobilization are greatly accelerated. Concomitant changes in amino acid metabolism include increased hepatic protein synthesis and, possibly, decreased amino acid catabolism, and increased peripheral mobilization of amino acids. Insulin resistance in adipose tissue and muscle, developed during late pregnancy, continues postpartum; adipose lipolytic responsiveness and sensitivity to adrenergic agents are increased postpartum beyond their levels during late pregnancy. Before parturition, these homeorhetic adjustments may be coordinated with lactogenesis by increased secretion of estradiol and prolactin. Their amplification and reinforcement at and soon after parturition may be regulated mostly by somatotropin.

Reduction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is due to decreased mRNA levels

We have previously shown that the rate of hepatic gluconeogenesis is reduced in TCDD-treated rats and that this decrease in carbohydrate production is associated with a dose-dependent reduction of the activity of PEPCK, the rate limiting enzyme of gluconeogenesis. This derailment of glucose metabolism has been suggested to be the critical lesion in acute TCDD toxicity. To further elucidate the mechanism of decreased PEPCK activity we performed Northern blot analyses using a cDNA probe complementary to a portion of the mRNA coding for PEPCK. We have demonstrated that 4 and 8 days after TCDD treatment (125 μg/kg, p.o.) liver PEPCK mRNA in Sprague-Dawley rats was decreased to very low levels as compared to vehicle-treated and pair-fed control animals. This decline of PEPCK mRNA was paralleled by decreased levels of PEPCK protein, as revealed by Western blot analyses and was accompanied by a reduction in the enzymatic activity of PEPCK. These results indicate thatthe decrease of PEPCK activity by TCDD is most likely the result of decreased expression of the PEPCK gene. These together with previous results also suggest that many of the physiological responses occurring in TCDD-treated animals (reduced feed intake, decreased insulin, increased corticosterone, increased glucagon and cAMP levels) which would normally stimulate PEPCK gene expression, are ineffective. Furthermore tryptophan 2,3-dioxygenase (TdO) activity, which is regulated in a very similar fashion to PEPCK activity, is also reduced after TCDD treatment, suggesting a common mechanism by which TCDD alters the regulation of these enzymes. P-450 1A1 mRNA and related EROD activity were maximally induced under the conditions of these experiments and represent a positive control for TCDD-related alterations of gene expression. However, because of differences in the dose-response characteristics of TCDD-induced reduction of PEPCK activity and induction of EROD activity an involvement of the Ah receptor in the reduction of PEPCK activity cannot be postulated.

Gluconeogenesis in hepatocytes determined with [2- 13C] acetate and quantitative 13C NMR spectroscopy

1. 1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using [2- 13C]acetate and 13C magnetic resonance spectroscopy. 2. 2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the 13C distribution in glucose where the overall 13C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean 13C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30. 3. 3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20. 4. 4. With this carbon exchange factor and the approximated 13C enrichment of acetyl-CoA the intramitochondrial 13C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated. 5. 5. Since acetate was metabolized solely in liver cells the 13C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate. 6. 6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively. 7. 7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the 13C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%. 8. 8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz [Katz J. (1985) Am. J. Physiol. 248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis. 9. 9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.

Ketone formation in the intestinal mucosa of infant rats

The intestinal mucosa of infant rats was found to produce ketones when incubated in Krebs-Ringer-Bicarbonate solution. No production was found in weaned rats. Ketogenesis could be inhibited by D-carnitine or tetradecylglycidic acid (TDGA) an inhibitor of long-chain acylcarnitine transferase, suggesting that ketone production is due to a large extent to break-down of long-chain fatty acids. It is considered possible that both ketones and glucose (also produced by the infant mucosa) serve as substrates for the muscular part of the intestine.The main energy source of the newborn rat is fat (1). Glucose is in short supply and hence the rate of hepatic gluconeogenesis is very high in the suckling period (2). Recently, we have been able to show that gluconeogenesis also occurs in the intestinal mucosa of infants (3). This surprising finding suggested that the mucosa in infancy behaves to some extent like the liver. Hence it seemed appropriate to examine whether the mucosa is capable of producing ketones, since, in the rat at least, hepatic ketogenesis rises about 50fold after birth and stays elevated throughout the suckling period (5). It has also been shown that gluconeogenesis in the liver is dependent on fatty acid oxidation and thus carnitine metabolism (4). The activity of the carnitine acyltransferases of the intestinal mucosa is highest in infant rats (6).

Contribution of hepatic fatty acid oxidation and exogenous galactose supply to the regulation of glucose homeostasis in the newborn rabbit.

The inhibition of hepatic gluconeogenesis by 3-mercaptopicolinic acid (3-MPA) leads to a profound hypoglycemia in both suckling and fasting 24-hour-old rabbits. This hypoglycemia is totally reversed 1 h after the intragastric injection of an amount of galactose corresponding to the one ingested daily by the suckling newborns. This results from an active gluconeogenesis from galactose, which bypasses the site of inhibition by 3-MPA. However, this amount of galactose is not sufficient to maintain a normal blood glucose concentration for a long time, since 3 h after galactose injection, the blood glucose concentrations of newborn rabbits return to hypoglycemic values. When hepatic fatty acid oxidation is inhibited by 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (POCA), 24-hour-old fasting rabbits become rapidly hypoglycemic secondary to a decrease in liver gluconeogenesis. The rate of hepatic gluconeogenesis is totally restored by giving medium-chain triglycerides, and the 24-hour-old rabbits become normoglycemic.

Conversion of Alanine, Aspartate and Lactate to Glucose and CO2 in Liver from Stress-Susceptible and Stress-Resistant Pigs

Rates of conversion of lactate, alanine and aspartate to glucose and oxidation of each to CO2 were determined in incubated liver slices from nine stress-susceptible (SS) and seven stress-resistant (SR) Yorkshire pigs ranging in body weight from 24 to 54 kg. Pigs were screened for stress susceptibility by exposure to halothane at 7 weeks of age. Stress was minimized before slaughter, and liver samples were obtained immediately after death. Rates of lactate and aspartate conversion to glucose were not significantly different between pig types. Mean rates of lactate conversion to glucose in livers of SS and SR pigs were 637 and 413 nmoles/(100 mg X 2 hours), respectively. Mean rates of aspartate conversion to glucose were 441 and 540 nmoles/(100 mg X 2 hours) in SS and SR pigs, respectively. Alanine conversion to glucose in livers of SS pigs was slower than that in SR pigs [527 and 813 nmoles/(100 mg X 2 hours), respectively]. Rates of hepatic gluconeogenesis from lactate probably do not predispose SS pigs to the lactic acidosis observed during the porcine stress syndrome. Rates of lactate, alanine and aspartate oxidation to CO2 in livers of SS pigs were 61, 59 and 76%, respectively, of the rates observed in SR pigs. Decreased rates of substrate oxidation to CO2 may contribute to the development of the syndrome in SS pigs.

Fluctuation of hepatic enzymes important in glucose metabolism in relation to thyroid function.

Enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate kinase and of mitochondrial glycerolphosphate dehydrogenase were studied in the livers of rats under the following states of thyroid function: 131I‐thyroidectomized rats, 131I‐thyroidectomized rats treated with triiodothyronine, normal rats treated with excess of triiodothyronine, and normal untreated rats. In the thyroidectomized group all of the enzyme activities listed were significantly diminished. After treatment with a single dose of triiodothyronine the activities of pyruvate carboxylase and of pyruvate kinase were restored to normal whereas phosphoenolpyruvate carboxykinase and — most markedly — glycerolphosphate dehydrogenase were raised above the normal level. After repeated administration of triiodothyronine to normal rats a further increase in the activity of pyruvate carboxylase, phosphoenolpyruvate carboxykinase and of glycerolphosphate dehydrogenase was observed.The activities of glycerolphosphate dehydrogenase and of pyruvate carboxylase 70 h following triiodothyronine injection into hypothyroid rats were more than 50% lower after treatment with actinomycin C suggesting that the increase in enzyme activities was due to increased synthesis of enzyme protein. Phosphoenolpyruvate carboxykinase activity was not changed significantly by actinomycin C.The possible significance of these thyroid hormone dependent enzyme fluctuations in controlling the overall rate of hepatic gluconeogenesis is discussed.