Contribution Of Gluconeogenesis Research Articles

Heptanoate Improves Compensatory Mechanism of Glucose Homeostasis in Mitochondrial Long-Chain Fatty Acid Oxidation Defect.

Defects in mitochondrial fatty acid β-oxidation (FAO) impair metabolic flexibility, which is an essential process for energy homeostasis. Very-long-chain acyl-CoA dehydrogenase (VLCADD; OMIM 609575) deficiency is the most common long-chain mitochondrial FAO disorder presenting with hypoglycemia as a common clinical manifestation. To prevent hypoglycemia, triheptanoin-a triglyceride composed of three heptanoates (C7) esterified with a glycerol backbone-can be used as a dietary treatment, since it is metabolized into precursors for gluconeogenesis. However, studies investigating the effect of triheptanoin on glucose homeostasis are limited. To understand the role of gluconeogenesis in the pathophysiology of long-chain mitochondrial FAO defects, we injected VLCAD-deficient (VLCAD-/-) mice with 13C3-glycerol in the presence and absence of heptanoate (C7). The incorporation of 13C3-glycerol into blood glucose was higher in VLCAD-/- mice than in WT mice, whereas the difference disappeared in the presence of C7. The result correlates with 13C enrichment of liver metabolites in VLCAD-/- mice. In contrast, the C7 bolus significantly decreased the 13C enrichment. These data suggest that the increased contribution of gluconeogenesis to the overall glucose production in VLCAD-/- mice increases the need for gluconeogenesis substrate, thereby avoiding hypoglycemia. Heptanoate is a suitable substrate to induce glucose production in mitochondrial FAO defect.

In Vivo Investigation of Glucose Metabolism in Idiopathic and PRKN-Related Parkinson's Disease.

Alterations in mitochondrial dysfunction have been implicated in the pathogenesis of Parkinson's disease (PD). Mitochondrial energy production is linked to glucose metabolism, and diabetes is associated with PD. However, studies investigating glucose metabolism in vivo in genetically stratified PD patients and controls have yet to be performed. The objectives of this study were to explore glucose production, gluconeogenesis, and the contribution of gluconeogenesis to glucose production in idiopathic and PRKN PD compared with healthy controls with state-of-the-art biochemical methods. We applied a dried-blood sampling/gas chromatography/mass spectrometry approach to monitor fluxes in the Cori cycle in vivo. The contribution of gluconeogenesis to total glucose production is increased in idiopathic PD patients (n=33), but not in biallelic PRKN mutation carriers (n=5) compared with healthy controls (n=13). We provide first-time in vivo evidence for alterations in glucose metabolism in idiopathic PD, in keeping with the epidemiological evidence for an association between PD and diabetes. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Insulin Pulse Characteristics and Insulin Action in Non-diabetic Humans.

Pulsatile insulin secretion is impaired in diseases such as type 2 diabetes that are characterized by insulin resistance. This has led to the suggestion that changes in insulin pulsatility directly impair insulin signaling. We sought to examine the effects of pulse characteristics on insulin action in humans, hypothesizing that a decrease in pulse amplitude or frequency is associated with impaired hepatic insulin action. We studied 29 nondiabetic subjects on two occasions. On 1 occasion, hepatic and peripheral insulin action was measured using a euglycemic clamp. The deuterated water method was used to estimate the contribution of gluconeogenesis to endogenous glucose production. On a separate study day, we utilized nonparametric stochastic deconvolution of frequently sampled peripheral C-peptide concentrations during fasting to reconstruct portal insulin secretion. In addition to measuring basal and pulsatile insulin secretion, we used approximate entropy to measure orderliness and Fourier transform to measure the average, and the dispersion of, insulin pulse frequencies. In univariate analysis, basal insulin secretion (R2 = 0.16) and insulin pulse amplitude (R2 = 0.09) correlated weakly with insulin-induced suppression of gluconeogenesis. However, after adjustment for age, sex, and weight, these associations were no longer significant. The other pulse characteristics also did not correlate with the ability of insulin to suppress endogenous glucose production (and gluconeogenesis) or to stimulate glucose disappearance. Overall, our data demonstrate that insulin pulse characteristics, considered independently of other factors, do not correlate with measures of hepatic and peripheral insulin sensitivity in nondiabetic humans.

1787-P: Insulin Pulse Characteristics Do Not Alter Hepatic Glucose Metabolism and Insulin Action

Insulin is secreted into the portal vein in a pulsatile manner. Portal vein insulin concentrations directly and rapidly alter endogenous glucose production (EGP). Pulse frequency has been inversely correlated with hepatic insulin action in some, but not all, human studies. Indeed, increased pulse disorderliness, decreased secretory burst mass and decreased pulse frequency have been observed in type 2 diabetes, in aging and in obesity. In dogs, disruption of pulsatile insulin secretion is associated with defects in hepatic insulin action. As part of a series of experiments examining the mechanisms underlying prediabetes, we studied 29 nondiabetic subjects (BMI 28 ± 1 Kg/m2) on 2 occasions. On one occasion, EGP was measured using [3-3H]glucose after an overnight fast (14.2 ± 0.5μmol/kg/min) and during a euglycemic clamp (6.8 ± 1.1μmol/kg/min). The deuterated water method was used to estimate the contribution of gluconeogenesis to EGP. On a separate study day we utilized nonparametric stochastic deconvolution of frequently sampled peripheral C-peptide concentrations during fasting to reconstruct portal insulin secretion. In addition to measuring basal and pulsatile insulin secretion, we used Approximate Entropy (ApEn) to measure orderliness and a Fourier transform to measure the average and the dispersion of insulin pulse frequency. The gradient of Loge EGP vs. Loge insulin in each individual (-1.2 ± 0.5) was used as a measure of hepatic insulin action. Basal secretion (126 ± 8 pmol/min, R2=0.04), pulse amplitude (41 ± 38 pmol/min, R2=0.02), ApEn (1.14 ± 0.03, R2=0.01), pulse interval (5.7 ± 0.4 min, R2<0.01) and frequency dispersion (0.47 ± 0.01, R2<0.01) did not correlate (all p > 0.10) with hepatic insulin action. Suppression of gluconeogenesis by insulin also did not correlate with fasting insulin pulse characteristics. Overall, our data demonstrate that insulin pulse characteristics do not directly alter the sensitivity of EGP and gluconeogenesis to insulin in humans. Disclosure M.C. Laurenti: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. R.T. Varghese: None. J. Jones: None. C. Barosa: None. R.A. Rizza: None. A. Matveyenko: None. G. De Nicolao: None. C. Cobelli: None. A. Vella: Advisory Panel; Self; vTv Therapeutics, Zealand Pharma A/S. Research Support; Self; Novo Nordisk A/S, Xeris Pharmaceuticals, Inc. Funding National Institutes of Health (DK78646, DK116231)

MON-159 Increased Gluconeogenesis and Lower Glycogenolysis in Patients with Non-Selective Compared Selective Insulin Resistance

Type 2 diabetes is characterized by increased hepatic glucose production and selective insulin resistance that occurs downstream of the insulin receptor. However, the differential pathways and exact molecules implicated in selective insulin signaling are unclear. To elucidate the hepatic mechanistic pathways affected by selective versus non-selective insulin resistance, we compared basal rates of hepatic glucose production and its components, gluconeogenesis and glycogenolysis, in patients with severe non-selective insulin resistance (insulin receptor mutations (INSR)) to patients with selective insulin resistance (lipodystrophy and type 2 diabetes). In 7 patients with INSR, 14 with lipodystrophy and 9 with type 2 diabetes, we measured rates of hepatic glucose production (via [6,6-2H2] glucose) and fractional gluconeogenesis (via 2H2O) after an overnight fast. Absolute rates of gluconeogenesis were calculated as the product of fractional gluconeogenesis and hepatic glucose production, and glycogenolysis were calculated as hepatic glucose production minus gluconeogenesis. In patients with INSR, lipodystrophy and type 2 diabetes, HbA1c (8.2 ± 2.8, 8.4 ± 1.9, 7.4 ± 0.9%) and fasting glucose (120 ± 70, 159 ± 55, 127 ± 32 mg/dl) were comparable (P>0.05). Fasting serum insulin was higher in INSR vs. lipodystrophy and type 2 diabetes (332 ± 355, 48 ± 50, 50 ± 16 μU/ml; P<0.01). Patients with INSR and lipodystrophy had higher hepatic glucose production vs. type 2 diabetes (24.0 ± 4.0, 24.1 ± 6.2, 15.8 ± 4.3 µmol/kgLEAN/min; P<0.01). Fractional gluconeogenesis was higher in INSR vs. lipodystrophy and type 2 diabetes (77.1 ± 9.4, 55.4 ± 11.5, 59.1 ± 5.9%; P<0.01). Absolute gluconeogenesis was highest in INSR, intermediate in lipodystrophy, and lowest in type 2 diabetes (19.1 ± 4.4, 13.5 ± 4.2, 9.5 ± 1.9 µmol/kgLEAN/min; P<0.01). Glycogenolysis was lowest in INSR, highest in lipodystrophy, and intermediate in type 2 diabetes (4.9 ± 2.2 vs. 10.6 ± 4.8, 6.3 ± 2.6 µmol/kgLEAN/min; P<0.01). Higher hepatic glucose production in lipodystrophy vs. type 2 diabetes is consistent with higher hepatic insulin resistance in patients with lipodystrophy, but comparable fractional rates of gluconeogenesis and glycogenolysis confirm similar mechanisms of selective post-receptor insulin resistance. By contrast, the markedly higher contribution of gluconeogenesis to hepatic glucose production in INSR may reflect complete loss of insulin signaling for glycogen accumulation and/or suppression of gluconeogenesis. Further studies should evaluate whether greater substrate and fuel availability for gluconeogenesis, and/or lower hepatic glycogen stores are important determinants of the components of glucose production in patients with non-selective insulin resistance due to INSR mutation.

Acute behavioural and physiological responses of sheep to S/C ivermectin injection

Acute behavioural and physiological responses of sheep to S/C ivermectin injection

Lysine post‐translational modification of glyceraldehyde‐3‐phosphate dehydrogenase regulates hepatic and systemic metabolism

Reciprocal regulation of hepatic glycolysis and gluconeogenesis contributes to systemic metabolic homeostasis. Recent evidence from lower order organisms has found that reversible post-translational modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), particularly acetylation, contributes to the reciprocal regulation of glycolysis/gluconeogenesis. However, whether this occurs in mammalian hepatocytes in vitro or in vivo is unknown. Several proteomics studies have identified 4 lysine residues in critical regions of mammalian GAPDH that are altered by multiple post-translational modifications. In FAO hepatoma cells, mutation of all 4 lysine residues (4K-R GAPDH) to mimic their unmodified state reduced GAPDH glycolytic activity and glycolytic flux and increased gluconeogenic GAPDH activity and glucose production. Hepatic expression of 4K-R GAPDH in mice increased GAPDH gluconeogenic activity and the contribution of gluconeogenesis to endogenous glucose production in the unfed state. Consistent with the increased reliance on the energy-consuming gluconeogenic pathway, plasma free fatty acids and ketones were elevated in mice expressing 4K-R GAPDH, suggesting enhanced lipolysis and hepatic fatty acid oxidation. In normal mice, food withholding and refeeding, as well as hormonal regulators of reciprocal glycolysis/gluconeogenesis, such as insulin, glucagon, and norepinephrine, had no effect on global GAPDH acetylation. However, GAPDH acetylation was reduced in obese and type 2 diabetic db/db mice. These findings show that post-translational modification of GAPDH lysine residues regulates hepatic and systemic metabolism, revealing an unappreciated role for hepatic GAPDH in substrate selection and utilization.-Bond, S. T., Howlett, K. F., Kowalski, G. M., Mason, S., Connor, T., Cooper, A., Streltsov, V., Bruce, C. R., Walder, K. R., McGee, S. L. Lysine post-translational modification of glyceraldehyde-3-phosphate dehydrogenase regulates hepatic and systemic metabolism.

Effect of renal sympathetic denervation on hepatic glucose metabolism and blood pressure in a rat model of insulin resistance.

Hypertension and diabetes are associated with impaired glucose metabolism and insulin resistance. Chronic activation of the sympathetic nervous system may contribute to either condition. The purpose of this study was to investigate the effect of renal denervation on glucose kinetics and insulin signal pathways in high-fat diet (HFD)-fed rats. We examined the effects of renal denervation on glucose kinetics and insulin sensitivity in HFD-fed rats with a hyperinsulinemic-euglycemic clamp technique combined with [3-H] glucose and [U-C]-lactate as a tracer. We also analyzed in vivo the flux through glucose-6-phosphatase and the relative contribution of gluconeogenesis and glycogenolysis in renal denervation rats. In addition, western blotting was used to identify the activities of insulin signaling proteins. Renal denervation in HFD-fed rats markedly decreased blood pressure and hepatic glucose production. Hepatic glucose production reduction in renal-denervation-treated rats includes decrease in glycogenolysis by 18.2% for left renal denervation or 31.9% for bilateral renal denervation and decrease in gluconeogenesis by 16.3% for left renal denervation or 42.8% for bilateral renal denervation. These changes were accompanied by decreased hepatic expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Importantly, renal denervation increased phosphorylation of insulin receptors, insulin receptor substrate-1 and Akt kinase in HFD-fed rats. These data corroborate the notion that renal denervation potentiates hepatic insulin sensitivity.

Mechanisms Underlying the Pathogenesis of Isolated Impaired Glucose Tolerance in Humans

Prediabetes is a heterogeneous disorder classified on the basis of fasting glucose concentrations and 2-hour glucose tolerance. We sought to determine the relative contributions of insulin secretion and action to the pathogenesis of isolated impaired glucose tolerance (IGT). The study consisted of an oral glucose tolerance test and a euglycemic clamp performed in two cohorts matched for anthropometric characteristics and fasting glucose but discordant for glucose tolerance. An inpatient clinical research unit at an academic medical center. Twenty-five subjects who had normal fasting glucose (NFG) and normal glucose tolerance (NGT) and 19 NFG/IGT subjects participated in this study. Subjects underwent a seven-sample oral glucose tolerance test and a 4-hour euglycemic, hyperinsulinemic clamp on separate occasions. Glucose turnover during the clamp was measured using tracers, and endogenous hormone secretion was inhibited by somatostatin. We sought to determine whether hepatic glucose metabolism, specifically the contribution of gluconeogenesis to endogenous glucose production, differed between subjects with NFG/NGT and those with NFG/IGT. Endogenous glucose production did not differ between groups before or during the clamp. Insulin-stimulated glucose disappearance was lower in NFG/IGT (24.6 ± 2.2 vs 35.0 ± 3.6 μmol/kg/min; P = .03). The disposition index was decreased in NFG/IGT (681 ± 102 vs 2231 ± 413 × 10-14 dL/kg/min2 per pmol/L; P < .001). We conclude that innate defects in the regulation of glycogenolysis and gluconeogenesis do not contribute to NFG/IGT. However, insulin-stimulated glucose disposal is impaired, exacerbating defects in β-cell function.

Diabetes-Associated Variation in TCF7L2 Is Not Associated With Hepatic or Extrahepatic Insulin Resistance.

A common genetic variation in TCF7L2 is associated with type 2 diabetes. However, the mechanism by which this occurs remains elusive. In addition to affecting insulin secretion, genetic variation at the TCF7L2 locus may alter insulin action or directly modify hepatic glucose metabolism. We sought to determine whether the diabetes-associated variant in this locus (the T allele of rs7903146) increases fasting endogenous glucose production (EGP), and impairs insulin-induced suppression of EGP and insulin-stimulated glucose disappearance. To address this, we studied individuals who were either homozygous for the diabetes-associated allele (TT) at rs7903146 or were homozygous for the protective allele (CC). Subjects were matched for other anthropometric characteristics and were studied using a euglycemic clamp. EGP and glucose uptake were measured using the tracer dilution technique, and the relative contribution of gluconeogenesis to EGP was quantitated using deuterated water corrected for transaldolase exchange. We report that the diabetes-associated variation in TCF7L2 did not associate with fasting EGP, insulin-induced suppression of EGP, and insulin-induced stimulation of glucose uptake. There was no association with the contribution of gluconeogenesis and glycogenolysis to EGP. These data indicate that genetic variation at TCF7L2 does not predispose an individual to type 2 diabetes by altering either hepatic or extrahepatic insulin action.

Malate as substrate for catabolism and gluconeogenesis during ripening in the pericarp of different grape cultivars

Malate is accumulated in grape pericarp until the start of ripening and then it is dissimilated. One aim of this study was to determine if the potential contribution of stored malate to the substrate requirements of metabolism in ripening grape pericarp is dependent on the cultivar. Two Vitis vinifera L. cultivars which accumulated different amounts of malate and had ripening periods of a different length were compared. The potential contribution of stored malate over the whole period of ripening was around 20 % in the cv. Sagrantino and 29 % in the cv. Pinot Noir. The contribution was higher in Pinot Noir because it contained more malate and had a shorter ripening period. A second aim of this study was to evaluate the contribution of gluconeogenesis to the amount of sugar accumulated in the pericarp. If all the dissimilated malate was utilized by gluconeogenesis, then the maximum contribution of stored malate to the total amount of sugar accumulated in the pericarp over the whole period of ripening was around 2.4 % in Sagrantino and 2.9 % in Pinot Noir. However, the actual contribution was only about 0.1–0.6 % in both cultivars because the majority of stored malate was not utilized by gluconeogenesis. However, it is likely that the actual contribution is much lower. This suggests that the function of gluconeogenesis is not to support accumulation of sugars in the fruits, but probably it plays other roles.

Increased gluconeogenesis in youth with newly diagnosed type 2 diabetes.

The role of increased gluconeogenesis as an important contributor to fasting hyperglycaemia at diabetes onset is not known. We evaluated the contribution of gluconeogenesis and glycogenolysis to fasting hyperglycaemia in newly diagnosed youths with type 2 diabetes following an overnight fast. Basal rates (μmol kg(FFM) (-1) min(-1)) of gluconeogenesis ((2)H2O), glycogenolysis and glycerol production ([(2)H5] glycerol) were measured in 18 adolescents (nine treatment naive diabetic and nine normal-glucose-tolerant obese adolescents). Type 2 diabetes was associated with higher gluconeogenesis (9.2 ± 0.6 vs 7.0 ± 0.3 μmol kg(FFM) (-1) min(-1), p < 0.01), plasma fasting glucose (7.0 ± 0.6 vs 5.0 ± 0.2 mmol/l, p = 0.004) and insulin (300 ± 30 vs 126 ± 31 pmol/l, p = 0.001). Glucose production and glycogenolysis were similar between the groups (15.4 ± 0.3 vs 12.4 ± 1.4 μmol kg(FFM) (-1) min(-1), p = 0.06; and 6.2 ± 0.8 vs 5.3 ± 0.7 μmol kg(FFM) (-1) min(-1), p = 0.5, respectively). After controlling for differences in adiposity, gluconeogenesis, glycogenolysis and glucose production were higher in diabetic youth (p ≤ 0.02). Glycerol concentration (84 ± 6 vs 57 ± 6 μmol/l, p = 0.01) and glycerol production (5.0 ± 0.3 vs 3.6 ± 0.5 μmol kg(FFM) (-1) min(-1), p = 0.03) were 40% higher in youth with diabetes. The increased glycerol production could account for only ~1/3 of substrate needed for the increased gluconeogenesis in diabetic youth. Increased gluconeogenesis was a major contributor to fasting hyperglycaemia and hepatic insulin resistance in newly diagnosed untreated adolescents and was an early pathological feature of type 2 diabetes. Increased glycerol availability may represent a significant source of new carbon substrates for increased gluconeogenesis but would not account for all the carbons required to sustain the increased rates.

Cancer cachexia and diabetes: similarities in metabolic alterations and possible treatment.

Cancer cachexia is a metabolic syndrome featuring many alterations typical of type 2 diabetes (T2D). While muscle wasting is a hallmark of cachexia, epidemiological evidence also supports an accelerated age-related muscle loss in T2D. Insulin resistance manifests in both conditions and impairs glucose disposal and protein anabolism by tissues. A greater contribution of gluconeogenesis to glucose production may limit amino acid availability for muscle protein synthesis, further aggravating muscle loss. In the context of inter-dependence between glucose and protein metabolism, the present review summarizes the current state of knowledge on alterations that may lead to muscle wasting in human cancer. By highlighting the similarities with T2D, a disease that has been more extensively studied, the objective of this review is to provide a better understanding of the pathophysiology of cancer cachexia and to consider potential treatments usually targeted for T2D. Nutritional approaches aimed at stimulating protein anabolism might include specially formulated food with optimal protein and amino acid composition. Because the gradual muscle loss in T2D may be attenuated by diabetes treatment, anti-diabetic drugs might be considered in cachexia treatment. Metformin emerges as a choice candidate as it acts both on reducing gluconeogenesis and improving insulin sensitivity, and has demonstrated tumour suppressor properties in multiple cancer types. Such a multimodal approach to slow or reverse muscle wasting in cachexia warrants further investigation.

A 13C NMR spectrometric method for the determination of intramolecular δ13C values in fructose from plant sucrose samples

Recent developments in (13) C NMR spectrometry have allowed the determination of intramolecular (13) C/(12) C ratios with high precision. However, the analysis of carbohydrates requires their derivatization to constrain the anomeric carbon. Fructose has proved to be particularly problematic because of a byproduct occurring during derivatization and the complexity of the NMR spectrum of the derivative. Here, we describe a method to determine the intramolecular (13) C/(12) C ratios in fructose by (13) C NMR analysis of the acetyl-isopropylidene derivative. We have applied this method to measure the intramolecular (13) C/(12) C distribution in the fructosyl moiety of sucrose and have compared this with that in the glucosyl moiety. Three prominent features stand out. First, in sucrose from both C(3) and C(4) plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C(3) and C(4) plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from Crassulacean acid metabolism (CAM) metabolism have a different profile. These contrasting values can be interpreted as a result of the isotopic selectivity of enzymes that break or make covalent bonds in glucose metabolism, whereas the distinctive (13) C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.

High protein diet maintains glucose production during exercise-induced energy deficit: a controlled trial

BackgroundInadequate energy intake induces changes in endogenous glucose production (GP) to preserve muscle mass. Whether addition provision of dietary protein modulates GP response to energy deficit is unclear. The objective was to determine whether exercise-induced energy deficit effects on glucose metabolism are mitigated by increased dietary protein.MethodsNineteen men ([mean ± SD] 23 ± 2 y, VO2peak 59 ± 5 ml·kg-1·min-1) were divided into three groups, two consuming moderate (MP; 0.9 g protein kg-1 d-1), and one high (HP; 1.8 g protein kg-1 d-1) protein diets (55% energy from carbohydrate) for 11 days. Following 4 days of energy balance (D1-4), energy expenditure was increased for 7 days (D5-12) in all groups. Energy intake was unchanged in two, creating a 1000 kcal d-1 deficit (DEF-MP, DEF-HP; n = 6, both groups), whereas energy balance was maintained in the third (BAL-MP, n = 7). Biochemical markers of substrate metabolism were measured during fasting rest on D4 and D12, as were GP and contribution of gluconeogenesis to endogenous glucose production (fgng) using 4-h primed, continuous infusions of [6,6-2H2]glucose (dilution-method) and [2-13C]glycerol (MIDA technique). Glycogen breakdown (GB) was derived from GP and fgng.ResultsPlasma β-hydroxybutyrate levels increased, and plasma glucose and insulin declined from D4 to D12, regardless of group. DEF-MP experienced decreased plasma GP from D4 to D12 ([mean change ± SD] 0.24 ± 0.24 mg·kg-1·min-1), due to reduced GB from D4 (1.40 ± 0.28 mg·kg-1·min-1) to D12 (1.16 ± 0.17 mg·kg-1·min-1), P < 0.05. Conversely, BAL-MP and DEF-HP sustained GP from D4 to D12 ([mean change ± SD] 0.1 ± 0.5 and 0.0 ± 0.2 mg·kg-1·min-1, respectively) by maintaining GB.ConclusionExercise-induced energy deficit decreased GP and additional dietary protein mitigated that effect.

A simplified mass isotopomer approach to estimate gluconeogenesis rate in vivo using deuterium oxide

We compare a new simplified (2)H enrichment mass isotopomer analysis (MIA) against the laborious hexamethylentetramine (HMT) method to quantify the contribution of gluconeogenesis (GNG) to total glucose production (GP) in calves. Both methods are based on the (2)H labeling of glucose after in vivo administration of deuterium oxide. The (2)H enrichments of plasma glucose at different C-H positions were measured as aldonitrile pentaacetate (AAc) and methyloxime-trimethylsilyl (MoxTMS) derivatives or HMT by gas chromatography/mass spectrometry (GC/MS). Two pre-ruminating fasted Holstein calves (51 kg body mass, BM, age 7 days) received two oral bolus doses of (2)H(2)O (10 g/kg BM, 70 atom% (2)H) at 7:00 h and 11:00 h after overnight food withdrawal. Blood samples for fractional GNG determination were collected at -24 and between 6 and 9 h after the first (2)H(2)O dose. The ratio of (2)H enrichments C5/C2 represents the contribution of GNG to GP. The (2)H enrichment at C2 was calculated based on the ion fragments at m/z 328 (C1-C6) - m/z 187 (C3-C6) of glucose AAc. The (2)H enrichment at C5 was approximated either by averaging the (2)H enrichment at C5-C6 using the ion fragment of glucose MoxTMS at m/z 205 or by conversion of the C5 of glucose into HMT. The fractional GNG calculated by the C5-C6 average (2)H enrichment method (41.4 +/- 6.9%) compared to the HMT method (34.3 +/- 11.4%) was not different (mean +/- SD, n = 6 replicates). In conclusion, GNG can be estimated with less laborious sample preparation by means of our new C5-C6 average (2)H enrichment method using AAc and MoxTMS glucose derivatives.

Impaired Glucose Tolerance and Predisposition to the Fasted State in Liver Glycogen Synthase Knock-out Mice

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.

Responses of the perfused liver of neonatal type 2 diabetic rats to gluconeogenic and ammoniogenic substrates

The responses of livers from rats with type 2 diabetes to alanine (gluconeogenesis and ammonia detoxification) and other gluconeogenic substrates were investigated. The experimental system was the isolated perfused rat liver. Neonatal type 2 diabetes was induced with streptozotocin. Ammoniogenesis from endogenous substrates was 610% higher in livers from diabetic rats when compared to the control condition. Alanine (2.5 mM) ammoniogenesis was 285% higher in livers of diabetic rats. Gluconeogenesis from the following substrates was smaller in the liver of diabetic rats: Alanine (?43.5%), lactate (?28.3%) and glycerol (?30.5%). Pyruvate gluconeogenesis was normal. The high rate of ammoniogenesis explains the moderate hyperammonemia of type 2 diabetic rats. The enzymatic machinery of the gluconeogenic pathway of type 2 diabetic rats seems to be adapted to low rates of glucose removal by extrahepatic tissues. A significant contribution of gluconeogenesis to the fasting hyperglycemia can be expected only by short-term up-regulation mechanisms.

Glyceroneogenesis Is the Dominant Pathway for Triglyceride Glycerol Synthesis in Vivo in the Rat

Triglyceride synthesis in mammalian tissues requires glycerol 3-phosphate as the source of triglyceride glycerol. In this study the relative contribution of glyceroneogenesis and glycolysis to triglyceride glycerol synthesis was quantified in vivo in adipose tissue, skeletal muscle, and liver of the rat in response to a chow diet (controls), 48-h fast, and lipogenic (high sucrose) diet. The rate of glyceroneogenesis was quantified using the tritium ([(3)H(2)]O) labeling of body water, and the contribution of glucose, via glycolysis, was determined using a [U-(14)C]glucose tracer. In epididymal and mesenteric adipose tissue of control rats, glyceroneogenesis accounted for approximately 90% of triglyceride glycerol synthesis. Fasting for 48 h did not alter glyceroneogenesis in adipose tissue, whereas the contribution of glucose was negligible. In response to sucrose feeding, the synthesis of triglyceride glycerol via both glyceroneogenesis and glycolysis nearly doubled (versus controls); however, glyceroneogenesis remained quantitatively higher as compared with the contribution of glucose. Enhancement of triglyceride-fatty acid cycling by epinephrine infusion resulted in a higher rate of glyceroneogenesis in adipose tissue, as compared with controls, whereas the contribution of glucose via glycolysis was not measurable. Glyceroneogenesis provided the majority of triglyceride glycerol in the gastrocnemius and soleus. In the liver the fractional contribution of glyceroneogenesis remained constant (approximately 60%) under all conditions and was higher than that of glucose. Thus, glyceroneogenesis, in contrast to glucose, via glycolysis, is quantitatively the predominant source of triglyceride glycerol in adipose tissue, skeletal muscle, and liver of the rat during fasting and high sucrose feeding.

Sources of hepatic glucose production by 2H2O ingestion and Bayesian analysis of 2H glucuronide enrichment

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after (2)H(2)O ingestion by Bayesian analysis of the position 2 and 5 (2)H-NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 +/- 1.0 kg/m(2); fasting glucose = 4.7 +/- 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 +/- 0.7 kg/m(2); fasting glucose = 7.1 +/- 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% +/- 5%. This uncertainty was associated with a mean signal-to-noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% +/- 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% +/- 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% +/- 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia.