Neonatal hypoglycemia triggered by maternal ritodrine administration is caused by inhibition of gluconeogenesis in the neonatal liver in rodents.

Invited review: Fueling milk production carbon by carbon-Regulation of hepatic glucose production in dairy cattle.

ABSTRACTMaintaining constant blood glucose levels is essential for energizing glucose‐dependent tissues. During the fed state, insulin lowers elevated blood glucose, while in the fasted state, glucagon maintains blood glucose levels through hepatic stimulation of fatty acid oxidation, glycogenolysis, and gluconeogenesis (GNG). The liver plays a crucial role in these metabolic adaptations. Deregulation of GNG is a hallmark of type 2 diabetes mellitus (T2DM), driven by hepatic insulin resistance, elevated glucagon levels, and excess circulating free fatty acids. The glucose metabolism of 8‐ to 12‐week‐old WT and Anxa6 knock‐out (Anxa6−/−) mice was analysed during regular feeding and fasting using indirect calorimetry, tolerance tests and biochemical analysis. Despite normal insulin‐sensitive control of glucose levels and effective glycogen mobilization, Anxa6−/− mice display rapid hypoglycaemia during fasting. This metabolic disarrangement, in particular during the early stages of fasting is characterized by a low respiratory exchange ratio (RER) and increased lipid oxidation during the diurnal period, indicating a reliance on lipid oxidation due to hypoglycaemia. Elevated glucagon levels during fasting suggest deficiencies in GNG. Further analysis reveals that Anxa6−/− mice are unable to utilize alanine for hepatic GNG, highlighting a specific impairment in the glucose‐alanine cycle in fasted Anxa6−/− mice, underscoring the critical role of ANXA6 in maintaining glucose homeostasis under metabolic stress. During fasting, slightly reduced expression levels of alanine aminotransferase 2 (Gpt2) and lactate dehydrogenase (Ldha2), enzymes converting alanine to pyruvate, and the hepatic alanine transporter SNAT4 might contribute to these observations in the Anxa6−/− mice. These findings identify that ANXA6 deficiency causes an inability to maintain glycolytic metabolism under fasting conditions due to impaired alanine‐dependent GNG.

Introduction and Objective: In Type 2 Diabetes (T2D) higher nocturnal glucose production (EGP) results from higher glycogenolysis (GGL) and gluconeogenesis (GNG). Appropriate medications are needed to restore nocturnal EGP. We hypothesized that Dorzagliatin (DG) IND-159103-Glucokinase activator), insulin glargine (IG) (inhibitor of GGL) and metformin (M) (inhibitor of GNG) may lower GGL and GNG thereby lowering EGP in T2D. Methods: Twenty-three T2D subjects (age ~62 yrs, BMI ~32.0Kg/m2, FPG ~7.0mmol/L, HbA1C ~7.0%, ~9 years average diabetes duration) insulin or long acting GLP-1 agonist naive received 6-week monotherapy with either DG=7 (75mg bid), IG=9 (once daily) or M=7 (1.5-2gm daily). All other antidiabetes medications were washed out. Studies were conducted overnight at baseline (BL) and post treatment (PT). EGP, GNG and GGL were estimated at 1,4,7 AM using infusion of [6,6-2H2] glucose (10 PM-7 AM) following ingestion of deuterium labeled water (2H2O) as previously established. Results: The trial is ongoing. EGP was lower with DG (BL vs PT:18.7±9.8vs.15.6±12.5 at 1am;15.4±4.9 vs 14.6±7.0 µmol/KgFFM/min at 7am) primarily due to lower GGL (BL vs PT:10.3±4.2 vs 9.1±7.5 at 1am; 10.4±3.4 vs 7.9±3.5 µmol/kgFFM/min at 7am with similar values at 4am). Similarly, EGP was lower with IG (BL vs PT:19.1±8.0 vs 18.5±10.4 at 1am, 17.9±5.6 vs 16.4±7.3 at 4am and 18.9±5.5 vs 17.1±8.1 µmol/kgFFM/min at 7am). Decrease in EGP was due to lower GGL overnight as hypothesized (9.8±4.2 vs 8.6±3.7 at 4am and 10.2±4.4 vs 9.3± 4.4 µmol/kgFFM/min at 7am). On the contrary, M therapy with the prescribed standard dose was insufficient to lower EGP overnight (BL vs PT: 14.3±8.8 vs 17.5±4.1 at 1am;18.0±5.0 vs 18.2±6.0 at 4am;12.2±4.6 vs 15.6±5.5 µmol/kgFFM/min at 7am). Conclusion: Both DG and IG lowered nocturnal EGP by reducing rates of GGL overnight with a smaller contribution of GNG. M used as monotherapy did not provide adequate lowering of nocturnal EGP. These drugs used in combination may target GGL and GNG overnight thereby reducing nighttime EGP. Disclosure A. Hodhod: None. U.S. Unni: None. B. Gran: None. A. Basu: None. R. Basu: Advisory Panel; Novo Nordisk, Boehringer-Ingelheim. Funding National Institutes of Health (R01 DK029953, R01 DK085516DK059637 (MMPC), and DK020593 (DRTC))

Introduction and Objective: Combination metformin (Met) and liraglutide (Lira) improves glycemia in African American (AA) youth with type 2 diabetes (Y-T2D), but factors influencing medication response are unknown. We constructed the first Met population pharmacokinetic (PK) model for AA Y-T2D and evaluated the relationship of pharmacogenetic (PG) variants of Met+Lira with glycemia. Methods: In a 3-month parallel arm trial of standard release Met (n=12) and Met+Lira (n=10), we measured HbA1c, fasting plasma glucose (FPG), and fractional gluconeogenesis (GNG) pre- and post-intervention. Plasma Met and Lira concentrations were analyzed by LC-MS and dose adjusted. Common PG variants were genotyped in OCT1 (rs628031 and rs622342, Met transporter) and TCF7L2 (rs7903146, transcription factor). A one-compartment first-order absorption and elimination Met PK model was constructed. Linear regression models assessed relationships of PK variables, glycemic outcomes, and PG variants, adjusted for age, sex, and treatment. Results: The Met PK parameters absorption rate constant, clearance (Cl), and volume of distribution were similar by group and not related to glycemia. However, Met+Lira reduced HbA1c more than Met (Δ 1.2±0.8 vs 0.1±0.6%, mean±SD, P < 0.01). OCT1 variants were associated with lower Met Cl (rs628031 G>A, β = -39 L/hr, Adj. R2 = 0.4, P < 0.02) and lower fractional rates of GNG (rs622342 A>C, β = -7.6%, Adj. R2 = 0.5, P < 0.003). TCF7L2 rs7903146 C>T was associated with lower Lira concentrations (β = -36 ng/mL * mg-1, Adj. R2 = 0.4, P = 0.03) and reduced FPG (β = -1.4 mmol/L, Adj. R2 = 0.8, P < 0.002). Conclusion: In AA Y-T2D, genetic variants modulated therapeutic response and glycemic outcomes. OCT1 variants explained up to 50% of the variability in Met Cl and GNG response but did not predict overall glycemia. TCF7L2 variant rs7903146 modulated Lira concentrations and glycemia. Genotyping OCT1 and TCF7L2 in Y-T2D may enable optimization of patient-specific Met and Lira medication regimens. Disclosure S.L. Cantor: None. Y. Zeng: None. F.S. Davis: None. G.P. Thota: None. S.B. Glaros: None. N.A. Macheret: None. I.N. Kacker: None. N. Malandrino: None. L. Mabundo: None. W.D. Figg: None. A.R. Bentley: None. S.T. Chung: None. Funding National Institute of Diabetes & Digestive & Kidney Diseases (ZIA DK 075133)

Higher gluconeogenesis (GNG) contributes to higher nocturnal endogenous glucose production (EGP) in type 2 diabetes (T2D). Studies using 13C magnetic resonance spectroscopy (MRS) have confirmed lower hepatic glycogen content in subjects with T2D than in subjects with no diabetes (ND). We determined the role of glycogen loading (GL) vs nonglycogen loading (NGL) on the contribution of GNG to nocturnal EGP in T2D. In total, 14 subjects with T2D and 15 matched subjects with ND were studied on 2 occasions, with GL (60% carbohydrate) vs NGL (40% carbohydrate) isocaloric meals for 3 days, in random order in the overnight state. [6,6-2H2] glucose was infused to measure EGP, deuterium labelled water was used to measure GNG, and 13C MRS scans were performed in fed and fasted states to measure hepatic glycogen content. Hepatic glycogen content and nocturnal EGP were higher (P < .05) in GL vs NGL in both cohorts. The % GNG to EGP averaged ∼50% in subjects with ND throughout the night after both meals. In contrast, % GNG to nocturnal EGP in T2D was lower with GL vs NGL and matched the pattern observed in subjects with ND with GL lowering overnight rates of GNG in subjects with T2D. Selective targeting of GNG at night with appropriate medications could reduce nocturnal and early morning fasting hyperglycemia and hepatic insulin resistance in people with T2D.

Hepatic gluconeogenesis (GNG) is essential for maintaining euglycemia during prolonged fasting. However, GNG becomes pathologically elevated and drives chronic hyperglycemia in type 2 diabetes (T2D). Lactate/pyruvate is a major GNG substrate known to be imported into mitochondria for GNG. Yet, the subsequent mitochondrial carbon export mechanisms required to supply the extra-mitochondrial canonical GNG pathway have not been genetically delineated. Here, we evaluated the role of the mitochondrial dicarboxylate carrier (DiC) in mediating GNG from lactate/pyruvate. We generated liver-specific DiC knockout (DiC LivKO) mice. During lactate/pyruvate tolerance tests, DiC LivKO decreased plasma glucose excursion and 13C-lactate/-pyruvate flux into hepatic and plasma glucose. In a Western diet (WD) feeding model of T2D, acute DiC LivKO after induction of obesity decreased lactate/pyruvate-driven GNG, hyperglycemia, and hyperinsulinemia. Our results show that mitochondrial carbon export through the DiC mediates GNG and that the DiC contributes to impaired glucose homeostasis in a mouse model of T2D.

The kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG. Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K + . Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters. On a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K + prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation. Renal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K + promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance. The kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.

Non-alcoholic steatohepatitis (NASH) is characterized from its early stages by a profound remodeling of the liver microenvironment, encompassing changes in the composition and activities of multiple cell types and associated gene expression patterns. Hyperpolarized (HP) 13C MRI provides a unique view of the metabolic microenvironment, with potential relevance for early diagnosis of liver disease. Previous studies have detected changes in HP 13C pyruvate to lactate conversion, catalyzed by lactate dehydrogenase (LDH), with experimental liver injury. HP ∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document}-ketobutyrate (∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document} KB) is a close molecular analog of pyruvate with modified specificity for LDH isoforms, specifically attenuated activity with their LDHA-expressed subunits that dominate liver parenchyma. Building on recent results with pyruvate, we investigated HP ∝\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\propto $$\\end{document} KB in methionine-choline deficient (MCD) diet as a model of early-stage NASH. Similarity of results between this new agent and pyruvate (~ 50% drop in cytoplasmic reducing capacity), interpreted together with gene expression data from the model, suggests that changes are mediated through broad effects on intermediary metabolism. Plausible mechanisms are depletion of the lactate pool by upregulation of gluconeogenesis (GNG) and pentose phosphate pathway (PPP) flux, and a possible shift toward increased lactate oxidation. These changes may reflect high levels of oxidative stress and/or shifting macrophage populations in NASH.

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG.

Background: Insulin promotes renal proximal tubule glucose reabsorption and suppresses gluconeogenesis (GNG). The kinase mTORC2 is critical for insulin signaling in multiple cell types. In the kidney tubules, mTORC2 knockout has recently been shown to cause glycosuria via reduced plasma membrane SGLT2 and SGLT1 as well as inappropriately increased renal GNG. Potassium (K+) also plays an important role in systemic glucose homeostasis. However, the overall importance of K+ for the regulation of renal glucose reabsorption and GNG is poorly understood. Methods: Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. To examine the role of dietary K+ on renal glucose homeostasis, TRKO mice and wild-type (WT) littermates were fed with either a normal 0.5% K+ diet or high 3% K+ diet while in balance cages. Kidney tissue was harvested after feeding and processed to study plasma membrane glucose transporters, gluconeogenic enzymes, and mTORC2-regulated targets by western blot and qPCR. Results: On a normal K+ diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane fractions showed decreased SGLT2 and SGLT1, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes. On a high K+ diet, glycosuria resolved and plasma membrane SGLT2 and SGLT1 were restored in TRKO mice. In addition, TRKO animals fed with a high K+ diet had suppressed gluconeogenic enzymes compared to WT mice. Regardless of dietary K+ content, TRKO animals had similar reductions in phosphorylation of mTORC2 substrates compared to WT controls. Conclusion: Renal tubule mTORC2 is critical for coordinated glucose reabsorption by SGLT2 and SGLT1 as well as suppression of renal GNG. High dietary K+ promotes sodium-glucose cotransport by restoring plasma membrane SGLT2 and SGLT1 in TRKO mice. High dietary K+ also prevents excessive GNG in TRKO mice. High K+ appears to regulate these processes independently of mTORC2 and its downstream substrates, such as Akt. High K+ could potentially provide an alternative signaling mechanism to regulate renal glucose reabsorption and GNG in states of insulin resistance. Grants from the NIH (T32, R01) and The James Hilton Manning and Emma Austin Manning Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Disclosure: E. Quaye: None. S. Chacko: None. M. Startzell: None. R.J. Brown: Other; Self; Metreleptin for the study was donated by Amryt Pharma. Leptin is an adipocyte-derived hormone that signals overall energy sufficiency. In patients with lipodystrophy, treatment with recombinant leptin (metreleptin) improved measures of glycemia and decreased energy expenditure. We hypothesized that metreleptin in patients with lipodystrophy would decrease gluconeogenesis (GNG) and gluconeogenic substrates through improvements in peripheral and hepatic insulin sensitivity. We conducted a single-arm prospective study of metreleptin administration in 9 patients with lipodystrophy from 2013-2018. The main outcomes of this study were basal and insulin-mediated suppression of GNG, carbon sources for GNG (glycerol rate of appearance (Ra) measured with isotope tracers, and plasma alanine and lactate), palmitate Ra (a driver of GNG, measured with isotope tracers), and peripheral and hepatic insulin sensitivity (measured as glucose disposal during a hyperinsulinemic clamp, and % suppression of glucose Ra during the clamp, respectively). Peripheral insulin sensitivity increased 2-fold (by 115±134%; P=0.001) after 6 months of metreleptin. Hepatic insulin sensitivity increased from 61% to 81% after 6 months of metreleptin with a trend toward statistical significance (P=0.08; 6 months vs. baseline). Basal GNG decreased by 16±12%, from 12.4±3.9 to 10.9±2.6 µmol/kgLBM/min, after 2 weeks of metreleptin (P=0.02) but did not change after 6 months (P=0.2). Metreleptin increased insulin-mediated suppression of GNG during the hyperinsulinemic clamp from 73% at baseline to 91% after 2 weeks (P&amp;lt;0.002; 2 weeks vs. baseline), and 94% after 6 months (P&amp;lt;0.001; 6 months vs. baseline). Alanine was unchanged after 2 weeks of metreleptin (P=0.1) but decreased by 21±22% after 6 months (P=0.005). There was a positive correlation between alanine and GNG at baseline (r=0.8, P=0.01), 2 weeks (r=0.8, P=0.03) and 6 months (r=0.9, P=0.002). Lactate decreased by 20±18% after 2 weeks (P=0.014) and 21±24% (P=0.001) after 6 months of metreleptin. There was no significant decrease in glycerol or palmitate Ra after 2 weeks of metreleptin, but palmitate Ra decreased after 6 months (P=0.04). Metreleptin treatment in patients with lipodystrophy reduced basal GNG and increased insulin mediated suppression of GNG. Reduced GNG may be mediated by improved insulin sensitivity leading to reductions in alanine and lactate, which are carbon sources for GNG. Associations between alanine and GNG persisted after metreleptin, suggesting that potential therapeutic interventions to reduce protein turnover in insulin-resistant states may improve glucose homeostasis. Presentation: Saturday, June 17, 2023

The gluconeogenesis pathway, which converts nonsugar molecules into glucose, is critical for maintaining glucose homeostasis. Techniques that measure flux through this pathway are invaluable for studying metabolic diseases such as diabetes that are associated with dysregulation of this pathway. We introduce a new method that measures fractional gluconeogenesis by heavy water labeling and gas chromatographic-mass spectrometric analysis. This technique circumvents cumbersome benchwork or inference of positionality from mass spectra. The enrichment and pattern of deuterium label on glucose is quantified by use of mass isotopomer distribution analysis, which informs on how much of glucose-6-phosphate-derived glucose comes from the gluconeogenesis (GNG) pathway. We use an invivo model of the GNG pathway that is based on previously published models but offers a new approach to calculating GNG pathway and subpathway contributions using combinatorial probabilities. We demonstrated that this method accurately quantifies fractional GNG through experiments that perturb flux through the pathway and by probing analytical sensitivity. While this method was developed in mice, the results suggest that it is translatable to humans in a clinical setting.

Gluconeogenesis (GNG) is the process of regenerating glucose and NAD+ that allows for continued ATP synthesis by glycolysis during fasting or in hypoxia. Recent data from C. elegans and crustaceans challenged with hypoxia show differential and tissue-specific expression of GNG-specific genes. Here we report differential expression of several GNG-specific genes in the head and body of a model organism, Daphnia magna, a planktonic crustacean, in normoxic and acute hypoxic conditions. We predict that GNG-specific transcripts will be enriched in the body, where most of the fat tissue is located, rather than in the head, where the tissues critical for survival in hypoxia, the central nervous system and locomotory muscles, are located. We measured the relative expression of GNG-specific transcripts in each body part by qRT-PCR and normalized them by either the expression of a reference gene or the rate-limiting glycolysis enzyme pyruvate kinase (PK). Our data show that of the three GNG-specific transcripts tested, pyruvate carboxylase (PC) showed no differential expression in either the head or body. Phosphoenolpyruvate carboxykinase (PEPCK-C), on the other hand, is upregulated in hypoxia in both body parts. Fructose-1,6-bisphosphatase (FBP) is upregulated in the body relative to the head and upregulated in hypoxia relative to normoxia, with a stronger body effect in hypoxia when normalized by PK expression. These results support our hypothesis that Daphnia can survive hypoxic conditions by implementing the Cori cycle, where body tissues supply glucose and NAD+ to the brain and muscles, enabling them to continuously generate ATP by glycolysis.

The effects of leptin, an adipocyte-derived hormone that signals overall energy sufficiency, can only be studied in leptin-deficient conditions. In patients with lipodystrophy, a rare disease and unique model of leptin deficiency, treatment with recombinant leptin (metreleptin) improves glycemia and decreases energy expenditure. We hypothesized that these improvements might be mediated by reduced gluconeogenesis (GNG), an energy-requiring process. To determine the effects of metreleptin on GNG and GNG substrates. This was a single-arm prospective study of metreleptin administration in 15 patients with lipodystrophy, 9 of whom had data on GNG (NIH, 2013-2018). We analyzed total GNG, insulin-mediated suppression of GNG, glycerol, palmitate, alanine, lactate, peripheral and hepatic insulin sensitivity, and markers of glycemia (eg, HbA1c, glucose, fasting insulin). Metreleptin administration decreased basal GNG, increased insulin-mediated suppression of GNG, and improved insulin sensitivity and markers of glycemic control. Metreleptin reduced carbon sources for GNG, including plasma alanine and lactate, and rate of appearance (Ra) of glycerol, and decreased Ra of palmitate, a driver of GNG. Glycerol and palmitate Ra correlated with GNG prior to but not during metreleptin administration. Alanine strongly correlated with GNG both before and during metreleptin administration. Metreleptin treatment in patients with lipodystrophy reduced GNG likely through decreased availability of carbon sources for gluconeogenesis, such as alanine, lactate, and glycerol. Associations between alanine and GNG persisted after metreleptin treatment while correlations with glycerol and palmitate Ra did not persist, suggesting reduced importance of lipolysis as a driver of GNG in the leptin-replete state.

Higher gluconeogenesis (GNG) during the latter part of the night contributes to higher endogenous glucose production (EGP) in type 2 diabetes (T2D). We have shown that glycogen synthesis is reduced in T2D due to a functional defect in the rate-limiting hepatic glucokinase activity. Studies using MRIs have confirmed lower net hepatic glycogen content in T2D than subjects without diabetes (ND) before and after dinner. We hypothesize that increasing post dinner hepatic glycogen content might restore overnight EGP in T2D by reducing GNG. 14 T2D and 15 anthropometrically matched ND were randomly assigned to glycogen loading (GL; 60% carb) vs. non glycogen loading (NGL; 40% carb) meals for 3 days. [6,6-2H2] Glucose was infused to measure EGP. GNG was estimated with deuterium labeled water and glycogen content measured via C13 NMR in fed and fasted state. Results are shown in Figure1. Liver glycogen content was higher with GL vs NGL in both cohorts. EGP was higher (p&amp;lt;0.05) in GL vs NGL throughout night in both cohorts. The percent contribution of GNG and glycogenolysis (GGL) to EGP averaged ~ 50% in ND throughout the night in both GL and NGL. In contrast, the percent contribution of GNG to EGP in T2D with GL was lower vs. NGL overnight (average ~48% vs. 60%) and matched the pattern observed in ND with GL. The results indicate that lower glycogen content in the liver contributes to increased nocturnal rates of GNG in T2D. Disclosure U.Unni: None. Y.Yadav: None. J.P.Mugler, iii: None. R.Carter: None. A.Basu: None. R.Basu: Consultant; Sparrow Pharmaceuticals Inc, Research Support; Abbott Diabetes, AstraZeneca. Funding National Institute for Health and Care Research (R01DK029953)

Fasting hyperglycemia in diabetes mellitus is caused by unregulated glucagon secretion that activates gluconeogenesis (GNG) and increases the use of pyruvate, lactate, amino acids, and glycerol. Studies of GNG in hepatocytes, however, tend to test a limited number of substrates at nonphysiologic concentrations. Therefore, we treated cultured primary hepatocytes with three identical substrate mixtures of pyruvate/lactate, glutamine, and glycerol at serum fasting concentrations, where a different U-13C- or 2-13C-labeled substrate was substituted in each mix. In the absence of glucagon stimulation, 80% of the glucose produced in primary hepatocytes incorporated either one or two 13C-labeled glycerol molecules in a 1:1 ratio, reflecting the high overall activity of this pathway. In contrast, glucose produced from 13C-labeled pyruvate/lactate or glutamine rarely incorporated two labeled molecules. While glucagon increased the glycerol and pyruvate/lactate contributions to glucose carbon by 1.6- and 1.8-fold, respectively, the glutamine contribution to glucose carbon was increased 6.4-fold in primary hepatocytes. To account for substrate 13C carbon loss during metabolism, we also performed a metabolic flux analysis, which confirmed that the majority of glucose carbon produced by primary hepatocytes was from glycerol. Invivo studies using a PKA-activation mouse model that represents elevated glucagon activity confirmed that most circulating lactate carbons originated from glycerol, but very little glycerol was derived from lactate carbons, reflecting glycerol's importance as a carbon donor to GNG. Given the diverse entry points for GNG substrates, hepatic glucagon action is unlikely to be due to a single mechanism.

Insulin resistant individuals display elevated fasting and post-prandial glucose levels which are mainly driven by inadequate inhibition of gluconeogenesis (GNG) and are rarely normalized. The aim of this study was to investigate whether direct inhibition of GNG could be a new therapeutical approach to improve glucose homeostasis using in vitro and in vivo models of insulin resistance. Fructose 1,6-bisphosphatase (FBP1) is key in controlling GNG in liver and kidney and loss of function patients display hypoglycaemia episodes and lactate acidosis. Systemic FBP1 inhibition using small molecules has shown to improve glucose homeostasis. To assess the clinical efficacy and safety potential of silencing FBP1 selectively in the liver we dosed insulin resistant DIO rats with a hepatocyte specific GalXC-FBP1 siRNA entity. GalXC-FBP1 siRNA markedly reduced FBP1 mRNA level in the liver by over 90%. This was associated with a complete lack of glucose excursion following a pyruvate challenge indicating inhibition of GNG. Blood glucose level decreased similarly in rats treated with GalXC-FBP1 siRNA or vehicle during an insulin challenge or a prolonged fast suggesting that hepatic FBP1 silencing is not inducing hypoglycaemia. Insulin sensitivity and hyperinsulinemia were improved. Plasma lactate and liver enzymes were not elevated, but a significant 2-fold increase in liver triglycerides was observed in the GalXC-FBP1 siRNA group. Using human hepatocytes in a Liver-on-Chip in vitro model FBP1 silencing reduced glucose production by 30% supporting the relevance of this approach in humans. Collectively these data suggest that liver specific FBP1 silencing has the potential to improve insulin sensitivity in DIO rats without inducing hypoglycaemia but may be associated with a risk of liver steatosis overtime. This concept highlights the difficulty of blocking liver GNG without re-directing the metabolic intermediates fluxes toward triglycerides accumulation in liver. Disclosure C. Fledelius: Employee; Novo Nordisk A/S. D. Demozay: Employee; Novo Nordisk A/S. H. Iversen: None. R. S. Ingvorsen: None. L. B. Eriksen: None. A. Blois: Employee; Novo Nordisk. Y. Montauban: None. R. Rijnbrand: Employee; Novo Nordisk. W. Han: None. J. F. Jeppesen: Employee; Novo Nordisk A/S.

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 &amp; Development, LLC, Merck &amp; 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. .

Exercise is an important lifestyle intervention for the prevention and treatment of NAFLD. Importantly, exercise can reduce hepatic steatosis independent of changes in body mass, which may stem from increased oxidative demand in liver during exercise. Increased hepatic gluconeogenesis (GNG) and tricarboxylic acid (TCA) cycle flux have been observed during exercise in rodents, but it is unclear whether this adaptation is chronically maintained outside of the exercise window. Liver metabolism is also altered by nutritional status. Fasting promotes hepatic fat oxidation, ketogenesis and GNG, while feeding promotes glycogen storage and fat synthesis. Hence, fasting and exercise may have a positive interaction, whereas exogenous nutrient intake or glucose infusion is known to reduce the induction of GNG during exercise. Here, we tested whether nutritional status impacts exercise induced hepatic metabolic adaptations. Sprague-Dawley rats were kept sedentary or provided voluntary wheel running (VWR) for 4 weeks in the presence or absence of food restriction during the dark phase when rats spontaneously run. Experiments were conducted one day following the last exercise bout. Hepatic metabolic flux was examined by in vivo stable isotope infusions of [U-13C3]propionate, [3,4-13C2]glucose and 2H2O in rats using mass spectrometry and computational analysis. Chronic VWR in the fed state had no effect on basal hepatic fluxes outside of the exercise window. However, exercise during food restriction chronically induced a 2-fold induction of TCA cycle turnover, increase in GNG from glycerol and lowered ketogenesis outside of the exercise window. Thus, VWR during food restriction triggered an increase in hepatic energy demand that was sustained for at least 24 hours, while the effects of exercise in the fed state dissipated by 24 hours. These data suggest that acute nutritional state may be an important factor in the long-term programming of liver metabolism by exercise. Disclosure S.Deja: None. B.Kucejova: None. A.Maurer: None. M.N.Mizerska: None. X.Fu: None. J.P.Thyfault: None. S.C.Burgess: n/a. Funding National Institutes of Health (R01DK121497, R01DK078184, P41EB015908)