Elevated Gluconeogenesis Research Articles

Integration of transcriptomics and metabolomics data revealed role of insulin resistant SNW1 gene in the pathophysiology of gestational diabetes.

Gestational Diabetes Mellitus (GDM) is an emerging maternal health problem with increasing incidences. The lack of complete understanding of its pathophysiological mechanisms and novel regulatory biomarkers makes early diagnosis difficult. High-throughput RNA sequencing and computational bioinformatics analyses were conducted to identify novel hub genes, and their regulatory mechanisms were validated through qRT-PCR, western blot, and siRNA-mediated knockdown studies. Intermediate metabolites and circulatory levels of amino acids in the serum of GDM patients and healthy controls were measured. Transcriptomic studies identified SNW1 as the most sensitive and specific biomarker, significantly up-regulated in GDM (fold change = 1.09; p < 0.001). Metabolomic studies indicated significantly elevated gluconeogenesis in GDM, evidenced by decreased levels of alanine and increased levels of pyruvate and glucose compared to controls. siRNA-mediated knockdown of SNW1 in PANC1 cells resulted in significant down-regulation of alanine aminotransferase (ALT/GPT) and insulin receptor substrate (IRS1), while glucose transporters (GLUT2/GLUT4) and insulin (INS) were significantly up-regulated at both mRNA and protein levels. This study identified SNW1 as a novel insulin-resistant gene that induces hyperglycemia by elevating gluconeogenesis and decreasing glucose uptake. SNW1 may be considered a potential therapeutic target with clinical utility for the management of GDM.

LGR4 is a key regulator of hepatic gluconeogenesis.

Emerging evidence underscored the significance of leucine-rich repeat-containing G protein-coupled receptor (LGR) 4 in endocrine and metabolic disorders. Despite this, its role in LGR4 in hepatic glucose metabolism remains poorly understood. In this study we set out to test whether LGR4 regulates glucose production in liver through a specific signaling pathway. Hepatic glucose production and gluconeogenic gene expressions were detected after silence of LGR4 in three obese mice models. Then, whole-body LGR4-deficient (LGR4 KO) mice, liver-specific LGR4 knockout (LGR4LKO) mice, and liver-specific LGR4 overexpression (LGR4LOV) mice were generated, in which we analyzed the effects of LGR4 on hepatic glucose metabolism upon HFD feeding, among which live imaging and quantitative analysis of hepatic phosphoenolpyruvate carboxykinase (PEPCK)-luciferase activity were conducted. LGR4 expression was significantly upregulated in the liver of three obese mouse models, and presented dynamic expression patterns in response to nutritional fluxes. We utilized global and liver-specific LGR4 knockouts (LGR4LKO), along with adenoviral-mediated LGR4 knockdown in mice, to show improved glucose tolerance and decreased hepatic gluconeogenesis. Specifically, the expression of rate-limiting gluconeogenic enzymes, PEPCK was significantly downregulated. Conversely, mouse model with adenovirus-mediated LGR4 overexpression (LGR4LOV) exhibited elevated gluconeogenesis and PEPCK expression and reversed the suppression observed in LGR4 knockout models. Notably, neither RANKL nor PKA signaling pathways, which were reported to take part in LGR4's function, were involved in the process of LGR4 regulating PEPCK. Instead, TopFlash reporter system and inhibitors application suggested that LGR4's influence on hepatic gluconeogenesis operates through the canonical Wnt/β-catenin/TCF7L2 signaling pathway. Overall, these findings underscore a novel mechanism by which LGR4 regulates hepatic gluconeogenesis, presenting a potential therapeutic target for diabetes management.

Effects of Acute Aerobic Exercise on Skeletal Muscle and Liver Glucose Metabolism in Male Rodents with Type 1 Diabetes.

Aerobic exercise (AE) is associated with a significant hypoglycemia risk in individuals with type 1 diabetes mellitus (T1DM). However, the mechanisms in the liver and skeletal muscle governing exercise-induced hypoglycemia in T1DM are poorly understood. This study examined the effects of a 60-minute bout of AE on hepatic and muscle glucose metabolism in T1DM rats. Nineteen male Sprague-Dawley rats were divided into sedentary (SC; n=5) and T1DM (DSC; n=14) groups. T1DM rats were subcategorized into pre-exercise (DPRE; n=6) and post-exercise (DPOST; n=8). DPOST were sacrificed immediately after 60-minute of AE. Results demonstrate that DPOST animals experienced reductions in BG following 30 and 60 minutes of AE compared to pre-exercise. Both DPRE and DPOST animals exhibited lower hepatic glycogen content, while muscle glycogen did not differ, suggesting impaired glycogenolysis in T1DM. Hepatic glycogen-6-phosphatase content, and muscle and hepatic protein kinase B phosphorylation were significantly greater in DPOST animals, suggesting elevated gluconeogenesis and insulin stimulation during exercise. Glycogen phosphorylase activity did not differ between groups. These data suggest that drops in BG during AE in T1DM were due to lower glycogen levels in the liver and muscle and a lack of muscle glycogen utilization; leading to a reliance on gluconeogenesis and BG.

Hepatic IDH2 regulates glycolysis and gluconeogenesis

Background and aimsThe liver plays a central role in controlling glucose and lipid metabolism. IDH2, a mitochondrial protein, controls TCA cycle flux. However, its role in regulating metabolism in obesity is still unclear. This study intends to investigate the impact of hepatic IDH2 expression on overnutrition-regulated glucose and lipid metabolism. MethodsHepatic IDH2 was knocked-out in mice by the approach of CRISPR-Cas9. Mice were subjected to starvation and refeeding for hepatic glucose and lipid studies in vivo. Primary hepatocytes and mouse normal liver cell line, AML12 cells were used for experiments in vitro. ResultsThis study found that IDH2 protein levels were elevated in the livers of obese people and mice with high-fat diet consumption or hepatic steatosis. Liver IDH2-deletion mice (IDH2LKO) were resistant to high-fat diet-induced body weight gain, with lower serum glucose and TG levels, increased insulin sensitivity, and higher FGF21 secretion, despite the higher TG content in the liver. Consistently, overexpression of IDH2 in hepatocytes promoted gluconeogenesis and enhanced glycogenesis. By performing mass spectrometry and proteomics analyses, we further demonstrated that IDH2-deficiency in hepatocytes accelerated ATP production by increasing forward TCA cycle flux, thus promoting glycolysis pathway and decreasing glycogen synthesis at refeeding state, and inhibiting hepatic gluconeogenesis, increasing β-oxidation during starvation. Moreover, experiments in vivo demonstrated that IDH2-knockout might not exacerbate hepatic inflammatory responses in the NASH model. ConclusionsElevated hepatic IDH2 under over-nutrition state contributes to elevated gluconeogenesis and glycogen synthesis. Inhibition of IDH2 in the liver could be a potential therapeutic target for obesity and diabetes.

Early Life Polychlorinated Biphenyl 126 Exposure Disrupts Gut Microbiota and Metabolic Homeostasis in Mice Fed with High-Fat Diet in Adulthood.

Evidence supports the potential influence of persistent organic pollutants (POPs) on the pathogenesis and progression of obesity and diabetes. Diet-toxicant interactions appear to be important in diet-induced obesity/diabetes; however, the factors influencing this interaction, especially the early life environmental exposure, are unclear. Herein, we investigated the metabolic effects following early life five-day exposure (24 μg/kg body weight per day) to 3,3′,4,4′,5-pentacholorobiphenyl (PCB 126) at four months after exposure in mice fed with control (CTRL) or high-fat diet (HFD). Activation of aryl hydrocarbon receptor (AHR) signaling as well as higher levels of liver nucleotides were observed at 4 months after PCB 126 exposure in mice, independent of diet status. Inflammatory responses including higher levels of serum cytokines and adipose inflammatory gene expression caused by early life PCB 126 were observed only in HFD-fed mice in adulthood. Notably, early life PCB 126 exposure worsened HFD-induced impaired glucose homeostasis characterized by glucose intolerance and elevated gluconeogenesis and tricarboxylic acid (TCA) cycle flux without worsening the effects of HFD related to adiposity in adulthood. Furthermore, early life PCB 126 exposure resulted in diet-dependent changes in bacterial community structure and function later in life, as indicated by metagenomic and metabolomic analyses. These data contribute to a more comprehensive understanding of the interactions between diet and early life environmental chemical exposure.

Impact of Dietary Empagliflozin on Renal Cortex Metabolome and Lipidome in Mice

Sodium glucose cotransporter, type 2 inhibitors (SGLT2i) have been demonstrated to be protective of kidney by unclear mechanisms. The aim of the current study was to determine how treatment with a common SGLT2i, Empagliflozin affected kidney cortical metabolome and lipidome in high‐fat‐fed, male TallyHo/Jng (TH) mice. TH mice are prone to obesity and insulin resistance progressing to type 2 diabetes (T2D). Adult mice (n = 7 or 8/group) were fed a control high‐milk‐fat diet (60% by weight) or this same diet supplemented with empagliflozin (0.01%) for 8‐weeks, then euthanized and kidney cortex dissected and rapidly frozen. Metabolomics (targeted and untargeted) and lipidomics (targeted) were conducted on separate sections of cortex in our core facility by liquid chromatography‐followed by tandem mass‐spectroscopy. Data pre‐processing included signal drift and inter‐batch correction, as well as, removal of species with high coefficient of variability on pooled or reference analysis. Metabolites and lipids passing quality control (QC) included 207 and 467 unique species, respectively, which were further analyzed by MetaboAnalyst 5.0 and lipids, additionally by LipidSig, both publically available software platforms. Volcano plotting of metabolites and lipids revealed oppositely skewed patterns in that metabolites tended to be relatively decreased and lipid species increased with Empagliflozin. Seventy‐four metabolites met our threshold of changed &gt; 20% change and/or p &lt; 0.05 and were analyzed for pathway enrichment. The top three pathways down regulated by Empagliflozin were urea cycle, spermine/spermidine biosynthesis, and aspartate metabolism. Analysis of metabolites of glycolysis/oxidative phosphorylation revealed a general 20‐45% reduction in several species including phosphoenolpyruvate (PEP), succinate, and malic acid. In contrast, in general, several lipid species were increased with hierarchical clustering showing greatest effect on phosphatidylglycerol (16:1/16:1), 40% increase, p &lt; 0.02, lysophosphatidylcholine (LPC, 20:4) 38% increase p &lt; 0.03, and 10 additional phosphatidylcholine (PC) species. Overall, these analyses suggest a greater abundance of lipid, relative to carbohydrate and protein‐based species, in the kidney cortex in response to chronic Empagliflozin treatment. It is unclear whether these differences are due to intracellular metabolic alterations or transport reduction in substrate (glucose and peptides) for metabolic pathways. In addition, we found no evidence of elevated gluconeogenesis with Empagliflozin observing a significant reduction in one key metabolite in this particular pathway, PEP. We may speculate oxidative phosphorylation and ATP generation is maintained relatively normal by greater oxidation of lipids (as opposed to proteins and carbohydrates) in the kidney proximal tubule of Empagliflozin‐treated mice.

Multitissue 2H/13C flux analysis reveals reciprocal upregulation of renal gluconeogenesis in hepatic PEPCK-C-knockout mice.

The liver is the major source of glucose production during fasting under normal physiological conditions. However, the kidney may also contribute to maintaining glucose homeostasis in certain circumstances. To test the ability of the kidney to compensate for impaired hepatic glucose production in vivo, we developed a stable isotope approach to simultaneously quantify gluconeogenic and oxidative metabolic fluxes in the liver and kidney. Hepatic gluconeogenesis from phosphoenolpyruvate was disrupted via liver-specific knockout of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; KO). 2H/13C isotopes were infused in fasted KO and WT littermate mice, and fluxes were estimated from isotopic measurements of tissue and plasma metabolites using a multicompartment metabolic model. Hepatic gluconeogenesis and glucose production were reduced in KO mice, yet whole-body glucose production and arterial glucose were unaffected. Glucose homeostasis was maintained by a compensatory rise in renal glucose production and gluconeogenesis. Renal oxidative metabolic fluxes of KO mice increased to sustain the energetic and metabolic demands of elevated gluconeogenesis. These results show the reciprocity of the liver and kidney in maintaining glucose homeostasis by coordinated regulation of gluconeogenic flux through PEPCK-C. Combining stable isotopes with mathematical modeling provides a versatile platform to assess multitissue metabolism in various genetic, pathophysiological, physiological, and pharmacological settings.

207-LB: Simultaneous 2H/13C Assessment of Liver and Kidney Metabolic Fluxes Reveals Reciprocal Upregulation of Renal Gluconeogenesis in Hepatic PEPCK-C Knockout Mice

The liver is the major source of glucose production during fasting under normal physiological conditions. However, the kidney may also contribute to maintaining glucose homeostasis in certain circumstances. To test the ability of the kidney to compensate for impaired hepatic glucose production in vivo, we developed a novel stable isotope approach to simultaneously quantify hepatic and renal gluconeogenic and oxidative metabolic fluxes. Hepatic gluconeogenesis from phosphoenolpyruvate was disrupted via liver-specific knockout of cytosolic PEPCK (KO). Experiments were performed in fasted (~18hr) KO and wild-type (WT) littermate mice (n≥4, per group) fitted with jugular vein and carotid artery catheters. A multi-compartment metabolic model was used to assess fluxes using the isotopic enrichment of plasma and tissue metabolites from mice infused with 2H/13C isotopes. Hepatic gluconeogenesis and glucose production were reduced in KO mice, yet whole-body glucose production and arterial glucose were unaffected. Glucose homeostasis was maintained by a compensatory rise in renal glucose production and gluconeogenesis of 17 and 30-fold, respectively. This was consistent with a ~2-fold rise in PEPCK-C and mitochondrial PEPCK protein expression in the kidneys. Renal oxidative metabolic fluxes of KO mice increased to sustain the energetic and metabolic demands of elevated gluconeogenesis. These results show the reciprocity of the liver and kidney in maintaining glucose homeostasis by regulating gluconeogenic flux through PEPCK-C. This reciprocity creates a layer of redundancy that protects glucose homeostasis in the event that hepatic glucose production is impaired. Combining stable isotopes with mathematical modeling provides a versatile platform to assess multi-tissue metabolism in various genetic, (patho)physiological, and pharmacological settings. Disclosure M. Rahim: None. C. M. Hasenour: Employee; Self; Eli Lilly and Company. T. Bednarski: None. C. C. Hughey: None. D. Wasserman: None. J. Young: Consultant; Self; Pfizer Inc., Stock/Shareholder; Self; Metalytics. Funding National Institutes of Health (R01DK106348, U01CA235508, T32DK101003, U24DK059637)

Intestinal stem cell-derived enteroids from morbidly obese patients preserve obesity-related phenotypes: Elevated glucose absorption and gluconeogenesis

ObjectiveThe mechanisms behind the efficacy of bariatric surgery (BS) for treating obesity and type 2 diabetes, particularly with respect to the influence of the small bowel, remain poorly understood. In vitro and animal models are suboptimal with respect to their ability to replicate the human intestinal epithelium under conditions induced by obesity. Human enteroids have the potential to accelerate the development of less invasive anti-obesity therapeutics if they can recapitulate the pathophysiology of obesity. Our aim was to determine whether adult stem cell-derived enteroids preserve obesity-characteristic patient-specific abnormalities in carbohydrate absorption and metabolism. MethodsWe established 24 enteroid lines representing 19 lean, overweight, or morbidly obese patients, including post-BS cases. Dietary glucose absorption and gluconeogenesis in enteroids were measured. The expression of carbohydrate transporters and gluconeogenic enzymes was assessed and a pharmacological approach was used to dissect the specific contribution of each transporter or enzyme to carbohydrate absorption and metabolism, respectively. ResultsFour phenotypes representing the relationship between patients’ BMI and intestinal dietary sugar absorption were found, suggesting that human enteroids retain obese patient phenotype heterogeneity. Intestinal glucose absorption and gluconeogenesis were significantly elevated in enteroids from a cohort of obese patients. Elevated glucose absorption was associated with increased expression of SGLT1 and GLUT2, whereas elevated gluconeogenesis was related to increased expression of GLUT5, PEPCK1, and G6Pase. ConclusionsObesity phenotypes preserved in human enteroids provide a mechanistic link to aberrant dietary carbohydrate absorption and metabolism. Enteroids can be used as a preclinical platform to understand the pathophysiology of obesity, study the heterogeneity of obesity mechanisms, and identify novel therapeutics.

Illuminated night alters behaviour and negatively affects physiology and metabolism in diurnal zebra finches

Light at night (LAN) negatively impacts the behaviour and physiology; however, very little is known about molecular correlates of LAN-induced effects in diurnal animals. Here, we assessed LAN-induced effects on behaviour and physiology, and examined molecular changes in the liver of diurnal zebra finches (Taeniopygia guttata). Birds were exposed to dim LAN (dLAN: 12L = 150 lux: 12D = 5 lux), with controls on 12L (150 lux): 12D (0 lux). dLAN altered daily activity-rest and eating patterns, induced nocturnal eating and caused body fattening and weight gain, and reduced nocturnal melatonin levels. Concomitant increased nighttime glucose levels, decreased daytime thyroxine and triglycerides levels, and hepatic lipid accumulation suggested the impairment of metabolism under dLAN. Transcriptional assays evidenced dLAN-induced negative effects on metabolism in the liver, the site of metabolic homeostasis. Particularly, increased g6pc and foxo1 mRNA expressions suggested an enhanced gluconeogenesis, while increased egr1 and star expressions suggested enhanced cholesterol biosynthesis and lipid metabolism, respectively. Similarly, overexpressed sirt1 indicated protection from the metabolic damage due to elevated gluconeogenesis and cholesterol biosynthesis under dLAN. However, no effect on genes involved in lipogenesis (fasn) and insulin signalling pathway (socs3 and insig1) might indicate for the post transcriptional/post translational modification effects or the involvement of other genetic pathways in LAN-induced effects. We also found daily rhythm in the hepatic expression of selected clock and clock-controlled genes (per2, bmal1 and reverb-beta), with an elevated mesor and amplitude of per2 oscillation, suggesting a role of per2 in the liver metabolism. These results demonstrate dLAN-induced negative effects on the behaviour and physiology, and provide molecular insights into metabolic risks of the exposure to illuminated nights to diurnal animals including humans in an urban setting.

Non-invasive detection of divergent metabolic signals in insulin deficiency vs. insulin resistance in\xa0vivo

The type 2 diabetic phenotype results from mixed effects of insulin deficiency and insulin resistance, but the relative contributions of these two distinct factors remain poorly characterized, as do the respective roles of the gluconeogenic organs. The purpose of this study was to investigate localized in vivo metabolic changes in liver and kidneys of contrasting models of diabetes mellitus (DM): streptozotocin (STZ)-treated wild-type Zucker rats (T1DM) and Zucker diabetic fatty (ZDF) rats (T2DM). Intermediary metabolism was probed using hyperpolarized (HP) [1-13C]pyruvate MRI of the liver and kidneys. These data were correlated with gene expression data for key mediators, assessed using rtPCR. Increased HP [1-13C]lactate was detected in both models, in association with elevated gluconeogenesis as reflected by increased expression of phosphoenolpyruvate carboxykinase. In contrast, HP [1-13C]alanine diverged between the two models, increasing in ZDF rats, while decreasing in the STZ-treated rats. The differences in liver alanine paralleled differences in key lipogenic mediators. Thus, HP [1-13C]alanine is a marker that can identify phenotypic differences in kidneys and liver of rats with T1DM vs. T2DM, non-invasively in vivo. This approach could provide a powerful diagnostic tool for characterizing tissue metabolic defects and responses to treatment in diabetic patients with ambiguous systemic manifestations.

112: Elevated hepatic gluconeogenesis by H19-mediated epigenetic regulation underlies altered metabolism in offspring prenatally exposed to metformin

112: Elevated hepatic gluconeogenesis by H19-mediated epigenetic regulation underlies altered metabolism in offspring prenatally exposed to metformin

Reduced Insulin Receptor Expression Enhances Proximal Tubule Gluconeogenesis.

Reduced insulin receptor protein levels have been reported in the kidney cortex from diabetic humans and animals. We recently reported that, targeted deletion of insulin receptor (IR) from proximal tubules (PT) resulted in hyperglycemia in non-obese mice. To elucidate the mechanism, we examined human proximal tubule cells (hPTC) and C57BL/6 mice fed with high-fat diet (HFD, 60% fat for 20 weeks). Immunoblotting revealed a significantly lower protein level of IR in HFD compare to normal chow diet (NCD). Furthermore, a blunted rise in p-AKT308 levels in the kidney cortex of HFD mice was observed in response to acute insulin (0.75 IU/kg body weight, i.p) relative to NCD n = 8/group, P < 0.05). Moreover, we found significantly higher transcript levels of phosphoenolpyruvate carboxykinase (PEPCK, a key gluconeogenic enzyme) in the kidney cortex from HFD, relative to mice on NCD. The higher level of PEPCK in HFD was confirmed by immunoblotting. However, no significant differences were observed in cortical glucose-6-phosphatase (G6Pase) or fructose-1,6, bisphosphosphatase (FBPase) enzyme transcript levels. Furthermore, we demonstrated insulin inhibited glucose production in hPTC treated with cyclic AMP and dexamethasone (cAMP/DEXA) to stimulate gluconeogenesis. Transcript levels of the gluconeogenic enzyme PEPCK were significantly increased in cAMP/DEXA-stimulated hPTC cells (n = 3, P < 0.05), and insulin attenuated this upregulation Furthermore, the effect of insulin on cAMP/DEXA-induced gluconeogenesis and PEPCK induction was significantly attenuated in IR (siRNA) silenced hPTC (n = 3, P < 0.05). Overall the above data indicate a direct role for IR expression as a determinant of PT-gluconeogenesis. Thus reduced insulin signaling of the proximal tubule may contribute to hyperglycemia in the metabolic syndrome via elevated gluconeogenesis. J. Cell. Biochem. 118: 276-285, 2017. © 2016 Wiley Periodicals, Inc.

Hepatic Mitochondrial Pyruvate Carrier 1 Is Required for Efficient Regulation of Gluconeogenesis and Whole-Body Glucose Homeostasis

Gluconeogenesis is critical for maintenance of euglycemia during fasting. Elevated gluconeogenesis during type 2 diabetes (T2D) contributes to chronic hyperglycemia. Pyruvate is a major gluconeogenic substrate and requires import into the mitochondrial matrix for channeling into gluconeogenesis. Here, we demonstrate that the mitochondrial pyruvate carrier (MPC) comprising the Mpc1 and Mpc2 proteins is required for efficient regulation of hepatic gluconeogenesis. Liver-specific deletion of Mpc1 abolished hepatic MPC activity and markedly decreased pyruvate-driven gluconeogenesis and TCA cycle flux. Loss of MPC activity induced adaptive utilization of glutamine and increased urea cycle activity. Diet-induced obesity increased hepatic MPC expression and activity. Constitutive Mpc1 deletion attenuated the development of hyperglycemia induced by a high-fat diet. Acute, virally mediated Mpc1 deletion after diet-induced obesity decreased hyperglycemia and improved glucose tolerance. We conclude that the MPC is required for efficient regulation of gluconeogenesis and that the MPC contributes to the elevated gluconeogenesis and hyperglycemia in T2D.

Discovery of systematic responses and potential biomarkers induced by ochratoxin A using metabolomics

Ochratoxin A (OTA) is known to be nephrotoxic and hepatotoxic in rodents when exposed orally. To understand the systematic responses to OTA exposure, GC-MS- and 1H-NMR-based metabolomic techniques together with histopathological assessments were applied to analyse the urine and plasma of OTA-exposed rats. It was found that OTA exposure caused significant elevation of amino acids (alanine, glycine, leucine etc.), pentose (ribose, glucitol, xylitol etc.) and nucleic acid metabolites (pseudouridine, adenosine, uridine). Moreover, myo-inositol, trimethylamine-oxide (TMAO), pseudouridine and leucine were identified as potential biomarkers for OTA toxicity. The primary pathways included the pentose phosphate pathway (PPP), the Krebs cycle (TCA), the creatine pathway and gluconeogenesis. The activated PPP was attributed to the high requirements for nicotinamide adenine dinucleotide phosphate (NADPH), which is involved in OTA metabolism through cytochrome P450. The elevated gluconeogenesis and TCA suggest that energy metabolism was involved. The up-regulated synthesis of creatinine reveals the elevated catabolism of proteins. These findings provide an overview of systematic responses to OTA exposure and metabolomic insight into the toxicological mechanism of OTA.

Elevated Gluconeogenesis in Aging and Lung Cancer is Related to Inflammation and Blunted Insulin‐Induced Protein Anabolism

Cancer and aging are both accompanied by metabolic alterations leading to muscle loss that may result from an increased use of amino acids for gluconeogenesis (GNG).Methodswe measured the fractional contributions (%) of glycogen, glycerol and GNG from phosphoenolpyruvate (PEP) to 17h‐fasting endogenous glucose production (EGP), using oral 2H20 and deuterium enrichment of plasma glucose, by NMR spectroscopy. Whole‐body protein and glucose kinetics (1‐13C‐leucine and 3H3‐glucose) were measured fasting and during a subsequent hyperinsulinemic, euglycemic, isoaminoacidemic clamp in: young (Y, n=11; 27±3 y), older (O, n=13; 65±2 y) and older men with lung cancer (CA, n=9; 66±2 y).ResultsMen with CA had greater serum CRP levels and resting energy expenditure (REE) than Y and O, and a lesser clamp glucose disposal, indicating insulin resistance. EGP was elevated with aging and cancer with greater contribution from GNG: Y:37±2, O:43±2 and CA:48±4% (ANOVA p&lt;0.05); % contribution from glycogen and glycerol did not differ. The resulting GNG flux (EGP × %GNG) correlated positively with CRP (r=0.69) and REE (r=0.61), and negatively with fat‐free mass index (r=−0.41), all p&lt;0.05. GNG flux was related to lesser glucose uptake and protein anabolic response (r= −0.54, p=0.001) in response to insulin.ConclusionsInflammation may underlie the greater production of glucose from GNG and blunted protein anabolism. (CIHR)

Pathogenesis of Prediabetes: Role of the Liver in Isolated Fasting Hyperglycemia and Combined Fasting and Postprandial Hyperglycemia

People with prediabetes are at high risk of developing diabetes. The objective of this study was to determine the pathogenesis of fasting and postprandial hyperglycemia in prediabetes. Glucose production, gluconeogenesis, glycogenolysis, and glucose disappearance were measured before and during a hyperinsulinemic clamp using [6,6-(2)H2]glucose and the deuterated water method corrected for transaldolase exchange. The study was conducted at the Mayo Clinic Clinical Research Unit. Subjects with impaired fasting glucose (IFG)/normal glucose tolerance (NGT) (n = 14), IFG/impaired glucose tolerance (IGT) (n = 18), and normal fasting glucose (NFG)/NGT (n = 16) were studied. A hyperinsulinemic clamp was used. Glucose production, glucose disappearance, gluconeogenesis, and glycogenolysis were measured. Fasting glucose production was higher (P < .0001) in subjects with IFG/NGT than in those with NFG/NGT because of increased rates of gluconeogenesis (P = .003). On the other hand, insulin-induced suppression of glucose production, gluconeogenesis, glycogenolysis, and stimulation of glucose disappearance all were normal. Although fasting glucose production also was increased (P = .0002) in subjects with IFG/IGT, insulin-induced suppression of glucose production, gluconeogenesis, and glycogenolysis and stimulation of glucose disappearance were impaired (P = .005). Fasting hyperglycemia is due to excessive glucose production in people with either IFG/NGT or IFG/IGT. Both insulin action and postprandial glucose concentrations are normal in IFG/NGT but abnormal in IFG/IGT. This finding suggests that hepatic and extrahepatic insulin resistance causes or exacerbates postprandial glucose intolerance in IFG/IGT. Elevated gluconeogenesis in the fasting state in IFG/NGT and impaired insulin-induced suppression of both gluconeogenesis and glycogenolysis in IFG/IGT suggest that alteration in the regulation of these pathways occurs early in the evolution of type 2 diabetes.

Elevated TCA cycle function in the pathology of diet-induced hepatic insulin resistance and fatty liver

The manner in which insulin resistance impinges on hepatic mitochondrial function is complex. Although liver insulin resistance is associated with respiratory dysfunction, the effect on fat oxidation remains controversial, and biosynthetic pathways that traverse mitochondria are actually increased. The tricarboxylic acid (TCA) cycle is the site of terminal fat oxidation, chief source of electrons for respiration, and a metabolic progenitor of gluconeogenesis. Therefore, we tested whether insulin resistance promotes hepatic TCA cycle flux in mice progressing to insulin resistance and fatty liver on a high-fat diet (HFD) for 32 weeks using standard biomolecular and in vivo (2)H/(13)C tracer methods. Relative mitochondrial content increased, but respiratory efficiency declined by 32 weeks of HFD. Fasting ketogenesis became unresponsive to feeding or insulin clamp, indicating blunted but constitutively active mitochondrial β-oxidation. Impaired insulin signaling was marked by elevated in vivo gluconeogenesis and anaplerotic and oxidative TCA cycle flux. The induction of TCA cycle function corresponded to the development of mitochondrial respiratory dysfunction, hepatic oxidative stress, and inflammation. Thus, the hepatic TCA cycle appears to enable mitochondrial dysfunction during insulin resistance by increasing electron deposition into an inefficient respiratory chain prone to reactive oxygen species production and by providing mitochondria-derived substrate for elevated gluconeogenesis.

Metformin ameliorates IL-6-induced hepatic insulin resistance via induction of orphan nuclear receptor small heterodimer partner (SHP) in mouse models

IL-6 is a proinflammatory cytokine associated with the pathogenesis of hepatic diseases. Metformin is an anti-diabetic drug used for the treatment of type 2 diabetes, and orphan nuclear receptor small heterodimer partner (SHP, also known as NR0B2), a transcriptional co-repressor, plays an important role in maintaining metabolic homeostasis. Here, we demonstrate that metformin-mediated activation of AMP-activated protein kinase (AMPK) increases SHP protein production and regulates IL-6-induced hepatic insulin resistance. We investigated metformin-mediated SHP production improved insulin resistance through the regulation of an IL-6-dependent pathway (involving signal transducer and activator of transcription 3 [STAT3] and suppressor of cytokine signalling 3 [SOCS3]) in both Shp knockdown and Shp null mice. IL-6-induced STAT3 transactivation and SOCS3 production were significantly repressed by metformin, adenoviral constitutively active AMPK (Ad-CA-AMPK), and adenoviral SHP (Ad-SHP), but not in Shp knockdown, or with the adenoviral dominant negative form of AMPK (Ad-DN-AMPK). Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP) and protein localisation studies showed that SHP inhibits DNA binding of STAT3 on the Socs3 gene promoter via interaction and colocalisation within the nucleus. Upregulation of inflammatory genes and downregulation of hepatic insulin signalling by acute IL-6 treatment were observed in wild-type mice but not in Shp null mice. Finally, chronic IL-6 exposure caused hepatic insulin resistance, leading to impaired insulin tolerance and elevated gluconeogenesis, and these phenomena were aggravated in Shp null mice. Our results demonstrate that SHP upregulation by metformin may prevent hepatic disorders by regulating the IL-6-dependent pathway, and that this pathway can help to ameliorate the pathogenesis of cytokine-mediated metabolic dysfunction.

A primary defect in glucose production alone cannot induce glucose intolerance without defects in insulin secretion

Increased glucose production is associated with fasting hyperglycaemia in type 2 diabetes but whether or not it causes glucose intolerance is unclear. This study sought to determine whether a primary defect in gluconeogenesis (GNG) resulting in elevated glucose production is sufficient to induce glucose intolerance in the absence of insulin resistance and impaired insulin secretion. Progression of glucose intolerance was assessed in phosphoenolpyruvate carboxykinase (PEPCK) transgenic rats, a genetic model with a primary increase in GNG. Young (4-5 weeks of age) and adult (12-14 weeks of age) PEPCK transgenic and Piebald Virol Glaxo (PVG/c) control rats were studied. GNG, insulin sensitivity, insulin secretion and glucose tolerance were assessed by intraperitoneal and intravascular substrate tolerance tests and hyperinsulinaemic/euglycaemic clamps. Despite elevated GNG and increased glucose appearance, PEPCK transgenic rats displayed normal glucose tolerance due to adequate glucose disposal and robust glucose-mediated insulin secretion. Glucose intolerance only became apparent in the PEPCK transgenic rats following the development of insulin resistance (both hepatic and peripheral) and defective glucose-mediated insulin secretion. Taken together, a single genetic defect in GNG leading to increased glucose production does not adversely affect glucose tolerance. Insulin resistance and impaired glucose-mediated insulin secretion are required to precipitate glucose intolerance in a setting of chronic glucose oversupply.