Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis.

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and ß-Hydroxybutyrate/Acetoacetate in Biological Samples.

The metabolic ratios lactate/pyruvate and ß-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, ß-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency's Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and ß-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

Reduced intestinal lipid absorption and body weight-independent improvements in insulin sensitivity in high-fat diet-fed Park2 knockout mice.

Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. PARK2 is a ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of PARK2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed a high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after 1-wk HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of PARK2 during metabolic stress.

Controlled-release mitochondrial protonophore (CRMP) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates.

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body ß-hydroxybutyrate (ß-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.

Mitochondrial GTP Links Nutrient Sensing to ß Cell Health, Mitochondrial Morphology, and Insulin Secretion Independent of OxPhos.

Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

Author Correction: Metformin inhibits gluconeogenesis via a redox-dependent mechanism in vivo.

In the version of this article originally published, the VPC and VCS flux data shown in Fig. 6e,f were inadvertently duplicated from Fig. 5j,k. The correct data are now shown in Fig. 6e,f. In these corrected data, VPC flux in response to chronic oral metformin treatment was still significantly decreased (Fig. 6e), and there was still no impact of metformin on VCS flux (Fig. 6f). Therefore, the text describing these data remains the same and this correction does not change the conclusion of this study.

Publisher Correction: Non-invasive assessment of hepatic mitochondrial metabolism by positional isotopomer NMR tracer analysis (PINTA).

The originally published version of this Article contained an error in Equation 30, which was inadvertently introduced during the production process. This has now been corrected in the PDF and HTML versions of the Article.

Metformin inhibits gluconeogenesis via a redox-dependent mechanism in vivo.

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin's antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.

Non-invasive assessment of hepatic mitochondrial metabolism by positional isotopomer NMR tracer analysis (PINTA).

Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (V CS) and pyruvate carboxylase flux (V PC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of V CS and V PC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-13C]lactate and glucose tracer. PINTA measures V CS and V PC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic V CS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the V PC/V CS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism.Liver mitochondrial metabolism plays an important role for glucose and lipid homeostasis and its alterations contribute to metabolic disorders, including fatty liver and diabetes. Here Perry et al. develop a method for the measurement of hepatic fluxes by using lactate and glucose tracers in combination with NMR spectroscopy.

Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.

MARCH1 regulates insulin sensitivity by controlling cell surface insulin receptor levels.

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.