MAD2-Dependent Insulin Receptor Endocytosis Regulates Metabolic Homeostasis.

Insulin activates insulin receptor (IR) signaling and subsequently triggers IR endocytosis to attenuate signaling. Cell division regulators MAD2, BUBR1, and p31comet promote IR endocytosis on insulin stimulation. Here, we show that genetic ablation of the IR-MAD2 interaction in mice delays IR endocytosis, increases IR levels, and prolongs insulin action at the cell surface. This in turn causes a defect in insulin clearance and increases circulating insulin levels, unexpectedly increasing glucagon levels, which alters glucose metabolism modestly. Disruption of the IR-MAD2 interaction increases serum fatty acid concentrations and hepatic fat accumulation in fasted male mice. Furthermore, disruption of the IR-MAD2 interaction distinctly changes metabolic and transcriptomic profiles in the liver and adipose tissues. Our findings establish the function of cell division regulators in insulin signaling and provide insights into the metabolic functions of IR endocytosis. ARTICLE HIGHLIGHTS: The physiological role of IR endocytosis in insulin sensitivity remains unclear. Disruption of the IR-MAD2 interaction delays IR endocytosis and prolongs insulin signaling. IR-MAD2 controls insulin clearance and glucose metabolism. IR-MAD2 maintains energy homeostasis.

Q-Flux: A method to assess hepatic mitochondrial succinate dehydrogenase, methylmalonyl-CoA mutase, and glutaminase fluxes in vivo.

The mammalian succinate dehydrogenase (SDH) complex has recently been shown as capable of operating bidirectionally. Here, we develop a method (Q-Flux) capable of measuring absolute rates of both forward (VSDH(F)) and reverse (VSDH(R)) flux through SDH in vivo while also deconvoluting the amount of glucose derived from four discreet carbon sources in the liver. In validation studies, a mitochondrial uncoupler increased net SDH flux by >100% in awake rodents but also increased SDH cycling. During hyperglucagonemia, attenuated pyruvate cycling enhances phosphoenolpyruvate carboxykinase efficiency to drive increased gluconeogenesis, which is complemented by increased glutaminase (GLS) flux, methylmalonyl-CoA mutase (MUT) flux, and glycerol conversion to glucose. During hyperinsulinemic-euglycemic clamp, both pyruvate carboxylase and GLS are suppressed, while VSDH(R) is increased. Unstimulated MUT is a minor anaplerotic reaction but is readily induced by small amounts of propionate, which elicits glucagon-like metabolic rewiring. Taken together, Q-Flux yields a comprehensive picture of hepatic mitochondrial metabolism and should be broadly useful to researchers.

Distinct subcellular localisation of intramyocellular lipids and reduced PKCε/PKCθ activity preserve muscle insulin sensitivity in exercise-trained mice.

AIMS/HYPOTHESIS: Athletes exhibit increased muscle insulin sensitivity, despite increased intramuscular triacylglycerol content. This phenomenon has been coined the 'athlete's paradox' and is poorly understood. Recent findings suggest that the subcellular distribution of sn-1,2-diacylglycerols (DAGs) in the plasma membrane leading to activation of novel protein kinase Cs (PKCs) is a crucial pathway to inducing insulin resistance. Here, we hypothesised that regular aerobic exercise would preserve muscle insulin sensitivity by preventing increases in plasma membrane sn-1,2-DAGs and activation of PKCε and PKCθ despite promoting increases in muscle triacylglycerol content. METHODS: C57BL/6J mice were allocated to three groups (regular chow feeding [RC]; high-fat diet feeding [HFD]; RC feeding and running wheel exercise [RC-EXE]). We used a novel LC-MS/MS/cellular fractionation method to assess DAG stereoisomers in five subcellular compartments (plasma membrane [PM], endoplasmic reticulum, mitochondria, lipid droplets and cytosol) in the skeletal muscle. RESULTS: We found that the HFD group had a greater content of sn-DAGs and ceramides in multiple subcellular compartments compared with the RC mice, which was associated with an increase in PKCε and PKCθ translocation. However, the RC-EXE mice showed, of particular note, a reduction in PM sn-1,2-DAG and ceramide content when compared with HFD mice. Consistent with the PM sn-1,2-DAG-novel PKC hypothesis, we observed an increase in phosphorylation of threonine1150 on the insulin receptor kinase (IRKT1150), and reductions in insulin-stimulated IRKY1162 phosphorylation and IRS-1-associated phosphoinositide 3-kinase activity in HFD compared with RC and RC-EXE mice, which are sites of PKCε and PKCθ action, respectively. CONCLUSIONS/INTERPRETATION: These results demonstrate that lower PKCθ/PKCε activity and sn-1,2-DAG content, especially in the PM compartment, can explain the preserved muscle insulin sensitivity in RC-EXE mice.

Systemic gene therapy for methylmalonic acidemia using the novel adeno-associated viral vector 44.9.

Methylmalonic acidemia (MMA) is a severe and potentially lethal autosomal recessive inborn error of metabolism most frequently caused by mutations in the methylmalonyl-CoA mutase (MMUT) gene. Proof-of-concept adeno-associated virus (AAV) gene therapy studies using mouse models of MMA have demonstrated promise for this therapeutic approach but translation to the clinic could be limited by preexisting capsid immunity and vector potency. Here we explore the efficacy of a novel clade E capsid, 44.9, as a serotype for systemic AAV gene therapy for MMA. An anti-AAV44.9 neutralizing antibody (NAb) survey in adult volunteers (n = 19) and a large cohort of MMA patients (n = 48) revealed a seroprevalence rate of ∼26% and 13%, respectively. The efficacy of AAV44.9 gene delivery was examined in two murine models of MMA, representing neonatal lethal and juvenile phenotypes of MMA. Systemic delivery of the AAV44.9-Mmut vector prevented lethality and lowered disease-related metabolites in MMA mice. Tissue biodistribution and transgene expression studies in treated MMA mice showed that AAV44.9 was efficient at transducing the liver and heart. In summary, we establish that AAV44.9 exhibits a low prevalence of preexisting NAb in humans, is highly efficacious in the treatment of clinically severe MMA mouse models and is therefore a promising vector for clinical translation.

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.

Promoterless, Nuclease-Free Genome Editing Confers a Growth Advantage for Corrected Hepatocytes in Mice With Methylmalonic Acidemia.

BACKGROUND AND AIMS: Adeno-associated viral (AAV) gene therapy has shown great promise as an alternative treatment for metabolic disorders managed using liver transplantation, but remains limited by transgene loss and genotoxicity. Our study aims to test an AAV vector with a promoterless integrating cassette, designed to provide sustained hepatic transgene expression and reduced toxicity in comparison to canonical AAV therapy. APPROACH AND RESULTS: Our AAV vector was designed to insert a methylmalonyl-CoA mutase (MMUT) transgene into the 3' end of the albumin locus and tested in mouse models of methylmalonic acidemia (MMA). After neonatal delivery, we longitudinally evaluated hepatic transgene expression, plasma levels of methylmalonate, and the MMA biomarker, fibroblast growth factor 21 (Fgf21), as well as integration of MMUT in the albumin locus. At necropsy, we surveyed for AAV-related hepatocellular carcinoma (HCC) in all treated MMA mice and control littermates. AAV-mediated genome editing of MMUT into the albumin locus resulted in permanent hepatic correction in MMA mouse models, which was accompanied by decreased levels of methylmalonate and Fgf21, and improved survival without HCC. With time, levels of transgene expression increased and methylmalonate progressively decreased, whereas the number of albumin-MMUT integrations and corrected hepatocytes in MMA mice increased, but not in similarly treated wild-type animals. Additionally, expression of MMUT in the setting of MMA conferred a selective growth advantage upon edited cells, which potentiates the therapeutic response. CONCLUSIONS: In conclusion, our findings demonstrate that AAV-mediated, promoterless, nuclease-free genome editing at the albumin locus provides safe and durable therapeutic benefit in neonatally treated MMA mice.

Systemic AAV9 gene therapy improves the lifespan of mice with Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 (NPC1) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. A murine model of NPC1 disease (Npc1-/-) displays a rapidly progressing form of NPC1 disease which is characterized by weight loss, ataxia, increased cholesterol storage, loss of cerebellar Purkinje neurons and early lethality. To test the potential efficacy of gene therapy for NPC1, we constructed adeno-associated virus serotype 9 (AAV9) vectors to deliver the NPC1 gene under the transcriptional control of the neuronal-specific (CamKII) or a ubiquitous (EF1a) promoter. The Npc1-/- mice that received a single dose of AAV9-CamKII-NPC1 as neonates (2.6 × 1011GC) or at weaning (1.3 × 1012GC), and the mice that received a single dose of AAV9-EF1a-NPC1 at weaning (1.2 × 1012GC), exhibited an increased life span, characterized by delayed weight loss and diminished motor decline. Cholesterol storage and Purkinje neuron loss were also reduced in the central nervous system of AAV9 treated Npc1-/- mice. Treatment with AAV9-EF1a-NPC1, as compared to AAV9-CamKII-NPC1, resulted in significantly increased survival (mean survival increased from 69 days to 166 and 97 days, respectively) and growth, and reduced hepatic-cholesterol accumulation. Our results provide the first demonstration that gene therapy may represent a therapeutic option for NPC1 patients and suggest that extraneuronal NPC1 expression can further augment the lifespan of the Npc1-/- mice after systemic AAV gene delivery.