Summary Reversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.

Aims/hypothesis The primary aim of this completed multicentre randomised, parallel, double-blind placebo-controlled study was to elucidate the mechanisms of glucose-lowering with colesevelam and secondarily to investigate its effects on lipid metabolism (hepatic de novo lipogenesis, cholesterol and bile acid synthesis). Methods Participants with type 2 diabetes (HbA 1c 6.7-10.0% [50-86 mmol/mol], fasting glucose <16.7 mmol/l, fasting triacylglycerols <3.9 mmol/l and LDL-cholesterol >1.55 mmol/l) treated with diet and exercise, sulfonylurea, metformin or a combination thereof, were randomised by a central coordinator to either 3.75 g/day colesevelam (n=30) or placebo (n=30) for 12 weeks at three clinical sites in the USA. The primary measure was the change from baseline in glucose kinetics with colesevelam compared to placebo treatment. Fasting and postprandial glucose, lipid and bile acid pathways were measured at baseline and post-treatment using stable isotope techniques. Plasma glucose, insulin, total glucagon-like peptide-1 (GLP-1), total glucose-dependent insulinotropic polypeptide (GIP), glucagon and fibroblast growth factor-19 (FGF-19) concentrations were measured during the fasting state and following a meal tolerance test. Data was collected by people blinded to treatment. Colesevelam increased cholesterol and bile acid synthesis and decreased FGF-19 concentrations. However, no effect was seen on fractional hepatic de novo lipogenesis. Conclusions/interpretation Colesevelam, a non-absorbed bile acid sequestrant, increased circulating incretins and improved tissue glucose metabolism in both the fasting and postprandial states in a manner different from other approved oral agents.Trial registration: ClinicalTrials.gov NCT00596427 Funding: The study was funded by Daiichi Sankyo.

Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid-based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water ( 2 H 2 O), distinct, newly synthesized 2 H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2 H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2 H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2 H 2 O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson's disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.

Type II methanotrophic bacteria are a promising production platform for PHA biopolymers. These bacteria are known to produce pure poly-3-hydroxybutyrate homopolymer (PHB). We isolated a strain, Methylocystis sp. WRRC1, that was capable of producing a wide range of polyhydroxybutyrate-co-hydroxyvalerate copolymers (PHB-co-HV) when co-fed methane and valerate or n-pentanol. The ratio of HB to HV monomer was directly related to the concentration of valeric acid in the PHA accumulation media. We observed increased incorporation of HV and total polymer under copper-free growth conditions. The PHB-co-HV copolymers produced had decreased melting temperatures and crystallinity compared with methanotroph-produced PHB.

OBJECTIVE -The purpose of this study was to compare an in vivo test of whole-body glycolysis, the deuterated-glucose disposal test ( 2 H-GDT), with insulin sensitivity measured by the euglycemic-hyperinsulinemic glucose clamp and the steady-state plasma glucose (SSPG) test. RESEARCH DESIGN AND METHODS -The2 H-GDT consists of an oral glucose challenge containing deuterated glucose, followed by measurement of heavy water ( 2 H 2 O) production, which represents whole-body glycolytic disposal of the glucose load.2 H 2 O production is corrected for ambient insulin concentration as an index of tissue insulin sensitivity. The 2 H-GDT was compared with euglycemic-hyperinsulinemic glucose clamps in healthy lean subjects (n ϭ 8) and subjects with the metabolic syndrome (n ϭ 9) and with the SSPG test in overweight (n ϭ 12) and obese (n ϭ 6) subjects.RESULTS -A strong correlation with the clamp was observed for the 75-g and 30-g 2 H-GDT (r ϭ 0.95, P Ͻ 0.0001 and r ϭ 0.88, P Ͻ 0.0001, respectively). The 2 H-GDT and clamp studies revealed marked insulin resistance in subjects with metabolic syndrome compared with lean control subjects. The correlation with the clamp was maintained in each group (lean, r ϭ 0.86, P Ͻ 0.01; metabolic syndrome, r ϭ 0.81, P Ͻ 0.01) for the 75-g test. The 2 H-GDT also correlated strongly with the SSPG test (r ϭ Ϫ0.87, P Ͻ 0.0001) in overweight and obese subjects. CONCLUSIONS -The2 H-GDT, which measures whole-body glycolysis in humans in a quantitative manner, correlates highly with the euglycemic-hyperinsulinemic glucose clamp and the SSPG test. Impaired insulin-mediated whole-body glycolysis is a feature of insulin resistance, which provides a means of assessing insulin sensitivity in vivo.