Publications by authors named "Jonas T Treebak"

65 Publications

Compound- and fiber type-selective requirement of AMPKγ3 for insulin-independent glucose uptake in skeletal muscle.

Mol Metab 2021 Mar 30:101228. Epub 2021 Mar 30.

Nestlé Research, Société des Produits Nestlé S.A., EPFL Innovation Park, Lausanne, 1015, Switzerland; Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, 2200, Denmark. Electronic address:

Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin-independent glucose uptake in muscle and regulates the metabolic activity of brown and beige adipose tissue (BAT). The regulatory AMPKγ3 isoform is uniquely expressed in skeletal muscle and also potentially in BAT. Here, we investigated the role that AMPKγ3 plays in mediating skeletal muscle glucose uptake and whole-body glucose clearance in response to small-molecule activators that act on AMPK via distinct mechanisms. We also assessed if γ3 plays a role in adipose thermogenesis and browning.

Methods: Global AMPKγ3 knockout (KO) mice were generated. A systematic whole-body, tissue and molecular phenotyping linked to glucose homeostasis was performed in γ3 KO and wild type (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood glucose clearance in response to small molecule AMPK activators that target nucleotide-binding domain of γ subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the interface of the α and β subunit (991, MK-8722) were assessed. Oxygen consumption, thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) treatment were performed to assess BAT thermogenesis, characteristics and function.

Results: Genetic ablation of γ3 did not affect body weight, body composition, physical activity, and parameters associated with glucose homeostasis under chow or high fat diet. γ3 deficiency had no effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated glucose uptake in skeletal muscle. Glycolytic muscles in γ3 KO mice showed a partial loss of AMPKα2 activity, which was associated with reduced levels of AMPKα2 and β2 subunit isoforms. Notably, γ3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected γ3 in BAT and found that it preferentially interacts with α2 and β2. We observed no differences in oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue (iWAT), or markers of BAT activity between WT and γ3 KO mice.

Conclusions: These results demonstrate that γ3 plays a key role in mediating AICAR- but not ADaM site binding drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal muscle. We also showed that γ3 is dispensable for β3-adrenergic receptor agonist-induced thermogenesis and browning of iWAT.
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http://dx.doi.org/10.1016/j.molmet.2021.101228DOI Listing
March 2021

Ablation of Nampt in AgRP neurons leads to neurodegeneration and impairs fasting- and ghrelin-mediated food intake.

FASEB J 2021 May;35(5):e21450

Integrative Metabolism and Environmental Influences, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus regulates food intake and whole-body metabolism. NAD regulates multiple cellular processes controlling energy metabolism. Yet, its role in hypothalamic AgRP neurons to control food intake is poorly understood. Here, we aimed to assess whether genetic deletion of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD production, affects AgRP neuronal function to impact whole-body metabolism and food intake. Metabolic parameters during fed and fasted states, and upon systemic ghrelin and leptin administration were studied in AgRP-specific Nampt knockout (ARNKO) mice. We monitored neuropeptide expression levels and density of AgRP neurons in ARNKO mice from embryonic to adult age. NPY cells were used to determine effects of NAMPT inhibition on neuronal viability, energy status, and oxidative stress in vitro. In these cells, NAD depletion reduced ATP levels, increased oxidative stress, and promoted cell death. Agrp expression in the hypothalamus of ARNKO mice gradually decreased after weaning due to progressive AgRP neuron degeneration. Adult ARNKO mice had normal glucose and insulin tolerance, but exhibited an elevated respiratory exchange ratio (RER) when fasted. Remarkably, fasting-induced food intake was unaffected in ARNKO mice when evaluated in metabolic cages, but fasting- and ghrelin-induced feeding and body weight gain decreased in ARNKO mice when evaluated outside metabolic cages. Collectively, deletion of Nampt in AgRP neurons causes progressive neurodegeneration and impairs fasting and ghrelin responses in a context-dependent manner. Our data highlight an essential role of Nampt in AgRP neuron function and viability.
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http://dx.doi.org/10.1096/fj.202002740RDOI Listing
May 2021

Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3RORγtIL-17 Tregs and improve metabolism.

Nat Commun 2021 02 17;12(1):1093. Epub 2021 Feb 17.

Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Oslo, Norway.

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration. Intake of the Methylococcu-based diet markedly boosts Parabacteroides abundances in a manner depending on adaptive immunity, and upregulates triple positive (Foxp3RORγtIL-17) regulatory T cells in the small and large intestine. Collectively, these data point to the potential for leveraging the use of McB lysates to improve immunometabolic homeostasis.
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http://dx.doi.org/10.1038/s41467-021-21408-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889900PMC
February 2021

An abundant biliary metabolite derived from dietary omega-3 polyunsaturated fatty acids regulates triglycerides.

J Clin Invest 2021 Mar;131(6)

Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Omega-3 fatty acids from fish oil reduce triglyceride levels in mammals, yet the mechanisms underlying this effect have not been fully clarified, despite the clinical use of omega-3 ethyl esters to treat severe hypertriglyceridemia and reduce cardiovascular disease risk in humans. Here, we identified in bile a class of hypotriglyceridemic omega-3 fatty acid-derived N-acyl taurines (NATs) that, after dietary omega-3 fatty acid supplementation, increased to concentrations similar to those of steroidal bile acids. The biliary docosahexaenoic acid-containing (DHA-containing) NAT C22:6 NAT was increased in human and mouse plasma after dietary omega-3 fatty acid supplementation and potently inhibited intestinal triacylglycerol hydrolysis and lipid absorption. Supporting this observation, genetic elevation of endogenous NAT levels in mice impaired lipid absorption, whereas selective augmentation of C22:6 NAT levels protected against hypertriglyceridemia and fatty liver. When administered pharmacologically, C22:6 NAT accumulated in bile and reduced high-fat diet-induced, but not sucrose-induced, hepatic lipid accumulation in mice, suggesting that C22:6 NAT is a negative feedback mediator that limits excess intestinal lipid absorption. Thus, biliary omega-3 NATs may contribute to the hypotriglyceridemic mechanism of action of fish oil and could influence the design of more potent omega-3 fatty acid-based therapeutics.
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http://dx.doi.org/10.1172/JCI143861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954602PMC
March 2021

ARDD 2020: from aging mechanisms to interventions.

Aging (Albany NY) 2020 12 30;12(24):24484-24503. Epub 2020 Dec 30.

Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7 Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1 to 4 of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8 ARDD meeting that is scheduled for the 31 of August to 3 of September, 2021, at Columbia University, USA.
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http://dx.doi.org/10.18632/aging.202454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803558PMC
December 2020

Glucagon acutely regulates hepatic amino acid catabolism and the effect may be disturbed by steatosis.

Mol Metab 2020 12 13;42:101080. Epub 2020 Sep 13.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; Department for Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Objective: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear.

Methods: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD).

Results: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis).

Conclusions: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.
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http://dx.doi.org/10.1016/j.molmet.2020.101080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560169PMC
December 2020

Dynamic changes in DICER levels in adipose tissue control metabolic adaptations to exercise.

Proc Natl Acad Sci U S A 2020 09 8;117(38):23932-23941. Epub 2020 Sep 8.

Program in Molecular Biology, Federal University of São Paulo, 04044-020 São Paulo, Brazil;

DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.
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http://dx.doi.org/10.1073/pnas.2011243117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7519274PMC
September 2020

Fasting- and ghrelin-induced food intake is regulated by NAMPT in the hypothalamus.

Acta Physiol (Oxf) 2020 04 14;228(4):e13437. Epub 2020 Jan 14.

Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Aim: Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signalling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied.

Methods: We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration.

Results: NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD , increased levels of ROS, and affected the expression of Agrp, Pomc and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake.

Conclusion: Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.
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http://dx.doi.org/10.1111/apha.13437DOI Listing
April 2020

Nicotinamide riboside does not alter mitochondrial respiration, content or morphology in skeletal muscle from obese and insulin-resistant men.

J Physiol 2020 02 26;598(4):731-754. Epub 2019 Dec 26.

Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Key Points: This is the first long-term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase.

Abstract: Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD ) precursor nicotinamide riboside (NR) boosts NAD levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle-aged, obese, insulin-resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo-controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high-resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady-state NAD levels as well as gene expression and protein abundance of other NAD biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin-resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people.
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http://dx.doi.org/10.1113/JP278752DOI Listing
February 2020

Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise.

Nat Commun 2019 10 11;10(1):4623. Epub 2019 Oct 11.

Department of Nutrition, Exercise and Sports, Section of Molecular Physiology, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen, Denmark.

Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise.
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http://dx.doi.org/10.1038/s41467-019-12523-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789013PMC
October 2019

Fiber type-specific effects of acute exercise on insulin-stimulated AS160 phosphorylation in insulin-resistant rat skeletal muscle.

Am J Physiol Endocrinol Metab 2019 12 1;317(6):E984-E998. Epub 2019 Oct 1.

Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, Michigan.

Muscle is a heterogeneous tissue composed of multiple fiber types. Earlier research revealed fiber type-selective postexercise effects on insulin-stimulated glucose uptake (ISGU) from insulin-resistant rats (increased for type IIA, IIB, IIBX, and IIX, but not type I). In whole muscle from insulin-resistant rats, the exercise increase in ISGU is accompanied by an exercise increase in insulin-stimulated AS160 phosphorylation (pAS160), an ISGU-regulating protein. We hypothesized that, in insulin-resistant muscle, the fiber type-selective exercise effects on ISGU would correspond to the fiber type-selective exercise effects on pAS160. Rats were fed a 2-wk high-fat diet (HFD) and remained sedentary (SED) or exercised before epitrochlearis muscles were dissected either immediately postexercise (IPEX) or at 3 h postexercise (3hPEX) using an exercise protocol that previously revealed fiber type-selective effects on ISGU. 3hPEX muscles and SED controls were incubated ± 100µU/mL insulin. Individual myofibers were isolated and pooled on the basis of myosin heavy chain (MHC) expression, and key phosphoproteins were measured. Myofiber glycogen and MHC expression were evaluated in muscles from other SED, IPEX, and 3hPEX rats. Insulin-stimulated pAkt and pAkt were unaltered by exercise in all fiber types. Insulin-stimulated pAS160 was greater for 3hPEX vs. SED on at least one phosphosite (Ser, Thr, and/or Ser) in type IIA, IIBX, and IIB fibers, but not in type I or IIX fibers. Both IPEX and 3hPEX glycogen were decreased versus SED in all fiber types. These results provided evidence that fiber type-specific pAS160 in insulin-resistant muscle may play a role in the previously reported fiber type-specific elevation in ISGU in some, but not all, fiber types.
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http://dx.doi.org/10.1152/ajpendo.00304.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957376PMC
December 2019

Effects of Nicotinamide Riboside on Endocrine Pancreatic Function and Incretin Hormones in Nondiabetic Men With Obesity.

J Clin Endocrinol Metab 2019 11;104(11):5703-5714

Department of Clinical Pharmacology, Aarhus University Hospital, Aarhus, Denmark.

Objective: Augmenting nicotinamide adenine dinucleotide (NAD+) metabolism through dietary provision of NAD+ precursor vitamins translates to improved glucose handling in rodent models of obesity and diabetes. Preclinical evidence suggests that the NAD+/SIRT1 axis may be implicated in modulating important gut-related aspects of glucose regulation. We sought to test whether NAD+ precursor supplementation with nicotinamide riboside (NR) affects β-cell function, α-cell function, and incretin hormone secretion as well as circulating bile acid levels in humans.

Design: A 12-week randomized, double-blind, placebo-controlled, parallel-group trial in 40 males with obesity and insulin resistance allocated to NR at 1000 mg twice daily (n = 20) or placebo (n = 20). Two-hour 75-g oral glucose tolerance tests were performed before and after the intervention, and plasma concentrations of glucose, insulin, C-peptide, glucagon, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were determined. β-Cell function indices were calculated based on glucose, insulin, and C-peptide measurements. Fasting plasma concentrations of bile acids were determined.

Results: NR supplementation during 12 weeks did not affect fasting or postglucose challenge concentrations of glucose, insulin, C-peptide, glucagon, GLP-1, or GIP, and β-cell function did not respond to the intervention. Additionally, no changes in circulating adipsin or bile acids were observed following NR supplementation.

Conclusion: The current study does not provide evidence to support that dietary supplementation with the NAD+ precursor NR serves to impact glucose tolerance, β-cell secretory capacity, α-cell function, and incretin hormone secretion in nondiabetic males with obesity. Moreover, bile acid levels in plasma did not change in response to NR supplementation.
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http://dx.doi.org/10.1210/jc.2019-01081DOI Listing
November 2019

Mitochondrial function in liver cells is resistant to perturbations in NAD salvage capacity.

J Biol Chem 2019 09 18;294(36):13304-13326. Epub 2019 Jul 18.

Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, DK2200 Copenhagen, Denmark. Electronic address:

Supplementation with NAD precursors such as nicotinamide riboside (NR) has been shown to enhance mitochondrial function in the liver and to prevent hepatic lipid accumulation in high-fat diet (HFD)-fed rodents. Hepatocyte-specific knockout of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT) reduces liver NAD levels, but the metabolic phenotype of deficient hepatocytes in mice is unknown. Here, we assessed role in maintaining mitochondrial and metabolic functions in the mouse liver. Using the Cre-LoxP system, we generated hepatocyte-specific knockout (HNKO) mice, having a 50% reduction of liver NAD levels. We screened the HNKO mice for signs of metabolic dysfunction following 60% HFD feeding for 20 weeks ± NR supplementation and found that NR increases hepatic NAD levels without affecting fat mass or glucose tolerance in HNKO or WT animals. High-resolution respirometry revealed that NR supplementation of the HNKO mice did not increase state III respiration, which was observed in WT mice following NR supplementation. Mitochondrial oxygen consumption and fatty-acid oxidation were unaltered in primary HNKO hepatocytes. Mitochondria isolated from whole-HNKO livers had only a 20% reduction in NAD, suggesting that the mitochondrial NAD pool is less affected by HNKO than the whole-tissue pool. When stimulated with tryptophan in the presence of [N]glutamine, HNKO hepatocytes had a higher [N]NAD enrichment than WT hepatocytes, indicating that HNKO mice compensate through NAD synthesis. We conclude that NAMPT-deficient hepatocytes can maintain substantial NAD levels and that the knockout has only minor consequences for mitochondrial function in the mouse liver.
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http://dx.doi.org/10.1074/jbc.RA118.006756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737221PMC
September 2019

Assessment of mouse liver [1-13C]pyruvate metabolism by dynamic hyperpolarized MRS.

J Endocrinol 2019 09;242(3):251-260

Department of Clinical Medicine, The MR Research Center, Aarhus University Hospital, Aarhus, Denmark.

Hyperpolarized [1-13C]pyruvate magnetic resonance (MR) spectroscopy has the unique ability to detect real-time metabolic changes in vivo owing to its high sensitivity compared with thermal MR and high specificity compared with other metabolic imaging methods. The aim of this study was to explore the potential of hyperpolarized MR spectroscopy for quantification of liver pyruvate metabolism during a hyperinsulinemic-isoglycemic clamp in mice. Hyperpolarized [1-13C]pyruvate was used for in vivo MR spectroscopy of liver pyruvate metabolism in mice. Mice were divided into two groups: (i) non-stimulated 5-h fasted mice and (ii) hyperinsulinemic-isoglycemic clamped mice. During clamp conditions, insulin and donor blood were administered at a constant rate, whereas glucose was infused to maintain isoglycemia. When steady state was reached, insulin-stimulated mice were rapidly infused with hyperpolarized [1-13C]pyruvate for real-time tracking of the dynamic distribution of metabolic derivatives from pyruvate, such as [1-13C]lactate, [1-13C]alanine and [13C]bicarbonate. Isotopomer analysis of plasma glucose confirmed 13C-incorporation from [1-13C]pyruvate into glucose was increased in fasted mice compared with insulin-stimulated mice, demonstrating an increased gluconeogenesis in fasted mice. The AUC ratios for [1-13C]alanine/[1-13C]pyruvate (38.2%), [1-13C]lactate/[1-13C]pyruvate (41.8%) and [13C]bicarbonate/[1-13C]pyruvate (169%) all increased significantly during insulin stimulation. Hyperpolarized [1-13C]pyruvate can be used for in vivo MR spectroscopy of liver pyruvate metabolism during hyperinsulinemic-isoglycemic clamp conditions. Under these conditions, insulin decreased gluconeogenesis and increased [1-13C]alanine, [1-13C]lactate and [13C]bicarbonate after a [1-13C]pyruvate bolus. This application of in vivo spectroscopy has the potential to identify impairments in specific metabolic pathways in the liver associated with obesity, insulin resistance and nonalcoholic fatty liver disease.
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http://dx.doi.org/10.1530/JOE-19-0159DOI Listing
September 2019

Aerobic and resistance exercise training reverses age-dependent decline in NAD salvage capacity in human skeletal muscle.

Physiol Rep 2019 07;7(12):e14139

Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Aging decreases skeletal muscle mass and strength, but aerobic and resistance exercise training maintains skeletal muscle function. NAD is a coenzyme for ATP production and a required substrate for enzymes regulating cellular homeostasis. In skeletal muscle, NAD is mainly generated by the NAD salvage pathway in which nicotinamide phosphoribosyltransferase (NAMPT) is rate-limiting. NAMPT decreases with age in human skeletal muscle, and aerobic exercise training increases NAMPT levels in young men. However, whether distinct modes of exercise training increase NAMPT levels in both young and old people is unknown. We assessed the effects of 12 weeks of aerobic and resistance exercise training on skeletal muscle abundance of NAMPT, nicotinamide riboside kinase 2 (NRK2), and nicotinamide mononucleotide adenylyltransferase (NMNAT) 1 and 3 in young (≤35 years) and older (≥55 years) individuals. NAMPT in skeletal muscle correlated negatively with age (r  = 0.297, P < 0.001, n = 57), and VO peak was the best predictor of NAMPT levels. Moreover, aerobic exercise training increased NAMPT abundance 12% and 28% in young and older individuals, respectively, whereas resistance exercise training increased NAMPT abundance 25% and 30% in young and in older individuals, respectively. None of the other proteins changed with exercise training. In a separate cohort of young and old people, levels of NAMPT, NRK1, and NMNAT1/2 in abdominal subcutaneous adipose tissue were not affected by either age or 6 weeks of high-intensity interval training. Collectively, exercise training reverses the age-dependent decline in skeletal muscle NAMPT abundance, and our findings highlight the value of exercise training in ameliorating age-associated deterioration of skeletal muscle function.
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http://dx.doi.org/10.14814/phy2.14139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6577427PMC
July 2019

Time of Exercise Specifies the Impact on Muscle Metabolic Pathways and Systemic Energy Homeostasis.

Cell Metab 2019 07 18;30(1):92-110.e4. Epub 2019 Apr 18.

Center for Epigenetics and Metabolism, INSERM U1233, Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, CA, USA. Electronic address:

While the timing of food intake is important, it is unclear whether the effects of exercise on energy metabolism are restricted to unique time windows. As circadian regulation is key to controlling metabolism, understanding the impact of exercise performed at different times of the day is relevant for physiology and homeostasis. Using high-throughput transcriptomic and metabolomic approaches, we identify distinct responses of metabolic oscillations that characterize exercise in either the early rest phase or the early active phase in mice. Notably, glycolytic activation is specific to exercise at the active phase. At the molecular level, HIF1α, a central regulator of glycolysis during hypoxia, is selectively activated in a time-dependent manner upon exercise, resulting in carbohydrate exhaustion, usage of alternative energy sources, and adaptation of systemic energy expenditure. Our findings demonstrate that the time of day is a critical factor to amplify the beneficial impact of exercise on both metabolic pathways within skeletal muscle and systemic energy homeostasis.
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http://dx.doi.org/10.1016/j.cmet.2019.03.013DOI Listing
July 2019

Underpowered or negative? A crucial distinction.

Diabetologia 2019 06 25;62(6):1094-1095. Epub 2019 Mar 25.

Steno Diabetes Center Aarhus (SDCA), Aarhus University Hospital and Central Denmark Region, Hedeager 3, DK-8200, Aarhus N, Denmark.

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http://dx.doi.org/10.1007/s00125-019-4853-xDOI Listing
June 2019

Voluntary wheel running in the late dark phase ameliorates diet-induced obesity in mice without altering insulin action.

J Appl Physiol (1985) 2019 04 7;126(4):993-1005. Epub 2019 Feb 7.

Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen , Copenhagen , Denmark.

Metabolic dysfunction and Type 2 diabetes are associated with perturbed circadian rhythms. However, exercise appears to ameliorate circadian disturbances, as it can phase-shift or reset the internal clock system. Evidence is emerging that exercise at a distinct time of day can correct misalignments of the circadian clock and influence energy metabolism. This suggests that timing of exercise training can be important for the prevention and management of metabolic dysfunction. In this study, obese, high-fat diet-fed mice were subjected to voluntary wheel running (VWR) at two different periods of the day to determine the effects of time-of-day-restricted VWR on basal and insulin-stimulated glucose disposal. VWR in the late dark phase reduced body weight gain compared with VWR in the beginning of the dark phase. Conversely, time-of-day-restricted VWR did not influence insulin action and glucose disposal, since skeletal muscle and adipose tissue glucose uptake and insulin signaling remained unaffected. Protein abundance of the core clock proteins, brain-muscle arnt-like 1 (BMAL1), and circadian locomotor output control kaput (CLOCK), were increased in skeletal muscle after VWR, independent of whether mice had access to running wheels in the early or late dark phase. Collectively, we provide evidence that VWR in the late dark phase ameliorates diet-induced obesity without altering insulin action or glucose homeostasis. NEW & NOTEWORTHY Exercise appears to ameliorate circadian disturbances as it can entrain the internal clock system. We provide evidence that voluntary wheel running increases core clock protein abundance and influences diet-induced obesity in mice in a time-of-day-dependent manner. However, the effect of time-of-day-restricted voluntary wheel running on body weight gain is not associated with enhanced basal- and insulin-stimulated glucose disposal, suggesting that time-of-day-restricted voluntary wheel running affects energy homeostasis rather than glucose homeostasis.
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http://dx.doi.org/10.1152/japplphysiol.00737.2018DOI Listing
April 2019

ADAMTS9 Regulates Skeletal Muscle Insulin Sensitivity Through Extracellular Matrix Alterations.

Diabetes 2019 03 9;68(3):502-514. Epub 2019 Jan 9.

Section for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

The rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.
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http://dx.doi.org/10.2337/db18-0418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385758PMC
March 2019

The aromatic amino acid sensor GPR142 controls metabolism through balanced regulation of pancreatic and gut hormones.

Mol Metab 2019 01 5;19:49-64. Epub 2018 Nov 5.

Section for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark; Laboratory for Molecular Pharmacology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Objectives: GPR142, which is highly expressed in pancreatic islets, has recently been deorphanized as a receptor for aromatic amino acids; however, its physiological role and pharmacological potential is unclear.

Methods And Results: We find that GPR142 is expressed not only in β- but also in α-cells of the islets as well as in enteroendocrine cells, and we confirm that GPR142 is a highly selective sensor of essential aromatic amino acids, in particular Trp and oligopeptides with N-terminal Trp. GPR142 knock-out mice displayed a very limited metabolic phenotype but demonstrated that L-Trp induced secretion of pancreatic and gut hormones is mediated through GPR142 but that the receptor is not required for protein-induced hormone secretion. A synthetic GPR142 agonist stimulated insulin and glucagon as well as GIP, CCK, and GLP-1 secretion. In particular, GIP secretion was sensitive to oral administration of the GPR142 agonist an effect which in contrast to the other hormones was blocked by protein load. Oral administration of the GPR142 agonist increased [H]-2-deoxyglucose uptake in muscle and fat depots mediated through insulin action while it lowered liver glycogen conceivably mediated through glucagon, and, consequently, it did not lower total blood glucose. Nevertheless, acute administration of the GPR142 agonist strongly improved oral glucose tolerance in both lean and obese mice as well as Zucker fatty rat. Six weeks in-feed chronic treatment with the GPR142 agonist did not affect body weight in DIO mice, but increased energy expenditure and carbohydrate utilization, lowered basal glucose, and improved insulin sensitivity.

Conclusions: GPR142 functions as a sensor of aromatic amino acids, controlling GIP but also CCK and GLP-1 as well as insulin and glucagon in the pancreas. GPR142 agonists could have novel interesting potential in modifying metabolism through a balanced action of gut hormones as well as both insulin and glucagon.
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http://dx.doi.org/10.1016/j.molmet.2018.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323244PMC
January 2019

Electrical pulse stimulation induces differential responses in insulin action in myotubes from severely obese individuals.

J Physiol 2019 01 2;597(2):449-466. Epub 2018 Dec 2.

Human Performance Laboratory, Ward Sports Medicine Building, East Carolina University, Greenville, NC, USA.

Key Points: Exercise/exercise training can enhance insulin sensitivity through adaptations in skeletal muscle, the primary site of insulin-mediated glucose disposal; however, in humans the range of improvement can vary substantially. The purpose of this study was to determine if obesity influences the magnitude of the exercise response in relation to improving insulin sensitivity in human skeletal muscle. Electrical pulse stimulation (EPS; 24 h) of primary human skeletal muscle myotubes improved insulin action in tissue from both lean and severely obese individuals, but responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and increased AMP accumulation and AMPK Thr phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise-responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects.

Abstract: Exercise/muscle contraction can enhance whole-body insulin sensitivity; however, in humans the range of improvements can vary substantially. In order, to determine if obesity influences the magnitude of the exercise response, this study compared the effects of electrical pulse stimulation (EPS)-induced contractile activity upon primary myotubes derived from lean and severely obese (BMI ≥ 40 kg/m ) women. Prior to muscle contraction, insulin action was compromised in myotubes from the severely obese as was evident from reduced insulin-stimulated glycogen synthesis, glucose oxidation, glucose uptake, insulin signal transduction (IRS1, Akt, TBC1D4), and insulin-stimulated GLUT4 translocation. EPS (24 h) increased AMP, IMP, AMPK Thr phosphorylation, PGC1α content, and insulin action in myotubes of both the lean and severely obese subjects. However, despite normalizing indices of insulin action to levels seen in the lean control (non-EPS) condition, responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and EPS increased AMP accumulation and AMPK Thr phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise-responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects.
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http://dx.doi.org/10.1113/JP276990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333093PMC
January 2019

Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice.

FASEB J 2019 02 10;33(2):2719-2731. Epub 2018 Oct 10.

Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Remodeling of the gut microbiota is implicated in various metabolic and inflammatory diseases of the gastrointestinal tract. We hypothesized that the gut microbiota affects the DNA methylation profile of intestinal epithelial cells (IECs) which could, in turn, alter intestinal function. In this study, we used mass spectrometry and methylated DNA capture to respectively investigate global and genome-wide DNA methylation of intestinal epithelial cells from germ-free (GF) and conventionally raised mice. In colonic IECs from GF mice, DNA was markedly hypermethylated. This was associated with a dramatic loss of ten-eleven-translocation activity, a lower DNA methyltransferase activity and lower circulating levels of the 1-carbon metabolite, folate. At the gene level, we found an enrichment for differentially methylated regions proximal to genes regulating the cytotoxicity of NK cells (false-discovery rate < 8.9E), notably genes regulating the cross-talk between NK cells and target cells, such as members of the NK group 2 member D ligand superfamily Raet. This distinct epigenetic signature was associated with a marked decrease in Raet1 expression and a loss of CD56/CD45 cells in the intestine of GF mice. Thus, our results indicate that altered activity of methylation-modifying enzymes in GF mice influences the IEC epigenome and modulates the crosstalk between IECs and NK cells. Epigenetic reprogramming of IECs may modulate intestinal function in diseases associated with altered gut microbiota.-Poupeau, A., Garde, C., Sulek, K., Citirikkaya, K., Treebak, J. T., Arumugam, M., Simar, D., Olofsson, L. E., Bäckhed, F., Barrès, R. Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice.
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http://dx.doi.org/10.1096/fj.201800787RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338647PMC
February 2019

Skeletal Muscle Insulin Sensitivity Show Circadian Rhythmicity Which Is Independent of Exercise Training Status.

Front Physiol 2018 28;9:1198. Epub 2018 Aug 28.

Section of Integrative Physiology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.

Circadian rhythms can be perturbed by shift work, travel across time zones, many occupational tasks, or genetic mutations. Perturbed circadian rhythms are associated with the increasing problem of obesity, metabolic dysfunction, and insulin resistance. We hypothesized that insulin sensitivity in skeletal muscle follows a circadian pattern and that this pattern is important for overall metabolic function. This hypothesis was verified using mice as a model system. We observed circadian rhythmicity in whole body insulin tolerance, as well as in signaling pathways regulating insulin- and exercise-induced glucose uptake in skeletal muscle, including AKT, 5'-adenosine monophosphate-activated protein kinase (AMPK) and TBC1 domain family member 4 (TBC1D4) phosphorylation. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissues also differed between day- and nighttime. However, the rhythmicity of glucose uptake differed from the rhythm of whole-body insulin tolerance. These results indicate that neither skeletal muscle nor adipose tissue play a major role for the circadian rhythmicity in whole-body insulin tolerance. To study the circadian pattern of insulin sensitivity directly in skeletal muscle, we determined glucose uptake under basal and submaximal insulin-stimulated conditions every sixth hour. Both insulin sensitivity and signaling of isolated skeletal muscle peaked during the dark period. We next examined the effect of exercise training on the circadian rhythmicity of insulin sensitivity. As expected, voluntary exercise training enhanced glucose uptake in skeletal muscle. Nevertheless, exercise training did not affect the circadian rhythmicity of skeletal muscle insulin sensitivity. Taken together, our results provide evidence that skeletal muscle insulin sensitivity exhibits circadian rhythmicity.
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http://dx.doi.org/10.3389/fphys.2018.01198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121032PMC
August 2018

A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects.

Am J Clin Nutr 2018 08;108(2):343-353

Department of Clinical Pharmacology, Aarhus University Hospital, Aarhus, Denmark.

Background: Animal studies suggest a positive role for nicotinamide riboside (NR) on insulin sensitivity and hepatic steatosis in models of obesity and type 2 diabetes. NR, an NAD+ precursor, is a member of the vitamin B-3 family now available as an over-the-counter supplement. Although data from preclinical trials appear consistent, potential effects and safety need to be evaluated in human clinical trials.

Objective: The aim of this study was to test the safety of dietary NR supplementation over a 12-wk period and potential to improve insulin sensitivity and other metabolic parameters in obese, insulin-resistant men.

Design: In an investigator-initiated randomized, placebo-controlled, double-blinded, and parallel-group designed clinical trial, forty healthy, sedentary men with a body mass index (BMI) > 30 kg/m2, age-range 40-70 y were randomly assigned to 12 wk of NR (1000 mg twice daily) or placebo. We determined the effects of NR supplementation on insulin sensitivity by a hyperinsulinemic euglycemic clamp and substrate metabolism by indirect calorimetry and labeled substrates of tritiated glucose and palmitate. Body composition and fat mass distribution were determined by whole-body dual-energy X-ray absorptiometry (DXA) and MRI scans, and measurements of intrahepatic lipid content were obtained by MR spectroscopy.

Results: Insulin sensitivity, endogenous glucose production, and glucose disposal and oxidation were not improved by NR supplementation. Similarly, NR supplementation had no effect on resting energy expenditure, lipolysis, oxidation of lipids, or body composition. No serious adverse events due to NR supplementation were observed and safety blood tests were normal.

Conclusion: 12 wk of NR supplementation in doses of 2000 mg/d appears safe, but does not improve insulin sensitivity and whole-body glucose metabolism in obese, insulin-resistant men. This trial was registered at clinicaltrials.gov as NCT02303483.
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http://dx.doi.org/10.1093/ajcn/nqy132DOI Listing
August 2018

Sevoflurane Impairs Insulin Secretion and Tissue-Specific Glucose Uptake In Vivo.

Basic Clin Pharmacol Toxicol 2018 Dec 24;123(6):732-738. Epub 2018 Aug 24.

Department of Biomedicine, Health, Aarhus University, Aarhus C, Denmark.

The use of anaesthetics severely influences substrate metabolism. This poses challenges for patients in clinical settings and for the use of animals in diabetes research. Sevoflurane can affect regulation of glucose homoeostasis at several steps, but the tissue-specific response remains to be determined. The aim of the study was to investigate the pharmacological effect of sevoflurane anaesthesia on glucose homoeostasis during hyperinsulinaemic clamp conditions, the gold standard method for assessment of whole-body insulin sensitivity. Conscious mice (n = 6) and mice under sevoflurane anaesthesia (n = 8) underwent a hyperinsulinaemic clamp where constant infusion of insulin and donor blood was administered during variable glucose infusion to maintain isoglycaemia. 2-[1- C]-deoxy-D-glucose was infused to determine tissue-specific uptake of glucose in adipose tissue, heart, brain and skeletal muscle. Sevoflurane anaesthesia severely impaired insulin-stimulated whole-body glucose uptake demonstrated by a 50% lower glucose infusion rate (GIR). This was associated with decreased glucose uptake in brain, soleus, triceps and gastrocnemius muscles in sevoflurane-anaesthetized mice compared to conscious mice. Plasma-free fatty acids (FFA), a potent inducer of insulin resistance, increased by 42% in mice during sevoflurane anaesthesia. In addition, insulin secretion from pancreatic β-cell was lower in fasted, anaesthetized mice. Sevoflurane anaesthesia impairs insulin secretion, induces insulin resistance in mice and reduces glucose uptake in non-insulin-sensitive tissue like the brain. The underlying mechanisms may involve sevoflurane-induced mobilization of FFA.
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http://dx.doi.org/10.1111/bcpt.13087DOI Listing
December 2018

NAMPT-mediated NAD biosynthesis is indispensable for adipose tissue plasticity and development of obesity.

Mol Metab 2018 05 7;11:178-188. Epub 2018 Mar 7.

Section for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, 2200 Copenhagen, Denmark; Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Objective: The ability of adipose tissue to expand and contract in response to fluctuations in nutrient availability is essential for the maintenance of whole-body metabolic homeostasis. Given the nutrient scarcity that mammals faced for millions of years, programs involved in this adipose plasticity were likely evolved to be highly efficient in promoting lipid storage. Ironically, this previously advantageous feature may now represent a metabolic liability given the caloric excess of modern society. We speculate that nicotinamide adenine dinucleotide (NAD) biosynthesis exemplifies this concept. Indeed NAD/NADH metabolism in fat tissue has been previously linked with obesity, yet whether it plays a causal role in diet-induced adiposity is unknown. Here we investigated how the NAD biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) supports adipose plasticity and the pathological progression to obesity.

Methods: We utilized a newly generated Nampt loss-of-function model to investigate the tissue-specific and systemic metabolic consequences of adipose NAD deficiency. Energy expenditure, glycemic control, tissue structure, and gene expression were assessed in the contexts of a high dietary fat burden as well as the transition back to normal chow diet.

Results: Fat-specific Nampt knockout (FANKO) mice were completely resistant to high fat diet (HFD)-induced obesity. This was driven in part by reduced food intake. Furthermore, HFD-fed FANKO mice were unable to undergo healthy expansion of adipose tissue mass, and adipose depots were rendered fibrotic with markedly reduced mitochondrial respiratory capacity. Yet, surprisingly, HFD-fed FANKO mice exhibited improved glucose tolerance compared to control littermates. Removing the HFD burden largely reversed adipose fibrosis and dysfunction in FANKO animals whereas the improved glucose tolerance persisted.

Conclusions: These findings indicate that adipose NAMPT plays an essential role in handling dietary lipid to modulate fat tissue plasticity, food intake, and systemic glucose homeostasis.
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http://dx.doi.org/10.1016/j.molmet.2018.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6001355PMC
May 2018

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity.

Mol Metab 2018 05 24;11:160-177. Epub 2018 Feb 24.

Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. Electronic address:

Objective: Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle.

Methods: We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses.

Results: We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2).

Conclusions: Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders.
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http://dx.doi.org/10.1016/j.molmet.2018.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6001359PMC
May 2018

Hepatic NAD levels and NAMPT abundance are unaffected during prolonged high-fat diet consumption in C57BL/6JBomTac mice.

Mol Cell Endocrinol 2018 09 2;473:245-256. Epub 2018 Feb 2.

Novo Nordisk Foundation Center for Basic Metabolic Research, Section of Integrative Physiology, University of Copenhagen, Copenhagen, Denmark. Electronic address:

Dietary supplementation of nicotinamide adenine dinucleotide (NAD) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD levels, but caused a decrease in NADP and NADPH levels. Decreased NADP content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD pool in HFD-challenged C57BL/6JBomTac male mice.
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http://dx.doi.org/10.1016/j.mce.2018.01.025DOI Listing
September 2018

Perturbations in the p53/miR-34a/SIRT1 pathway in the R6/2 Huntington's disease model.

Mol Cell Neurosci 2018 04 28;88:118-129. Epub 2017 Dec 28.

Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Electronic address:

The three factors, p53, the microRNA-34 family and Sirtuin 1 (SIRT1), interact in a positive feedback loop involved in cell cycle progression, cellular senescence and apoptosis. Each factor in this triad has roles in metabolic regulation, maintenance of mitochondrial function, and regulation of brain-derived neurotrophic factor (BDNF). Thus, this regulatory network holds potential importance for the pathophysiology of Huntington's disease (HD), an inherited neurodegenerative disorder in which both mitochondrial dysfunction and impaired neurotrophic signalling are observed. We investigated expression of the three members of this regulatory triad in the R6/2 HD mouse model. Compared to wild-type littermates, we found decreased levels of miR-34a-5p, increased SIRT1 mRNA and protein levels, and increased levels of p53 protein in brain tissue from R6/2 mice. The upregulation of SIRT1 did not appear to lead to an increased activity of the enzyme, as based on measures of p53 acetylation. In other words, the observed changes did not reflect the known interactions between these factors, indicating a general perturbation of the p53, miR-34a and SIRT1 pathway in HD. This is the first study investigating the entire triad during disease progression in an HD model. Given the importance of these three factors alone and within the triad, our results indicate that outside factors are regulating - or dysregulating - this pathway in HD.
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http://dx.doi.org/10.1016/j.mcn.2017.12.009DOI Listing
April 2018

Age-dependent alterations of glucose clearance and homeostasis are temporally separated and modulated by dietary fat.

J Nutr Biochem 2018 04 18;54:66-76. Epub 2017 Oct 18.

Laboratory of Genomics and Molecular Biomedicine, Department of Biology, Faculty of Science, University of Copenhagen, Denmark. Electronic address:

Diet- and age-dependent changes in glucose regulation in mice occur, but the temporal development, mechanisms and influence of dietary fat source remain to be defined. We followed metabolic changes in three groups of mice including a low-fat diet (LFD) reference group and two high-fat, high-sucrose diets based on either fish oil (FOD) or soybean oil (SOD), rich in ω3- and ω6-polyunsaturated fatty acids, respectively, to closely monitor the age-dependent development in glucose regulation in both obese (SOD-fed) and lean (LFD- and FOD-fed) mice. We assessed glucose homeostasis and glucose clearance at week 8, 12, 16, 24, 31, and 39 and performed an insulin tolerance test at week 40. We further analyzed correlations between the gut microbiota and key metabolic parameters. Interestingly, alterations in glucose homeostasis and glucose clearance were temporally separated, while 16S ribosomal gene amplicon sequencing revealed that gut microbial alterations formed correlation clusters with fat mass and either glucose homeostasis or glucose clearance, but rarely both. Importantly, effective glucose clearance was maintained in FOD- and even increased in LFD-fed mice, whereas SOD-fed mice rapidly developed impaired glucose clearance followed by a gradual improvement from week 8 to week 39. All groups had similar responses to insulin 40 weeks post diet initiation despite severe nonalcoholic steatohepatitis in SOD-fed mice. We conclude that age-related alterations in glucose regulation may occur in both lean and obese mice and are modulated by dietary fat as indicated by the sustained metabolic homeostasis observed in mice fed ω3-polyunsaturated fatty acids.
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http://dx.doi.org/10.1016/j.jnutbio.2017.09.026DOI Listing
April 2018