Publications by authors named "Gerald I Shulman"

367 Publications

Mechanisms and disease consequences of nonalcoholic fatty liver disease.

Cell 2021 May;184(10):2537-2564

Departments of Internal Medicine and Cellular & Molecular Physiology, Yale Diabetes Research Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Nonalcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide. Its more advanced subtype, nonalcoholic steatohepatitis (NASH), connotes progressive liver injury that can lead to cirrhosis and hepatocellular carcinoma. Here we provide an in-depth discussion of the underlying pathogenetic mechanisms that lead to progressive liver injury, including the metabolic origins of NAFLD, the effect of NAFLD on hepatic glucose and lipid metabolism, bile acid toxicity, macrophage dysfunction, and hepatic stellate cell activation, and consider the role of genetic, epigenetic, and environmental factors that promote fibrosis progression and risk of hepatocellular carcinoma in NASH.
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http://dx.doi.org/10.1016/j.cell.2021.04.015DOI Listing
May 2021

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

Int J Mol Sci 2021 Apr 30;22(9). Epub 2021 Apr 30.

Service of Biochemistry and Molecular Genetics, Hospital Clinic Universitari, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Carrer de Villarroel 170, 08036 Barcelona, Spain.

The metabolic ratios lactate/pyruvate and β-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, β-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 β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.
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http://dx.doi.org/10.3390/ijms22094752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8125771PMC
April 2021

Point: An alternative hypothesis for why exposure to static magnetic and electric fields treats type 2 diabetes.

Am J Physiol Endocrinol Metab 2021 May 12;320(5):E999-E1000. Epub 2021 Apr 12.

Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut.

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http://dx.doi.org/10.1152/ajpendo.00657.2020DOI Listing
May 2021

Reply to Carter et al.: An alternative hypothesis for why exposure to static magnetic and electric fields treats type 2 diabetes.

Am J Physiol Endocrinol Metab 2021 May 12;320(5):E1003. Epub 2021 Apr 12.

Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut.

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http://dx.doi.org/10.1152/ajpendo.00120.2021DOI Listing
May 2021

Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake.

Nat Metab 2021 03 8;3(3):378-393. Epub 2021 Mar 8.

Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA.

TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.
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http://dx.doi.org/10.1038/s42255-021-00359-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990718PMC
March 2021

Therapeutic potential of mitochondrial uncouplers for the treatment of metabolic associated fatty liver disease and NASH.

Mol Metab 2021 Apr 3;46:101178. Epub 2021 Feb 3.

Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Background: Mitochondrial uncouplers shuttle protons across the inner mitochondrial membrane via a pathway that is independent of adenosine triphosphate (ATP) synthase, thereby uncoupling nutrient oxidation from ATP production and dissipating the proton gradient as heat. While initial toxicity concerns hindered their therapeutic development in the early 1930s, there has been increased interest in exploring the therapeutic potential of mitochondrial uncouplers for the treatment of metabolic diseases.

Scope Of Review: In this review, we cover recent advances in the mechanisms by which mitochondrial uncouplers regulate biological processes and disease, with a particular focus on metabolic associated fatty liver disease (MAFLD), nonalcoholic hepatosteatosis (NASH), insulin resistance, and type 2 diabetes (T2D). We also discuss the challenges that remain to be addressed before synthetic and natural mitochondrial uncouplers can successfully enter the clinic.

Major Conclusions: Rodent and non-human primate studies suggest that a myriad of small molecule mitochondrial uncouplers can safely reverse MAFLD/NASH with a wide therapeutic index. Despite this, further characterization of the tissue- and cell-specific effects of mitochondrial uncouplers is needed. We propose targeting the dosing of mitochondrial uncouplers to specific tissues such as the liver and/or developing molecules with self-limiting properties to induce a subtle and sustained increase in mitochondrial inefficiency, thereby avoiding systemic toxicity concerns.
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http://dx.doi.org/10.1016/j.molmet.2021.101178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085597PMC
April 2021

An update on brown adipose tissue biology: a discussion of recent findings.

Am J Physiol Endocrinol Metab 2021 03 18;320(3):E488-E495. Epub 2021 Jan 18.

Department of Health Sciences, Laboratory of Molecular Biology of Exercise, School of Applied Sciences, University of Campinas, São Paulo, Brazil.

Brown adipose tissue (BAT) has been encouraged as a potential treatment for obesity and comorbidities due to its thermogenic activity capacity and contribution to energy expenditure. Some interventions such as cold and β-adrenergic drugs are able to activate BAT thermogenesis as well as promote differentiation of white adipocytes into brown-like cells (browning), enhancing the thermogenic activity of these cells. In this mini-review, we discuss new mechanisms related to BAT and energy expenditure. In this regard, we will also discuss recent studies that have revealed the existence of important secretory molecules from BAT "batokines" that act in autocrine, paracrine, and endocrine mechanisms, which in turn may explain some of the beneficial roles of BAT on whole body glucose and fat metabolism. Finally, we will discuss new insights related to BAT thermogenesis with an additional focus on the distinct features of BAT metabolism between rodents and humans.
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http://dx.doi.org/10.1152/ajpendo.00310.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988785PMC
March 2021

Short-term overnutrition induces white adipose tissue insulin resistance through sn-1,2-diacylglycerol/PKCε/insulin receptor Thr1160 phosphorylation.

JCI Insight 2021 Feb 22;6(4). Epub 2021 Feb 22.

Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut, USA.

White adipose tissue (WAT) insulin action has critical anabolic function and is dysregulated in overnutrition. However, the mechanism of short-term high-fat diet-induced (HFD-induced) WAT insulin resistance (IR) is poorly understood. Based on recent evidences, we hypothesize that a short-term HFD causes WAT IR through plasma membrane (PM) sn-1,2-diacylglycerol (sn-1,2-DAG) accumulation, which promotes protein kinase C-ε (PKCε) activation to impair insulin signaling by phosphorylating insulin receptor (Insr) Thr1160. To test this hypothesis, we assessed WAT insulin action in 7-day HFD-fed versus regular chow diet-fed rats during a hyperinsulinemic-euglycemic clamp. HFD feeding caused WAT IR, reflected by impaired insulin-mediated WAT glucose uptake and lipolysis suppression. These changes were specifically associated with PM sn-1,2-DAG accumulation, higher PKCε activation, and impaired insulin-stimulated Insr Tyr1162 phosphorylation. In order to examine the role of Insr Thr1160 phosphorylation in mediating lipid-induced WAT IR, we examined these same parameters in InsrT1150A mice (mouse homolog for human Thr1160) and found that HFD feeding induced WAT IR in WT control mice but not in InsrT1150A mice. Taken together, these data demonstrate the importance of the PM sn-1,2-DAG/PKCε/Insr Thr1160 phosphorylation pathway in mediating lipid-induced WAT IR and represent a potential therapeutic target to improve WAT insulin sensitivity.
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http://dx.doi.org/10.1172/jci.insight.139946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934919PMC
February 2021

A feed-forward regulatory loop in adipose tissue promotes signaling by the hepatokine FGF21.

Genes Dev 2021 Jan 17;35(1-2):133-146. Epub 2020 Dec 17.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

The cJun NH-terminal kinase (JNK) signaling pathway is activated by metabolic stress and promotes the development of metabolic syndrome, including hyperglycemia, hyperlipidemia, and insulin resistance. This integrated physiological response involves cross-talk between different organs. Here we demonstrate that JNK signaling in adipocytes causes an increased circulating concentration of the hepatokine fibroblast growth factor 21 (FGF21) that regulates systemic metabolism. The mechanism of organ crosstalk is mediated by a feed-forward regulatory loop caused by JNK-regulated FGF21 autocrine signaling in adipocytes that promotes increased expression of the adipokine adiponectin and subsequent hepatic expression of the hormone FGF21. The mechanism of organ cross-talk places circulating adiponectin downstream of autocrine FGF21 expressed by adipocytes and upstream of endocrine FGF21 expressed by hepatocytes. This regulatory loop represents a novel signaling paradigm that connects autocrine and endocrine signaling modes of the same hormone in different tissues.
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http://dx.doi.org/10.1101/gad.344556.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778269PMC
January 2021

Mitophagy-mediated adipose inflammation contributes to type 2 diabetes with hepatic insulin resistance.

J Exp Med 2021 Mar;218(3)

Department of Pathology, Yale School of Medicine, New Haven, CT.

White adipose tissues (WAT) play crucial roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to hepatic insulin resistance and type 2 diabetes mellitus (T2DM). However, the mechanisms underlying these alterations remain unknown. By analyzing the transcriptome landscape in human adipocytes based on available RNA-seq datasets from lean, obese, and T2DM patients, we reveal elevated mitochondrial reactive oxygen species (ROS) pathway and NF-κB signaling with altered fatty acid metabolism in T2DM adipocytes. Mice with adipose-specific deletion of mitochondrial redox Trx2 develop hyperglycemia, hepatic insulin resistance, and hepatic steatosis. Trx2-deficient WAT exhibited excessive mitophagy, increased inflammation, and lipolysis. Mechanistically, mitophagy was induced through increasing ROS generation and NF-κB-dependent accumulation of autophagy receptor p62/SQSTM1, which recruits damaged mitochondria with polyubiquitin chains. Importantly, administration of ROS scavenger or NF-κB inhibitor ameliorates glucose and lipid metabolic disorders and T2DM progression in mice. Taken together, this study reveals a previously unrecognized mechanism linking mitophagy-mediated adipose inflammation to T2DM with hepatic insulin resistance.
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http://dx.doi.org/10.1084/jem.20201416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927432PMC
March 2021

Mechanisms by which adiponectin reverses high fat diet-induced insulin resistance in mice.

Proc Natl Acad Sci U S A 2020 12 8;117(51):32584-32593. Epub 2020 Dec 8.

Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520;

Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2-associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane -1,2-DAG-induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.
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http://dx.doi.org/10.1073/pnas.1922169117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768680PMC
December 2020

Hepatic Insulin Resistance Is Not Pathway Selective in Humans With Nonalcoholic Fatty Liver Disease.

Diabetes Care 2021 Feb 8;44(2):489-498. Epub 2020 Dec 8.

Department of Endocrinology and Metabolism, Amsterdam University Medical Center, Amsterdam, the Netherlands

Objective: Both glucose and triglyceride production are increased in type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). For decades, the leading hypothesis to explain these paradoxical observations has been selective hepatic insulin resistance wherein insulin drives de novo lipogenesis (DNL) while failing to suppress glucose production. Here, we aimed to test this hypothesis in humans.

Research Design And Methods: We recruited obese subjects who met criteria for bariatric surgery with ( = 16) or without ( = 15) NAFLD and assessed ) insulin-mediated regulation of hepatic and peripheral glucose metabolism using hyperinsulinemic-euglycemic clamps with [6,6-H]glucose, ) fasting and carbohydrate-driven hepatic DNL using deuterated water (HO), and ) hepatocellular insulin signaling in liver biopsy samples collected during bariatric surgery.

Results: Compared with subjects without NAFLD, those with NAFLD demonstrated impaired insulin-mediated suppression of glucose production and attenuated-not increased-glucose-stimulated/high-insulin lipogenesis. Fructose-stimulated/low-insulin lipogenesis was intact. Hepatocellular insulin signaling, assessed for the first time in humans, exhibited a proximal block in insulin-resistant subjects: Signaling was attenuated from the level of the insulin receptor through both glucose and lipogenesis pathways. The carbohydrate-regulated lipogenic transcription factor was increased in subjects with NAFLD.

Conclusions: Acute increases in lipogenesis in humans with NAFLD are not explained by altered molecular regulation of lipogenesis through a paradoxical increase in lipogenic insulin action; rather, increases in lipogenic substrate availability may be the key.
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http://dx.doi.org/10.2337/dc20-1644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818337PMC
February 2021

Effect of a Low-Fat Vegan Diet on Body Weight, Insulin Sensitivity, Postprandial Metabolism, and Intramyocellular and Hepatocellular Lipid Levels in Overweight Adults: A Randomized Clinical Trial.

JAMA Netw Open 2020 11 2;3(11):e2025454. Epub 2020 Nov 2.

Physicians Committee for Responsible Medicine, Washington, DC.

Importance: Excess body weight and insulin resistance lead to type 2 diabetes and other major health problems. There is an urgent need for dietary interventions to address these conditions.

Objective: To measure the effects of a low-fat vegan diet on body weight, insulin resistance, postprandial metabolism, and intramyocellular and hepatocellular lipid levels in overweight adults.

Design, Setting, And Participants: This 16-week randomized clinical trial was conducted between January 2017 and February 2019 in Washington, DC. Of 3115 people who responded to flyers in medical offices and newspaper and radio advertisements, 244 met the participation criteria (age 25 to 75 years; body mass index of 28 to 40) after having been screened by telephone.

Interventions: Participants were randomized in a 1:1 ratio. The intervention group (n = 122) was asked to follow a low-fat vegan diet and the control group (n = 122) to make no diet changes for 16 weeks.

Main Outcomes And Measures: At weeks 0 and 16, body weight was assessed using a calibrated scale. Body composition and visceral fat were measured by dual x-ray absorptiometry. Insulin resistance was assessed with the homeostasis model assessment index and the predicted insulin sensitivity index (PREDIM). Thermic effect of food was measured by indirect calorimetry over 3 hours after a standard liquid breakfast (720 kcal). In a subset of participants (n = 44), hepatocellular and intramyocellular lipids were quantified by proton magnetic resonance spectroscopy. Repeated measure analysis of variance was used for statistical analysis.

Results: Among the 244 participants in the study, 211 (87%) were female, 117 (48%) were White, and the mean (SD) age was 54.4 (11.6) years. Over the 16 weeks, body weight decreased in the intervention group by 5.9 kg (95% CI, 5.0-6.7 kg; P < .001). Thermic effect of food increased in the intervention group by 14.1% (95% CI, 6.5-20.4; P < .001). The homeostasis model assessment index decreased (-1.3; 95% CI, -2.2 to -0.3; P < .001) and PREDIM increased (0.9; 95% CI, 0.5-1.2; P < .001) in the intervention group. Hepatocellular lipid levels decreased in the intervention group by 34.4%, from a mean (SD) of 3.2% (2.9%) to 2.4% (2.2%) (P = .002), and intramyocellular lipid levels decreased by 10.4%, from a mean (SD) of 1.6 (1.1) to 1.5 (1.0) (P = .03). None of these variables changed significantly in the control group over the 16 weeks. The change in PREDIM correlated negatively with the change in body weight (r = -0.43; P < .001). Changes in hepatocellular and intramyocellular lipid levels correlated with changes in insulin resistance (both r = 0.51; P = .01).

Conclusions And Relevance: A low-fat plant-based dietary intervention reduces body weight by reducing energy intake and increasing postprandial metabolism. The changes are associated with reductions in hepatocellular and intramyocellular fat and increased insulin sensitivity.

Trial Registration: ClinicalTrials.gov Identifier: NCT02939638.
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http://dx.doi.org/10.1001/jamanetworkopen.2020.25454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705596PMC
November 2020

A MicroRNA Linking Human Positive Selection and Metabolic Disorders.

Cell 2020 10 14;183(3):684-701.e14. Epub 2020 Oct 14.

Departments of Internal Medicine and Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA.

Positive selection in Europeans at the 2q21.3 locus harboring the lactase gene has been attributed to selection for the ability of adults to digest milk to survive famine in ancient times. However, the 2q21.3 locus is also associated with obesity and type 2 diabetes in humans, raising the possibility that additional genetic elements in the locus may have contributed to evolutionary adaptation to famine by promoting energy storage, but which now confer susceptibility to metabolic diseases. We show here that the miR-128-1 microRNA, located at the center of the positively selected locus, represents a crucial metabolic regulator in mammals. Antisense targeting and genetic ablation of miR-128-1 in mouse metabolic disease models result in increased energy expenditure and amelioration of high-fat-diet-induced obesity and markedly improved glucose tolerance. A thrifty phenotype connected to miR-128-1-dependent energy storage may link ancient adaptation to famine and modern metabolic maladaptation associated with nutritional overabundance.
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http://dx.doi.org/10.1016/j.cell.2020.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092355PMC
October 2020

Dissociation of Muscle Insulin Resistance from Alterations in Mitochondrial Substrate Preference.

Cell Metab 2020 11 8;32(5):726-735.e5. Epub 2020 Oct 8.

Department of Medicine, Yale School of Medicine, New Haven, CT, USA; Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT, USA. Electronic address:

Alterations in muscle mitochondrial substrate preference have been postulated to play a major role in the pathogenesis of muscle insulin resistance. In order to examine this hypothesis, we assessed the ratio of mitochondrial pyruvate oxidation (V) to rates of mitochondrial citrate synthase flux (V) in muscle. Contrary to this hypothesis, we found that high-fat-diet (HFD)-fed insulin-resistant rats did not manifest altered muscle substrate preference (V/V) in soleus or quadriceps muscles in the fasting state. Furthermore, hyperinsulinemic-euglycemic (HE) clamps increased V/V in both muscles in normal and insulin-resistant rats. We then examined the muscle V/V flux in insulin-sensitive and insulin-resistant humans and found similar relative rates of V/V, following an overnight fast (∼20%), and similar increases in V/V fluxes during a HE clamp. Altogether, these findings demonstrate that alterations in mitochondrial substrate preference are not an essential step in the pathogenesis of muscle insulin resistance.
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http://dx.doi.org/10.1016/j.cmet.2020.09.008DOI Listing
November 2020

Obesity-Linked PPARγ S273 Phosphorylation Promotes Insulin Resistance through Growth Differentiation Factor 3.

Cell Metab 2020 10 16;32(4):665-675.e6. Epub 2020 Sep 16.

Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA. Electronic address:

The thiazolidinediones (TZDs) are ligands of PPARγ that improve insulin sensitivity, but their use is limited by significant side effects. Recently, we demonstrated a mechanism wherein TZDs improve insulin sensitivity distinct from receptor agonism and adipogenesis: reversal of obesity-linked phosphorylation of PPARγ at serine 273. However, the role of this modification hasn't been tested genetically. Here we demonstrate that mice encoding an allele of PPARγ that cannot be phosphorylated at S273 are protected from insulin resistance, without exhibiting differences in body weight or TZD-associated side effects. Indeed, hyperinsulinemic-euglycemic clamp experiments confirm insulin sensitivity. RNA-seq in these mice reveals reduced expression of Gdf3, a BMP family member. Ectopic expression of Gdf3 is sufficient to induce insulin resistance in lean, healthy mice. We find Gdf3 inhibits BMP signaling and insulin signaling in vitro. Together, these results highlight the diabetogenic role of PPARγ S273 phosphorylation and focus attention on a putative target, Gdf3.
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http://dx.doi.org/10.1016/j.cmet.2020.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543662PMC
October 2020

Membrane-bound -1,2-diacylglycerols explain the dissociation of hepatic insulin resistance from hepatic steatosis in MTTP knockout mice.

J Lipid Res 2020 12 9;61(12):1565-1576. Epub 2020 Sep 9.

Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA. Electronic address:

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane -1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L- mice against the development of hepatic steatosis, increased plasma membrane -1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of -1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L- mice as well as the development of lipid-induced hepatic insulin resistance in aged L- mice.
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http://dx.doi.org/10.1194/jlr.RA119000586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707176PMC
December 2020

Myosteatosis in the Context of Skeletal Muscle Function Deficit: An Interdisciplinary Workshop at the National Institute on Aging.

Front Physiol 2020 7;11:963. Epub 2020 Aug 7.

The Maine Medical Center Research Institute, Scarborough, ME, United States.

Skeletal muscle fat infiltration (known as myosteatosis) is an ectopic fat depot that increases with aging and is recognized to negatively correlate with muscle mass, strength, and mobility and disrupt metabolism (insulin resistance, diabetes). An interdisciplinary workshop convened by the National Institute on Aging Division of Geriatrics and Clinical Gerontology on September 2018, discussed myosteatosis in the context of skeletal muscle function deficit (SMFD). Its purpose was to gain a better understanding of the roles of myosteatosis in aging muscles and metabolic disease, particularly its potential determinants and clinical consequences, and ways of properly assessing it. Special attention was given to functional status and standardization of measures of body composition (including the value of D-creatine dilution method) and imaging approaches [including ways to better use dual-energy X-ray absorptiometry (DXA) through the shape and appearance modeling] to assess lean mass, sarcopenia, and myosteatosis. The workshop convened innovative new areas of scientific relevance to light such as the effect of circadian rhythms and clock disruption in skeletal muscle structure, function, metabolism, and potential contribution to increased myosteatosis. A muscle-bone interaction perspective compared mechanisms associated with myosteatosis and bone marrow adiposity. Potential preventive and therapeutic approaches highlighted ongoing work on physical activity, myostatin treatment, and calorie restriction. Myosteatosis' impact on cancer survivors raised new possibilities to identify its role and to engage in cross-disciplinary collaboration. A wide range of research opportunities and challenges in planning for the most appropriate study design, interpretation, and translation of findings into clinical practice were discussed and are presented here.
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http://dx.doi.org/10.3389/fphys.2020.00963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7438777PMC
August 2020

Cellular and Molecular Mechanisms of Metformin Action.

Endocr Rev 2021 Jan;42(1):77-96

Department of Internal Medicine, Yale School of Medicine, New Haven, Connecticut.

Metformin is a first-line therapy for the treatment of type 2 diabetes, due to its robust glucose-lowering effects, well-established safety profile, and relatively low cost. While metformin has been shown to have pleotropic effects on glucose metabolism, there is a general consensus that the major glucose-lowering effect in patients with type 2 diabetes is mostly mediated through inhibition of hepatic gluconeogenesis. However, despite decades of research, the mechanism by which metformin inhibits this process is still highly debated. A key reason for these discrepant effects is likely due to the inconsistency in dosage of metformin across studies. Widely studied mechanisms of action, such as complex I inhibition leading to AMPK activation, have only been observed in the context of supra-pharmacological (>1 mM) metformin concentrations, which do not occur in the clinical setting. Thus, these mechanisms have been challenged in recent years and new mechanisms have been proposed. Based on the observation that metformin alters cellular redox balance, a redox-dependent mechanism of action has been described by several groups. Recent studies have shown that clinically relevant (50-100 μM) concentrations of metformin inhibit hepatic gluconeogenesis in a substrate-selective manner both in vitro and in vivo, supporting a redox-dependent mechanism of metformin action. Here, we review the current literature regarding metformin's cellular and molecular mechanisms of action.
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http://dx.doi.org/10.1210/endrev/bnaa023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846086PMC
January 2021

A Membrane-Bound Diacylglycerol Species Induces PKCϵ-Mediated Hepatic Insulin Resistance.

Cell Metab 2020 10 2;32(4):654-664.e5. Epub 2020 Sep 2.

Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06510, USA; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address:

Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance (HIR); however, the key lipid species and molecular mechanisms linking these conditions are widely debated. We developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum (ER), mitochondria, plasma membrane (PM), lipid droplets, and cytosol. Acute knockdown (KD) of diacylglycerol acyltransferase-2 in liver induced HIR in rats. This was due to PM sn-1,2-DAG accumulation, which promoted PKCϵ activation and insulin receptor kinase (IRK)-T1160 phosphorylation, resulting in decreased IRK-Y1162 phosphorylation. Liver PM sn-1,2-DAG content and IRK-T1160 phosphorylation were also higher in humans with HIR. In rats, liver-specific PKCϵ KD ameliorated high-fat diet-induced HIR by lowering IRK-T1160 phosphorylation, while liver-specific overexpression of constitutively active PKCϵ-induced HIR by promoting IRK-T1160 phosphorylation. These data identify PM sn-1,2-DAGs as the key pool of lipids that activate PKCϵ and that hepatic PKCϵ is both necessary and sufficient in mediating HIR.
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http://dx.doi.org/10.1016/j.cmet.2020.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544641PMC
October 2020

Sodium-glucose cotransporter-2 inhibitors: Understanding the mechanisms for therapeutic promise and persisting risks.

J Biol Chem 2020 10 12;295(42):14379-14390. Epub 2020 Aug 12.

Departments of Cellular and Molecular Physiology and Internal Medicine (Endocrinology), Yale School of Medicine, New Haven, Connecticut, USA

In a healthy person, the kidney filters nearly 200 g of glucose per day, almost all of which is reabsorbed. The primary transporter responsible for renal glucose reabsorption is sodium-glucose cotransporter-2 (SGLT2). Based on the impact of SGLT2 to prevent renal glucose wasting, SGLT2 inhibitors have been developed to treat diabetes and are the newest class of glucose-lowering agents approved in the United States. By inhibiting glucose reabsorption in the proximal tubule, these agents promote glycosuria, thereby reducing blood glucose concentrations and often resulting in modest weight loss. Recent work in humans and rodents has demonstrated that the clinical utility of these agents may not be limited to diabetes management: SGLT2 inhibitors have also shown therapeutic promise in improving outcomes in heart failure, atrial fibrillation, and, in preclinical studies, certain cancers. Unfortunately, these benefits are not without risk: SGLT2 inhibitors predispose to euglycemic ketoacidosis in those with type 2 diabetes and, largely for this reason, are not approved to treat type 1 diabetes. The mechanism for each of the beneficial and harmful effects of SGLT2 inhibitors-with the exception of their effect to lower plasma glucose concentrations-is an area of active investigation. In this review, we discuss the mechanisms by which these drugs cause euglycemic ketoacidosis and hyperglucagonemia and stimulate hepatic gluconeogenesis as well as their beneficial effects in cardiovascular disease and cancer. In so doing, we aim to highlight the crucial role for selecting patients for SGLT2 inhibitor therapy and highlight several crucial questions that remain unanswered.
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http://dx.doi.org/10.1074/jbc.REV120.008387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573269PMC
October 2020

OGT suppresses S6K1-mediated macrophage inflammation and metabolic disturbance.

Proc Natl Acad Sci U S A 2020 07 29;117(28):16616-16625. Epub 2020 Jun 29.

Program in Integrative Cell Signaling and Neurobiology of Metabolism, Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520;

Enhanced inflammation is believed to contribute to overnutrition-induced metabolic disturbance. Nutrient flux has also been shown to be essential for immune cell activation. Here, we report an unexpected role of nutrient-sensing -linked β--acetylglucosamine (-GlcNAc) signaling in suppressing macrophage proinflammatory activation and preventing diet-induced metabolic dysfunction. Overnutrition stimulates an increase in -GlcNAc signaling in macrophages. -GlcNAc signaling is down-regulated during macrophage proinflammatory activation. Suppressing -GlcNAc signaling by -GlcNAc transferase (OGT) knockout enhances macrophage proinflammatory polarization, promotes adipose tissue inflammation and lipolysis, increases lipid accumulation in peripheral tissues, and exacerbates tissue-specific and whole-body insulin resistance in high-fat-diet-induced obese mice. OGT inhibits macrophage proinflammatory activation by catalyzing ribosomal protein S6 kinase beta-1 (S6K1) -GlcNAcylation and suppressing S6K1 phosphorylation and mTORC1 signaling. These findings thus identify macrophage -GlcNAc signaling as a homeostatic mechanism maintaining whole-body metabolism under overnutrition.
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http://dx.doi.org/10.1073/pnas.1916121117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368321PMC
July 2020

Metabolic control analysis of hepatic glycogen synthesis in vivo.

Proc Natl Acad Sci U S A 2020 04 18;117(14):8166-8176. Epub 2020 Mar 18.

Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06520;

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 [C]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.
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http://dx.doi.org/10.1073/pnas.1921694117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149488PMC
April 2020

One-leg inactivity induces a reduction in mitochondrial oxidative capacity, intramyocellular lipid accumulation and reduced insulin signalling upon lipid infusion: a human study with unilateral limb suspension.

Diabetologia 2020 06 17;63(6):1211-1222. Epub 2020 Mar 17.

NUTRIM, School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands.

Aims/hypothesis: Physical inactivity, low mitochondrial function, increased intramyocellular lipid (IMCL) deposition and reduced insulin sensitivity are common denominators of chronic metabolic disorders, like obesity and type 2 diabetes. Yet, whether low mitochondrial function predisposes to insulin resistance in humans is still unknown.

Methods: Here we investigated, in an intervention study, whether muscle with low mitochondrial oxidative capacity, induced by one-legged physical inactivity, would feature stronger signs of lipid-induced insulin resistance. To this end, ten male participants (age 22.4 ± 4.2 years, BMI 21.3 ± 2.0 kg/m) underwent a 12 day unilateral lower-limb suspension with the contralateral leg serving as an active internal control.

Results: In vivo, mitochondrial oxidative capacity, assessed by phosphocreatine (PCr)-recovery half-time, was lower in the inactive vs active leg. Ex vivo, palmitate oxidation to CO was lower in the suspended leg vs the active leg; however, this did not result in significantly higher [C]palmitate incorporation into triacylglycerol. The reduced mitochondrial function in the suspended leg was, however, paralleled by augmented IMCL content in both musculus tibialis anterior and musculus vastus lateralis, and by increased membrane bound protein kinase C (PKC) θ. Finally, upon lipid infusion, insulin signalling was lower in the suspended vs active leg.

Conclusions/interpretation: Together, these results demonstrate, in a unique human in vivo model, that a low mitochondrial oxidative capacity due to physical inactivity directly impacts IMCL accumulation and PKCθ translocation, resulting in impaired insulin signalling upon lipid infusion. This demonstrates the importance of mitochondrial oxidative capacity and muscle fat accumulation in the development of insulin resistance in humans.

Trial Registration: ClinicalTrial.gov NCT01576250.

Funding: PS was supported by a 'VICI' Research Grant for innovative research from the Netherlands Organization for Scientific Research (Grant 918.96.618).
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http://dx.doi.org/10.1007/s00125-020-05128-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228997PMC
June 2020

Effect of a ketogenic diet on hepatic steatosis and hepatic mitochondrial metabolism in nonalcoholic fatty liver disease.

Proc Natl Acad Sci U S A 2020 03 16;117(13):7347-7354. Epub 2020 Mar 16.

Minerva Foundation Institute for Medical Research, Helsinki 00290, Finland;

Weight loss by ketogenic diet (KD) has gained popularity in management of nonalcoholic fatty liver disease (NAFLD). KD rapidly reverses NAFLD and insulin resistance despite increasing circulating nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). To explore the underlying mechanism, we quantified hepatic mitochondrial fluxes and their regulators in humans by using positional isotopomer NMR tracer analysis. Ten overweight/obese subjects received stable isotope infusions of: [D]glucose, [C]β-hydroxybutyrate and [3-C]lactate before and after a 6-d KD. IHTG was determined by proton magnetic resonance spectroscopy (H-MRS). The KD diet decreased IHTG by 31% in the face of a 3% decrease in body weight and decreased hepatic insulin resistance (-58%) despite an increase in NEFA concentrations (+35%). These changes were attributed to increased net hydrolysis of IHTG and partitioning of the resulting fatty acids toward ketogenesis (+232%) due to reductions in serum insulin concentrations (-53%) and hepatic citrate synthase flux (-38%), respectively. The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state (+167%) and decreased plasma leptin (-45%) and triiodothyronine (-21%) concentrations. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by KD: That is, markedly altered hepatic mitochondrial fluxes and redox state to promote ketogenesis rather than synthesis of IHTG.
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http://dx.doi.org/10.1073/pnas.1922344117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132133PMC
March 2020

Leptin mediates postprandial increases in body temperature through hypothalamus-adrenal medulla-adipose tissue crosstalk.

J Clin Invest 2020 04;130(4):2001-2016

Departments of Internal Medicine.

Meal ingestion increases body temperature in multiple species, an effect that is blunted by obesity. However, the mechanisms responsible for these phenomena remain incompletely understood. Here we show that refeeding increases plasma leptin concentrations approximately 8-fold in 48-hour-fasted lean rats, and this normalization of plasma leptin concentrations stimulates adrenomedullary catecholamine secretion. Increased adrenal medulla-derived plasma catecholamines were necessary and sufficient to increase body temperature postprandially, a process that required both fatty acids generated from adipose tissue lipolysis and β-adrenergic activation of brown adipose tissue (BAT). Diet-induced obese rats, which remained relatively hyperleptinemic while fasting, did not exhibit fasting-induced reductions in temperature. To examine the impact of feeding-induced increases in body temperature on energy balance, we compared rats fed chronically by either 2 carbohydrate-rich boluses daily or a continuous isocaloric intragastric infusion. Bolus feeding increased body temperature and reduced weight gain compared with continuous feeding, an effect abrogated by treatment with atenolol. In summary, these data demonstrate that leptin stimulates a hypothalamus-adrenal medulla-BAT axis, which is necessary and sufficient to induce lipolysis and, as a result, increase body temperature after refeeding.
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http://dx.doi.org/10.1172/JCI134699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108915PMC
April 2020

Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis.

Nature 2020 03 4;579(7798):279-283. Epub 2020 Mar 4.

Department of Internal Medicine, Yale School of Medicine, New Haven, CT, USA.

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 diabetes, 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.
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http://dx.doi.org/10.1038/s41586-020-2074-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101062PMC
March 2020

The omentum of obese girls harbors small adipocytes and browning transcripts.

JCI Insight 2020 03 26;5(6). Epub 2020 Mar 26.

Department of Pediatrics.

Severe obesity (SO) affects about 6% of youth in the United States, augmenting the risks for cardiovascular disease and type 2 diabetes. Herein, we obtained paired omental adipose tissue (omVAT) and abdominal subcutaneous adipose tissue (SAT) biopsies from girls with SO undergoing sleeve gastrectomy (SG), to test whether differences in cellular and transcriptomic profiles between omVAT and SAT depots affect insulin sensitivity differently. Following weight loss, these analyses were repeated in a subgroup of subjects having a second SAT biopsy. We found that omVAT displayed smaller adipocytes compared with SAT, increased lipolysis through adipose triglyceride lipase phosphorylation, reduced inflammation, and increased expression of browning/beiging markers. Contrary to omVAT, SAT adipocyte diameter correlated with insulin resistance. Following SG, both weight and insulin sensitivity improved markedly in all subjects. SAT adipocytes' size became smaller, showing increased lipolysis through perilipin 1 phosphorylation, decreased inflammation, and increased expression in browning/beiging markers. In summary, in adolescent girls with SO, both omVAT and SAT depots showed distinct cellular and transcriptomic profiles. Following weight loss, the SAT depot changed its cellular morphology and transcriptomic profiles into more favorable ones. These changes in the SAT depot may play a fundamental role in the resolution of insulin resistance.
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http://dx.doi.org/10.1172/jci.insight.135448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213797PMC
March 2020

Slc20a1/Pit1 and Slc20a2/Pit2 are essential for normal skeletal myofiber function and survival.

Sci Rep 2020 02 20;10(1):3069. Epub 2020 Feb 20.

Department of Internal Medicine, Section Endocrinology, Yale University School of Medicine, New Haven, CT, USA.

Low blood phosphate (Pi) reduces muscle function in hypophosphatemic disorders. Which Pi transporters are required and whether hormonal changes due to hypophosphatemia contribute to muscle function is unknown. To address these questions we generated a series of conditional knockout mice lacking one or both house-keeping Pi transporters Pit1 and Pit2 in skeletal muscle (sm), using the postnatally expressed human skeletal actin-cre. Simultaneous conditional deletion of both transporters caused skeletal muscle atrophy, resulting in death by postnatal day P13. smPit1, smPit2 and three allele mutants are fertile and have normal body weights, suggesting a high degree of redundance for the two transporters in skeletal muscle. However, these mice show a gene-dose dependent reduction in running activity also seen in another hypophosphatemic model (Hyp mice). In contrast to Hyp mice, grip strength is preserved. Further evaluation of the mechanism shows reduced ERK1/2 activation and stimulation of AMP kinase in skeletal muscle from smPit1; smPit2 mice consistent with energy-stress. Similarly, C2C12 myoblasts show a reduced oxygen consumption rate mediated by Pi transport-dependent and ERK1/2-dependent metabolic Pi sensing pathways. In conclusion, we here show that Pit1 and Pit2 are essential for normal myofiber function and survival, insights which may improve management of hypophosphatemic myopathy.
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http://dx.doi.org/10.1038/s41598-020-59430-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033257PMC
February 2020