Irwin J. Kurland

The Histone Deacetylase Sirt6 Regulates Glucose Homeostasis via Hif1α

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F1F0 ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism

Hepatic glucose production is crucial for glucose homeostasis, and its dysregulation contributes to the pathogenesis of diabetes. Here, we show that members of the NR4A family of ligand-independent orphan nuclear receptors are downstream mediators of cAMP action in the hormonal control of gluconeogenesis. Hepatic expression of Nur77, Nurr1 and NOR1 is induced by the cAMP axis in response to glucagon and fasting in vivo and is increased in diabetic mice that exhibit elevated gluconeogenesis. Adenoviral expression of Nur77 induces genes involved in gluconeogenesis, stimulates glucose production both in vitro and in vivo, and raises blood glucose levels. Conversely, expression of an inhibitory mutant Nur77 receptor antagonizes gluconeogenic gene expression and lowers blood glucose levels in db/db mice. These results outline a previously unrecognized role for orphan nuclear receptors in the transcriptional control of glucose homeostasis.

SIRT4 Coordinates the Balance between Lipid Synthesis and Catabolism by Repressing Malonyl CoA Decarboxylase

Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis.

Hepatic Insulin Signaling Is Required for Obesity-Dependent Expression of SREBP-1c mRNA but Not for Feeding-Dependent Expression

Dissecting the role of insulin in the complex regulation of triglyceride metabolism is necessary for understanding dyslipidemia and steatosis. Liver insulin receptor knockout (LIRKO) mice show that in the physiological context of feeding, hepatic insulin signaling is not required for the induction of mTORC1, an upstream activator of the lipogenic regulator, SREBP-1c. Feeding induces SREBP-1c mRNA in LIRKO livers, though not to the extent observed in controls. A high fructose diet also partially induces SREBP-1c and lipogenic gene expression in LIRKO livers. Insulin signaling becomes more important in the pathological context of obesity, as knockdown of the insulin receptor in ob/ob mice, a model of Type 2 diabetes, using antisense oligonucleotides, abolishes the induction of SREBP-1c and its targets by obesity and ameliorates steatosis. Thus, insulin-independent signaling pathways can partially compensate for insulin in the induction of SREBP-1c by feeding but the further induction by obesity/Type 2 diabetes is entirely dependent upon insulin.

Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1.

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding-induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis.

Fyn-Dependent Regulation of Energy Expenditure and Body Weight Is Mediated by Tyrosine Phosphorylation of LKB1

Fyn null mice display reduced adiposity associated with increased fatty acid oxidation, energy expenditure, and activation of the AMP-dependent protein kinase (AMPK) in skeletal muscle and adipose tissue. The acute pharmacological inhibition of Fyn kinase activity with SU6656 in wild-type mice reproduces these metabolic effects and induced a specific reduction in fat mass with no change in lean mass. LKB1, the main upstream AMPK kinase (AMPKK) in peripheral tissues, was redistributed from the nucleus into the cytoplasm of cells treated with SU6656 and in cells expressing a kinase-deficient, but not a constitutively kinase-active, Fyn mutant. Moreover, Fyn kinase directly phosphorylated LKB1 on tyrosine 261 and 365 residues, and mutations of these sites resulted in LKB1 export into the cytoplasm and increased AMPK phosphorylation. These data demonstrate a crosstalk between Fyn tyrosine kinase and the AMPK energy-sensing pathway, through Fyn-dependent regulation of the AMPK upstream activator LKB1.

Integrated control of hepatic lipogenesis versus glucose production requires FoxO transcription factors

Insulin integrates hepatic glucose and lipid metabolism, directing nutrients to storage as glycogen and triglyceride. In type 2 diabetes, levels of the former are low and the latter are exaggerated, posing a pathophysiologic and therapeutic conundrum. A branching model of insulin signaling, with FoxO1 presiding over glucose production and Srebp–1c regulating lipogenesis, provides a potential explanation. Here we illustrate an alternative mechanism that integrates glucose production and lipogenesis under the unifying control of FoxO. Liver–specific ablation of three FoxOs (L–FoxO1,3,4) prevents the induction of glucose–6–phosphatase and the repression of glucokinase during fasting, thus increasing lipogenesis at the expense of glucose production. We document a similar pattern in the early phases of diet-induced insulin resistance, and propose that FoxOs are required to enable the liver to direct nutritionally derived carbons to glucose vs. lipid metabolism. Our data underscore the heterogeneity of hepatic insulin resistance during progression from the metabolic syndrome to overt diabetes, and the conceptual challenge of designing therapies that curtail glucose production without promoting hepatic lipid accumulation.

The Fasted/Fed Mouse Metabolic Acetylome: N6-Acetylation Differences Suggest Acetylation Coordinates Organ-Specific Fuel Switching

The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the cosubstrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this, we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MS/MS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de)acetylases in insulin-sensitive versus -insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes.

Insulin Resistance in Striated Muscle-specific Integrin Receptor β1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin β1 subunit in striated muscle results in a near complete loss of integrin β1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin β1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR·Rictor·LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin β1 signaling events in mediating cross-talk to that of insulin action.