Yasuhiko Minokoshi

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5′-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase.

Leptin is a hormone secreted by adipocytes that plays a pivotal role in regulating food intake, energy expenditure and neuroendocrine function. Leptin stimulates the oxidation of fatty acids and the uptake of glucose, and prevents the accumulation of lipids in nonadipose tissues, which can lead to functional impairments known as “lipotoxicity”. The signalling pathways that mediate the metabolic effects of leptin remain undefined. The 5′-AMP-activated protein kinase (AMPK) potently stimulates fatty-acid oxidation in muscle by inhibiting the activity of acetyl coenzyme A carboxylase (ACC). AMPK is a heterotrimeric enzyme that is conserved from yeast to humans and functions as a ‘fuel gauge’ to monitor the status of cellular energy. Here we show that leptin selectively stimulates phosphorylation and activation of the α2 catalytic subunit of AMPK (α2 AMPK) in skeletal muscle, thus establishing a previously unknown signalling pathway for leptin. Early activation of AMPK occurs by leptin acting directly on muscle, whereas later activation depends on leptin functioning through the hypothalamic-sympathetic nervous system axis. In parallel with its activation of AMPK, leptin suppresses the activity of ACC, thereby stimulating the oxidation of fatty acids in muscle. Blocking AMPK activation inhibits the phosphorylation of ACC stimulated by leptin. Our data identify AMPK as a principal mediator of the effects of leptin on fatty-acid metabolism in muscle.

AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus.

Obesity is an epidemic in Western society, and causes rapidly accelerating rates of type 2 diabetes and cardiovascular disease. The evolutionarily conserved serine/threonine kinase, AMP-activated protein kinase (AMPK), functions as a ‘fuel gauge’ to monitor cellular energy status. We investigated the potential role of AMPK in the hypothalamus in the regulation of food intake. Here we report that AMPK activity is inhibited in arcuate and paraventricular hypothalamus (PVH) by the anorexigenic hormone leptin, and in multiple hypothalamic regions by insulin, high glucose and refeeding. A melanocortin receptor agonist, a potent anorexigen, decreases AMPK activity in PVH, whereas agouti-related protein, an orexigen, increases AMPK activity. Melanocortin receptor signalling is required for leptin and refeeding effects on AMPK in PVH. Dominant negative AMPK expression in the hypothalamus is sufficient to reduce food intake and body weight, whereas constitutively active AMPK increases both. Alterations of hypothalamic AMPK activity augment changes in arcuate neuropeptide expression induced by fasting and feeding. Furthermore, inhibition of hypothalamic AMPK is necessary for leptins effects on food intake and body weight, as constitutively active AMPK blocks these effects. Thus, hypothalamic AMPK plays a critical role in hormonal and nutrient-derived anorexigenic and orexigenic signals and in energy balance.

PTP1B Regulates Leptin Signal Transduction In Vivo

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.

ATP-sensitive K + channels in the hypothalamus are essential for the maintenance of glucose homeostasis

Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6.2 is the pore-forming subunit of KATP channels in many cell types, including pancreatic β-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+ (KATP) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6.2−/− mice. Although pancreatic α-cells were functional in Kir6.2−/−, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that KATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.

A liver-derived secretory protein, selenoprotein P, causes insulin resistance.

The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin, by way of the production of secretory proteins, termed hepatokines. Here, we demonstrate that selenoprotein P (SeP), a liver-derived secretory protein, causes insulin resistance. Using serial analysis of gene expression (SAGE) and DNA chip methods, we found that hepatic SeP mRNA levels correlated with insulin resistance in humans. Administration of purified SeP impaired insulin signaling and dysregulated glucose metabolism in both hepatocytes and myocytes. Conversely, both genetic deletion and RNA interference-mediated knockdown of SeP improved systemic insulin sensitivity and glucose tolerance in mice. The metabolic actions of SeP were mediated, at least partly, by inactivation of adenosine monophosphate-activated protein kinase (AMPK). In summary, these results demonstrate a role of SeP in the regulation of glucose metabolism and insulin sensitivity and suggest that SeP may be a therapeutic target for type 2 diabetes.

Regulation of Pancreatic β Cell Mass by Neuronal Signals from the Liver

Metabolic regulation in mammals requires communication between multiple organs and tissues. The rise in the incidence of obesity and associated metabolic disorders, including type 2 diabetes, has renewed interest in interorgan communication. We used mouse models to explore the mechanism whereby obesity enhances pancreatic β cell mass, pathophysiological compensation for insulin resistance. We found that hepatic activation of extracellular regulated kinase (ERK) signaling induced pancreatic β cell proliferation through a neuronal-mediated relay of metabolic signals. This metabolic relay from the liver to the pancreas is involved in obesity-induced islet expansion. In mouse models of insulin-deficient diabetes, liver-selective activation of ERK signaling increased β cell mass and normalized serum glucose levels. Thus, interorgan metabolic relay systems may serve as valuable targets in regenerative treatments for diabetes.

Tissue-specific ablation of the GLUT4 glucose transporter or the insulin receptor challenges assumptions about insulin action and glucose homeostasis.

The prevalence of type 2 diabetes mellitus is growing worldwide. Most forms of type 2 diabetes are polygenic with complex inheritance patterns strongly influenced by environmental factors. The specific gene defects are unknown, but they affect both insulin action and insulin secretion. Glucose homeostasis is maintained by the fine orchestration of insulin secretion and insulin action to promote glucose transport into muscle and adipocytes and to inhibit hepatic glucose output. Resistance to these effects of insulin is a classic pathogenic feature of obesity and type 2 diabetes. Insulin action on lipid metabolism also has important effects on glucose homeostasis. Recent studies using tissue-specific gene targeting of the GLUT4 glucose transporter or the insulin receptor in mice reveal intercommunication among insulin target tissues which can modify the impact of genetic defects in individual tissues. These studies provide new concepts regarding the importance of adipose tissue versus muscle in whole-body insulin sensitivity and the role of proximal versus distal components of the insulin action cascade in the pathogenesis of obesity and type 2 diabetes. The insulin receptor (IR) is present in virtually all mammalian cells, and insulin binding results in activation of several phosphorylation-dephosphorylation cascades (1). The phosphoinositide 3-kinase cascade is necessary, albeit insufficient, for stimulation of glucose transport, and the mitogen-activated protein kinase cascade underlies the mitogenic effects of insulin. These insulin signaling cascades have pleiotrophic metabolic and growth-promoting effects (see Fig. 1 in Supplemental Material). Insulin stimulation of glucose transport in muscle and adipose cells is essential for maintenance of glucose homeostasis. This is mediated by sodium-independent, facilitated-diffusion glucose transporters (GLUTs). In tissues with insulin-sensitive glucose transport (i.e. skeletal and cardiac muscle and adipose cells), GLUT4 is the predominant glucose transporter and GLUT1 plays a minor role. The large stimulatory effect of insulin in these tissues results from the translocation of GLUT4-containing vesicles from intracellular storage sites to the plasma membrane where they dock and fuse with the membrane (2, 3), markedly augmenting glucose transport into the cell. GLUT4 is present primarily in white and brown adipocytes, skeletal muscle, and cardiac muscle, with expression in discrete areas of other tissues (e.g. brain and kidney) that are not traditionally thought to play a major role in glucose homeostasis (4). In insulin-resistant states including obesity and type 2 diabetes, GLUT4 expression is reduced in adipocytes but not in skeletal muscle (4, 5). This down-regulation in adipocytes was not thought to be important because skeletal muscle accounts for up to 85% of glucose disposal following a glucose infusion (6) and at least 50% following glucose ingestion (7), whereas adipose tissue accounts for much less. Glucose transport is the rate-controlling step in skeletalmuscle glucose metabolism in normal and type 2 diabetic subjects (8). Impaired glucose uptake in skeletal muscle is present even in nondiabetic relatives of type 2 diabetic subjects and is a risk factor for developing diabetes (9, 10). Defects in GLUT4 trafficking or function in skeletal muscle are thought to be most important in the development of insulin resistance. To understand the role of IR and GLUT4 in glucose homeostasis, mice were engineered to destroy the function of these genes. Genetic ablation of IR in all tissues results in lethality at 4–5 days after birth due to severe diabetic ketoacidosis (11). When GLUT4 is “knocked out” of all tissues (GLUT4-null), mice are growth-retarded, with markedly reduced fat mass, cardiomegaly, and shortened lifespan but no diabetes (12). In contrast, at least 50% of heterozygous GLUT4-null mice developed diabetes by 6 months of age (13). Hence, to distinguish the role of the insulin receptor and GLUT4 in adipose tissue and muscle in glucose homeostasis, diabetes, and adiposity, tissue-specific knock-out mice were made using Cre/loxP gene targeting (14). These mice challenge long held concepts about the control of glucose homeostasis (Fig. 1).

Hypothalamic Orexin Stimulates Feeding-Associated Glucose Utilization in Skeletal Muscle via Sympathetic Nervous System

Hypothalamic neurons containing orexin (hypocretin) are activated during motivated behaviors and active waking. We show that injection of orexin-A into the ventromedial hypothalamus (VMH) of mice or rats increased glucose uptake and promoted insulin-induced glucose uptake and glycogen synthesis in skeletal muscle, but not in white adipose tissue, by activating the sympathetic nervous system. These effects of orexin were blunted in mice lacking beta-adrenergic receptors but were restored by forced expression of the beta(2)-adrenergic receptor in both myocytes and nonmyocyte cells of skeletal muscle. Orexin neurons are activated by conditioned sweet tasting and directly excite VMH neurons, thereby increasing muscle glucose metabolism and its insulin sensitivity. Orexin and its receptor in VMH thus play a key role in the regulation of muscle glucose metabolism associated with highly motivated behavior by activating muscle sympathetic nerves and beta(2)-adrenergic signaling.

Leptin Stimulates Fatty Acid Oxidation and Peroxisome Proliferator-Activated Receptor α Gene Expression in Mouse C2C12 Myoblasts by Changing the Subcellular Localization of the α2 Form of AMP-Activated Protein Kinase

ABSTRACT Leptin stimulates fatty acid oxidation in skeletal muscle through the activation of AMP-activated protein kinase (AMPK) and the induction of gene expression, such as that for peroxisome proliferator-activated receptor α (PPARα). We now show that leptin stimulates fatty acid oxidation and PPARα gene expression in the C2C12 muscle cell line through the activation of AMPK containing the α2 subunit (α2AMPK) and through changes in the subcellular localization of this enzyme. Activated α2AMPK containing the β1 subunit was shown to be retained in the cytoplasm, where it phosphorylated acetyl coenzyme A carboxylase and thereby stimulated fatty acid oxidation. In contrast, α2AMPK containing the β2 subunit transiently increased fatty acid oxidation but underwent rapid translocation to the nucleus, where it induced PPARα gene transcription. A nuclear localization signal and Thr172 phosphorylation of α2 were found to be essential for nuclear translocation of α2AMPK, whereas the myristoylation of β1 anchors α2AMPK in the cytoplasm. The prevention of α2AMPK activation and the change in its subcellular localization inhibited the metabolic effects of leptin. Our data thus suggest that the activation of and changes in the subcellular localization of α2AMPK are required for leptin-induced stimulation of fatty acid oxidation and PPARα gene expression in muscle cells.