G Protein�CCoupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity

OBJECTIVE

Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulin responses.

RESEARCH DESIGN AND METHODS

We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed.

RESULTS

Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity.

CONCLUSIONS

Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders.Insulin resistance, a diminished ability of cells to respond to the action of insulin, is a key feature associated with the pathogenesis of metabolic disorders such as type 2 diabetes and obesity (1). Alterations in any of the key components of the insulin-signaling cascade, including negative regulators, have been proposed to contribute to insulin resistance (1,2). However, the origin and precise mechanisms mediating insulin resistance in physiopathological conditions are not fully understood (3).Both aging and obesity are associated with increased risk of developing type 2 diabetes and cardiovascular disease. An increase in proinflammatory and a decrease in anti-inflammatory factors is found in the obese state and may influence glucose homeostasis and insulin sensitivity (4,5). Peripheral tissues exposed to these proinflammatory cytokines develop an insulin-resistant state (6). In fact, obesity is now being considered a chronic state of low-intensity inflammation. In this regard, the cytokine tumor necrosis factor-α (TNF-α) is highly expressed in adipose tissue of obese animals and humans, and obese mice lacking either TNF-α or its receptors show protection against developing insulin resistance. The molecular mechanisms underlying TNF-α–mediated insulin resistance have been studied in models of murine and human myocytes and adipocytes and in vivo (7–11).Insulin suppresses hepatic glucose production and regulates glucose uptake in muscle and fat through translocation of GLUT4 to the cell surface (12,13). Insulin-induced GLUT4 translocation requires at least two signals, one mediated through phosphatidylinositol 3-kinase (PI3K) and another via Gαq/11 (14) in 3T3L1 adipocytes. The activated insulin receptor can phosphorylate the G protein subunit Gαq/11, leading to activation of cdc42 and PI3K, which triggers glucose transport stimulation (14–16). Signaling of receptors via G proteins is regulated by G protein–coupled receptor kinases (GRKs), a family of seven serine/threonine protein kinases that specifically recognize and phosphorylate agonist-activated G protein–coupled receptors (GPCRs). This recruits arrestin proteins that uncouple receptors from G proteins and promote internalization. The ubiquitous GRK2 isoform has been reported to regulate other pathways independently of its GPCR phosphorylation ability (17,18). GRK2 can act as an inhibitor of insulin-mediated glucose transport stimulation in 3T3L1 adipocytes by interacting with Gαq/11 function independently of its kinase activity (19). GRK2 also inhibits basal and insulin-stimulated glycogen synthesis in mouse liver FL83B cells (20). In this context, we have investigated whether GRK2 may play a relevant physiological role in the modulation of insulin responses in vivo. GRK2 expression is increased in key tissues in different experimental models of insulin resistance, and a 50% downregulation of GRK2 levels in hemizygous GRK2^+/− mice is sufficient to protect against TNF-α, aging, or high-fat diet (HFD)–induced alterations in glucose homeostasis and insulin signaling, strongly arguing for a key role for GRK2 in the modulation of insulin sensitivity in physiological and pathological conditions.     

Dual Inhibition of Classical Protein Kinase C-α and Protein Kinase C-β Isoforms Protects Against Experimental Murine Diabetic Nephropathy

Activation of protein kinase C (PKC) has been implicated in the pathogenesis of diabetic nephropathy with proteinuria and peritubular extracellular matrix production. We have previously shown that the PKC isoforms α and β mediate different cellular effects. PKC-β contributes to hyperglycemia-induced renal matrix production, whereby PKC-α is involved in the development of albuminuria. We further tested this hypothesis by deletion of both isoforms and used a PKC inhibitor. We analyzed the phenotype of nondiabetic and streptozotocin (STZ)-induced diabetic homozygous PKC-α/β double-knockout mice (PKC-α/β^−/−). After 8 weeks of diabetes mellitus, the high-glucose–induced renal and glomerular hypertrophy as well as transforming growth factor-β1) and extracellular matrix production were diminished in the PKC-α/β^−/− mice compared with wild-type controls. Urinary albumin/creatinine ratio also was significantly reduced, however, it was not completely abolished in diabetic PKC-α/β^−/− mice. Treatment with {"type":"entrez-protein","attrs":{"text":"CGP41252","term_id":"812271292","term_text":"CGP41252"}}CGP41252, which inhibits PKC-α and PKC-β, is able to prevent the development of albuminuria and to reduce existing albuminuria in type 1 (STZ model) or type 2 (db/db model) diabetic mice. These results support our hypothesis that PKC-α and PKC-β contribute to the pathogenesis of diabetic nephropathy, and that dual inhibition of the classical PKC isoforms is a suitable therapeutic strategy in the prevention and treatment of diabetic nephropathy.Diabetic nephropathy is the most common cause of progressive chronic kidney disease and end-stage renal disease in the Western world (1). It is postulated that diabetic nephropathy may result from a local interplay of metabolic and hemodynamic factors either through direct effects of high-glucose levels or autocrine and paracrine actions of various vasoactive substances in the diabetic kidney (2). More than 20 years ago, it was described that activation of the protein kinase C (PKC) system by hyperglycemia may represent an important mediator of glucotoxicity in diabetic nephropathy (3,4). PKC constitutes a family of homologous serine/threonine kinases that are involved in many signaling events (5). In mammals, a gene family of nine independent gene loci is distributed over the whole genome (6). Because of biochemical properties and sequence homologies, the PKC family is divided into classical (α, β I, β II, γ), novel (δ, ɛ, η, θ), and atypical (ζ, ι/λ) isoforms. The functional role of distinct PKC isoforms in the development of diabetic nephropathy has recently been further elucidated by means of single isoform-specific knockout mice (7–13). We and others have revealed that activation of the PKC-β isoform contributes to high-glucose–induced, transforming growth factor (TGF)-β₁–mediated renal hypertrophy and extracellular matrix expansion (9,14), whereas perlecan and vascular endothelial growth factor (VEGF) as well as nephrin expression are regulated by a PKC-α–dependent signaling pathway leading to diabetic albuminuria (7,8).To further understand the role of PKC isoforms in the development of diabetic nephropathy, we characterized the renal phenotype of homozygous PKC-α/β double knockout (KO) mice and tested the hypothesis that deletion of both classical PKC isoforms, PKC-α and PKC-β, is able to completely abolish the development of experimental diabetic nephropathy in the streptozotocin (STZ)-induced diabetic stress model. Furthermore, we also tested if pharmacological inhibition of the classical PKC isoforms with the classical PKC inhibitor {"type":"entrez-protein","attrs":{"text":"CGP41251","term_id":"875035598","term_text":"CGP41251"}}CGP41251, the N-Benzoyl derivative of the naturally occurring alkaloid staurosporine, is safely achievable and beneficial in type 1 (STZ model) and type 2 diabetic (db/db mice) animal models. {"type":"entrez-protein","attrs":{"text":"CGP41251","term_id":"875035598","term_text":"CGP41251"}}CGP41251 previously has been used in several phase I–III cancer trials, showing an IC50 for the classical PKC isoforms of ∼20–30 nmol/L and for the novel isoforms between 160 and 1,250 nmol/L (15).     

The Extracellular Matrix Protein MAGP1 Supports Thermogenesis and Protects Against Obesity and Diabetes Through Regulation of TGF-β

Microfibril-associated glycoprotein 1 (MAGP1) is a component of extracellular matrix microfibrils. Here we show that MAGP1 expression is significantly altered in obese humans, and inactivation of the MAGP1 gene (Mfap2^−/−) in mice results in adipocyte hypertrophy and predisposition to metabolic dysfunction. Impaired thermoregulation was evident in Mfap2^−/− mice prior to changes in adiposity, suggesting a causative role for MAGP1 in the increased adiposity and predisposition to diabetes. By 5 weeks of age, Mfap2^−/− mice were maladaptive to cold challenge, uncoupling protein-1 expression was attenuated in the brown adipose tissue, and there was reduced browning of the subcutaneous white adipose tissue. Levels of transforming growth factor-β (TGF-β) activity were elevated in Mfap2^−/− adipose tissue, and the treatment of Mfap2^−/− mice with a TGF-β–neutralizing antibody improved their body temperature and prevented the increased adiposity phenotype. Together, these findings indicate that the regulation of TGF-β by MAGP1 is protective against the effects of metabolic stress, and its absence predisposes individuals to metabolic dysfunction.     

Loss-of-Function Mutation in Myostatin Reduces Tumor Necrosis Factor �� Production and Protects Liver Against Obesity-Induced Insulin Resistance

OBJECTIVE

Insulin resistance develops in tandem with obesity. Ablating myostatin (Mstn) prevents obesity, so we investigated if Mstn deficiency could improve insulin sensitivity. A loss-of-function mutation (Mstn^Ln) in either one or both alleles of the Mstn gene shows how Mstn deficiency protects whole-body insulin sensitivity.

RESEARCH DESIGN AND METHODS

Mstn^Ln/Ln mice were weaned onto a high-fat diet (HFD) or standard diet. HFD-fed Mstn^Ln/Ln mice exhibited high lean, low-fat body compositions compared with wild types. Wild-type and heterozygous and homozygous mutant mice were bled to determine basal levels of insulin, glucose, and homeostasis model assessment of insulin resistance. To evaluate postprandial insulin sensitivity between animals of a similar size, glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamp studies were performed with heterozygous and homozygous mutant mice. Quantitative RT-PCR quantified TNF∝, IL-6, IL-1β, F4/80, GPR43, and CD36 expression in muscle, fat, and liver. Histological analysis measured hepatosteatosis.

RESULTS

Homozygous mutants were glucose tolerant and protected against overall insulin resistance compared with heterozygous mice. Hyperinsulinemic-euglycemic clamp studies revealed a dramatically improved glucose infusion rate, glucose disposal rate, and hepatic glucose production in 11-month-old Mstn^Ln/Ln mice on an HFD. Improvements to muscle and liver insulin sensitivity (∼200–400%) correlated with 50–75% decreased tumor necrosis factor (TNF)α production and coincided with severe Mstn deficiency. Hepatosteatosis appeared to be ameliorated. Short-term treatment of Mstn^Ln/Ln mice with recombinant Mstn led to increased plasma TNFα and insulin resistance.

CONCLUSIONS

We find that severe Mstn deficiency caused by Ln (lean) mutations in HFD-fed mice protects muscle and liver against obesity-induced insulin resistance.Myostatin (Mstn; also called Gdf-8) is a transforming growth factor-β family member that is predominantly expressed in skeletal muscle tissue (1,2). Mstn negatively regulates muscle mass (2,3) and is a potent regulator of adipogenesis (4,5). Knockout of the Mstn gene (Mstn) from agouti lethal yellow (A^y) and obese (Lep^ob/ob) mice improves glucose tolerance, increases muscle mass, and decreases adiposity (6). Obesity-prone A^y and Lep^ob/ob Mstn null mice have body sizes roughly equal to those of their littermate controls due to a diametrical change in muscle and fat characterized by fewer adipocytes that are reduced in size and hyperplasia and hypertrophy of individual muscle fibers. In addition, Mstn knockout mice exhibit an ability to fend off age-related expansion of adipose tissue mass by keeping their fat cells small, unlike wild types, which have enlarged adipocytes contributing to increasing adiposity with age. Evidently, knockout mice experience continued protection against obesity over the later part of their lifespan as they age compared with wild-type mice, yet they have similar total and resting metabolic rates.Here, we describe a novel, loss-of-function allele of Mstn (Mstn^Ln) identified from a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-mutagenized mice challenged with a high-fat diet (HFD). HFD-fed mice with the Ln (lean) mutations displayed altered body compositions consistent with the in vivo effects on growth produced by Mstn deficiency. The loss-of-function Mstn mutation prevented obesity, decreased plasma insulin, and normalized the body weights of heterozygous and homozygous mice. Therefore, we studied relationships among partial Mstn deficiency, lipid utilization, and whole-body insulin sensitivity by comparing animals of a similar size to avoid the complicating factor of differences in body weight. Hyperinsuliemic-euglycemic clamp studies performed in aged HFD-fed Mstn^Ln/Ln and Mstn^+/Ln mice showed that there was a marked improvement to muscle and liver insulin sensitivity, and this was associated with reduced steatosis and decreased tumor necrosis factor (TNF)α and interleukin (IL)-6 gene expression in muscle and fat but not in liver.     

SIRT3 Is Crucial for Maintaining Skeletal Muscle Insulin Action and Protects Against Severe Insulin Resistance in High-Fat–Fed Mice

Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate–based respiration is decreased while fatty acid–based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function.     

Overexpression of Kinase-Negative Protein Kinase C�� in Pancreatic ��-Cells Protects Mice From Diet-Induced Glucose Intolerance and ��-Cell Dysfunction

OBJECTIVE

In vitro models suggest that free fatty acid–induced apoptotic β-cell death is mediated through protein kinase C (PKC)δ. To examine the role of PKCδ signaling in vivo, transgenic mice overexpressing a kinase-negative PKCδ (PKCδKN) selectively in β-cells were generated and analyzed for glucose homeostasis and β-cell survival.

RESEARCH DESIGN AND METHODS

Mice were fed a standard or high-fat diet (HFD). Blood glucose and insulin levels were determined after glucose loads. Islet size, cleaved caspase-3, and PKCδ expression were estimated by immunohistochemistry. In isolated islet cells apoptosis was assessed with TUNEL/TO-PRO3 DNA staining and the mitochondrial potential by rhodamine-123 staining. Changes in phosphorylation and subcellular distribution of forkhead box class O1 (FOXO1) were analyzed by Western blotting and immunohistochemistry.

RESULTS

PKCδKN mice were protected from HFD-induced glucose intolerance. This was accompanied by increased insulin levels in vivo, by an increased islet size, and by a reduced staining of β-cells for cleaved caspase-3 compared with wild-type littermates. In accordance, long-term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild-type but not from PKCδKN mice. PKCδKN overexpression protected islet cells from palmitate-induced mitochondrial dysfunction and inhibited nuclear accumulation of FOXO1 in mouse islet and INS-1E cells. The inhibition of nuclear accumulation of FOXO1 by PKCδKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein.

CONCLUSIONS

Overexpression of PKCδKN in β-cells protects from HFD-induced β-cell failure in vivo by a mechanism that involves inhibition of fatty acid–mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.Obesity is associated with high plasma concentrations of free fatty acids (FFAs). Especially, saturated FFAs like palmitate have been described to induce apoptotic cell death in insulin-secreting cells (1–4). Previous data suggest that the protein kinase C (PKC)δ is activated by FFAs and plays a crucial role in β-cell survival (5). In particular, overexpression of kinase-negative PKCδ (PKCδKN) in insulin-secreting RINm5F cells protected cells from palmitate-induced cell death by a mechanism involving nuclear translocation of PKCδ and probably stimulation of a phospholipase C (6). Overexpression of PKCδKN in INS-1 cells inhibited interleukin-1β–induced cell death (7). In contrast, downregulation of PKCδ by long-term treatment with phorbol myristate acetate (PMA), a synthetic analog of diacylglycerol, did not protect against palmitate-induced cell death (8). Furthermore, PKCδKO mice displayed reduced glucose-induced insulin secretion and developed glucose intolerance as they aged (9).Due to these controversial observations, our previous study showing that PKCδ mediates FFA-induced apoptotic cell death needs further in vivo evidence (6). For this purpose, we generated a transgenic mouse model overexpressing PKCδKN selectively in insulin-secreting β-cells. This mouse model was used to test whether PKCδKN protects against high-fat diet (HFD)-induced glucose intolerance in vivo and to analyze molecular changes due to PKCδKN overexpression in comparison with wild-type littermate controls.     

Mitogen-Activated Protein Kinase Phosphatase 3 (MKP-3)–Deficient Mice Are Resistant to Diet-Induced Obesity

Mitogen-activated protein kinase phosphatase 3 (MKP-3) is a negative regulator of extracellular signal–related kinase signaling. Our laboratory recently demonstrated that MKP-3 plays an important role in obesity-related hyperglycemia by promoting hepatic glucose output. This study shows that MKP-3 deficiency attenuates body weight gain induced by a high-fat diet (HFD) and protects mice from developing obesity-related hepatosteatosis. Triglyceride (TG) contents are dramatically decreased in the liver of MKP-3^−/− mice fed an HFD compared with wild-type (WT) controls. The absence of MKP-3 also reduces adiposity, possibly by repressing adipocyte differentiation. In addition, MKP-3^−/− mice display increased energy expenditure, enhanced peripheral glucose disposal, and improved systemic insulin sensitivity. We performed global phosphoproteomic studies to search for downstream mediators of MKP-3 action in liver lipid metabolism. Our results revealed that MKP-3 deficiency increases the phosphorylation of histone deacetylase (HDAC) 1 on serine 393 by 3.3-fold and HDAC2 on serine 394 by 2.33-fold. Activities of HDAC1 and 2 are increased in the livers of MKP-3^−/− mice fed an HFD. Reduction of HDAC1/2 activities is sufficient to restore TG content of MKP-3^−/− primary hepatocytes to a level similar to that in WT cells.     

Glucose Deprivation Regulates KATP Channel Trafficking via AMP-Activated Protein Kinase in Pancreatic ��-Cells

OBJECTIVE

AMP-activated protein kinase (AMPK) and the ATP-sensitive K⁺ (K_ATP) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the K_ATP channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood.

RESEARCH DESIGN AND METHODS

Rat pancreatic β-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum K_ATP conductance (G_max) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. K_ATP channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of K_ATP channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay.

RESULTS

G_max and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in G_max and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on K_ATP channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C.

CONCLUSIONS

K_ATP channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.ATP-sensitive K⁺ (K_ATP) channels are metabolic sensors that couple cellular energy status to electrical activity and play key roles in energy-dependent insulin secretion in pancreatic β-cells (1). The molecular mechanisms underlying the regulation of K_ATP channel activity have been investigated extensively. Adenine nucleotides are well known to induce K_ATP channel closure by binding to the pore-forming subunit Kir6.2 (2), yet activate channel opening by interacting with the regulatory subunit SUR in a Mg²⁺-dependent manner (3,4). Therefore, energy-dependent regulations of K_ATP currents are believed to be because of the direct effects of these nucleotides on K_ATP channel gating. However, the total conductance of an ion channel is determined not only by open probabilities but also by the available channel numbers. Our work addresses the latter, focusing on whether K_ATP channel numbers at the surface membrane can be regulated by cellular energy status.The importance of the trafficking mechanism for K_ATP channels was first recognized in studies on mutant channels involved in insulin secretion disorders. For some mutations causing congenital hyperinsulinism the forward trafficking is impaired (5,6), whereas mutations that affect the signaling motif responsible for endocytic trafficking cause neonatal diabetes (7). The trafficking of normal K_ATP channels has been reported to be regulated in several recent studies. High-glucose conditions have led to the recruitment of K_ATP channels to the β-cell plasma membrane in a Ca²⁺ and PKA-dependent manner, resulting in an increase in K_ATP currents (8), whereas a protein kinase C activator facilitated endocytic trafficking of K_ATP, resulting in decreased K_ATP currents (9). These studies suggest that regulation of the surface density of K_ATP channels is a dynamic process involving various steps of trafficking and that each step is subject to regulation by various cellular signaling mechanisms. However, the involvement of energy-dependent signaling mechanisms in the regulation of K_ATP channel trafficking has not been fully studied.AMP-activated protein kinase (AMPK) is an evolutionarily conserved metabolic sensor that is activated under conditions of energy deficiency and plays key roles as a regulator of energy metabolism (10). Recent studies have found that AMPK also plays important roles in coupling membrane transport to cellular metabolism (11). AMPK has been shown to upregulate glucose transporters and fatty acid translocase (12) but downregulate ion-transport proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels (13) and epithelial Na⁺ channels (14). Although the mechanisms involved in these effects are not fully understood, AMPK-dependent downregulation of CFTR has been shown to be associated with decreased CFTR surface expression in colonic epithelium (15), whereas AMPK increases GLUT4 translocation to the sarcolemma in skeletal and cardiac muscle (16,17). These results may suggest that AMPK regulates the mechanisms involved in the trafficking of surface proteins.Pancreatic β-cells are a key player in the regulation of whole-body energy balance. They are specialized to synthesize and secrete insulin, a key anabolic hormone of the body. Insulin secretion is controlled tightly by blood glucose concentration, and the ability of the K_ATP channel to couple its activity to cellular energy status is generally believed to be responsible for glucose-dependent insulin secretion. AMPK activity is also controlled by glucose concentration in insulin-secreting cells (18), but little is known about the roles of AMPK in pancreatic β-cells. In the present study, we investigated whether AMPK activation contributes to the activation of K_ATP channels in pancreatic β-cells and INS-1 cells. We found that the activation of AMPK by glucose deprivation induces an increase in the surface levels of K_ATP channels, and this increase contributes to the increased K_ATP conductance.     

AMP-Activated Protein Kinase�CDeficient Mice Are Resistant to the Metabolic Effects of Resveratrol

OBJECTIVE

Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5′ AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKα1 or -α2 to determine whether the metabolic effects of resveratrol are mediated by AMPK.

RESEARCH DESIGN AND METHODS

Mice deficient in the catalytic subunit of AMPK (α1 or α2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro.

RESULTS

Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKα1^−/− mice. In the absence of either AMPKα1 or -α2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly.

CONCLUSIONS

We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.Resveratrol is a natural polyphenolic compound found in grapes and red wine and has been shown to extend lifespan in many organisms, including yeast (1), flies (2), and worms (2–4). Resveratrol extended lifespan in mice on a high-fat diet (5) but not a regular diet (6). In mice with diet-induced obesity, resveratrol reduced fat accumulation and improved glucose tolerance and insulin sensitivity (5,7). In addition, resveratrol increases mitochondrial biogenesis and physical endurance. A resveratrol derivative with higher bioavailability is being tested in clinical trials for treating type 2 diabetes.Given its potential as a lead molecule for the development of drugs that treat metabolic disorders, it is critical to understand how resveratrol modulates metabolism. It is widely accepted that Sirt1, the founding member of the Sirtuin family (8) of NAD-dependent deacetylase, is the target of resveratrol (1,5,7). However, whether the putative Sirt1 activators such as resveratrol actually target Sirt1 in vivo is controversial because resveratrol increases Sirt1 activity in vitro only if the substrate is modified with a fluorescent tag (9,10). Resveratrol appears to increase the deacetylation rate by enhancing the affinity of Sirt1 for fluorescent-tagged peptides.Resveratrol also has a number of indirect effects (11), including stimulation of 5′ AMP-activated protein kinase (AMPK) (5,12,13). AMPK is a heterotrimeric protein consisting of an α-catalytic subunit and two regulatory subunits, β and γ (14). AMPK is a fuel-sensing kinase, which is activated by ATP-depleting conditions such as physical exercise, ischemia, and glucose deprivation. The catalytic subunit of AMPK has two isoforms, α1 and α2, which have different tissue expression patterns. Muscle expresses predominantly the α2-isoform (15), whereas fat and brain express predominantly the α1 isoform (16,17), and liver expresses both α1 and α2 isoforms (18). AMPKα1 and AMPKα2 knockout mice are viable, but AMPKα1/α2 double knockout causes embryonic lethality. Like resveratrol, activation of AMPK has been shown to reduce fat accumulation and increase glucose tolerance, insulin sensitivity, mitochondrial biogenesis, and physical endurance (19–23). Therefore, it is possible that the metabolic effects of resveratrol are mediated by AMPK. Supporting this possibility, resveratrol-mediated extension of lifespan in worms requires AMPK (24).Resveratrol may activate AMPK in several different ways. Resveratrol, as well as other polyphenols, can reduce ATP levels by inhibiting ATP synthase (25). Resveratrol can also activate AMPK without altering the AMP-to-ATP ratio. Dasgupta et al. (12) showed that, at lower doses, resveratrol can activate AMPK through a Sirt1-independent manner. Interestingly, Hou et al. (26) and Lan et al. (27) reported that the activity of liver kinase B (LKB)-1, one of the AMPK kinases that is important for AMPK activity, is activated by resveratrol in a Sirt1-dependent manner.     

Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

The urea transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC-α regulates UT-A1 urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC-α promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC-α–deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC-α–induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.     

Overexpression of Sphingosine Kinase 1 Prevents Ceramide Accumulation and Ameliorates Muscle Insulin Resistance in High-Fat Diet–Fed Mice

The sphingolipids sphingosine-1-phosphate (S1P) and ceramide are important bioactive lipids with many cellular effects. Intracellular ceramide accumulation causes insulin resistance, but sphingosine kinase 1 (SphK1) prevents ceramide accumulation, in part, by promoting its metabolism into S1P. Despite this, the role of SphK1 in regulating insulin action has been largely overlooked. Transgenic (Tg) mice that overexpress SphK1 were fed a standard chow or high-fat diet (HFD) for 6 weeks before undergoing several metabolic analyses. SphK1 Tg mice fed an HFD displayed increased SphK activity in skeletal muscle, which was associated with an attenuated intramuscular ceramide accumulation compared with wild-type (WT) littermates. This was associated with a concomitant reduction in the phosphorylation of c-jun amino-terminal kinase, a serine threonine kinase associated with insulin resistance. Accordingly, skeletal muscle and whole-body insulin sensitivity were improved in SphK1 Tg, compared with WT mice, when fed an HFD. We have identified that the enzyme SphK1 is an important regulator of lipid partitioning and insulin action in skeletal muscle under conditions of increased lipid supply.Obesity is associated with the development of insulin resistance and type 2 diabetes. The pathogenesis of insulin resistance is a well-investigated area, yet the precise interplay between the molecular pathways that leads to this disorder is not fully understood (1). Extensive evidence, however, suggests that defects in fatty acid (FA) metabolism and subsequent lipid accumulation in liver and skeletal muscle play a major role (2,3). Although the increase in lipid manifests as an increase in triacylglycerol (TAG), it is likely that this is a marker of dysfunctional FA metabolism and that accumulation of bioactive lipids, such as ceramide and diacylglycerol (DAG), impair insulin action. DAG accumulation in muscle is associated with insulin resistance in humans (4), whereas mice with DAG kinase delta haploinsufficiency display increased DAG content and reduced peripheral insulin sensitivity (5).Ceramide is a potent lipid-signaling molecule that can cause insulin resistance by inhibiting the ability of insulin to activate Akt (6) and/or via the activation of c-jun amino terminal kinase (JNK) (7,8). Importantly, preventing ceramide accumulation by inhibiting de novo ceramide synthesis protects against the development of insulin resistance (9,10). These observations support the hypothesis that increases in ceramide are an important mechanism underlying the development of muscle insulin resistance, and therefore, targeting pathways to prevent ceramide accumulation may be a viable therapeutic approach.One such approach is to increase ceramide degradation and clearance. Two important enzymes in this pathway are ceramidase and sphingosine kinase (SphK). Ceramidase is responsible for converting ceramide to sphingosine, and SphK phosphorylates sphingosine to sphingosine 1 phosphate (S1P). Because breakdown of S1P is the only way for cellular lipids to exit the sphingolipid pathway, SphK is important in regulating sphingolipid metabolism (11). SphK exists in two isoforms, SphK1 and SphK2. Despite clear evidence that SphK1 activation reduces ceramide (12,13), the role of SphK1 in regulating insulin action has been largely overlooked. Activation of SphK1 prevents ceramide accumulation by promoting its metabolism into S1P. SIP is a molecule with many complex functions: it not only activates five specific G-coupled protein receptors that subsequently activate many downstream signaling pathways but also has important second messenger actions (14). SIP is generally thought to promote activation of inhibitor of κ kinase-β (IKK-β) and JNK via upstream activation of transforming growth factor-β–activated kinase 1 (14), however, SphK1 can block JNK activation (13,15) and prevent tissue inflammation (15), which is linked to insulin resistance (16). In contrast, inhibiting SphK1 leads to JNK activation (17). Interestingly, S1P itself opposes the effects of ceramide. For example, S1P has been shown to counteract ceramide-induced activation of JNK (18). Thus, it has been proposed that the ceramide-to-S1P ratio may function as an intracellular rheostat (19,20).Although not well studied in the context of metabolic disease, evidence is emerging to suggest that the sphingolipid rheostat is important in regulating insulin action. Adiponectin has been thought to exert insulin-sensitizing effects via activation of AMP-activated protein kinase (AMPK) (21). However, Scherer and colleagues (22) recently provided compelling evidence that adiponectin, by stimulating ceramidase activity, decreases intracellular ceramide and concomitantly increases S1P. Hence, the dynamic balance between the levels of ceramide and S1P may have implications for the development of obesity-induced insulin resistance. The aim of the current study was, therefore, to examine the role of SphK in regulating skeletal muscle ceramide content and insulin sensitivity in conditions of lipid oversupply. We hypothesized that SphK1 overexpression would promote flux through the sphingolipid degradative pathway, which would prevent high-fat diet (HFD)–induced ceramide accumulation and, therefore, enhance muscle insulin action.     

Disruption of the Cereblon Gene Enhances Hepatic AMPK Activity and Prevents High-Fat Diet–Induced Obesity and Insulin Resistance in Mice

A nonsense mutation in cereblon (CRBN) causes a mild type of mental retardation in humans. An earlier study showed that CRBN negatively regulates the functional activity of AMP-activated protein kinase (AMPK) in vitro by binding directly to the α₁-subunit of the AMPK complex. However, the in vivo role of CRBN was not studied. For elucidation of the physiological functions of Crbn, a mouse strain was generated in which the Crbn gene was deleted throughout the whole body. In Crbn-deficient mice fed a normal diet, AMPK in the liver showed hyperphosphorylation, which indicated the constitutive activation of AMPK. Since Crbn-deficient mice showed significantly less weight gain when fed a high-fat diet and their insulin sensitivity was considerably improved, the functions of Crbn in the liver were primarily investigated. These results provide the first in vivo evidence that Crbn is a negative modulator of AMPK, which suggests that Crbn may be a potential target for metabolic disorders of the liver.Initially, cereblon (CRBN) was identified as a target gene for a mild type of mental retardation in humans (1) and was subsequently characterized in several different functional contexts. CRBN interacts directly with large-conductance calcium-activated potassium channels and regulates their surface expression (2). Later, CRBN was identified as a primary target for thalidomide-induced teratogenicity and as a substrate receptor for the E3 ligase complex (3). More recently, we reported that CRBN interacts directly with the α₁-subunit of AMP-activated protein kinase (AMPK) and inhibits activation of the enzyme in vitro (4).AMPK is a metabolic master switch in response to variations in cellular energy homeostasis (5). The activity of AMPK can be modulated by the phosphorylation of a threonine at position 172 (Thr172) in the α-subunit by upstream kinases such as LKB1 (6). AMPK inactivates acetyl-CoA carboxylase (ACC) via direct protein phosphorylation and suppresses expression of lipogenic genes, including fatty acid synthase (FAS), thereby inhibiting fatty acid synthesis (7,8). AMPK is implicated in the regulation of hepatic glucose and lipid metabolism, thereby affecting the energy status of the whole body (7,9). Moreover, AMPK was identified as a major pharmacological target protein for the treatment of metabolic diseases. For example, experimental animal models of type 2 diabetes and obesity show that activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide ribonucleoside reduces blood glucose levels and improves lipid metabolism (10–12).Our recent study found that CRBN interacted directly with the AMPK α₁-subunit both in cultured cell lines and in vitro, and the binding sites within the two proteins were localized (4). The levels of the AMPK γ-subunit and CRBN in the AMPK complex varied in a reciprocal manner; i.e., a higher CRBN content corresponded to lower γ-subunit content. AMPK activation was reduced as its γ-subunit content was decreased by CRBN. Thus, it was proposed that CRBN may act as a negative regulator of AMPK in vivo (4). The aims of the current study were to test this hypothesis and to understand the physiological role(s) of CRBN by generating Crbn knockout (KO) mice. The results showed that AMPK activity was activated constitutively in Crbn KO mice under normal conditions and that Crbn KO mice fed a long-term high-fat diet (HFD) showed a marked improvement in their metabolic status.