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).     

Regulation of MAP Kinase�CDirected Mitogenic and Protein Kinase B�CMediated Signaling by Cannabinoid Receptor Type 1 in Skeletal Muscle Cells

OBJECTIVE

The endogenous cannabinoid (or endocannabinoid) system (ECS) is part of a central neuromodulatory system thought to play a key role in the regulation of feeding behavior and energy balance. However, increasing evidence suggests that modulation of the ECS may also act to regulate peripheral mechanisms involved in these processes, including lipogenesis in adipose tissue and liver, insulin release from pancreatic β-cells, and glucose uptake into skeletal muscle. It was recently shown that cannabinoid receptor type 1 (CB1) and type 2 (CB2), both key components of the ECS, are expressed in human and rodent skeletal muscle. However, their role in modulating insulin sensitivity in this metabolically active tissue has yet to be determined. Our aim was to establish the role, if any, of these receptors in modulating insulin sensitivity in skeletal muscle cells.

RESEARCH DESIGN AND METHODS

Cultured skeletal muscle cells were exposed to CB1 and/or CB2 pharmacological agonists/antagonists/inverse agonists, and the resulting effects on insulin-regulated phosphatidylinositol 3 kinase (PI 3-kinase)–protein kinase B (PKB) and extracellular signal–related kinases 1/2 (ERK1/2)-directed signaling were determined.

RESULTS

Here, we report that modulating the activity of the ECS in skeletal muscle regulates both insulin-dependent mitogen-activated protein (MAP) kinase (ERK1/2) and the canonical PI 3-kinase/PKB signaling pathways. We show that pharmacological activation or inhibition of CB1 receptor activity exerts a differential effect with regard to MAP kinase– and PKB-directed signaling.

CONCLUSIONS

Our study provides evidence that signaling via cannabinoid receptors can significantly modulate mitogenic and metabolic signaling in skeletal muscle with important implications for muscle growth and differentiation as well as the regulation of glucose and lipid metabolism.The increasing occurrence of obesity continues to pose major health problems globally and is linked with the growing incidence of conditions such as type 2 diabetes and cardiovascular disease (1,2). The mechanisms involved in the pathogenesis of type 2 diabetes are not yet fully understood and therefore are the focus of intense study. Insulin resistance probably develops due to unfavorable alterations in the insulin signal transduction pathways that are important for controlling glucose homeostasis in target tissues, such as liver, adipose tissue, and skeletal muscle (3,4). Initiation of these pathways stems from the activated insulin receptor kinase that can bind and regulate numerous downstream intracellular targets, such as the insulin receptor substrate (IRS) proteins, phosphatidylinositol 3 kinase (PI 3-kinase), and growth factor receptor–bound protein 2 (5–7).The endocannabinoid signaling system (ECS) has been shown to influence multiple metabolic pathways, via both its central and peripheral actions (8–10). Key components of this system include the 7-transmembrane endocannabinoid receptors (cannabinoid [CB] receptors) and endogenous lipid-derived endocannabinoid ligands such as anandamide and 2-arachidonoylglycerol (2-AG) (9). From work carried out in neurons, it is widely acknowledged that these endocannabinoids are not stored in cells but are produced “on-demand” from lipid precursors in response to elevated levels of intracellular calcium (11). The mechanisms by which endocannabinoids are synthesized in peripheral tissues have yet to be established.CB receptors belong to the superfamily of G-protein–coupled receptors, where the two principle subtypes, type 1 (CB1) and type 2 (CB2), are established as the mediators of the majority of the biological effects of cannabinoid ligands. Although these receptors are both G_i/o coupled, they do display very different pharmacological profiles and patterns of expression (8). The CB1 receptor, in particular, has been detected in adipose tissue, liver, muscle, and pancreas and is the most abundantly expressed G-protein–coupled receptor in the brain (8,12–14). CB2 receptors, on the other hand, are expressed primarily in spleen and leukocytes (8,15).The ECS is known to regulate energy balance and initially this was thought to occur via its central effects on feeding behavior, although more recent evidence suggests it may also regulate lipid and glucose metabolism by direct actions on peripheral targets (8,9,12). The generation of genetic mouse knockout models and the discovery of SR141716, a CB1 selective inverse agonist, have greatly contributed to our understanding of the role of the ECS in the regulation of energy metabolism (16,17). Centrally, antagonism of the CB1 receptor is found to suppress appetite and promote weight loss, effects that are also seen in mice lacking the CB1 receptor (12,17). On the other hand, with regard to peripheral effects, SR141716 acts to suppress lipogenesis in both liver and adipose tissue promoted by endocannabinoids acting via the CB1 receptor (9,18,19). In addition, pharmacological inhibition of the CB1 receptor increases the expression of adiponectin, an insulin-sensitizing adipokine (20). Overall, longer-term SR141716 treatment has been shown to reduce fasting glucose and insulin levels as well as improve lipid profile in both animals and humans (17,21,22). Emphasizing these important regulatory roles of CB1 signaling in metabolism, there is also increasing evidence that the ECS becomes dysregulated during hyperglycemia and obesity (9).Skeletal muscle plays a crucial role in glucose homeostasis by being the primary site of glucose disposal and fatty acid oxidation, processes that are acutely regulated by insulin (23,24). It is now known that CB1 receptors are expressed in human skeletal muscle (13), and preliminary evidence suggests that the ECS may have a role in regulating pathways involved in oxygen consumption and oxidation as well as glucose metabolism (25–27). However, as yet, little is known with regard to the effects of manipulating ECS activity on muscle insulin sensitivity. Here we demonstrate that the CB1 receptor is expressed in cultured rat skeletal muscle cells in a differentiation-dependent manner and show for the first time that whereas activating CB1 receptor activity pharmacologically produces differential effects on the insulin-dependent regulation of mitogen-activated protein (MAP) kinase (extracellular signal–related kinases 1/2 [ERK1/2]) and canonical PI 3-kinase/protein kinase B (PKB) signaling, CB1 receptor inverse agonism leads to the insulin sensitization of both pathways.     

Myocardial activation of the β-Adrenergic Receptor Kinase (βARK1) by Protein Kinase C (PKC) in vivo

Body. Chronic stimulation of Gq-coupled receptors in the heart leads to PKC activation and myocyte hypertrophy, a precursor to heart failure. βARK1 is a G-protein-coupled receptor kinase (GRK) which phosphorylates and desensitizes agonist-occupied β-adrenergic receptors (βARs), which are critical for cardiac function. Our objective was to determine if myocardial PKC can activate βARK1 in vivo. We studied βARK1 expression by protein immunoblotting and βARK1 activity in the hearts of transgenic mice with cardiac-specific expression of a peptide which increases αPKC activity in the heart by 50% (αPKCact) versus non-transgenic controls. Data are expressed as mean ± SEM. Results were compared using Student’s t-test. Protein expression of βARK1 in myocardial extracts was not significantly different between αPKCact and control: n = 7 in each group, P > 0.05. In contrast, βARK1 activity, as measured by its ability to phosphorylate rhodopsin, was elevated 3-fold in cardiac membrane preparations from αPKCact mice versus control: 3100 ± 214 versus 1058 ± 149 densitometry units, P < 0.05, n = 6 in each group. Based on these data, it appears that αPKC can activate βARK1 in the heart. This may be an important mechanism of βAR dysfunction in the development of myocardial hypertrophy as βARK1 is the primary GRK expressed in the heart.     

Specific Tyrosine Kinase Inhibitors Regulate Human Osteosarcoma Cells In vitro

Inhibitors of specific tyrosine kinases are attractive lead compounds for development of targeted chemotherapies for many tumors, including osteosarcoma. We asked whether inhibition of specific tyrosine kinases would decrease the motility, colony formation, and/or invasiveness by human osteosarcoma cell lines (TE85, MNNG, 143B, SAOS-2, LM-7). An EGF-R inhibitor reduced motility of all five cell lines by 50% to 80%. In contrast, an IGF-1R inhibitor preferentially reduced motility by 42% in LM-7 cells and a met inhibitor preferentially reduced motility by 80% in MNNG cells. The inhibitors of EGF-R, IGF-1R, and met reduced colony formation by more than 80% in all tested cell lines (TE85, MNNG, 143B). The EGF-R inhibitor reduced invasiveness by 62% in 143B cells. The JAK inhibitor increased motility of SAOS-2 and LM7 cells without affecting colony formation or invasiveness. Inhibitors of HER-2, NGF-R, and PDGF-Rs did not affect motility, invasiveness, or colony formation. These results support the hypothesis that specific tyrosine kinases regulate tumorigenesis and/or metastasis in osteosarcoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. One of the authors (PJM) received funding through an Allen Research Fellowship; one of the authors (REB) received funding through a Silber Student Fellowship from the Ohio Division of the American Cancer Society.     

AMP-Activated Protein Kinase α1 Protects Against Diet-Induced Insulin Resistance and Obesity

AMP-activated protein kinase (AMPK) is an essential sensor of cellular energy status. Defects in the α2 catalytic subunit of AMPK (AMPKα1) are associated with metabolic syndrome. The current study investigated the role AMPKα1 in the pathogenesis of obesity and inflammation using male AMPKα1-deficent (AMPKα1^−/−) mice and their wild-type (WT) littermates. After being fed a high-fat diet (HFD), global AMPKα1^−/− mice gained more body weight and greater adiposity and exhibited systemic insulin resistance and metabolic dysfunction with increased severity in their adipose tissues compared with their WT littermates. Interestingly, upon HFD feeding, irradiated WT mice that received the bone marrow of AMPKα1^−/− mice showed increased insulin resistance but not obesity, whereas irradiated AMPKα1^−/− mice with WT bone marrow had a phenotype of metabolic dysregulation that was similar to that of global AMPKα1^−/− mice. AMPKα1 deficiency in macrophages markedly increased the macrophage proinflammatory status. In addition, AMPKα1 knockdown enhanced adipocyte lipid accumulation and exacerbated the inflammatory response and insulin resistance. Together, these data show that AMPKα1 protects mice from diet-induced obesity and insulin resistance, demonstrating that AMPKα1 is a promising therapeutic target in the treatment of the metabolic syndrome.AMP-activated protein kinase (AMPK) is a major cellular energy sensor and plays a major role in regulating metabolic homeostasis (1,2). In mammals, AMPK is a heterotrimeric complex with a catalytic subunit (α1 or α2) and two regulatory subunits (β1 or β1 and γ1, γ2, or γ3) (1,2). AMPKα2 is the predominant catalytic form of AMPK in the liver, muscle, and hypothalamus. There is evidence that AMPKα2 is important for the regulation of systemic insulin sensitivity and metabolic homeostasis. In the hypothalamus, AMPKα2 signals regulate food intake and body weight gain (3). Mice globally deficient in AMPKα2 display different metabolic phenotypes when fed different diets (4,5). A lack of AMPKα2 activity in skeletal muscle exacerbates glucose intolerance and the insulin resistance that is caused by high-fat diets (HFDs) (6). In addition, AMPKα2 is required for the effects of many physiologic regulators or pharmaceutical modalities that maintain insulin sensitivity and metabolic homeostasis (7–10).Mice deficient in AMPKα1 had an increased inflammatory response in an experimental autoimmune encephalomyelitis model (11). AMPKα1 deficiency elevated the levels of reactive oxygen species and oxidized proteins, thereafter shortening the erythrocyte life span in mice (12). Macrophage AMPKα1 has been characterized as a key regulator of inflammatory function (13,14). Its role in protecting against diet-induced metabolic disorders has been hypothesized but not demonstrated (14). The activation of AMPK in adipocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) suppresses adipocyte differentiation and diet-induced obesity (15). However, the activation of AMPK is able to reduce isoproterenol-induced lipolysis; this result is supported by a decrease in adipocyte size and adipose mass in globally deficient in AMPKα1 (AMPKα1^−/−) mice (16). To define the physiologic role of AMPKα1 in energy homeostasis, we administered an HFD to AMPKα1^−/− mice and then evaluated diet-induced obesity and insulin resistance. We also used bone marrow (BM) transplantation (BMT) to characterize the specific roles of AMPKα1 in macrophages and adipocytes in the regulation of the diet-induced inflammatory response, adiposity, and systemic insulin resistance.     

Isoflurane-enhanced Recovery of Canine Stunned Myocardium: Role for Protein Kinase C?

Background: Isoflurane enhances the functional recovery of postischemic, reperfused myocardium by activating adenosine A1 receptors and adenosine triphosphate-regulated potassium channels. Whether protein kinase C is involved in this process is unknown. The authors tested the hypothesis that inhibition of protein kinase C, using the selective antagonist bisindolylmaleimide, attenuates isoflurane-enhanced recovery of stunned myocardium in dogs.

Methods: Fifty dogs were randomly assigned to receive intracoronary vehicle or bisindolylmaleimide (2 or 8 [mu]g/min) in the presence or absence of isoflurane (1 minimum alveolar concentration). Five brief (5 min) coronary artery occlusions interspersed with 5-min reperfusion periods followed by 180 min of final reperfusion were used to produce myocardial stunning. Hemodynamics, regional segment shortening, and myocardial blood flow (radioactive microspheres) were measured at selected intervals.

Results: There were no differences in baseline hemodynamics, segment shortening, or coronary collateral blood flow between groups. Isoflurane significantly (P < 0.05) decreased heart rate, mean arterial pressure, rate pressure product, and the maximum rate of increase of left ventricular pressure (+dP/dtmax) in the presence or absence of bisindolylmaleimide. Sustained contractile dysfunction was observed in dogs that received vehicle (recovery of segment shortening to 12 +/- 8% of baseline), in contrast to those that received isoflurane (75 +/- 7% recovery). Bisindolylmaleimide at a dose of 2 [mu]g/min alone enhanced recovery of segment shortening (50 +/- 7% of baseline) compared with vehicle-pretreated dogs, and isoflurane in the presence of 2 [mu]g/min bisindolylmaleimide further enhanced recovery of contractile function (79 +/- 8% of baseline). In contrast, 8 [mu]g/min bisindolylmaleimide alone (32 +/- 12%) or combined with isoflurane (37 +/- 17%) did not enhance recovery of segment shortening compared with vehicle-pretreated dogs.     

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.     

Tpl2 Kinase Is Upregulated in Adipose Tissue in Obesity and May Mediate Interleukin-1�� and Tumor Necrosis Factor-�� Effects on Extracellular Signal�CRegulated Kinase Activation and Lipolysis

OBJECTIVE

Activation of extracellular signal–regulated kinase-(ERK)-1/2 by cytokines in adipocytes is involved in the alterations of adipose tissue functions participating in insulin resistance. This study aims at identifying proteins regulating ERK1/2 activity, specifically in response to inflammatory cytokines, to provide new insights into mechanisms leading to abnormal adipose tissue function.

RESEARCH DESIGN AND METHODS

Kinase activities were inhibited with pharmacological inhibitors or siRNA. Lipolysis was monitored through glycerol production. Gene expression in adipocytes and adipose tissue of obese mice and subjects was measured by real-time PCR.

RESULTS

IκB kinase-(IKK)-β inhibition prevented mitogen-activated protein (MAP) kinase kinase (MEK)/ERK1/2 activation in response to interleukin (IL)-1β and tumor necrosis factor (TNF)-α but not insulin in 3T3-L1 and human adipocytes, suggesting that IKKβ regulated a MAP kinase kinase kinase (MAP3K) involved in ERK1/2 activation induced by inflammatory cytokines. We show that the MAP3K8 called Tpl2 was expressed in adipocytes and that IL-1β and TNF-α activated Tpl2 and regulated its expression through an IKKβ pathway. Pharmacological inhibition or silencing of Tpl2 prevented MEK/ERK1/2 activation by these cytokines but not by insulin, demonstrating its involvement in ERK1/2 activation specifically in response to inflammatory stimuli. Importantly, Tpl2 was implicated in cytokine-induced lipolysis and in insulin receptor substrate-1 serine phosphorylation. Tpl2 mRNA expression was upregulated in adipose tissue of obese mice and patients and correlated with TNF-α expression.

CONCLUSIONS

Tpl2 is selectively involved in inflammatory cytokine–induced ERK1/2 activation in adipocytes and is implicated in their deleterious effects on adipocyte functions. The deregulated expression of Tpl2 in adipose tissue suggests that Tpl2 may be a new actor in adipose tissue dysfunction in obesity.Obesity and type 2 diabetes are characterized by an insulin-resistant state that could be due to the development of an inflammatory state in the adipose tissue (1,2). Indeed, adipose tissue from obese subjects is infiltrated by bone marrow–derived macrophages that largely contribute to the increased level of proinflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β. These cytokines could act locally to impinge insulin signaling and action in adipocytes and could alter insulin action in liver and muscles (2). Furthermore, TNF-α and IL-1β exert lipolytic effects on adipocytes that participate in the increased free fatty acid (FFA) level during obesity. A paracrine loop involving FFAs and inflammatory cytokines between adipocytes and macrophages would establish a vicious circle that aggravates inflammatory changes in adipose tissue and that worsens insulin resistance (3).Although the exact mechanisms by which increased inflammatory cytokines contribute to insulin resistance and lipolysis are still unknown, it is now accepted that activation of protein kinases such as IκB kinase (IKK) and mitogen-activated protein (MAP) kinases including extracellular signal–regulated kinase (ERK)-1/2 plays an important role (2,4,5). Elevated activity of ERK is found in adipose tissue or muscles of obese and insulin-resistant rodents and humans (6,7). The ERK signaling pathway is activated by various inflammatory cytokines including TNF-α and IL-1β and is involved in insulin resistance in adipocytes through an increase in insulin receptor substrate (IRS)-1 serine phosphorylation and/or a decrease in its expression (7–9). The ERK pathway is also involved in cytokine-induced lipolysis in adipocytes (10–12). An important clue for the physiological importance of the ERK pathway in insulin resistance came from the study of genetically modified mice. Indeed, mice lacking the MAP kinase ERK1 are protected from obesity and insulin resistance when challenged on a high-fat diet (13), and overexpression of the MAP kinase phosphatase-4/dual-specificity phosphatase (MKP-4/DUSP-9) that dephosphorylates ERK1/2 protects against stress-induced insulin resistance (14). Conversely, mice deficient in p62, an ERK inhibitor, have a high basal level of ERK activity and develop mature-onset obesity and insulin resistance (15). However, depending on the stimuli, the ERK outcome response is totally different, and this pathway is involved in numerous effects in addition to inflammation and insulin resistance. Thus, the identification of regulatory proteins that govern the activity of ERK specifically in response to inflammatory cytokines may provide important insights into mechanisms that promote metabolic diseases, and these proteins could be potential targets to alleviate these diseases.MAP kinase and IKK/nuclear factor (NF)-κB pathways often act synergistically to mediate cytokine action (16). It is therefore possible that in adipocytes, proteins that control cytokine-induced ERK activation are regulated by the IKK/NF-κB pathway. One interesting candidate could be MAP kinase kinase kinase (MAP3K), which regulates ERK through the phosphorylation and activation of MAP kinase kinase (MEK) (17), because some of these pathways have been involved in ERK activation selectively downstream of innate immunoreceptors (18).Therefore, the aim of the present study was to identify kinases specifically involved in ERK activation by inflammatory cytokines in adipocytes and to address their implication in the alteration in adipocyte biology in obesity. We report for the first time that the MAP3K8 called tumor progression locus 2 (Tpl2) in mouse or Cancer Osaka thyroid (Cot) in human (19) is expressed in adipocytes and is specifically involved in ERK pathway activation by IL-1β and TNF-α, whereas it is dispensable for ERK activation by insulin. We provide the first evidence that the Tpl2 signaling pathway is implicated in cytokine-induced lipolysis and IRS-1 serine phosphorylation. We showed that Tpl2 mRNA expression is upregulated in adipose tissue of obese subjects and rodents and that inflammatory stimuli regulated Tpl2 expression.     

Overexpression of Nuclear Protein Kinase CK2 β Subunit and Prognosis in Human Gastric Carcinoma

Background  

Gastric carcinoma is one of the most common malignancies in the world, yet little is known about the molecular process of its development and progression. The aims of this study are to correlate the expression of nuclear protein kinase CK2 β subunit (CK2β) with clinicopathologic parameters and patient survival.     

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.     

Identification of Sucrose Non-Fermenting–Related Kinase (SNRK) as a Suppressor of Adipocyte Inflammation

In this study, the role of sucrose non-fermenting–related kinase (SNRK) in white adipocyte biology was investigated. SNRK is abundantly expressed in adipose tissue, and the expression level is decreased in obese mice. SNRK expression is repressed by inflammatory signals but increased by insulin sensitizer in cultured adipocytes. In vivo, adipose tissue SNRK expression can be decreased by lipid injection but enhanced by macrophage ablation. Knocking down SNRK in cultured adipocytes activates both JNK and IKKβ pathways as well as promotes lipolysis. Insulin-stimulated Akt phosphorylation and glucose uptake are impaired in SNRK knockdown adipocytes. Phosphoproteomic analysis with SNRK knockdown adipocytes revealed significantly decreased phosphorylation of 49 proteins by 25% or more, which are involved in various aspects of adipocyte function with a clear indication of attenuated mTORC1 signaling. Phosphorylation of 43 proteins is significantly increased by onefold or higher, among which several proteins are known to be involved in inflammatory pathways. The inflammatory responses in SNRK knockdown adipocytes can be partially attributable to defective mTORC1 signaling, since rapamycin treatment activates IKKβ and induces lipolysis in adipocytes. In summary, SNRK may act as a suppressor of adipocyte inflammation and its presence is necessary for maintaining normal adipocyte function.Sedentary lifestyle and excessive energy intake have caused obesity epidemics. Extensive studies have demonstrated that obesity-related insulin resistance and type 2 diabetes are associated with a low degree of inflammation in adipose tissue (1). Obese adipose tissue secretes a variety of inflammatory markers, cytokines, and chemokines at elevated levels. Some of these factors, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and MCP-1, have been reported to impair insulin signaling (2–5). Dysregulation of adipocyte lipolysis, induced by increased expression of adipose inflammatory cytokines, contributes to systemic insulin resistance through elevated circulating free fatty acid (FFA) levels. Multiple types of immune cells have been identified to regulate inflammatory pathways in obese adipose tissue, such as macrophages, neutrophils, T cells, and mast cells (6,7). Potent anti-inflammatory effects, such as suppression of adipose macrophage gene expression in vitro and in vivo and inhibition of proinflammatory mononuclear cells, have been reported for the only class of insulin sensitizer, thiazolidinediones (8–11). Curbing inflammation of obese patients with salsalate, a prodrug of salicylate for treating arthritis, has been reported to improve glycemic control (12). These results indicate that obesity-related adipose inflammation plays an important role in the development of insulin resistance. However, the molecular pathways involved in the development of adipose inflammation in response to overnutrition are not fully understood.Being the most extensively studied member of the family, AMP kinase (AMPK) has been described as a master energy-sensing enzyme activated by increased AMP-to-ATP ratio. Activation of AMPK turns on catabolic pathways that generate ATP while switching off ATP-consuming anabolic pathways. Recent studies also indicate an anti-inflammatory role for AMPKα1 in macrophages through diminishing inflammasome formation and activation of sirtuin 1 (SIRT1) (13,14). Extensive research efforts have established a clear role for AMPK in energy metabolism in muscle, liver, and macrophages. In contrast, limited and contradictory information is available on the role of AMPK in adipocytes. AMPK phosphorylates Ser⁵⁶⁵ of hormone-sensitive lipase, which was proposed to exert an antilipolytic effect through preventing phosphorylation of Ser⁵⁶³ by protein kinase A (PKA) (15). However, the significance of this mechanism has been questioned because of the recent finding that Ser⁵⁶³ is not essential for hormone-sensitive lipase activation (16). In addition, both pro- and antilipolytic effects have been described for AMPK in adipocytes (17,18). Although the AMPK activator AICAR stimulates glucose uptake in both muscle cells and adipocytes, AMPK only appears to mediate AICAR-induced glucose uptake in muscle cells—not in adipocytes (19). AMPKα2 knockout mice have unchanged fat mass despite impaired glucose tolerance when fed a normal chow diet, suggesting that AMPKα2 is not important for energy metabolism in adipose tissue of normal mice (20). Interestingly, AMPKα2 knockout mice develop adipocyte hypertrophy when fed a high-fat diet, indicating that AMPKα2 may be able to prevent excess lipogenesis in states of nutrition surplus (21). However, it is unclear whether this is because of the deficiency of AMPKα2 in adipocyte or because of the secondary effects of AMPKα2 deficiency in liver or muscle because AMPKα2 is expressed at a low level in fat. AMPKα1 has been reported to be the major isoform of AMPK in adipose tissue; yet, global AMPKα1 knockout mice derived from the same founders were reported to increase adiposity from one laboratory and decrease adiposity from another (18,22). These results raise questions regarding the importance of AMPKα1 in adipocyte function.The biological functions of many AMPK-related family members have not been clearly defined. In this study, we describe the potential role of an AMPK-related kinase, sucrose non-fermenting–related kinase (SNRK), as a potential suppressor of inflammation in adipocytes. As a family member of AMPK-related kinases, SNRK and its function have not previously been studied in adipose tissue. SNRK was initially cloned from fat cell cDNA library (23). The NH₂-terminal catalytic domain has a low homology to AMPKα, but the noncatalytic domain is unique (23). SNRK can be activated by liver kinase B (LKB)1, the same upstream kinase that activates AMPK (24). Gene knockdown study in zebrafish indicates that SNRK may play a role in angioblast development (25,26). A recent study reported that SNRK inhibits colon cancer cell proliferation (27). It is worthy to note that SNRK is a completely different protein from sucrose nonfermenting AMPK-related kinase (SNARK), which is also a member of the AMPK/SNF1 family and can be activated by LKB1 (28,29).     

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.     

Cleavage of Protein Kinase D After Acute Hypoinsulinemia Prevents Excessive Lipoprotein Lipase�CMediated Cardiac Triglyceride Accumulation

OBJECTIVE

During hypoinsulinemia, when cardiac glucose utilization is impaired, the heart rapidly adapts to using more fatty acids. One means by which this is achieved is through lipoprotein lipase (LPL). We determined the mechanisms by which the heart regulates LPL after acute hypoinsulinemia.

RESEARCH DESIGN AND METHODS

We used two different doses of streptozocin (55 [d-55] and 100 [d-100] mg/kg) to induce moderate and severe hypoinsulinemia, respectively, in rats. Isolated cardiomyocytes were also used for transfection or silencing of protein kinase D (PKD) and caspase-3.

RESULTS

There was substantial increase in LPL in d-55 hearts, an effect that was absent in severely hypoinsulinemic d-100 animals. Measurement of PKD, a key element involved in increasing LPL, revealed that only d-100 hearts showed an increase in proteolysis of PKD, an effect that required activation of caspase-3 together with loss of 14-3-3ζ, a binding protein that protects enzymes against degradation. In vitro, phosphomimetic PKD colocalized with LPL in the trans-golgi. PKD, when mutated to prevent its cleavage by caspase-3 and silencing of caspase-3, was able to increase LPL activity. Using a caspase inhibitor (Z-DEVD) in d-100 animals, we effectively lowered caspase-3 activity, prevented PKD cleavage, and increased LPL vesicle formation and translocation to the vascular lumen. This increase in cardiac luminal LPL was associated with a striking accumulation of cardiac triglyceride in Z-DEVD–treated d-100 rats.

CONCLUSIONS

After severe hypoinsulinemia, activation of caspase-3 can restrict LPL translocation to the vascular lumen. When caspase-3 is inhibited, this compensatory response is lost, leading to lipid accumulation in the heart.Cardiac muscle has a high demand for energy and can use multiple substrates (1). Among these, glucose (∼30%) and fatty acid (∼70%) are the major sources from which the heart derives most of its energy (2). Fatty acid delivery and utilization by the heart involves 1) release from adipose tissue and transport to the heart after complexing with albumin (3), 2) provision through the breakdown of endogenous cardiac triglyceride (4), 3) internalization of whole lipoproteins (5), and 4) hydrolysis of circulating triglyceride-rich lipoproteins to fatty acids by lipoprotein lipase (LPL) positioned at the endothelial surface of the coronary lumen (6). The molar concentration of fatty acids bound to albumin is ∼10-fold less than that of fatty acids in lipoprotein triglycerides, (7) and LPL-mediated hydrolysis of triglyceride-rich lipoproteins is suggested to be the principal source of fatty acids for cardiac utilization (8). Coronary endothelial cells do not synthesize LPL (9). In the heart, this enzyme is produced in cardiomyocytes and subsequently secreted onto heparan sulfate proteoglycan (HSPG) binding sites on the myocyte cell surface (10). From here, LPL is transported onto comparable binding sites on the luminal surface of endothelial cells (11). At the lumen, LPL actively metabolizes the triglyceride core of lipoproteins; the released fatty acids are then transported into the heart.The earliest change that occurs in the type 1 diabetic heart is altered energy metabolism where in the presence of lower glucose utilization, the heart switches to using more fatty acids for energy supply (12). One means by which this is possible is through an increase in LPL at the coronary lumen. Using retrograde perfusion of the heart with heparin to displace vascular LPL, we found elevated LPL following diabetes (13–15). We determined that the increased enzyme is 1) not the result of increased gene expression (13), 2) unrelated to an increase in the number of endothelial HSPG binding sites (13), 3) associated with an acute reduction in insulin (within 60 min) (16), and 4) functionally relevant and capable of hydrolyzing lipoprotein triglycerides (17). More recently, we examined the contributions of the cardiomyocyte and endothelial cell in enabling this increased enzyme at the vascular lumen. Within the myocyte, LPL vesicle fission was regulated by protein kinase D (PKD) (18), whereas recruitment of LPL to the cardiomyocyte surface was controlled by stress kinases like AMP-activated protein kinase (AMPK) (19) and p38 mitogen-activated protein kinase (MAPK) that allowed for provision of an actin network that facilitated LPL movement (20). Translocation of LPL from the cardiomyocyte surface to the apical side of endothelial cells is then dependent on the ability of the endothelium to release heparanase (21,22), which enables myocyte HSPG cleavage and transfer of LPL toward the coronary lumen.Selective β-cell death and an ensuing diabetic state can be produced after a single intravenous dose of streptozotocin (STZ) (23). In Wistar rats, a dose-dependent increase in severity of diabetes is produced by 25–100 mg/kg STZ (24). After injection of 55 mg/kg (d-55), stable hyperglycemia develops within 24–48 h in concert with a ∼50% reduction in plasma insulin (16,24). Although these animals were insulin deficient, they did not require insulin supplementation for survival and did not develop ketoacidosis. In the absence of any changes in plasma fatty acids and triglycerides, these animals demonstrated an increase in coronary vascular LPL (13–15). Rats administered 100 mg/kg STZ (d-100) demonstrated intense β-cell necrosis, loss of 98% pancreatic insulin stores, and severely reduced plasma insulin (16). Compared to d-55 diabetic rats, these d-100 animals show a remarkable elevation of plasma fatty acids and triglycerides. Importantly, LPL activity decreases (14,25) in d-100 hearts, suggesting a potential mechanism to restrain LPL-derived fatty acids when the supply of this substrate from other reservoirs is in surplus. The objective of the present study was to determine the mechanisms by which the hypoinsulinemic heart limits its LPL-derived fatty acids under conditions of hyperlipidemia. Our data demonstrate that caspase-3 activation, by cleaving PKD, attempts to restrict the hydrolysis of circulating triglyceride by LPL to limit fatty acid provision and cardiac triglyceride overload. Thus, although caspase-3 inhibition could be protective in reducing cell death, its augmentation of LPL may induce profound cardiac triglyceride accumulation.