Gary Sweeney

Cardiac Remodeling in Obesity

The dramatic increase in the prevalence of obesity and its strong association with cardiovascular disease have resulted in unprecedented interest in understanding the effects of obesity on the cardiovascular system. A consistent, but puzzling clinical observation is that obesity confers an increased susceptibility to the development of cardiac disease, while at the same time affording protection against subsequent mortality (termed the obesity paradox). In this review we focus on evidence available from human and animal model studies and summarize the ways in which obesity can influence structure and function of the heart. We also review current hypotheses regarding mechanisms linking obesity and various aspects of cardiac remodeling. There is currently great interest in the role of adipokines, factors secreted from adipose tissue, and their role in the numerous cardiovascular complications of obesity. Here we focus on the role of leptin and the emerging promise of adiponectin as a cardioprotective agent. The challenge of understanding the association between obesity and heart failure is complicated by the multifaceted interplay between various hemodynamic, metabolic, and other physiological factors that ultimately impact the myocardium. Furthermore, the end result of obesity-associated changes in the myocardial structure and function may vary at distinct stages in the progression of remodeling, may depend on the individual pathophysiology of heart failure, and may even remain undetected for decades before clinical manifestation. Here we summarize our current knowledge of this complex yet intriguing topic.

Globular adiponectin increases GLUT4 translocation and glucose uptake but reduces glycogen synthesis in rat skeletal muscle cells

Aims/hypothesisThe aim of this study was to determine whether adiponectin elicits glucose uptake via increased GLUT4 translocation and to investigate the metabolic fate of glucose in skeletal muscle cells treated with globular adiponectin.Materials and methodsBasal and insulin-stimulated 2-deoxy-d-[3H]glucose uptake, cell surface myc-tagged GLUT4 content, production of 14CO2 by oxidation of d-[U-14C]glucose and [1-14C]oleate, and incorporation of d-[U-14C]glucose into glycogen and lactate were measured in the presence and absence of globular adiponectin.ResultsRT-PCR and Western blot analysis revealed that L6 cells and rat skeletal muscle cells express AdipoR1 mRNA and protein. Globular adiponectin increased both GLUT4 translocation and glucose uptake by increasing the transport Vmax of glucose without altering the Km. Interestingly, the incorporation of d-[U-14C]glucose into glycogen under basal and insulin-stimulated conditions was significantly decreased by globular adiponectin, whereas lactate production was increased. Furthermore, globular adiponectin did not affect glucose oxidation, but enhanced phosphorylation of AMP kinase and acetyl-CoA carboxylase, and fatty acid oxidation.Conclusions/interpretationThe present study is the first to show that globular adiponectin increases glucose uptake in skeletal muscle cells via GLUT4 translocation and subsequently reduces the rate of glycogen synthesis and shifts glucose metabolism toward lactate production. These effects are consistent with the increased phosphorylation of AMP kinase and acetyl-CoA carboxylase and oxidation of fatty acids induced by globular adiponectin.

Analysis of paradoxical observations on the association between leptin and insulin resistance

Obesity is commonly associated with the development of insulin resistance and diabetes in humans and rodents. Insulin resistance and diabetes are observed in lipoatrophic individuals or rodent models of lipoatrophy. Here we focus on the role of leptin, the product of the obesity (ob) gene, in the development of insulin resistance and diabetes associated with obesity and lipoatrophy. We review the reported effects of leptin on whole body glucose metabolism and compare and contrast these with direct effects on skeletal muscle, fat and liver. This summary of paradoxical observations on the effects of leptin on glucose homeostasis and the ability of leptin to induce or improve insulin resistance suggests that a complex interplay exists between direct peripheral and centrally mediated effects of the hormone. Evidence suggesting that leptin acts as a mediator of insulin release from pancreatic β cells is reviewed. Finally, intracellular signaling mechanisms stimulated by both leptin and insulin are discussed, with potential points of cross‐talk suggested.—Ceddia, R. B., Koistinen, H. A., Zierath, J. R., Sweeney, G. Analysis of paradoxical observations on the association between leptin and insulin resistance. FASEB J. 16, 1163–1176 (2002)

Lipocalin-2 Deficiency Attenuates Insulin Resistance Associated With Aging and Obesity

OBJECTIVE The proinflammatory cytokines/adipokines produced from adipose tissue act in an autocrine and/or endocrine manner to perpetuate local inflammation and to induce peripheral insulin resistance. The present study investigates whether lipocalin-2 deficiency or replenishment with this adipokine has any impact on systemic insulin sensitivity and the underlying mechanisms. METHODS AND RESULTS Under conditions of aging or dietary-/genetic-induced obesity, lipocalin-2 knockout (Lcn2-KO) mice show significantly decreased fasting glucose and insulin levels and improved insulin sensitivity compared with their wild-type littermates. Despite enlarged fat mass, inflammation and the accumulation of lipid peroxidation products are significantly attenuated in the adipose tissues of Lcn2-KO mice. Adipose fatty acid composition of these mice varies significantly from that in wild-type animals. The amounts of arachidonic acid (C20:4 n6) are elevated by aging and obesity and are paradoxically further increased in adipose tissue, but not skeletal muscle and liver of Lcn2-KO mice. On the other hand, the expression and activity of 12-lipoxygenase, an enzyme responsible for metabolizing arachidonic acid, and the production of tumor necrosis factor-α (TNF-α), a critical insulin resistance–inducing factor, are largely inhibited by lipocalin-2 deficiency. Lipocalin-2 stimulates the expression and activity of 12-lipoxygenase and TNF-α production in fat tissues. Cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), an arachidonate lipoxygenase inhibitor, prevents TNF-α expression induced by lipocalin-2. Moreover, treatment with TNF-α neutralization antibody or CDC significantly attenuated the differences of insulin sensitivity between wild-type and Lcn2-KO mice. CONCLUSIONS Lipocalin-2 deficiency protects mice from developing aging- and obesity-induced insulin resistance largely by modulating 12-lipoxygenase and TNF-α levels in adipose tissue.

Membrane-permeant Esters of Phosphatidylinositol 3,4,5-Trisphosphate

Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4,5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor’s action.

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase.

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t(1/2)=2.5 min) preceded the stimulation of 2-deoxyglucose uptake (t(1/2)=6 min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22 degrees C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40-60%. Pretreatment with SB203580 lowered the apparent transport V(max) of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent K(m) for either hexose. The IC(50) values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2 microM respectively, and correlated with the IC(50) for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4 microM, respectively). Insulin caused a dose- (EC(50)=15 nM) and time- (t(1/2)=3 min) dependent increase in p38 MAPK phosphorylation, which peaked at 10 min (2.3+/-0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38 alpha (2.6+/-0.3-fold) and p38 beta (2.3+/-0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

Adiponectin-induced ERK and Akt Phosphorylation Protects against Pancreatic Beta Cell Apoptosis and Increases Insulin Gene Expression and Secretion

The functional impact of adiponectin on pancreatic beta cells is so far poorly understood. Although adiponectin receptors (AdipoR1/2) were identified, their involvement in adiponectin-induced signaling and other molecules involved is not clearly defined. Therefore, we investigated the role of adiponectin in beta cells and the signaling mediators involved. MIN6 beta cells and mouse islets were stimulated with globular (2.5 μg/ml) or full-length (5 μg/ml) adiponectin under serum starvation, and cell viability, proliferation, apoptosis, insulin gene expression, and secretion were measured. Lysates were subjected to Western blot analysis to determine phosphorylation of AMP-activated protein kinase (AMPK), Akt, or ERK. Functional significance of signaling was confirmed using dominant negative mutants or pharmacological inhibitors. Participation of AdipoRs was assessed by overexpression or siRNA. Adiponectin failed to activate AMPK after 10 min or 1- and 24-h stimulation. ERK was significantly phosphorylated after 24-h treatment with adiponectin, whereas Akt was activated at all time points examined. 24-h stimulation with adiponectin significantly increased cell viability by decreasing cellular apoptosis, and this was prevented by dominant negative Akt, wortmannin (PI3K inhibitor), and U0126 (MEK inhibitor). Moreover, adiponectin regulated insulin gene expression and glucose-stimulated insulin secretion, which was also prevented by wortmannin and U0126 treatment. Interestingly, the data also suggest adiponectin-induced changes in Akt and ERK phosphorylation and caspase-3 may occur independent of the level of AdipoR expression. This study demonstrates a lack of AMPK involvement and implicates Akt and ERK in adiponectin signaling, leading to protection against apoptosis and stimulation of insulin gene expression and secretion in pancreatic beta cells.

Regulation of the Na+/K+- ATPase by insulin: Why and how?

The sodium-potassium ATPase (Na+/K+-ATPase or Na+/K+-pump) is an enzyme present at the surface of all eukaryotic cells, which actively extrudes Na+ from cells in exchange for K+ at a ratio of 3:2, respectively. Its activity also provides the driving force for secondary active transport of solutes such as amino acids, phosphate, vitamins and, in epithelial cells, glucose. The enzyme consists of two subunits (α and β) each expressed in several isoforms. Many hormones regulate Na+/K+ -ATPase activity and in this review we will focus on the effects of insulin. The possible mechanisms whereby insulin controls Na+/K+-ATPase activity are discussed. These are tissue- and isoform-specific, and include reversible covalent modification of catalytic subunits, activation by a rise in intracellular Na+ concentration, altered Na+ sensitivity and changes in subunit gene or protein expression. Given the recent escalation in knowledge of insulin-stimulated signal transduction systems, it is pertinent to ask which intracellular signalling pathways are utilized by insulin in controlling Na+/K+-ATPase activity. Evidence for and against a role for the phosphatidylinositol-3-kinase and mitogen activated protein kinase arms of the insulin-stimulated intracellular signalling networks is suggested. Finally, the clinical relevance of Na+/K+-ATPase control by insulin in diabetes and related disorders is addressed.

Cardiovascular effects of leptin

A wealth of investigations, ranging from clinical and animal model studies to in vitro analyses, have generated great interest in the cardiovascular effects of leptin. Accordingly, many studies have examined the contribution of leptin to cardiac remodeling in heart failure and whether the effects of leptin on metabolism, apoptosis, extracellular matrix remodeling, and hypertrophy could explain the so-called obesity paradox. Furthermore, obesity and hyperleptinemia have often been associated with hypertension, and regulation of sympathetic tone or direct effects of leptin on contributors such as atherosclerosis, endothelial dysfunction, and thrombosis have been documented. Unfortunately, translating basic research studies in vitro, or in animal models, to human physiology has proven difficult. The degree of leptin resistance in obesity is one intriguing issue that must be resolved. Furthermore, the importance of autocrine and paracrine effects of leptin derived from the heart and perivascular adipose tissue must be further studied. Carefully planned and executed research to conclusively establish distinct effects of leptin on the cardiovascular system in normal and diseased states will be essential to harness any therapeutic potential associated with leptins effects.

Adiponectin is expressed by skeletal muscle fibers and influences muscle phenotype and function

Adiponectin (Ad) is linked to various disease states and mediates antidiabetic and anti-inflammatory effects. While it was originally thought that Ad expression was limited to adipocytes, we demonstrate here that Ad is expressed in mouse skeletal muscles and within differentiated L6 myotubes, as assessed by RT-PCR, Western blot, and immunohistochemical analyses. Serial muscle sections stained for fiber type, lipid content, and Ad revealed that muscle fibers with elevated intramyocellular Ad expression were consistently type IIA and IID fibers with detectably higher intramyocellular lipid (IMCL) content. To determine the effect of Ad on muscle phenotype and function, we used an Ad-null [knockout (KO)] mouse model. Body mass increased significantly in 24-wk-old KO mice [+5.5 +/- 3% relative to wild-type mice (WT)], with no change in muscle mass observed. IMCL content was significantly increased (+75.1 +/- 25%), whereas epididymal fat mass, although elevated, was not different in the KO mice compared with WT (+35.1 +/- 23%; P = 0.16). Fiber-type composition was unaltered, although type IIB fiber area was increased in KO mice (+25.5 +/- 6%). In situ muscle stimulation revealed lower peak tetanic forces in KO mice relative to WT (-47.5 +/- 6%), with no change in low-frequency fatigue rates. These data demonstrate that the absence of Ad expression causes contractile dysfunction and phenotypical changes in skeletal muscle. Furthermore, we demonstrate that Ad is expressed in skeletal muscle and that its intramyocellular localization is associated with elevated IMCL, particularly in type IIA/D fibers.