Brian J. DeBosch

Akt1 Is Required for Physiological Cardiac Growth

Background— Postnatal growth of the heart chiefly involves nonproliferative cardiomyocyte enlargement. Cardiac hypertrophy exists in a “physiological” form that is an adaptive response to long-term exercise training and as a “pathological” form that often is a maladaptive response to provocative stimuli such as hypertension and aortic valvular stenosis. A signaling cascade that includes the protein kinase Akt regulates the growth and survival of many cell types, but the precise role of Akt1 in either form of cardiac hypertrophy is unknown. Methods and Results— To evaluate the role of Akt1 in physiological cardiac growth, akt1−/− adult murine cardiac myocytes (AMCMs) were treated with IGF-1, and akt1−/− mice were subjected to exercise training. akt1−/− AMCMs were resistant to insulin-like growth factor-1–stimulated protein synthesis. The akt1−/− mice were found to be resistant to swimming training–induced cardiac hypertrophy. To evaluate the role of Akt in pathological cardiac growth, akt1−/− AMCMs were treated with endothelin-1, and akt1−/− mice were subjected to pressure overload by transverse aortic constriction. Surprisingly, akt1−/− AMCMs were sensitized to endothelin-1–induced protein synthesis, and akt1−/− mice developed an exacerbated form of cardiac hypertrophy in response to transverse aortic constriction. Conclusions— These results establish Akt1 as a pivotal regulatory switch that promotes physiological cardiac hypertrophy while antagonizing pathological hypertrophy.

Akt2 Regulates Cardiac Metabolism and Cardiomyocyte Survival

The Akt family of serine-threonine kinases participates in diverse cellular processes, including the promotion of cell survival, glucose metabolism, and cellular protein synthesis. All three known Akt family members, Akt1, Akt2 and Akt3, are expressed in the myocardium, although Akt1 and Akt2 are most abundant. Previous studies demonstrated that Akt1 and Akt3 overexpression results in enhanced myocardial size and function. Yet, little is known about the role of Akt2 in modulating cardiac metabolism, survival, and growth. Here, we utilize murine models with targeted disruption of the akt2 or the akt1 genes to demonstrate that Akt2, but not Akt1, is required for insulin-stimulated 2-[3H]deoxyglucose uptake and metabolism. In contrast, akt2-/- mice displayed normal cardiac growth responses to provocative stimulation, including ligand stimulation of cultured cardiomyocytes, pressure overload by transverse aortic constriction, and myocardial infarction. However, akt2-/- mice were found to be sensitized to cardiomyocyte apoptosis in response to ischemic injury, and apoptosis was significantly increased in the peri-infarct zone of akt2-/- hearts 7 days after occlusion of the left coronary artery. These results implicate Akt2 in the regulation of cardiomyocyte metabolism and survival.

Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis

The disaccharide trehalose blocks glucose uptake in hepatocytes and induces autophagy that prevents fatty liver disease. A sugary inhibitor of liver disease The accumulation of lipids in hepatocytes that occurs in nonalcoholic fatty liver disease (NAFLD) can result in liver failure or liver cancer. Trehalose is a ubiquitous sugar that is present in the food consumed by animals. DeBosch et al. determined that trehalose blocked glucose uptake into cells by inhibiting glucose transporters in the plasma membrane, which induced a “starvation”-like response that activated autophagy even in the presence of adequate nutrients and glucose. Furthermore, providing trehalose to mice that are a model of NAFLD prevented lipid accumulation in the liver. As noted by Mardones et al. in the associated Focus, trehalose, which has been previously under investigation to treat neurodegenerative diseases characterized by toxic protein aggregates, may be a “silver bullet” for treating diseases resulting from inadequate cellular degradative metabolism. Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5′-monophosphate–activated protein kinase)–dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.

TRB3 Function in Cardiac Endoplasmic Reticulum Stress

Rationale: Tribbles (TRB)3 is an intracellular pseudokinase that modulates the activity of several signal transduction cascades. TRB3 has been reported to inhibit the activity of Akt protein kinases. TRB3 gene expression is highly regulated in many cell types, and amino acid starvation, hypoxia, or endoplasmic reticulum (ER) stress promotes TRB3 expression in noncardiac cells. Objective: The objective of this work was to examine TRB3 expression and function in cultured cardiac myocytes and in mouse heart. Methods and Results: Agents that induced ER stress increased TRB3 expression in cultured cardiac myocytes while blocking insulin-stimulated Akt activation in these cells. Knockdown of TRB3 in cultured cardiac myocytes reversed the effects of ER stress on insulin signaling. Experimental myocardial infarction led to increased TRB3 expression in murine heart tissue in the infarct border zone suggesting that ER stress may play a role in pathological cardiac remodeling. Transgenic mice with cardiac-specific overexpression of TRB3 were generated and they exhibited normal contractile function but altered cardiac signal transduction and metabolism with reduced cardiac glucose oxidation rates. Transgenic TRB3 mice were also sensitized to infarct expansion and cardiac myocyte apoptosis in the infarct border zone after myocardial infarction. Conclusions: These results demonstrate that TRB3 induction is a significant aspect of the ER stress response in cardiac myocytes and that TRB3 antagonizes cardiac glucose metabolism and cardiac myocyte survival.

Early-onset metabolic syndrome in mice lacking the intestinal uric acid transporter SLC2A9

Excess circulating uric acid, a product of hepatic glycolysis and purine metabolism, often accompanies metabolic syndrome. However, whether hyperuricaemia contributes to the development of metabolic syndrome or is merely a by-product of other processes that cause this disorder has not been resolved. In addition, how uric acid is cleared from the circulation is incompletely understood. Here we present a genetic model of spontaneous, early-onset metabolic syndrome in mice lacking the enterocyte urate transporter Glut9 (encoded by the SLC2A9 gene). Glut9-deficient mice develop impaired enterocyte uric acid transport kinetics, hyperuricaemia, hyperuricosuria, spontaneous hypertension, dyslipidaemia and elevated body fat. Allopurinol, a xanthine oxidase inhibitor, can reverse the hypertension and hypercholesterolaemia. These data provide evidence that hyperuricaemia per se could have deleterious metabolic sequelae. Moreover, these findings suggest that enterocytes may regulate whole-body metabolism, and that enterocyte urate metabolism could potentially be targeted to modulate or prevent metabolic syndrome.

Glucose transporter 8 (GLUT8) regulates enterocyte fructose transport and global mammalian fructose utilization.

Enterocyte fructose absorption is a tightly regulated process that precedes the deleterious effects of excess dietary fructose in mammals. Glucose transporter (GLUT)8 is a glucose/fructose transporter previously shown to be expressed in murine intestine. The in vivo function of GLUT8, however, remains unclear. Here, we demonstrate enhanced fructose-induced fructose transport in both in vitro and in vivo models of enterocyte GLUT8 deficiency. Fructose exposure stimulated [(14)C]-fructose uptake and decreased GLUT8 protein abundance in Caco2 colonocytes, whereas direct short hairpin RNA-mediated GLUT8 knockdown also stimulated fructose uptake. To assess GLUT8 function in vivo, we generated GLUT8-deficient (GLUT8KO) mice. GLUT8KO mice exhibited significantly greater jejunal fructose uptake at baseline and after high-fructose diet (HFrD) feeding vs. wild-type mice. Strikingly, long-term HFrD feeding in GLUT8KO mice exacerbated fructose-induced increases in blood pressure, serum insulin, low-density lipoprotein and total cholesterol vs. wild-type controls. Enhanced fructose uptake paralleled with increased abundance of the fructose and glucose transporter, GLUT12, in HFrD-fed GLUT8KO mouse enterocytes and in Caco2 cultures exposed to high-fructose medium. We conclude that GLUT8 regulates enterocyte fructose transport by regulating GLUT12, and that disrupted GLUT8 function has deleterious long-term metabolic sequelae. GLUT8 may thus represent a modifiable target in the prevention and treatment of malnutrition or the metabolic syndrome.

Glucose Transporter 8 (GLUT8) Mediates Fructose-induced de Novo Lipogenesis and Macrosteatosis

Background: GLUT8 is a facilitative fructose and glucose transporter expressed in liver. Results: GLUT8-deficient mice are resistant to fructose-induced fatty liver disease. Conclusion: Hexose transporters can mediate fructose-induced fatty liver disease. Significance: Hepatic hexose transporters represent a novel class of targets to prevent or modulate non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.

Effects of insulin-like growth factor-1 on retinal endothelial cell glucose transport and proliferation

Insulin‐like growth factor‐1 (IGF‐1) plays important roles in the developing and mature retina and in pathological states characterized by retinal neovascularization, such as diabetic retinopathy. The effects of IGF‐1 on glucose transport and proliferation and the signal transduction pathways underlying these effects were studied in a primary bovine retinal endothelial cell (BREC) culture model. IGF‐1 stimulated uptake of the glucose analog 2‐deoxyglucose in a dose‐dependent manner, with a maximal uptake at 25 ng/mL (3.3 nm) after 24 h. Increased transport occurred in the absence of an increase in total cellular GLUT1 transcript or protein. IGF‐1 stimulated activity of both protein kinase C (PKC) and phosphatidylinositol‐3 kinase (PI3 kinase), and both pathways were required for IGF‐1‐mediated BREC glucose transport and thymidine incorporation. Use of a selective inhibitor of the β isoform of PKC, LY379196, revealed that IGF‐1 stimulation of glucose transport was mediated by PKC‐β; however, inhibition of PKC‐β had no effect on BREC proliferation. Taken together, these data suggest that the actions of IGF‐1 in retinal endothelial cells couple proliferation with delivery of glucose, an essential metabolic substrate. The present studies extend our general understanding of the effects of IGF‐1 on vital cellular activities within the retina in normal physiology and in pathological states such as diabetic retinopathy.

Insulin Signaling Pathways and Cardiac Growth

The development, growth, function and metabolism of the heart are regulated by extracellular growth factors, cytokines and ligands. In this review, the role of insulin and insulin-like growth factors in the regulation of cardiac growth will be discussed. In addition, the role of insulin- and insulin-like growth factor-stimulated intracellular signaling proteins in cardiac growth will be reviewed.

The 14-3-3τ phosphoserine-binding protein is required for cardiomyocyte survival

ABSTRACT 14-3-3 family members are intracellular dimeric phosphoserine-binding proteins that regulate signal transduction, cell cycle, apoptotic, and metabolic cascades. Previous work with global 14-3-3 protein inhibitors suggested that these proteins play a critical role in antagonizing apoptotic cell death in response to provocative stimuli. To determine the specific role of one family member in apoptosis, mice were generated with targeted disruption of the 14-3-3τ gene. 14-3-3τ−/− mice did not survive embryonic development, but haploinsufficient mice appeared normal at birth and were fertile. Cultured adult cardiomyocytes derived from 14-3-3τ+/− mice were sensitized to apoptosis in response to hydrogen peroxide or UV irradiation. 14-3-3τ+/− mice were intolerant of experimental myocardial infarction and developed pathological ventricular remodeling with increased cardiomyocyte apoptosis. ASK1, c-jun NH2-terminal kinase, and p38 mitogen-activated protein kinase (MAPK) activation was increased, but extracellular signal-regulated kinase MAPK activation was reduced, in 14-3-3τ+/− cardiac tissue. Inhibition of p38 MAPK increased survival in 14-3-3τ+/− mice subjected to myocardial infarction. These results demonstrate that 14-3-3τ plays a critical antiapoptotic function in cardiomyocytes and that therapeutic agents that increase 14-3-3τ activity may be beneficial to patients with myocardial infarction.