Stefan Krauss

Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes

Abstract β cells sense glucose through its metabolism and the resulting increase in ATP, which subsequently stimulates insulin secretion. Uncoupling protein-2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. In the present study, we assessed UCP2s role in regulating insulin secretion. UCP2-deficient mice had higher islet ATP levels and increased glucose-stimulated insulin secretion, establishing that UCP2 negatively regulates insulin secretion. Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. These results establish UCP2 as a key component of β cell glucose sensing, and as a critical link between obesity, β cell dysfunction, and type 2 diabetes.

BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis

Glycolysis and apoptosis are considered major but independent pathways that are critical for cell survival. The activity of BAD, a pro-apoptotic BCL-2 family member, is regulated by phosphorylation in response to growth/survival factors. Here we undertook a proteomic analysis to assess whether BAD might also participate in mitochondrial physiology. In liver mitochondria, BAD resides in a functional holoenzyme complex together with protein kinase A and protein phosphatase 1 (PP1) catalytic units, Wiskott–Aldrich family member WAVE-1 as an A kinase anchoring protein, and glucokinase (hexokinase IV). BAD is required to assemble the complex in that Bad-deficient hepatocytes lack this complex, resulting in diminished mitochondria-based glucokinase activity and blunted mitochondrial respiration in response to glucose. Glucose deprivation results in dephosphorylation of BAD, and BAD-dependent cell death. Moreover, the phosphorylation status of BAD helps regulate glucokinase activity. Mice deficient for BAD or bearing a non-phosphorylatable BAD(3SA) mutant display abnormal glucose homeostasis including profound defects in glucose tolerance. This combination of proteomics, genetics and physiology indicates an unanticipated role for BAD in integrating pathways of glucose metabolism and apoptosis.

The mitochondrial uncoupling-protein homologues

Uncoupling protein(UCP)1 is an integral membrane protein that is located in the mitochondrial inner membrane of brown adipocytes. Its physiological role is to mediate a regulated, thermogenic proton leak. UCP2 and UCP3 are recently identified UCP1 homologues. They also mediate regulated proton leak, and might function to control the production of superoxide and other downstream reactive oxygen species. However, their role in normal physiology remains unknown. Recent studies have shown that UCP2 has an important part in the pathogenesis of type-2 diabetes. The obscure roles of the UCP homologues in normal physiology, together with their emerging role in pathophysiology, provide exciting potential for further investigation.

A significant portion of mitochondrial proton leak in intact thymocytes depends on expression of UCP2

The uncoupling protein homologue UCP2 is expressed in a variety of mammalian cells. It is thought to be an uncoupler of oxidative phosphorylation. Uncoupling proteins previously have been shown to be capable of translocating protons across phospholipid bilayers in proteoliposome systems. Furthermore, studies in mitochondria from yeast overexpressing the proteins have led to suggestions that they may act as uncouplers in cells. However, this issue is controversial, and to date, definitive experimental evidence is lacking as to whether UCP2 mediates part or all of the basal mitochondrial proton leak in mammalian cells in situ. In the present study, by using thymocytes isolated from UCP2-deficient and wild-type (WT) mice, we addressed the question whether UCP2 is directly involved in catalyzing proton leak in intact cells. Over a range of mitochondrial membrane potentials (ΔΨm), proton leak activity was lower in thymocytes from UCP2-deficient mice compared with WT mice. At physiological levels of ΔΨm, a significant portion (50%) of basal proton leak in resting cells depended on UCP2. Of note, proton leak in whole cells from WT mice, but not UCP2-deficient mice, responded to stimulation by 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoic acid (TTNPB), a known activator of UCP2 activity. Consistent with the observed changes in proton leak, ΔΨm and ATP levels were increased in untreated thymocytes from UCP2-deficient mice. Interestingly, resting respiration was unaltered, suggesting that UCP2 function in resting cells may be concerned with the control of ATP production rather than substrate oxidation. This study establishes that UCP2, expressed at endogenous levels, mediates proton leak in intact cells.

Signaling Takes a Breath – New Quantitative Perspectives on Bioenergetics and Signal Transduction

Immune cells are in constant need of energy for both basic housekeeping and specific immune functions. Increased energy demand during lymphocyte stimulation is coordinated by signal transduction pathways. This review explores the interface between lymphocyte signaling and energy metabolism. In particular, it discusses recent work that allows weighing signaling routes with respect to their role in the regulation of energy metabolism during lymphocyte activation.

Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry–based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

PGC-1α Is a Key Regulator of Glucose-Induced Proliferation and Migration in Vascular Smooth Muscle Cells

Background Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose. Methodology/Principal Findings PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation. Conclusions/Significance These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis.

Control of cell growth and survival by enzymes of the fatty acid synthesis pathway in HCT-116 colon cancer cells.

Purpose: For many tumor cells, de novo lipogenesis is a requirement for growth and survival. A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. Experimental Design: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. Results: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1 and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. Conclusions: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.