Acrp30 is a circulating protein synthesized in adipose tissue. A single injection in mice of purified recombinant Acrp30 leads to a 2-3-fold elevation in circulating Acrp30 levels, which triggers a transient decrease in basal glucose levels. Similar treatment in ob/ob, NOD (non-obese diabetic) or streptozotocin-treated mice transiently abolishes hyperglycemia. This effect on glucose is not associated with an increase in insulin levels. Moreover, in isolated hepatocytes, Acrp30 increases the ability of sub-physiological levels of insulin to suppress glucose production. We thus propose that Acrp30 is a potent insulin enhancer linking adipose tissue and whole-body glucose metabolism.

Acrp30/adiponectin is an adipocyte-specific secretory protein that has recently been implicated as a mediator of systemic insulin sensitivity with liver and muscle as target organs. Acrp30 is found as two forms in serum, as a lower molecular weight trimer-dimer and a high molecular weight complex. Little is know about the regulation and significance of these Acrp30 complexes in serum and about the events that lead to the generation of the bioactive ligand. Here, we show that there is a profound sexual dimorphism of Acrp30 levels and complex distribution in serum. Female mice display significantly higher levels of the high molecular weight complex in serum than males. In both females and males, levels of the high molecular weight complex are significantly reduced in response to a systemic increase of insulin. The ratio of the two complexes is restored upon normalization of glucose levels. Structurally, we show that oligomer formation of Acrp30 critically depends on disulfide bond formation mediated by Cys-39. Mutation of Cys-39 results in trimers that are subject to proteolytic cleavage in the collagenous domain. Surprisingly, Acrp30(C39S) or wild-type Acrp30 treated with dithiothreitol are significantly more bioactive than the higher order oligomeric forms of the protein with respect to reduction of serum glucose levels. Furthermore, treatment of primary hepatocytes with trimeric and higher order forms of Acrp30 confirms that the increased bioactivity seen in vivo is reflected in an augmented potency to reduce glucose output in the presence of gluconeogenic stimuli. Combined, these results shed new light on the regulation of this complex protein and suggest a new model for in vivo activation of the protein, implicating a serum reductase activity.Adipose has been under appreciated as an endocrine tissue for decades because of the prevalent opinion that it served merely as storage for lipids. Recently, however, the importance of adipocytes to whole body energy homeostasis and metabolism has been underscored by several reports focusing on secreted products of adipocytes (1-4). There has been increased interest in adipose tissue as an endocrine organ, and several of these secreted proteins, termed adipokines, are currently undergoing extensive study regarding roles as divergent as feeding behavior to cardiovascular protection. For instance, leptin, the gene disrupted in ob/ob mice, has central roles in the hypothalamus, as well as peripheral effects in liver, muscle, and endothelial cells (5). Other adipose-secreted products, such as tumor necrosis factor ␣ and adipsin (complement factor D), have well established functions in innate immunity (6 -9). The recently identified adipokine resistin has been implicated as a modulator of insulin sensitivity and is also being studied for its effects on metabolism (4, 10).Acrp30 (also known as adiponectin, AdipoQ, and GBP28) is an adipokine exclusively synthesized and secreted by adipocytes (11-14). Acrp30 has recently been shown to influence glucose homeostasis and insulin se...

Three of the major biochemical pathways implicated in the pathogenesis of hyperglycemia induced vascular damage (the hexosamine pathway, the advanced glycation end product (AGE) formation pathway and the diacylglycerol (DAG)-protein kinase C (PKC) pathway) are activated by increased availability of the glycolytic metabolites glyceraldehyde-3-phosphate and fructose-6-phosphate. We have discovered that the lipid-soluble thiamine derivative benfotiamine can inhibit these three pathways, as well as hyperglycemia-associated NF-kappaB activation, by activating the pentose phosphate pathway enzyme transketolase, which converts glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates and other sugars. In retinas of diabetic animals, benfotiamine treatment inhibited these three pathways and NF-kappaB activation by activating transketolase, and also prevented experimental diabetic retinopathy. The ability of benfotiamine to inhibit three major pathways simultaneously might be clinically useful in preventing the development and progression of diabetic complications.

IntroductionDiabetes causes a variety of pathologic changes in capillaries, arteries, and peripheral nerves. Large prospective clinical studies in both type 1 and type 2 diabetic patients have shown that there is a strong relationship between the level of hyperglycemia and both the onset and progression of diabetic microvascular complications in the retina, kidney, and peripheral nerve (1, 2). Hyperglycemia also appears to have an important role in the pathogenesis of diabetic macrovascular disease (2, 3).Four major molecular mechanisms have been implicated in hyperglycemia-induced tissue damage: activation of PKC isoforms via de novo synthesis of the lipid second messenger diacylglycerol, increased hexosamine pathway flux, increased advanced glycation end product (AGE) formation, and increased polyol pathway flux. In aortic endothelial cells, hyperglycemia also activates the proinflammatory transcription factor NF-κB. Recently, it has been shown that all of these mechanisms reflect a single hyperglycemiainduced process: overproduction of superoxide by the mitochondrial electron transport chain (4, 5). However, the molecular mechanism by which this hyperglycemia-induced overproduction of superoxide activates these different pathways of hyperglycemic damage has not been elucidated.Because hyperglycemia-induced overproduction of superoxide significantly inhibits GAPDH activity (6), we hypothesized that this inhibition would activate all the pathways of hyperglycemic damage by diverting upstream glycolytic metabolites into these signaling pathways. To test this hypothesis, we first evaluated the effect of inhibition of GAPDH activity by antisense oligonucleotide (ODN) on the activity of each of these pathways in aortic endothelial cells cultured in 5 mM glucose. Because aldose reductase In this report, we show that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain activates the three major pathways of hyperglycemic damage found in aortic endothelial cells by inhibiting GAPDH activity. In bovine aortic endothelial cells, GAPDH antisense oligonucleotides activated each of the pathways of hyperglycemic vascular damage in cells cultured in 5 mM glucose to the same extent as that induced by culturing cells in 30 mM glucose. Hyperglycemia-induced GAPDH inhibition was found to be a consequence of poly(ADP-ribosyl)ation of GAPDH by poly(ADP-ribose) polymerase (PARP), which was activated by DNA strand breaks produced by mitochondrial superoxide overproduction. Both the hyperglycemia-induced decrease in activity of GAPDH and its poly(ADP-ribosyl)ation were prevented by overexpression of either uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD), which decrease hyperglycemia-induced superoxide. Overexpression of UCP-1 or MnSOD also prevented hyperglycemiainduced DNA strand breaks and activation of PARP. Hyperglycemia-induced activation of each of the pathways of vascular damage was abolished by blocking PARP activity with the competitive PARP inhibitors PJ34 or...

Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Here, we analyze adipocytes exposed to hyperglycemia both in vitro as well as in vivo. Comparing cells differentiated at 4 mM to cells differentiated at 25 mM glucose (the standard differentiation protocol) reveals severe insulin resistance in cells exposed to 25 mM glucose. A global assessment of transcriptional changes shows an up-regulation of a number of mitochondrial proteins. Exposure to hyperglycemia is associated with a significant induction of reactive oxygen species (ROS), both in vitro as well as in vivo in adipocytes isolated from streptozotocin-treated hyperglycemic mice. Furthermore, hyperglycemia for a few hours in a clamped setting will trigger the induction of a pro-inflammatory response in adipose tissue from rats that can effectively be reduced by co-infusion of N-acetylcysteine (NAC). ROS levels in 3T3-L1 adipocytes can be reduced significantly with pharmacological agents that lower the mitochondrial membrane potential, or by overexpression of uncoupling protein 1 or superoxide dismutase. In parallel with ROS, interleukin-6 secretion from adipocytes is significantly reduced. On the other hand, treatments that lead to a hyperpolarization of the mitochondrial membrane, such as overexpression of the mitochondrial dicarboxylate carrier result in increased ROS formation and decreased insulin sensitivity, even under normoglycemic conditions. Combined, these results highlight the importance ROS production in adipocytes and the associated insulin resistance and inflammatory response.Many genetic and environmental factors can lead to the development of insulin resistance. Once a degree of insulin resistance is established, decreased glucose tolerance arises and occasional bouts of hyperglycemia ensue. Hyperglycemia can in turn cause a further deterioration of insulin sensitivity in a number of tissues, such as the vascular endothelium, muscle, and adipocytes (1).In the vascular endothelium, hyperglycemia has been shown to activate protein kinase C isoforms, give rise to increased levels of glucose-derived advanced glycation end products, and to cause an increased glucose flux through the aldose reductase pathway. Normalization of mitochondrial reactive oxygen species by a number of different approaches prevents these phenomena (2). In adipocytes, Tang and colleagues (3) have shown that a combination of hyperglycemia and hyperinsulinemia results in reduced insulin-stimulated glucose uptake that was in part because of reduced insulin receptor dephosphorylation.Gagnon and Sorisky (4) have previously assessed the effects of low and high glucose levels on 3T3-L1 adipocytes and reported effects on insulin-mediated IRS-1 1 phosphorylation and associated phosphatidylinositol kinase activity. Lu and coll...

SummaryStudies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C . elegans : progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C . elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C . elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C . elegans lifespan.

OBJECTIVEEstablishing Caenorhabditis elegans as a model for glucose toxicity–mediated life span reduction.RESEARCH DESIGN AND METHODSC. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied.RESULTSHigh glucose conditions reduced mean life span from 18.5 ± 0.4 to 16.5 ± 0.6 days and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal–detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 ± 0.6 to 20.6 ± 0.4 days) and maximum life span (23.2 ± 0.4 to 27.7 ± 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 ± 0.6 to 13.9 ± 0.7 days) and maximum life span (23.2 ± 0.4 to 20.3 ± 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner.CONCLUSIONSC. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1–dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.

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