The effects of a high concentration of glucose on the insulin receptor-down signaling were investigated in human hepatoma (HepG2) cells in vitro to delineate the molecular mechanism of insulin resistance under glucose toxicity. Treatment of the cells with high concentrations of glucose (15-33 mM) caused phosphorylation of serine residues of the insulin receptor substrate 1 (IRS-1), leading to reduced electrophoretic mobility of it. The phosphorylation of IRS-1 with high glucose treatment was blocked only by protein kinase C (PKC) inhibitors. The high glucose treatment attenuated insulininduced association of IRS-1 and phosphatidylinositol 3-kinase and insulin-stimulated phosphorylation of Akt. A metabolic effect of insulin, stimulation of glycogen synthesis, was also inhibited by the treatment. In contrast, insulin-induced association of Shc and Grb2 was not inhibited. Treatment of the cells with high glucose promoted the translocation of PKC⑀ and PKC␦ from the cytosol to the plasma membrane but not that of other PKC isoforms. Finally, PKC⑀ and PKC␦ directly phosphorylated IRS-1 under cell-free conditions. We conclude that a high concentration of glucose causes phosphorylation of IRS-1, leading to selective attenuation of metabolic signaling of insulin. PKC⑀ and PKC␦ are involved in the down-regulation of insulin signaling, and they may lie in a pathway regulating the phosphorylation of IRS-1.

Cyclic AMP potentiates glucose-stimulated insulin release by actions predominantly at a site, or sites, distal to the elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). Glucose also acts at a site, or sites, distal to the elevation of [Ca2+]i via the ATP-sensitive K+ channel (K+ATP channel)-independent signaling pathway. Accordingly, using rat pancreatic islets, we studied the location of the action of cAMP and its interaction with the glucose pathway. Forskolin, an activator of adenylyl cyclase, raised intracellular cAMP levels and enhanced KCl-induced (Ca2+ -stimulated) insulin release in the presence, but not in the absence, of glucose. Thus, cAMP has no direct effect on Ca2+ -stimulated insulin release. The interaction between cAMP and glucose occurs at a step distal to the elevation of [Ca2+]i because forskolin enhancement of KCl-induced insulin release, in the presence of glucose, was demonstrated in the islets treated with diazoxide, a K+ATP channel opener. The enhancement of insulin release was not associated with any increase in [Ca2+]i. Furthermore, the interaction between cAMP and glucose was unequivocally observed even under stringent Ca2+ -free conditions, indicating the Ca2+ -independent action of cAMP. This action of cAMP is physiologically relevant, because not only forskolin but also glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, and pituitary adenylyl cyclase activating polypeptide exerted similar actions. In conclusion, the cAMP/protein kinase A pathway has no direct effect on Ca2+ -stimulated insulin exocytosis. Rather, it strongly potentiates insulin release by increasing the effectiveness of the K+ATP channel-independent action of glucose.

Recent studies revealed favorable para- and/or autocrine effects of IGF-1 in the pathogenesis of diabetic complications. On the other hand, hyperglycemia is a risk factor for the development of diabetic vascular complications. In this study we examined the effects of high glucose and/or IGF-1 on cell migration and angiogenesis (tubular formation) by using human endothelial cells (EC) in vitro. First we examined cell migration by the two-chamber method. Chronic treatment with a high concentration of D-glucose strongly stimulated the cell migration, which was mimicked by PMA, a protein kinase C (PKC) agonist. The cell migration was also induced by IGF-1. The glucose-induced cell migration was blocked by PKC inhibitor, H7. IGF-1-induced cell migration was not blocked by PD98059, MAPK/ERK kinase (MEK) inhibitor or wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor. Next we examined the effects of high glucose and/or IGF-1 on the tubular formation of EC. The tubular formation was induced only when the cells were exposed to a combination of high glucose and IGF-1. The tubular formation was blocked by MEK inhibitor and PI 3-kinase inhibitor but not by PKC inhibitor. These results indicate that hyperglycemia and IGF-1, respectively, stimulate the EC migration, and tubular formation is induced by a combination of IGF-1 and hyperglycemia.

The nature of ATP-sensitive K+ (K+ATP) channel-independent, insulinotropic action of glucose was investigated using non-glucose-primed pancreatic islets. When the beta-cell was depolarized with K+, glucose dose dependently stimulated insulin release despite inhibition of the K+ATP channel closure by diazoxide. K+ depolarization could be replaced with BAY K 8644, a calcium channel agonist. Prior fasting of rats and lowering ambient temperature greatly suppressed glucose oxidation and utilization by the islet cells and abolished insulin release in response to high glucose alone. However, under these conditions, the K+ATP channel-independent, glucose-induced insulin release was clearly demonstrable. p-Nitrophenyl-alpha-D-glucopyranoside (sweet taste inhibitor) but not its beta-isomer, neomycin (phospholipase C inhibitor) and staurosporine (C kinase blocker) inhibited the K+ATP channel-independent, insulinotropic action of glucose. For the K+ATP channel-independent glucose-induced insulin release 1) elevation of cytosolic calcium is required, 2) minute glucose metabolism is enough, if glucose metabolism is necessary, and 3) direct recognition of glucose molecule, phospholipase C, and protein kinase C appear to be involved.

Nutrients such as glucose stimulate insulin release from pancreatic ␤-cells through both ATP-sensitive K + channel-independent and -dependent mechanisms, which are most likely interrelated. Although little is known of the molecular basis of ATP-sensitive K + channel-independent insulinotropic nutrient actions, mediation by cytosolic long-chain acyl-CoA has been implicated. Because protein acylation might be a sequel of cytosolic long-chain acyl-CoA accumulation, we examined if this reaction is engaged in nutrient stimulation of insulin release, using cerulenin, an inhibitor of protein acylation. In isolated rat pancreatic islets, cerulenin inhibited the glucose augmentation of Ca 2+ -stimulated insulin release evoked by a depolarizing concentration of K + in the presence of diazoxide and Ca 2+ -independent insulin release triggered by a combination of forskolin and phorbol ester under stringent Ca 2+ -free conditions. Cerulenin inhibition of glucose effects was concentration dependent, with a 50% inhibitory concentration (IC 50 ) of 5 µg/ml and complete inhibition at 100 µg/ml. Cerulenin also inhibited augmentation of insulin release by ␣-ketoisocaproate, a mitochondrial fuel. Furthermore, cerulenin abolished augmentation of both Ca 2+ -stimulated and Ca 2+ -independent insulin release by 10 µmol/l palmitate, which causes palmitoylation of cellular proteins. In contrast, cerulenin did not attenuate insulin release elicited by nonnutrient secretagogues, such as a depolarizing concentration of K + , activators of protein kinases A and C, and mastoparan. Glucose oxidation, ATP content in islets, and palmitate oxidation were not affected by cerulenin. In conclusion, cerulenin inhibits nutrient augmentation of insulin release with a high selectivity. The finding is consistent with a prominent role of protein acylation in the process of ␤-cell nutrient sensing. Diabetes 49:712-717, 2000

Mammalian poly(ADP-ribose) polymerase (PARP) is a nuclear chromatin-associated protein with a molecular mass of 114 kDa that catalyzes the transfer of ADP-ribose units from NAD؉ to nuclear proteins that are located within chromatin. We report here the identification of a novel property of PARP as a modulator of nuclear receptor signalling. PARP bound directly to retinoid X receptors (RXR) and repressed liganddependent transcriptional activities mediated by heterodimers of RXR and thyroid hormone receptor (TR). The interacting surface is located in the DNA binding domain of RXR␣. Gel shift assays demonstrated that PARP bound to TR-RXR heterodimers on the response element. Overexpression of wild-type PARP selectively blocked nuclear receptor function in transient transfection experiments, while enzyme-defective mutant PARP did not show significant inhibition, suggesting that the essential role of poly(ADP-ribosyl) enzymatic activity is in gene regulation by nuclear receptors. Furthermore, PARP fused to the Gal4 DNA binding domain suppressed the transcriptional activity of the promoter harboring the Gal4 binding site. Thus, PARP has transcriptional repressor activity when recruited to the promoter. These results indicates that poly(ADPribosyl)ation is a negative cofactor in gene transcription, regulating a member of the nuclear receptor superfamily.Nuclear hormone receptors for steroids, retinoids, thyroid hormone, vitamin D 3 , and prostanoids comprise a large family of sequence-specific transcription factors. They play diverse roles in development, differentiation, and homeostasis (18) by modulating gene transcription. Retinoid X receptors (RXR) are members of a superfamily of nuclear hormone receptors and heterodimerize with a variety of other family members, including all-trans-retinoic acid receptor (RAR), thyroid hormone receptor (TR), and vitamin D receptor (VDR), indicating that RXR play a central role in ligand-dependent transcriptional regulation by nuclear receptors (15,35,37). These heterodimers bind to specific DNA sequences and directly regulate transcription of target genes in response to specific ligands. Nuclear receptors are thought to mediate their transcriptional effects in concert with coregulator proteins that modulate receptor interactions with components of the basal transcription machinery (3, 5, 6, 9, 11-14, 16, 30-32).The mechanism of transcriptional regulation by nuclear receptors has been a focus of intense study. The demonstration of direct interactions of receptors with basal transcription factors, such as TFIIB and TBP (19,20,22,23,34), suggests that liganded receptors may directly influence the function of the basal transcription machinery. However, these direct interaction models do not explain transcriptional squelching between receptors or the roles of receptor-associated cofactors. Negative transcriptional regulation by TR and RAR is mediated, in part, by their association with a class of silencing mediators termed 21,24,25,27). In addition, at least three distinct classes of re...

OBJECTIVE -To clarify mortality and morbidity of intensively managed elderly diabetic individuals and to explore factors predicting mortality and diabetes-related end points. RESEARCH DESIGN AND METHODS-A total of 390 elderly (Ն65 years of age) outpatients with type 2 diabetes ( 173 men and 217 women, mean age 73.0 years) were analyzed. The mean HbA 1c upon entry was 6.8% (332 receiving oral hypoglycemics and/or insulin) and blood pressure upon entry was 136/74 mmHg (219 receiving antihypertensive drugs). The patients have been followed-up for 3 years with HbA 1c Ͻ7.0% and blood pressure Ͻ145/80 mmHg as targets, with mortality and an aggregate of fatal and nonfatal diabetes-related events as end points. Mortality rate and causes of mortality, as well as risk factors for mortality and morbidity, were determined. RESULTS -The mortality rate, 2.9% per year, was comparable to that of the age-and sex-matched general population. Stroke was a leading cause of mortality after malignancy. By the univariate Cox proportional hazards model, only high serum creatinine and prior stroke were highly significant and strong risks for both end points. In those without prior stroke and receiving antihypertensive agents, the incidence of the diabetes-related end point based on their systolic blood pressure (SBP) quartile was U-shaped, with the nadir at the 3rd (SBP,(137)(138)(139)(140)(141)(142)(143)(144)(145)(146)(147) and the peak at the 1st (SBP Յ 125 mmHg) quartile.CONCLUSIONS -In well-controlled elderly diabetic subjects, there was no excessive mortality compared to the age-and sex-matched general population. Renal dysfunction and prior stroke were independent risks for mortality and morbidity. In those without prior stroke, a risk of too much lowering of blood pressure was suggested. Diabetes Care 26:638 -644, 2003

Peroxisome proliferators (e.g. clofibric acid) and thyroid hormone play an important role in the metabolism of lipids. These effectors display their action through their own nuclear receptors, peroxisome proliferatoractivated receptor (PPAR) and thyroid hormone receptor (TR). PPAR and TR are ligand-dependent, DNA binding, trans-acting transcriptional factors belonging to the erbA-related nuclear receptor superfamily. The present study focused on the convergence of the effectors on the peroxisome proliferator response element (PPRE). Transcriptional activation induced by PPAR through a PPRE was significantly suppressed by cotransfection of TR in transient transfection assays. The inhibition, however, was not affected by adding 3,5,3-triiodo-L-thyronine (T3). Furthermore, the inhibition was not observed in cells cotransfected with retinoic acid receptor or vitamin D3 receptor. The inhibitory action by TR was lost by introducing a mutation in the DNA binding domain of TR, indicating that competition for DNA binding is involved in the molecular basis of this functional interaction. Gel shift assays revealed that TRs, expressed in insect cells, specifically bound to the 32 P-labeled PPRE as heterodimers with the retinoid X receptor (RXR). Both PPAR and TR bind to PPRE, although only PPAR mediates transcriptional activation via PPRE. TR⅐RXR heterodimers are potential competitors with PPAR•RXR for binding to PPREs. It is concluded that PPAR-mediated gene expression is negatively controlled by TR at the level of PPAR binding to PPRE. We report here the novel action of thyroid hormone receptor in controlling gene expression through PPREs.Peroxisomes are cytoplasmic organelles that are important in mammalian lipid homeostasis (1). The structurally diverse xenobiotic peroxisome proliferators (PPs), 1 such as clofibrate, nafenopin, and WY-14,643 stimulate the proliferation of peroxisomes (2-5) and cause tumorigenic transformation of hepatic cells in rodents (6, 7). Some of these compounds have been used in man as hypolipidemic agents. PPs have been shown to induce peroxisomal and microsomal enzymes involved in lipid metabolism through activation of the peroxisome proliferatoractivated receptor (PPAR) (8, 9). The PPAR is a member of the nuclear receptor superfamily of ligand-dependent transcriptional factors and is structurally related to the subfamily of receptors that includes the thyroid hormone receptor (TR), retinoic acid receptor (RAR), and vitamin D3 receptor (VDR) (10). To date, three subtypes of PPARs have been identified in amphibians, rodents, and humans, PPAR␣,9,[11][12][13][14]. Further investigation revealed that natural fatty acids are also potent activators of PPAR␣ (14, 15), although no direct interaction of PPAR␣ with either PPs or fatty acids has been described so far. Recently, ligands for PPAR␥ have been identified that are potent inducers of adipogenesis in vivo. These include thiazolidine diones, a class of anti-diabetic drugs, and the arachidonic acid derivative 15-deoxy-D12, 14-prostaglandin J2 (16 ...