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Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.

Peroxisome proliferator-activated receptor (PPAR)-gamma plays an important role in adipogenesis. However, the functions of PPAR-gamma in differentiated adipocytes have remained unclear. The role of PPAR-gamma in mature 3T3-L1 adipocytes was therefore investigated by overexpression of a dominant negative mutant of this protein (PPAR-gamma-DeltaC) that lacks the 16 COOH-terminal amino acids and that has been shown to prevent the thiazolidinedione-induced differentiation of 3T3-L1 cells into adipocytes. Overexpression of PPAR-gamma-DeltaC in mature 3T3-L1 adipocytes by adenovirus gene transfer resulted in a decrease in both cell size and intracellular triglyceride content, an increase in the extent of lipolysis, and a reduction in the rate of free fatty acid uptake. Furthermore, overexpression of this mutant reduced the abundance of mRNAs for several key enzymes that contribute to triglyceride and free fatty acid metabolism as well as the amounts of GLUT4, insulin receptor, insulin receptor substrate (IRS), and C/EBPalpha mRNAs. It also reduced both the concentration of IRS2 and the insulin-stimulated glucose uptake. These results suggest that PPAR-gamma plays an important role in mature 3T3-L1 adipocytes at least in part by maintaining the expression of genes that confer the characteristics of mature adipocytes.

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. Previously, we developed NPY-overexpressing mice by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but we failed to observe the obese phenotype in the heterozygote. Here we report that in the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibited significantly increased body weight gain with transiently increased food intake after 50% sucrose–loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of our observation period.

The allele frequencies for a Pro 12 →Ala substitution in peroxisome proliferator–activated receptor-γ differ among ethnic groups, and its relationship with diabetes and associated diseases is controversial. The prevalence of this polymorphism and its effects on clinical characteristics have now been evaluated with a large number of Japanese individuals with type 2 diabetes ( n = 2,201) and normal control subjects ( n = 1,212) recruited by 10 institutions located in seven different cities in Japan. The allele frequency for the Ala 12 variant was significantly lower in the type 2 diabetic group than in the control group (2.39 vs. 4.13%, P = 0.000054). However, compared with subjects without the Ala 12 variant, the diabetic subjects with this variant exhibited a significantly higher serum concentration of total cholesterol ( P = 0.001), manifested a reduced capacity for insulin secretion as evaluated by homeostasis model assessment ( P = 0.007), and tended to possess a higher level of HbA 1c . These data suggest that the Ala 12 variant is associated with a reduced risk for the development of diabetes in the general population, but that it may be also a risk factor for insulin deficiency and disease severity in individuals with type 2 diabetes.

A corpus of evidence suggests that T-helper type 1 (Th1)-dependent cellular immunity plays a pivotal role in the pathogenesis of autoimmune diabetes. This study was intended to find ways to prevent the development of NOD diabetes using a neutralizing anti-interleukin (IL)-12 antibody (C17.8) that inhibits Thl cell differentiation. When C17.8 was administered from 5 to 30 weeks of age, NOD mice exhibited suppression of both insulitis and diabetes. However, when C17.8 administration ceased at 15 weeks of age, 8 of 13 recipients showed diabetes at 30 weeks of age. These results suggest that IL-12 plays an important role not only in the development of effector cells but also in their activation. In contrast, when C17.8 was injected into 2-week-old female NOD mice for 6 consecutive days, all 16 recipients showed diabetes at 30 weeks of age, whereas 12 of 20 control mice became diabetic. This result suggests that depletion of endogenous IL-12 at a young age results in the enhancement of diabetes. Flow cytometric analysis indicated that activated memory T-cells were present in higher numbers after C17.8 treatment. Transfer of spleen cells from 15-week-old C17.8-treated NOD mice to NOD-scid mice resulted in an earlier onset and a higher incidence of diabetes. Furthermore, administration of C17.8 to 2-week-old NOD mice also resulted in a much earlier onset of diabetes. These results suggest that short-term treatment with anti-IL-12 antibody prohibits IL-2 production at a young age, which may influence the expansion and apoptosis of pathogenic T-cells, resulting in the acceleration of autoimmune diabetes.

Formation of advanced glycation end products (AGEs) is accelerated in diabetic subjects along with hyperglycemia. Although several lines of evidence indicate that AGEs stimulate macrophages to secrete several cytokines and growth factors, little is known about the effect of AGEs on the primary function of macrophages, such as phagocytosis. On the other hand, impairment of the phagocytic function of monocytes/macrophages is suggested to contribute to the low resistance to infection in diabetic subjects. In the present study, we examined the effect of AGEs on the phagocytic function of macrophages. Using flow cytometric analysis of mouse resident peritoneal macrophages, we showed that AGEs suppress phagocytosis of fluorescent microspheres by cultured macrophages. In addition, experiments using streptozotocin-induced diabetic mice demonstrated a significant decrease in the phagocytic activity of resident peritoneal macrophages 12 weeks after induction of diabetes compared with age-matched control mice. The phagocytic activity of peritoneal macrophages correlated inversely with AGE content in the adjacent peritoneal tissue. Furthermore, reduced phagocytic activity of macrophages was associated with a reduction in intracellular ATP content. Because phagocytosis is an important component of the defense system, suppression of such activity by AGEs may explain, at least in part, the increased susceptibility of diabetic patients to infection.

In the liver, signal transducer and activator of transcription 3 (STAT3) plays an important role in the suppression of gluconeogenic enzyme expression. While obesity-associated endoplasmic reticulum (ER) stress has been shown to increase hepatic gluconeogenic enzyme expression, the role of ER stress in STAT3-dependent regulation of such expression is unclear. The current study aimed to elucidate the effect of ER stress on the STAT3-dependent regulation of hepatic gluconeogenic enzyme expression. Genetically obese/diabetic db/db mice and db/db mouse–derived isolated hepatocytes were used as ER stress models. A tyrosine phosphatase inhibitor, a deacetylation inhibitor, and an acetylated mutant of STAT3 were used to examine the effect of ER stress on hepatic STAT3 action. ER stress inhibited STAT3-dependent suppression of gluconeogenic enzyme gene expression by suppressing hepatic Janus kinase (JAK)2 and STAT3 phosphorylation. A tyrosine phosphatase inhibitor restored ER stress–induced suppression of JAK2 phosphorylation but exhibited no improving effect on suppressed STAT3 phosphorylation. STAT3 acetylation is known to correlate with its phosphorylation. ER stress also decreased STAT3 acetylation. An acetylated mutant of STAT3 was resistant to ER stress–induced inhibition of STAT3-phosphorylation and STAT3-dependent suppression of hepatic gluconeogenic enzyme gene expression in vitro and in vivo. Trichostatin A, a histone deacetylase (HDAC) inhibitor, ameliorated ER stress–induced inhibition of STAT3 acetylation and phosphorylation. The current study revealed that ER stress inhibits STAT3-dependent suppression of hepatic gluconeogenic enzymes via JAK2 dephosphorylation and HDAC-dependent STAT3 deacetylation, playing an important role in the increase of hepatic glucose production in obesity and diabetes.

Tyrosine-phosphorylated proteins in Triton X-100–solubilized fractions of rat livers were examined by immunoblotting with anti-phosphotyrosine antibodies. After 2 min of insulin injection via the portal vein into livers, three major bands of 170,000, 140,000, and 95,000 Mr were stimulated. Because the incubation of nitrocellulose membrane with anti-phosphotyrosine antibodies in the presence of 40 mM phosphotyrosine completely abolished these bands, the anti-phosphotyrosine antibodies appear to recognize the phosphotyrosine residues of these proteins. Insulin injection (2–2000 μg) very quickly stimulated the tyrosine phosphorylation of these proteins in a dose-dependent fashion. In contrast, insulinlike growth factor I or epidermal growth factor injection had little effect in stimulating the tyrosine phosphorylation of these proteins. Because anti-insulin-receptor antibodies immunoprecipitated a tyrosine-phosphorylated 95,000-Mr protein, this protein must be the β-subunit of the insulin receptor; i.e., the β-subunit of the insulin receptor and two other proteins were phosphorylated at tyrosine residues in vivo by insulin injection. These data suggest that the tyrosine phosphorylation and tyrosine kinase activity of the insulin receptor may have important roles in in vivo insulin action.

OBJECTIVE—Resistin is an adipocyte-secreted cytokine associated with insulin resistance in mice. We previously reported that the G/G genotype of a resistin single nucleotide polymorphism (SNP) at −420 increases type 2 diabetes susceptibility by enhancing its promoter activity. The aim of the present study was to determine the relevance of SNP −120 in a large number of subjects. RESEARCH DESIGN AND METHODS— We examined 2,610 type 2 diabetic case and 2,502 control subjects. The relation between SNP −420 and the age of type 2 diabetes onset was further analyzed by adding 237 type 2 diabetic subjects with age of onset ≤40 years. RESULTS—When analyzed without considering subject age, the SNP −420 genotype was not associated with type 2 diabetes. Since we reported that the onset of type 2 diabetes was earlier in G/G genotype, we analyzed the data using a trend test for age intervals of 10 years. The frequency of G/G genotype differed among age grades in type 2 diabetes (P = 0.037) and appeared to be higher in younger grades. In type 2 diabetes, G/G genotype was more frequent in subjects aged CONCLUSIONS—The G/G genotype frequency of resistin SNP −420 appears to be increased in younger-onset type 2 diabetic subjects.

Phosphoinositide-dependent kinase-1 (PDK1) is implicated in the metabolic effects of insulin as a key mediator of phosphoinositide 3-kinase–dependent signaling. Here we show that mice with liver-specific PDK1 deficiency manifest various defects in the metabolic actions of insulin in the liver as well as a type 2 diabetes–like phenotype characterized by marked hyperinsulinemia and postprandial hyperglycemia. The hepatic abundance of glucokinase, an important determinant of glucose flux and glucose-evoked signaling in hepatocytes, was substantially reduced in these mice. Restoration of hepatic glucokinase expression, with the use of an adenoviral vector, induced insulin-like effects in the liver and almost completely normalized the fasting hyperinsulinemia and postprandial hyperglycemia in these animals. These results indicate that, if the hepatic abundance of glucokinase is maintained, ingested glucose is normally disposed of even in the absence of acute activation of proximal insulin signaling, such as the activation of Akt, in the liver.

AMP-activated protein kinase (AMPK) acts as a fuel gauge for glucose and lipid metabolism. The gene encoding the α2 isoform of the catalytic subunit of AMPK ( PRKAA2 ) is located at one of the Japanese type 2 diabetes loci mapped by our previous genome scan (1p36-32). PRKAA2 is, therefore, a good candidate gene for insulin resistance and type 2 diabetes. We screened all nine exons, their exon-intron boundaries, and the 5′ and 3′ flanking regions of PRKAA2 to identify single nucleotide polymorphisms (SNPs), and we genotyped 192 type 2 diabetic patients and 272 nondiabetic subjects to assess possible associations between genotypes or haplotypes and type 2 diabetes. None of the 10 SNPs genotyped was associated with type 2 diabetes, but the haplotype analysis, consisting of six representative SNPs, revealed one haplotype, with the A (minor) allele for rs2051040 and a major allele for the other five SNPs, to be associated with type 2 diabetes ( P = 0.009). This finding was confirmed in two larger replication samples (657 case and 360 control subjects, P = 0.021; and 356 case and 192 control subjects from the same area in Japan, P = 0.007) and a significant P value was obtained in the joint haplotype analysis of all samples (1,205 case and 824 control subjects, P = 0.0001). Furthermore, insulin resistance was associated with rs2051040 in nondiabetic subjects, and those with the A (minor) allele had a higher homeostasis model assessment of insulin resistance index than those who did not (initial control subjects [ n = 272], P = 0.002; and joint replication control subjects [ n = 552], P = 0.037). We speculate that the PRKAA2 gene influences insulin resistance and susceptibility to type 2 diabetes in the Japanese population.

The mechanism by which insulin induces the expression of the sterol regulatory element binding protein 1c (SREBP-1c) and glucokinase genes was investigated in cultured rat hepatocytes. Overexpression of an NH2-terminal fragment of IRS-1 that contains the pleckstrin homology and phosphotyrosine binding domains (insulin receptor substrate-1 NH2-terminal fragment [IRS-1N]) inhibited insulin-induced tyrosine phosphorylation of IRS-1 as well as the association of IRS-1 with phosphatidylinositol (PI) 3-kinase activity, whereas the tyrosine phosphorylation of IRS-2 and its association with PI 3-kinase activity were slightly enhanced. The equivalent fragment of IRS-2 (IRS-2N) prevented insulin-induced tyrosine phosphorylation of both IRS-1 and IRS-2, although that of IRS-1 was inhibited more efficiently. The insulin-induced increases in the abundance of SREBP-1c and glucokinase mRNAs, both of which were sensitive to a dominant-negative mutant of PI 3-kinase, were blocked in cells in which the insulin-induced tyrosine phosphorylation of IRS-1 was inhibited by IRS-1N or IRS-2N. A dominant-negative mutant of Akt enhanced insulin-induced tyrosine phosphorylation of IRS-1 (but not that of IRS-2) and its association with PI 3-kinase activity, suggesting that Akt contributes to negative feedback regulation of IRS-1. The Akt mutant also promoted the effects of insulin on the accumulation of SREBP-1c and glucokinase mRNAs. These results suggest that the IRS-1–PI 3-kinase pathway is essential for insulin-induced expression of SREBP-1c and glucokinase genes.

Apoptosis via Fas/Fas ligand (FasL) interactions has been proposed to be a major T-cell-mediated effector mechanism in autoimmune diabetes. To elucidate the role of Fas/FasL interactions in NOD diabetes, the effects of neutralizing anti-FasL antibody on autoimmune responses were evaluated. Islet-specific CD8(+) and CD4(+) T-cells expressed FasL upon activation and mediated FasL-dependent cytotoxicity against Fas-expressing target cells in vitro, although their cytotoxicity against islet cells was not blocked by anti-FasL antibody. Moreover, administration of anti-FasL antibody failed to inhibit diabetes in vivo in the CD8(+) T-cell adoptive transfer model. On the other hand, blockade of Fas/FasL interactions significantly inhibited CD4(+) T-cell-dependent diabetes in adoptive transfer models. These results suggest a substantial contribution of Fas/FasL interactions to CD4(+), but not CD8(+), T-cell-mediated destruction of pancreatic beta-cells. When anti-FasL antibody was administered to NOD mice between 5 and 15 weeks of age, the onset of diabetes was slightly delayed but the incidence was not decreased. However, administration of anti-FasL antibody at 2-4 weeks of age completely prevented insulitis and diabetes. These results suggest that Fas/FasL interactions contribute to CD4(+) T-cell-mediated beta-cell destruction and play an essential role in the initiation of autoimmune NOD diabetes.

The mouse obese (ob) gene has recently been isolated through the positional cloning technique and has been proved to result in the obese and NIDDM phenotype in mice when mutated (Nature 372:425–432, 1994). More recently, it has been demonstrated, by experiments with recombinant ob protein, that ob gene product can cause mice, including ob/ob mice, diet-induced obesity mice, and normal mice, to lower their food intake and body weight (Science 269:540–549, 1995). To investigate the genetic and/or environmental influences underlying the development of NIDDM associated with obesity, we isolated and partially sequenced the human obese (OB) gene. The human OB gene isolated in this study encoded 167 amino acids and its open reading frame was revealed to be divided into two parts with an intermediate intron of ∼2.4 kb. Using the single-strand conformation polymorphism (SSCP) technique, we screened Japanese and Asian Indian subjects for mutations in the protein coding regions of the OB gene. A total of 75 NIDDM patients with obesity (54 Japanese and 21 Asian Indians), 40 NIDDM patients without obesity (34 Japanese and 6 Asian Indians), and 34 Japanese patients with simple obesity showed no abnormal SSCP patterns in either component of the coding sequences. These results suggested that mutations in the coding regions of the OB gene are not likely to be commonly identifiable and that there would likely be a kind of obesity-associated NIDDM not caused by mutations of the OB gene.

Intercellular adhesion molecule 1 (ICAM-1) plays an important role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) by being involved in the extravasation of lymphocytes from the circulation into the inflamed pancreas. However, the mechanism of β-cell destruction by which expression of ICAM-1 on β-cells may facilitate adhesion of effector cells still remains to be elucidated. Several lines of evidence suggest that this adhesion molecule is involved in the destruction of pancreatic β-cells by killer lymphocytes in the NOD mouse, which shows an autoimmune diabetic syndrome similar to that of human IDDM. Immunohistochemical study under light microscopy demonstrated that all of the mononuclear cells infiltrating the islets strongly expressed ICAM-1 and leukocyte function-associated antigen 1 (LFA-1), a counterreceptor of ICAM-1, whereas ICAM-1 expression on islet cells was not apparent. However, immunohistochemical staining under electron microscopy revealed that islet β-cells adjacent to infiltrating lymphocytes were clearly stained by an anti-ICAM-1 monoclonal antibody (mAb). Flow cytometric analysis showed that the ICAM-1 expression on NOD islet cells and NOD-derived insulinoma cells (MIN6N8a) was inducible by interferon (IFN)-γ or tumor necrosis factor-α. These cytokines had an additive effect on the ICAM-1 induction. Susceptibility of MIN6N8a cells to lysis by a NOD islet-derived CD8+ cytotoxic T-cell clone was greatly enhanced by IFN-γ pretreatment, and this enhancement was abolished by anti-ICAM-1 and anti-LFA-1 mAbs. When both mAbs were administered into NOD mice with spontaneous or adoptively transferred diabetes, the development of diabetes was significantly prevented. These results suggest that cytokines secreted by isletinfiltrating mononuclear cells could induce the ICAM-1 expression on γ-cells, which accelerates the γ-cell destruction by cytotoxic T-cells. Therefore, immunointervention of the ICAM-l/LFA-1 pathway would be an excellent strategy to prevent human IDDM.

The insulin receptors from erythrocytes of 50 patients with non-insulin-dependent diabetes mellitus were tested for their ability to autophosphorylate. The assay was performed by a new enzyme-linked immunosorbent assay system that used monoclonal anti-insulin receptor antibodies absorbed to microtiter plates as a first antibody and polyclonal antiphosphotyrosine antibody as a labeled second antibody. By this assay, 3 patients were identified with defects in their insulin receptor kinase, although their defects appeared heterogeneous. Patient 1 had 85% less maximal autophosphorylation with a normal ED50 (1.6 × 10−9 M insulin). Patient 2, who had polycystic ovary disease, had a 49.2% decrease in maximal autophosphorylation of insulin receptors, and the ED50 was shifted to the right (5.6 × 10−8 M). Patient 3 with acanthosis nigricans had a normal maximal autophosphorylation, but the ED50 shifted to the right (2.9 × 10−8 M). The mechanisms for the diversity detected in this assay is not known, but this technique has sufficient specificity and sensitivity to be used to screen for insulin-resistant patients who have a lack of kinase activity.

Specific binding sites for insulin have been identified and characterized for the human erythroleukemia cell line K-562. The binding of [125I]-insulin to the cells increased as a function of time, reaching a maximum at 20 min when incubation was performed at 37°C. The binding of [125I]-insulin was dose-dependently inhibited by insulin or proinsulin. Scatchard plot of the binding data was curvilinear, and the number of insulin receptors was approximately 39,000. Insulin at concentrations of 0.05–10.0 ng/ml stimulated CO2 production and DNA and protein synthesis in K-562 cells in a dose-dependent manner, indicating that the insulin binding sites are functionally important in mediating these biochemical events induced by insulin. Maximal insulin responses were elicited at concentrations of

We studied the effect of sera from two patients who had an unusual form of diabetic syndrome with extreme insulin resistance on the metabolism of human adipocytes in vitro. The IgG fractions from sera A and B, which were obtained from two patients (1 and 2) with insulin-resistant diabetes, inhibited [125I]insulin binding to human adipocytes and, at the same time, stimulated glucose oxidation and inhibited the lipolysis induced by levarterenol in human adipocytes. On the other hand, the IgG fraction from the C serum, which was obtained from patient 2 after her diabetic syndrome had completely disappeared as a result of immunosuppressive therapy, did not inhibit [125I]insulin binding to human adipocytes, stimulate glucose oxidation, or inhibit lipolysis in human adipocytes. These facts suggest that these IgG fractions bind to or near the insulin receptor of human adipocytes, that they exhibit their insulin-like effect by binding to the insulin receptor in vitro, and, furthermore, that they are responsible for the extremely insulin-resistant diabetes. However, the apparent discrepancy between the effects of these IgG fractions on man in vitro and in vivo is puzzling and needs to be explained.