A Role for iNOS in Fasting Hyperglycemia and Impaired Insulin Signaling in the Liver of Obese Diabetic Mice
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糖尿病学杂志 2005年第5期
1 Department of Anesthesia and Critical Care, Massachusetts General Hospital, Shriners Hospital for Children, Harvard Medical School, Boston, Massachusetts
2 Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan
ABSTRACT
Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat dieteCinduced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, L-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5- and 2-fold, respectively, and enhanced IRS-1eCand IRS-2eCmediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice.
Chronic low-grade inflammation has been proposed to be involved in the pathogenesis in obesity-related insulin resistance and type 2 diabetes. The expression of proinflammatory cytokines, including tumor necrosis factor- (1) and interleukin-6 (2), is upregulated in animal models of and patients with type 2 diabetes. However, limited knowledge is thus far available about the molecular mechanisms by which chronic inflammation mediates insulin resistance and type 2 diabetes.
The activation of inhibitor B kinase (IKK)eCnuclear factor-B (NF-B), a crucial signaling cascade for inflammatory response, has been highlighted as a mediator of insulin resistance. The pharmacological inhibition or gene disruption of IKK reversed obesity-related insulin resistance and fasting hyperglycemia in rodents and humans (3eC5). However, little is known about genes that function as downstream effectors of the IKKbeCNF-B pathway to mediate insulin resistance.
Inducible nitric oxide synthase (iNOS; also termed NOS2), whose expression is regulated by IKKeCNF-B (6), is assumed to be one of the candidates that mediate inflammation-involved insulin resistance. Accumulating evidence indicates a close link between iNOS and insulin resistance. Although iNOS was originally identified in macrophages, it is now known that it is widely expressed in many tissues, including insulin-sensitive organs such as skeletal muscle, adipose tissue, and liver, in normal rodents and humans. The expression of iNOS is upregulated by most, if not all, inducers of insulin resistance, including proinflammatory cytokines, obesity (7), free fatty acids (8), hyperglycemia (9,10), endotoxins (6,11), and oxidative stress. In fact, elevated expression of iNOS was observed in skeletal muscle of high-fat dieteCfed mice (12), in heart of Zucker diabetic fatty rats (13), and in skeletal muscle (14) and platelets of patients with type 2 diabetes (15). Nitrosative protein modifications, such as tyrosine nitration often associated with iNOS expression, were elevated in plasma (16), skeletal muscle (14), vasculature (17,18), and retina (19) of patients with and rodent models of type 2 or obesity-related diabetes. Furthermore, iNOS induction resulted in attenuated insulin-stimulated glucose uptake in cultured skeletal muscle cells (20). Thiazolidinediones, a class of insulin sensitizer, suppress iNOS expression in cultured cells and in vivo in rodents (21,22). Thus, inhibition of iNOS expression has been recently proposed to be a new mechanism of actions of insulin sensitizers (23,24).
Recently, we and others have shown that iNOS plays an important role in the pathogenesis of insulin resistance in vivo. We demonstrated that iNOS inhibitor prevented fasting hyperglycemia and mitigated insulin resistance in lipopolysaccharide (LPS)-administered rats (11). Perreault and Marette (12) reported that iNOS deficiency protected against high-fat dieteCinduced insulin resistance. With respect to a role of iNOS in insulin resistance, our previous findings in LPS-administered rats appear to agree with the data of Perreault and Marette in high-fat dieteCfed mice. However, differences also exist in these previous studies (11,12), particularly in regard to the tissues in which iNOS inhibition or deficiency exerts its insulin-sensitizing effects. We found that iNOS expression was elevated in the liver by LPS and that iNOS inhibitor blocked LPS-induced increase in hepatic glucose output in Sprague-Dawley rats (11). By contrast, Perreault and Marette observed that iNOS expression was increased in skeletal muscle and adipose tissue but not in the liver of C57BL/6X129SvEv mice fed a high-fat diet and that iNOS disruption restored high-fat dieteCinduced defects in insulin signaling in skeletal muscle but not in adipose tissue or liver (12). Moreover, in their study, iNOS deficiency did not alter blood glucose levels in mice fed a high-fat diet, whereas whole-body insulin resistance was alleviated, as judged by an insulin tolerance test. A question, therefore, remains to be answered whether a role of iNOS in obesity-related insulin resistance is restricted to skeletal muscle or whether iNOS also plays a role in the liver, which is the primary organ to determine fasting blood glucose levels (25eC27). To address this issue, we examined the effects of iNOS inhibitor on fasting hyperglycemia and hepatic insulin signaling in genetically obese diabetic (ob/ob) mice.
RESEARCH DESIGN AND METHODS
Male ob/ob mice and lean wild-type C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The ob/ob mice were backcrossed onto wild-type C57BL/6J mice for at least 30 generations. The institutional animal care committee approved the study protocol. The mice were housed in mesh cages in a room maintained at 25°C and illuminated in 12:12-h light-dark cycles; they were provided with standard rodent diet and water ad libitum.
Treatment with iNOS inhibitor.
Male ob/ob mice at 10 weeks of age were treated with a specific inhibitor of iNOS, L-NIL (60 mg/kg body wt, i.p., b.i.d.; Cayman Chemical, Ann Arbor, MI), or PBS for 10 days. Following 10 days of treatment, the animals treated with L-NIL (n = 17) or PBS (n = 18) were fasted overnight and then received an injection of insulin (Humulin R; Eli Lilly, Indianapolis, IN; 15 units/kg body wt in PBS with 0.1% BSA) or vehicle (PBS with 0.1% BSA) via the portal vein under anesthesia with pentobarbital sodium (70 mg/kg body wt, i.p.). At 5 min after insulin injection, the liver, gastrocnemius muscle, and epididymal fat were taken for biochemical analyses. Male ob/ob mice at 10 weeks of age were also treated with L-NIL or PBS for 10 days under a pair-fed condition. After 10 days of treatment, the liver was taken from pair-fed mice under fed condition or after overnight fasting. Twenty-four mice were randomly assigned into four groups (L-NIL fasted, PBS fasted, L-NIL fed, and PBS fed) of six animals. The average food intake of both L-NIL and PBS-treated animals under pair-fed condition was 5.6 g/day.
Insulin tolerance test.
After 10 days of treatment, the animals treated with L-NIL (n = 10) or PBS (n = 10) were fasted for 4 h and then received an injection of insulin (2.5 units/kg body wt in PBS with 0.1% BSA, i.p.). At 0, 15, 30, 60, 90, and 120 min after insulin injection, blood samples were collected to measure glucose level.
Immunoprecipitation and immunoblotting.
Liver samples were homogenized as previously described (28). Immunoprecipitation and immunoblotting were performed (29) using anti-IR, IRS-1 and -2, p85 phosphatidylinositol 3(PI3)-kinase, iNOS, endothelial NOS, neuronal NOS (Upstate, Lake Placid, NY), Akt, phosphorylated Akt (Ser473) (Cell Signaling, Beverly, MA), and phosphotyrosine antibodies (Santa Cruz, Santa Cruz, CA) (online appendix available at http://diabetes.diabetesjournals.org).
Partial purification of iNOS.
Partial purification of iNOS was performed using immobilized 1400W beads (Calbiochem, San Diego, CA), which selectively bind to iNOS, according to the manufacturer’s instruction (online appendix).
PI3-kinase assay.
PI3-kinase activities in the immunoprecipitates with antieCIRS-1 or -2 antibody were measured in vitro (30).
Detection of tyrosine nitration.
Immunostaining with anti-nitrotyrosine antibody (Upstate) was performed according to the manufacturer’s instruction (online appendix).
RNase protection assay.
RNase protection assay was performed as previously described (11) using the RPA III kit (Ambion, Austin, TX). mRNA levels of IRS-1 and -2 and sterol regulatory elementeCbinding protein (SREBP)-1c were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (online appendix).
Cell culture.
Mouse Hepa1c1c7 and human HepG2 cells (American Type Culture Collection, Manassas, VA) were maintained in -minimum essential medium and minimum essential medium supplemented with 10% fetal bovine serum, respectively. After 8 h serum starvation, the cells were treated with the indicated concentrations of S-nitrosoglutathione (GSNO) or 3-morpholino-sydnonimine (Cayman Chemical, Ann Arbor, MI) in the presence or absence of ODQ (1 eol/l; Sigma) or 8-bromoguanosine-3',5'-cyclic monophosphorothioate (Br-cGMP; 100 eol/l; Axxora, San Diego, CA) for 24 h, unless otherwise indicated. Hepa1c1c7 cells were transfected with pCDNA3/iNOS or pCDNA3 using Lipofectamin 2000 (Invitrogen). pCDNA3/iNOS was kindly provided by Dr. Kone. At 48 h after transfection, the cells were harvested.
Measurement of blood glucose, insulin, triglycerides, glycogen, adiponectin, and free fatty acids.
Blood glucose level and plasma insulin concentration were determined by the glucose oxidase method (Ascensia Elite Glucometer; Bayer, Elkhart, IN) and enzyme-linked immunosorbent assay (Crystal Chem, Chicago, IL), respectively. Glycogen content was measured using a glucose hexokinase assay kit (Sigma) (31). A Glycerol phosphate oxidase trinder reagent (Sigma) was used to measure triglyceride (32). The concentrations of adiponectin and free fatty acids in serum from fasted animals were determined using an enzyme-linked immunosorbent assay kit (Linco Research, St. Charles, MO) and an enzymatic colorimetric assay kit (Wako Chemicals, Richmond, VA), respectively.
Statistical analysis.
Statistical analyses were performed using Student’s t test or one-way ANOVA followed by Fisher’s protected least significant differences test. Values of P < 0.05 were considered statistically significant. Data are expressed as means ± SE.
RESULTS
Increased expression of iNOS in the liver of diabetic mice.
We found a 2.5-fold increase in iNOS protein expression in the liver of ob/ob mice compared with wild-type mice (Fig. 1A). No difference was found in the protein expression of endothelial or neuronal NOS between ob/ob and wild-type mice (online appendix Fig. 1). iNOS expression was also increased in skeletal muscle and adipose tissue of ob/ob mice compared with wild-type mice to the extent comparable to that in the liver (online appendix Fig. 2).
Reduced nitrotyrosine immunoreactivity by iNOS inhibitor.
The immunoreactivity for nitrotyrosine, a marker of nitrosative stress, was also elevated in the liver of ob/ob mice compared with wild-type mice (Fig. 1B). Treatment with iNOS inhibitor (60 mg/kg body wt, i.p., b.i.d., for 10 days) reversed the elevated nitrotyrosine immunoreactivity in the liver of ob/ob mice compared with PBS. L-NIL treatment, however, did not alter the expression of iNOS and endothelial and neuronal NOS (data not shown). These results suggest that L-NIL, a competitive inhibitor for iNOS, effectively reduced the activity of iNOS in the liver of ob/ob mice without altering iNOS expression level, as expected.
Reversal of fasting hyperglycemia and amelioration of hyperinsulinemia by iNOS inhibitor in diabetic mice.
At the inception of the treatment, there was no difference in fed blood glucose level (L-NIL: 292.8 ± 21.8; PBS: 290.3 ± 18.7 mg/dl), plasma insulin concentration (L-NIL: 57.0 ± 0.5; PBS: 57.0 ± 0.5 ng/ml), or body weight (L-NIL: 57.0 ± 0.5; PBS: 56.8 ± 0.6 g) between L-NIL eCand PBS-administered animals. Treatment with L-NIL did not affect body weight at the time of its termination (L-NIL: 57.2 ± 0.5; PBS: 57.0 ± 0.6 g). There was no statistical difference in food intake between L-NIL eCand PBS-treated animals (L-NIL: 5.8 ± 0.2; PBS: 6.4 ± 0.2 g/day), although food intake in L-NILeCtreated animals appeared to be lower than in PBS-treated animals. However, L-NIL treatment reversed fasting hyperglycemia and significantly ameliorated fasting hyperinsulinemia in ob/ob mice (Fig. 2A and B). The insulin sensitivity index (fasting blood glucose x plasma insulin concentration) was also significantly improved by L-NIL (L-NIL: 16.9 ± 4.0; PBS: 51.0 ± 12.6 mg2/l2, P < 0.05). Improved insulin sensitivity by L-NIL treatment was confirmed by insulin tolerance test. ob/ob mice treated with L-NIL responded to insulin with a more profound decrease in blood glucose level than in PBS-administered ob/ob mice (Fig. 2C). Area under the curve analysis in response to the insulin tolerance test also revealed the beneficial effects of L-NIL (Fig. 2D).
To exclude a possible influence of food intake, the effects of L-NIL treatment were also examined in pair-fed ob/ob mice. The beneficial effects of L-NIL were corroborated in pair-fed mice. In pair-fed ob/ob mice, L-NIL treatment also led to the reversal of fasting hyperglycemia (L-NIL: 95.3 ± 13.4; PBS: 210.8 ± 18.1 mg/dl, P < 0.05) and significant amelioration of fasting hyperinsulinemia (L-NIL: 11.4 ± 3.2; PBS: 19.4 ± 1.9 ng/ml, P < 0.05) and insulin sensitivity index (L-NIL: 10.3 ± 2.8; PBS: 40.8 ± 5.9 mg2/l2, P < 0.05). Under fed condition, L-NIL treatment appeared to mitigate hyperglycemia in ob/ob mice (L-NIL: 195.7 ± 29.1; PBS: 272.2 ± 24.0, P < 0.10), although no statistical significance was found. Plasma insulin concentration under fed condition tended to be higher in L-NILeCtreated animals than PBS-treated animals (L-NIL: 86.4 ± 7.0; PBS: 57.5 ± 12.4 ng/ml, P < 0.10), although there was no statistical difference.
iNOS inhibitor improved IRS-1eCand IRS-2eCmediated insulin signaling in the liver of diabetic mice.
iNOS inhibitor, L-NIL, did not alter the protein expression of insulin receptor (IR). However, the protein expression of IRS-1 and -2 under a fasted condition was increased by 1.5- and 2-fold, respectively, compared with PBS-treated animals (Fig. 3A). Increased protein expression of IRS-1 and -2 by L-NIL was further confirmed in pair-fed mice. In pair-fed ob/ob mice, the protein expression of IRS-1 and -2 in L-NILeCtreated animals was greater than in PBS-treated animals under both fasted and fed conditions (Fig. 3B and online appendix Fig. 3). L-NIL treatment resulted in upregulated mRNA level of IRS-2 under both fasted and fed conditions (Fig. 3C and D). However, the mRNA level of IRS-1 was not altered by L-NIL treatment. L-NIL did not affect GAPDH mRNA level (data not shown). No difference was found in the protein expression of p85, p55, and p50 PI3-kinase in the liver between ob/ob mice treated with L-NIL and PBS (online appendix Fig. 4).
Insulin-stimulated tyrosine phosphorylation of IRS-1 and -2 in the liver was upregulated 1.5- and 3-fold by iNOS inhibitor, respectively, compared with PBS (Fig. 4). However, iNOS inhibitor did not alter insulin-stimulated tyrosine phosphorylation of IR. L-NIL treatment did not significantly alter basal (insulin-unstimulated) tyrosine phosphorylation of IR and IRS-1 and -2. The ratio of insulin-stimulated tyrosine phosphorylation to protein abundance for IR and IRS-1 and -2 was not significantly altered by L-NIL treatment (online appendix Fig. 5). These results suggest that the improvement by iNOS inhibitor in insulin-stimulated tyrosine phosphorylation of IRS-1 and -2 may be mainly attributable to the increased expression of IRS-1 and -2. Insulin-stimulated IRS-1eCand IRS-2eCassociated PI3-kinase activity was also enhanced by L-NIL up to 1.5- and 2.5-fold, respectively (Fig. 4D and E). L-NIL treatment also increased insulin-stimulated phosphorylation of Akt/protein kinase B, a downstream signaling molecule, while the expression of Akt/protein kinase B was not altered (Fig. 4F and G).
Glycogen content in the liver of fasted animals was significantly increased by L-NIL treatment (L-NIL: 14.9 ± 1.8; PBS: 7.3 ± 0.6 mg/g liver wt, P < 0.01). Triglyceride content in the liver of fasted animals appeared to be lower in L-NILeCtreated animals than in PBS-treated animals (L-NIL: 52.9 ± 5.6; PBS: 65.7 ± 4.0 e/g protein, P < 0.10), although there was no statistical difference. In skeletal muscle of ob/ob mice, L-NIL treatment increased IRS-1 protein expression, while IRS-2 abundance was unaltered. In adipose tissue, neither IRS-1 nor -2 expression was affected by L-NIL treatment (online appendix Fig. 6). We did not find a significant difference in circulating adiponectin (L-NIL: 6.32 ± 0.6; PBS: 6.00 ± 0.45 ng/ml) and free fatty acid (L-NIL: 1.83 ± 0.1; PBS: 1.71 ± 0.12 mEq/l) concentration between L-NILeCand PBS-administered ob/ob mice.
NO donor and iNOS reduced the expression of IRS-1 and -2 in cultured hepatocytes.
Next, we asked whether iNOS can downregulate IRS-1 and -2 expression in cultured hepatocytes. Exposure to NO donor, GSNO (1 mmol/l), for 24 h decreased the protein expression of IRS-1 and -2 in Hepalc1c7 and HepG2 cells (Fig. 5 and online appendix Fig. 7), whereas the expression of IR and p85 PI3-kinase was not affected by NO donor. Treatment with another NO donor, 3-morpholino-sydnonimine-1 (0.5 mmol/l), for 24 h also decreased the expression of IRS-1 and -2 (online appendix Fig. 7). Ectopically expressed iNOS reduced the expression of IRS-1 and -2 without altering p85 expression. The inhibitory effects of GSNO on IRS-1 and -2 expression were dose and time dependent (Fig. 6). In accordance with the greater increase in IRS-2 expression than in IRS-1 by L-NIL treatment in the liver of ob/ob mice (Fig. 3), the inhibitory effects of NO donor or iNOS transfection appeared to be more prominent on IRS-2 than -1 (Figs. 5 and 6). Treatment with 1 mmol/l GSNO for 24 h did not affect viability of the cells, as judged by the trypan blue exclusion test. Consistent with the effects of iNOS inhibitor on IRS-1 and -2 mRNA in the liver of ob/ob mice, 1 mmol/l GSNO decreased IRS-2 mRNA level but did not affect IRS-1 mRNA level in cultured Hepa1c1c7 cells (Fig. 7).
To further characterize the effects of NO donor on IRS-1 and -2, we examined the effects of ODQ, a soluble guanylate cyclase inhibitor, and Br-cGMP, a cell-permeable cGMP analog in Hepa1c1c7 cells. ODQ (1 eol/l) failed to inhibit GSNO-induced reduction in IRS-1 and -2 expression. cGMP analog, Br-cGMP (100 eol/l), did not affect the expression of IRS-1 and -2, either. These observations suggest that the inhibitory effects of GSNO are cGMP independent.
Since a recent study (33) revealed that SREBPs suppress the transcription of IRS-2 in the liver, we examined the effects of L-NIL and GSNO on SREBP-1c expression. SREBP-1c mRNA level was increased by GSNO (1 mmol/l) in cultured Hepa1c1c7 cells (Fig. 7C) and was reduced by L-NIL treatment in the liver of ob/ob mice under both fasted and fed conditions (Fig. 7D and E).
DISCUSSION
We found that iNOS expression was significantly elevated in the liver as well as skeletal muscle and adipose tissue in ob/ob mice compared with wild-type mice (Fig. 1) and that iNOS inhibitor reversed fasting hyperglycemia and ameliorated whole-body insulin resistance in ob/ob mice (Fig. 2). The improved glycemic control was accompanied by increased protein expression of IRS-1 and -2 and enhanced IRS-1eCand IRS-2eCmediated insulin signaling in the liver of ob/ob mice (Figs. 3 and 4). Moreover, we found that exposure to an NO donor and ectopically expressed iNOS reduced the protein expression of IRS-1 and -2 in cultured hepatocytes (Figs. 5 and 6). These results indicate that increased expression of iNOS may play an important role in obesity-induced fasting hyperglycemia and suggest that iNOS may contribute to the defects in hepatic insulin signaling, in particular, depressed expression of IRS-1 and -2 in ob/ob mice.
Fasting blood glucose levels are determined primarily by glucose output from the liver, and hepatic glucose output is suppressed by insulin in insulin-sensitive animals (21,22,25). Therefore, fasting hyperglycemia is accounted for by the failure of insulin to inhibit the production and release of glucose by the liver. A predominant role for defects in hepatic insulin signaling in fasting hyperglycemia is also supported by previous studies in mice with tissue-specific gene targeting of IR. Tissue-specific gene disruption of IR in skeletal muscle (34) or adipose tissue (35) was not associated with hyperglycemia, whereas liver-specific knockout of IR did exhibit hyperglycemia (36,37). Taken together, our findings suggest that increased iNOS expression may contribute to fasting hyperglycemia, in part, by exacerbating deranged insulin signaling in the liver of ob/ob mice.
In the liver, IRS-2 rather than IRS-1 plays a prominent role in metabolic actions of insulin, including the inhibition of hepatic glucose output, whereas IRS-1 has a major role in skeletal muscle (38eC41). Previous studies (42eC44) showed a marked reduction in IRS-2 expression in the liver of ob/ob mice, whereas IRS-1 expression was unaltered or modestly decreased. These findings suggest that defective IRS-2eCmediated insulin signaling is a major component of obesity-related hepatic insulin resistance.
iNOS inhibitor improved the protein expression of both IRS-1 and -2 in the liver of ob/ob mice. However, the beneficial effect of iNOS inhibitor on IRS-2 seemed more prominent compared with that on IRS-1 in the liver. Similarly, the reduction in protein expression induced by NO donor or iNOS transfection was more profound in IRS-2 compared with IRS-1 in Hepa1c1c7 and HepG2 cells. These findings suggest that iNOS may downregulate preferentially the expression of IRS-2 and, to a lesser extent, that of IRS-1 in the liver.
To date, limited knowledge is available about the regulatory mechanisms of IRS-2 expression in normal and disease states such as insulin resistance and diabetes. Previous studies (25,45,46) showed that insulin downregulates IRS-2 expression. Therefore, one can hypothesize that the improved expression of IRS-2 might be due to amelioration of hyperinsulinemia by L-NIL via as yet determined mechanisms rather than direct effects of iNOS inhibitor in the liver. However, our data argue against this hypothesis. NO donor and iNOS transfection reduced IRS-2 expression in cultured hepatocytes, suggesting that increased iNOS expression without hyperinsulinemia can reduce IRS-2 expression in the liver. Moreover, L-NIL increased IRS-2 expression at both protein and mRNA levels under fed conditions as well as in fasted animals, although the effects of L-NIL on plasma insulin level appeared to differ between fasted and fed ob/ob mice. Fasting plasma insulin concentration was significantly lower in L-NILeCtreated ob/ob mice than in PBS-treated animals, while plasma insulin concentration under fed condition tended to be higher in L-NILeCtreated animals, albeit with no statistical significance.
Recently, SREBPs were shown to negatively regulate the transcription of IRS-2 in the liver (33). We found that SREBP-1c expression was reduced by L-NIL in the liver in vivo and was elevated by NO donor in cultured hepatocytes (Fig. 7). In aggregate, therefore, it is possible that increased expression of IRS-2 may be associated with reduced SREBP-1 expression by iNOS inhibitor in the liver of ob/ob mice.
Protein expression of IRS-1 was increased by iNOS inhibitor in the liver of ob/ob mice, while NO donor and ectopically expressed iNOS decreased IRS-1 in cultured hepatocytes. However, mRNA of IRS-1 was not altered by iNOS inhibitor or NO donor. This is consistent with our preliminary observation that iNOS and NO donor decreased IRS-1 expression via proteasome-dependent proteolysis in skeletal muscle cells (H. Sugita, M.K., unpublished observations).
The effects of L-NIL on the expression of IRS-1 and -2 in skeletal muscle and adipose tissue were distinct from those in the liver. In contrast to the preferential improvement in IRS-2 expression in the liver, L-NIL treatment was associated with increased expression of IRS-1, but not of IRS-2, in skeletal muscle. In adipose tissue, L-NIL failed to modulate the expression of either IRS-1 or -2. These results in skeletal muscle are in agreement with our aforementioned unpublished observation that NO donor and iNOS transfection reduced IRS-1 expression but did not affect IRS-2 expression in skeletal muscle cells. Based on the predominant role of hepatic insulin signaling in fasting hyperglycemia, one can reasonably speculate that ameliorated expression of IRS-2 in the liver may be the more important contributor to the reversal of fasting hyperglycemia by L-NIL rather than improved IRS-1 expression in skeletal muscle. It is conceivable, however, that the protective effects of iNOS inhibitor in the skeletal muscle as well as in the liver may contribute in concert to improve whole-body insulin sensitivity in ob/ob mice.
The study of Perreault and Marette (12) showed that high-fat dieteCinduced elevation in iNOS expression was restricted to skeletal muscle and adipose tissue, but not observed in the liver in mice, in contrast to our results in ob/ob mice (Fig. 1). This discrepancy might be attributable to the methodological difference to induce obesity, namely leptin mutation in ob/ob mice versus high-fat diet. It is also possible that the difference in mouse strains could influence the results. We used ob/ob mice that were backcrossed onto C57BL/6J background, while C57BL/6X129SvEv mice were used by Perreault and Marette. Moreover, in contrast to the glucose-lowering effects of iNOS inhibitor (Fig. 2), iNOS disruption did not significantly alter high-fat dieteCinduced fasting hyperglycemia (12). The lack of iNOS induction in the liver might account for the ineffectiveness of iNOS disruption to mitigate fasting hyperglycemia in high-fat dieteCfed mice, although iNOS inhibitor effectively reversed fasting hyperglycemia in ob/ob mice in which iNOS expression was elevated in the liver.
Notably, there is a marked difference in the severity of fasting hyperglycemia between ob/ob mice and high-fat dieteCfed mice (12). In ob/ob mice, overt fasting hyperglycemia was associated with increased iNOS expression in the liver. But in mice fed a high-fat diet, fasting hyperglycemia was mild, and iNOS expression did not increase in the liver (12). Thus, iNOS induction in the liver seems to correlate with the magnitude of fasting hyperglycemia. Collectively, one can speculate that increased expression of iNOS in the liver might be a culprit for overt fasting hyperglycemia via suppressing IRS-1 and -2 expression. Further studies will be required to clarify this point.
In summary, the present data show that iNOS inhibitor reversed fasting hyperglycemia in parallel with the concomitant improvement in hepatic IRS-1eCand IRS-2eCmediated insulin signaling in ob/ob mice. The present study sheds a new light on a therapeutic potential of iNOS inhibitor to improve glycemic control in obesity-related diabetes.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health (R01DK058127 to M.K. and R01GM031569, R01GM055082, and R01GM061411 to J.A.J.M.) and grants from the Shriners Hospital for Children to J.A.J.M.
FOOTNOTES
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
Br-cGMP, 8-bromoguanosine-3',5'-cyclic monophosphorothioate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSNO, S-nitrosoglutathione; IKK, inhibitor B kinase; iNOS, inducible NO synthase; IR, insulin receptor; IRS, IR substrate; LPS, lipopolysaccharide; NF-B, nuclear factor-B; PI3, phosphatidylinositol-3; SREBP, sterol regulatory elementeCbinding protein
REFERENCES
Moller DE: Potential role of TNF-alpha in the pathogenesis of insulin resistance and type 2 diabetes. Trends Endocrinol Metab11 :212 eC217,2000
Febbraio MA, Pedersen BK: Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J16 :1335 eC1347,2002
Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE: Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikk-. Science293 :1673 eC1677,2001
Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI: Prevention of fat-induced insulin resistance by salicylate. J Clin Invest108 :437 eC446,2001
Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI: Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest109 :1321 eC1326,2002
Xie QW, Kashiwabara Y, Nathan C: Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem269 :4705 eC4708,1994
Elizalde M, Rydeen M, van Harmelen V, Eneroth P, Gyllenhammar H, Holm C, Ramel S, lund A, Arner P, Andersson K: Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans. J Lipid Res41 :1244 eC1251,2000
Shimabukuro M, Higa M, Zhou YT, Wang MY, Newgard CB, Unger RH: Lipoapoptosis in beta-cells of obese prediabetic fa/fa rats: role of serine palmitoyltransferase overexpression. J Biol Chem273 :32487 eC32490,1998
Sharma K, Danoff TM, DePiero A, Ziyadeh FN: Enhanced expression of inducible nitric oxide synthase in murine macrophages and glomerular mesangial cells by elevated glucose levels: possible mediation via protein kinase C. Biochem Biophys Res Commun207 :80 eC88,1995
Ceriello A, Quagliaro L, D’Amico M, Di Filippo C, Marfella R, Nappo F, Berrino L, Rossi F, Giugliano D: Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat. Diabetes51 :1076 eC1082,2002
Sugita H, Kaneki M, Tokunaga E, Sugita M, Koike C, Yasuhara S, Tompkins RG, Martyn JA: Inducible nitric oxide synthase plays a role in LPS-induced hyperglycemia and insulin resistance. Am J Physiol Endocrinol Metab282 :E386 eCE394,2002
Perreault M, Marette A: Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Nat Med7 :1138 eC1143,2001
Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, Orci L, Unger RH: Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci U S A97 :1784 eC1789,2000
Torres SH, De Sanctis JB, de L Briceo M, Hernandez N, Finol HJ: Inflammation and nitric oxide production in skeletal muscle of type 2 diabetic patients. J Endocrinol181 :419 eC427,2004
Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B, Mazzanti L, Mutus B: Evidence for iNOS-dependent peroxynitrite production in diabetic platelets. Diabetologia42 :539 eC544,1999
Ceriello A, Mercuri F, Quagliaro L, Assaloni R, Motz E, Tonutti L, Taboga C: Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia44 :834 eC838,2001
Frisbee JC, Maier KG, Stepp DW: Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats. Am J Physiol Heart Circ Physiol283 :H2160 eCH2168,2002
Szabe?C, Zanchi A, Komjei K, Pacher P, Krolewski AS, Quist WC, LoGerfo FW, Horton ES, Veves A: Poly(ADP-ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity. Circulation106 :2680 eC2686,2002
Ellis EA, Guberski DL, Hutson B, Grant MB: Time course of NADH oxidase, inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in the BBZ/WOR rat. Nitric Oxide6 :295 eC304,2002
Beedard S, Marcotte B, Marette A: Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase. Biochem J325 :487 eC493,1997
Dobashi K, Asayama K, Nakane T, Kodera K, Hayashibe H, Nakazawa S: Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo: study in 3T3eCL1 cells and Otsuka Long-Evans Tokushima Fatty rats. Life Sci67 :2093 eC2101,2000
Maggi LB Jr, Sadeghi H, Weigand C, Scarim AL, Heitmeier MR, Corbett JA: Anti-inflammatory actions of 15-deoxy-12,14-prostaglandin J2 and troglitazone: evidence for heat shockeCdependent and eCindependent inhibition of cytokine-induced inducible nitric oxide synthase expression. Diabetes49 :346 eC355,2000
Da Ros R, Assaloni R, Ceriello A: The preventive anti-oxidant action of thiazolidinediones: a new therapeutic prospect in diabetes and insulin resistance. Diabet Med21 :1249 eC1252,2004
Pilon G, Dallaire P, Marette A: Inhibition of inducible nitric-oxide synthase by activators of AMP-activated protein kinase: a new mechanism of action of insulin- sensitizing drugs. J Biol Chem279 :20767 eC20774,2004
Saltiel AR: New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell104 :517 eC529,2001
Taylor SI: Deconstructing type 2 diabetes. Cell97 :9 eC12,1999
Ekberg K, Landau BR, Wajngot A, Chandramouli V, Efendic S, Brunengraber H, Wahren J: Contributions by kidney and liver to glucose production in the postabsorptive state and after 60 h of fasting. Diabetes48 :292 eC298,1999
Hirose M, Kaneki M, Sugita H, Yasuhara S, Ibebunjo C, Martyn JAJ: Long-term denervation impairs insulin receptor substrate-1-mediated insulin signaling in skeletal muscle. Metabolism50 :216 eC222,2001
Kaneki M, Kharbanda S, Pandey P, Yoshida K, Takekawa M, Liou JR, Stone R, Kufe D: Functional role for protein kinase C as a regulator of stress-activated protein kinase activation and monocytic differentiation of myeloid leukemia cells. Mol Cell Biol19 :461 eC470,1999
Hirose M, Kaneki M, Sugita H, Yasuhara S, Martyn JA: Immobilization depresses insulin signaling in skeletal muscle. Am J Physiol Endocrinol Metab279 :E1235 eCE1241,2000
Inoue Y, Inoue J, Lambert G, Yim SH, Gonzalez FJ: Disruption of hepatic C/EBPalpha results in impaired glucose tolerance and age-dependent hepatosteatosis. J Biol Chem279 :44740 eC44748,2004
Lan H, Rabaglia ME, Stoehr JP, Nadler ST, Schueler KL, Zou F, Yandell BS, Attie AD: Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility. Diabetes52 :688 eC700,2003
Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T, Nakagawa Y, Takahashi A, Suzuki H, Sone H, Toyoshima H, Fukamizu A, Yamada N: SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat Cell Biol6 :351 eC357,2004
Wojtaszewski JF, Higaki Y, Hirshman MF, Michael MD, Dufresne SD, Kahn CR, Goodyear LJ: Exercise modulates postreceptor insulin signaling and glucose transport in muscle-specific insulin receptor knockout mice. J Clin Invest104 :1257 eC1264,1999
Bluher M, Michael MD, Peroni OD, Ueki K, Carter N, Kahn BB, Kahn CR: Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev Cell3 :25 eC38,2002
Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR: Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell6 :87 eC97,2000
Cohen SE, Tseng YH, Michael MD, Kahn CR: Effects of insulin-sensitising agents in mice with hepatic insulin resistance. Diabetologia47 :407 eC411,2004
Kido Y, Burks DJ, Withers D, Bruning JC, Kahn CR, White MF, Accili D: Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2. J Clin Invest105 :199 eC205,2000
Kubota N, Tobe K, Terauchi Y, Eto K, Yamauchi T, Suzuki R, Tsubamoto Y, Komeda K, Nakano R, Miki H, Satoh S, Sekihara H, Sciacchitano S, Lesniak M, Aizawa S, Nagai R, Kimura S, Akanuma Y, Taylor SI, Kadowaki T: Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory -cell hyperplasia. Diabetes49 :1880 eC1889,2000
Valverde AM, Burks DJ, Fabregat I, Fisher TL, Carretero J, White MF, Benito M: Molecular mechanisms of insulin resistance in IRS-2eCdeficient hepatocytes. Diabetes52 :2239 eC2248,2003
Suzuki R, Tobe K, Aoyama M, Inoue A, Sakamoto K, Yamauchi T, Kamon J, Kubota N, Terauchi Y, Yoshimatsu H, Matsuhisa M, Nagasaka S, Ogata H, Tokuyama K, Nagai R, Kadowaki T: Both insulin signaling defects in the liver and obesity contribute to insulin resistance and cause diabetes in IRS2(-/-) mice. J Biol Chem279 :25039 eC25049,2004
Kerouz NJ, Hrsch D, Pons S, Kahn CR: Differential regulation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinase isoforms in liver and muscle of the obese diabetic (ob/ob) mouse. J Clin Invest100 :3164 eC3172,1997
Anai M, Funaki M, Ogihara T, Terasaki J, Inukai K, Katagiri H, Fukushima Y, Yazaki Y, Kikuchi M, Oka Y, Asano T: Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes47 :13 eC23,1998
Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL: Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell6 :77 eC86,2000
Rui L, Fisher TL, Thomas J, White MF: Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2. J Biol Chem276 :40362 eC40367,2001
Hirashima Y, Tsuruzoe K, Kodama S, Igata M, Toyonaga T, Ueki K, Kahn CR, Araki E: Insulin downregulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway. J Endocrinol179 :253 eC266,2003(Masaki Fujimoto, Nobuyuki)
2 Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan
ABSTRACT
Chronic inflammation has been postulated to play an important role in the pathogenesis of insulin resistance. Inducible nitric oxide synthase (iNOS) has been implicated in many human diseases associated with inflammation. iNOS deficiency was shown to prevent high-fat dieteCinduced insulin resistance in skeletal muscle but not in the liver. A role for iNOS in fasting hyperglycemia and hepatic insulin resistance, however, remains to be investigated in obesity-related diabetes. To address this issue, we examined the effects of a specific inhibitor for iNOS, L-NIL, in obese diabetic (ob/ob) mice. iNOS expression was increased in the liver of ob/ob mice compared with wild-type mice. Treatment with iNOS inhibitor reversed fasting hyperglycemia with concomitant amelioration of hyperinsulinemia and improved insulin sensitivity in ob/ob mice. iNOS inhibitor also increased the protein expression of insulin receptor substrate (IRS)-1 and -2 1.5- and 2-fold, respectively, and enhanced IRS-1eCand IRS-2eCmediated insulin signaling in the liver of ob/ob mice. Exposure to NO donor and ectopically expressed iNOS decreased the protein expression of IRS-1 and -2 in cultured hepatocytes. These results suggest that iNOS plays a role in fasting hyperglycemia and contributes to hepatic insulin resistance in ob/ob mice.
Chronic low-grade inflammation has been proposed to be involved in the pathogenesis in obesity-related insulin resistance and type 2 diabetes. The expression of proinflammatory cytokines, including tumor necrosis factor- (1) and interleukin-6 (2), is upregulated in animal models of and patients with type 2 diabetes. However, limited knowledge is thus far available about the molecular mechanisms by which chronic inflammation mediates insulin resistance and type 2 diabetes.
The activation of inhibitor B kinase (IKK)eCnuclear factor-B (NF-B), a crucial signaling cascade for inflammatory response, has been highlighted as a mediator of insulin resistance. The pharmacological inhibition or gene disruption of IKK reversed obesity-related insulin resistance and fasting hyperglycemia in rodents and humans (3eC5). However, little is known about genes that function as downstream effectors of the IKKbeCNF-B pathway to mediate insulin resistance.
Inducible nitric oxide synthase (iNOS; also termed NOS2), whose expression is regulated by IKKeCNF-B (6), is assumed to be one of the candidates that mediate inflammation-involved insulin resistance. Accumulating evidence indicates a close link between iNOS and insulin resistance. Although iNOS was originally identified in macrophages, it is now known that it is widely expressed in many tissues, including insulin-sensitive organs such as skeletal muscle, adipose tissue, and liver, in normal rodents and humans. The expression of iNOS is upregulated by most, if not all, inducers of insulin resistance, including proinflammatory cytokines, obesity (7), free fatty acids (8), hyperglycemia (9,10), endotoxins (6,11), and oxidative stress. In fact, elevated expression of iNOS was observed in skeletal muscle of high-fat dieteCfed mice (12), in heart of Zucker diabetic fatty rats (13), and in skeletal muscle (14) and platelets of patients with type 2 diabetes (15). Nitrosative protein modifications, such as tyrosine nitration often associated with iNOS expression, were elevated in plasma (16), skeletal muscle (14), vasculature (17,18), and retina (19) of patients with and rodent models of type 2 or obesity-related diabetes. Furthermore, iNOS induction resulted in attenuated insulin-stimulated glucose uptake in cultured skeletal muscle cells (20). Thiazolidinediones, a class of insulin sensitizer, suppress iNOS expression in cultured cells and in vivo in rodents (21,22). Thus, inhibition of iNOS expression has been recently proposed to be a new mechanism of actions of insulin sensitizers (23,24).
Recently, we and others have shown that iNOS plays an important role in the pathogenesis of insulin resistance in vivo. We demonstrated that iNOS inhibitor prevented fasting hyperglycemia and mitigated insulin resistance in lipopolysaccharide (LPS)-administered rats (11). Perreault and Marette (12) reported that iNOS deficiency protected against high-fat dieteCinduced insulin resistance. With respect to a role of iNOS in insulin resistance, our previous findings in LPS-administered rats appear to agree with the data of Perreault and Marette in high-fat dieteCfed mice. However, differences also exist in these previous studies (11,12), particularly in regard to the tissues in which iNOS inhibition or deficiency exerts its insulin-sensitizing effects. We found that iNOS expression was elevated in the liver by LPS and that iNOS inhibitor blocked LPS-induced increase in hepatic glucose output in Sprague-Dawley rats (11). By contrast, Perreault and Marette observed that iNOS expression was increased in skeletal muscle and adipose tissue but not in the liver of C57BL/6X129SvEv mice fed a high-fat diet and that iNOS disruption restored high-fat dieteCinduced defects in insulin signaling in skeletal muscle but not in adipose tissue or liver (12). Moreover, in their study, iNOS deficiency did not alter blood glucose levels in mice fed a high-fat diet, whereas whole-body insulin resistance was alleviated, as judged by an insulin tolerance test. A question, therefore, remains to be answered whether a role of iNOS in obesity-related insulin resistance is restricted to skeletal muscle or whether iNOS also plays a role in the liver, which is the primary organ to determine fasting blood glucose levels (25eC27). To address this issue, we examined the effects of iNOS inhibitor on fasting hyperglycemia and hepatic insulin signaling in genetically obese diabetic (ob/ob) mice.
RESEARCH DESIGN AND METHODS
Male ob/ob mice and lean wild-type C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The ob/ob mice were backcrossed onto wild-type C57BL/6J mice for at least 30 generations. The institutional animal care committee approved the study protocol. The mice were housed in mesh cages in a room maintained at 25°C and illuminated in 12:12-h light-dark cycles; they were provided with standard rodent diet and water ad libitum.
Treatment with iNOS inhibitor.
Male ob/ob mice at 10 weeks of age were treated with a specific inhibitor of iNOS, L-NIL (60 mg/kg body wt, i.p., b.i.d.; Cayman Chemical, Ann Arbor, MI), or PBS for 10 days. Following 10 days of treatment, the animals treated with L-NIL (n = 17) or PBS (n = 18) were fasted overnight and then received an injection of insulin (Humulin R; Eli Lilly, Indianapolis, IN; 15 units/kg body wt in PBS with 0.1% BSA) or vehicle (PBS with 0.1% BSA) via the portal vein under anesthesia with pentobarbital sodium (70 mg/kg body wt, i.p.). At 5 min after insulin injection, the liver, gastrocnemius muscle, and epididymal fat were taken for biochemical analyses. Male ob/ob mice at 10 weeks of age were also treated with L-NIL or PBS for 10 days under a pair-fed condition. After 10 days of treatment, the liver was taken from pair-fed mice under fed condition or after overnight fasting. Twenty-four mice were randomly assigned into four groups (L-NIL fasted, PBS fasted, L-NIL fed, and PBS fed) of six animals. The average food intake of both L-NIL and PBS-treated animals under pair-fed condition was 5.6 g/day.
Insulin tolerance test.
After 10 days of treatment, the animals treated with L-NIL (n = 10) or PBS (n = 10) were fasted for 4 h and then received an injection of insulin (2.5 units/kg body wt in PBS with 0.1% BSA, i.p.). At 0, 15, 30, 60, 90, and 120 min after insulin injection, blood samples were collected to measure glucose level.
Immunoprecipitation and immunoblotting.
Liver samples were homogenized as previously described (28). Immunoprecipitation and immunoblotting were performed (29) using anti-IR, IRS-1 and -2, p85 phosphatidylinositol 3(PI3)-kinase, iNOS, endothelial NOS, neuronal NOS (Upstate, Lake Placid, NY), Akt, phosphorylated Akt (Ser473) (Cell Signaling, Beverly, MA), and phosphotyrosine antibodies (Santa Cruz, Santa Cruz, CA) (online appendix available at http://diabetes.diabetesjournals.org).
Partial purification of iNOS.
Partial purification of iNOS was performed using immobilized 1400W beads (Calbiochem, San Diego, CA), which selectively bind to iNOS, according to the manufacturer’s instruction (online appendix).
PI3-kinase assay.
PI3-kinase activities in the immunoprecipitates with antieCIRS-1 or -2 antibody were measured in vitro (30).
Detection of tyrosine nitration.
Immunostaining with anti-nitrotyrosine antibody (Upstate) was performed according to the manufacturer’s instruction (online appendix).
RNase protection assay.
RNase protection assay was performed as previously described (11) using the RPA III kit (Ambion, Austin, TX). mRNA levels of IRS-1 and -2 and sterol regulatory elementeCbinding protein (SREBP)-1c were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (online appendix).
Cell culture.
Mouse Hepa1c1c7 and human HepG2 cells (American Type Culture Collection, Manassas, VA) were maintained in -minimum essential medium and minimum essential medium supplemented with 10% fetal bovine serum, respectively. After 8 h serum starvation, the cells were treated with the indicated concentrations of S-nitrosoglutathione (GSNO) or 3-morpholino-sydnonimine (Cayman Chemical, Ann Arbor, MI) in the presence or absence of ODQ (1 eol/l; Sigma) or 8-bromoguanosine-3',5'-cyclic monophosphorothioate (Br-cGMP; 100 eol/l; Axxora, San Diego, CA) for 24 h, unless otherwise indicated. Hepa1c1c7 cells were transfected with pCDNA3/iNOS or pCDNA3 using Lipofectamin 2000 (Invitrogen). pCDNA3/iNOS was kindly provided by Dr. Kone. At 48 h after transfection, the cells were harvested.
Measurement of blood glucose, insulin, triglycerides, glycogen, adiponectin, and free fatty acids.
Blood glucose level and plasma insulin concentration were determined by the glucose oxidase method (Ascensia Elite Glucometer; Bayer, Elkhart, IN) and enzyme-linked immunosorbent assay (Crystal Chem, Chicago, IL), respectively. Glycogen content was measured using a glucose hexokinase assay kit (Sigma) (31). A Glycerol phosphate oxidase trinder reagent (Sigma) was used to measure triglyceride (32). The concentrations of adiponectin and free fatty acids in serum from fasted animals were determined using an enzyme-linked immunosorbent assay kit (Linco Research, St. Charles, MO) and an enzymatic colorimetric assay kit (Wako Chemicals, Richmond, VA), respectively.
Statistical analysis.
Statistical analyses were performed using Student’s t test or one-way ANOVA followed by Fisher’s protected least significant differences test. Values of P < 0.05 were considered statistically significant. Data are expressed as means ± SE.
RESULTS
Increased expression of iNOS in the liver of diabetic mice.
We found a 2.5-fold increase in iNOS protein expression in the liver of ob/ob mice compared with wild-type mice (Fig. 1A). No difference was found in the protein expression of endothelial or neuronal NOS between ob/ob and wild-type mice (online appendix Fig. 1). iNOS expression was also increased in skeletal muscle and adipose tissue of ob/ob mice compared with wild-type mice to the extent comparable to that in the liver (online appendix Fig. 2).
Reduced nitrotyrosine immunoreactivity by iNOS inhibitor.
The immunoreactivity for nitrotyrosine, a marker of nitrosative stress, was also elevated in the liver of ob/ob mice compared with wild-type mice (Fig. 1B). Treatment with iNOS inhibitor (60 mg/kg body wt, i.p., b.i.d., for 10 days) reversed the elevated nitrotyrosine immunoreactivity in the liver of ob/ob mice compared with PBS. L-NIL treatment, however, did not alter the expression of iNOS and endothelial and neuronal NOS (data not shown). These results suggest that L-NIL, a competitive inhibitor for iNOS, effectively reduced the activity of iNOS in the liver of ob/ob mice without altering iNOS expression level, as expected.
Reversal of fasting hyperglycemia and amelioration of hyperinsulinemia by iNOS inhibitor in diabetic mice.
At the inception of the treatment, there was no difference in fed blood glucose level (L-NIL: 292.8 ± 21.8; PBS: 290.3 ± 18.7 mg/dl), plasma insulin concentration (L-NIL: 57.0 ± 0.5; PBS: 57.0 ± 0.5 ng/ml), or body weight (L-NIL: 57.0 ± 0.5; PBS: 56.8 ± 0.6 g) between L-NIL eCand PBS-administered animals. Treatment with L-NIL did not affect body weight at the time of its termination (L-NIL: 57.2 ± 0.5; PBS: 57.0 ± 0.6 g). There was no statistical difference in food intake between L-NIL eCand PBS-treated animals (L-NIL: 5.8 ± 0.2; PBS: 6.4 ± 0.2 g/day), although food intake in L-NILeCtreated animals appeared to be lower than in PBS-treated animals. However, L-NIL treatment reversed fasting hyperglycemia and significantly ameliorated fasting hyperinsulinemia in ob/ob mice (Fig. 2A and B). The insulin sensitivity index (fasting blood glucose x plasma insulin concentration) was also significantly improved by L-NIL (L-NIL: 16.9 ± 4.0; PBS: 51.0 ± 12.6 mg2/l2, P < 0.05). Improved insulin sensitivity by L-NIL treatment was confirmed by insulin tolerance test. ob/ob mice treated with L-NIL responded to insulin with a more profound decrease in blood glucose level than in PBS-administered ob/ob mice (Fig. 2C). Area under the curve analysis in response to the insulin tolerance test also revealed the beneficial effects of L-NIL (Fig. 2D).
To exclude a possible influence of food intake, the effects of L-NIL treatment were also examined in pair-fed ob/ob mice. The beneficial effects of L-NIL were corroborated in pair-fed mice. In pair-fed ob/ob mice, L-NIL treatment also led to the reversal of fasting hyperglycemia (L-NIL: 95.3 ± 13.4; PBS: 210.8 ± 18.1 mg/dl, P < 0.05) and significant amelioration of fasting hyperinsulinemia (L-NIL: 11.4 ± 3.2; PBS: 19.4 ± 1.9 ng/ml, P < 0.05) and insulin sensitivity index (L-NIL: 10.3 ± 2.8; PBS: 40.8 ± 5.9 mg2/l2, P < 0.05). Under fed condition, L-NIL treatment appeared to mitigate hyperglycemia in ob/ob mice (L-NIL: 195.7 ± 29.1; PBS: 272.2 ± 24.0, P < 0.10), although no statistical significance was found. Plasma insulin concentration under fed condition tended to be higher in L-NILeCtreated animals than PBS-treated animals (L-NIL: 86.4 ± 7.0; PBS: 57.5 ± 12.4 ng/ml, P < 0.10), although there was no statistical difference.
iNOS inhibitor improved IRS-1eCand IRS-2eCmediated insulin signaling in the liver of diabetic mice.
iNOS inhibitor, L-NIL, did not alter the protein expression of insulin receptor (IR). However, the protein expression of IRS-1 and -2 under a fasted condition was increased by 1.5- and 2-fold, respectively, compared with PBS-treated animals (Fig. 3A). Increased protein expression of IRS-1 and -2 by L-NIL was further confirmed in pair-fed mice. In pair-fed ob/ob mice, the protein expression of IRS-1 and -2 in L-NILeCtreated animals was greater than in PBS-treated animals under both fasted and fed conditions (Fig. 3B and online appendix Fig. 3). L-NIL treatment resulted in upregulated mRNA level of IRS-2 under both fasted and fed conditions (Fig. 3C and D). However, the mRNA level of IRS-1 was not altered by L-NIL treatment. L-NIL did not affect GAPDH mRNA level (data not shown). No difference was found in the protein expression of p85, p55, and p50 PI3-kinase in the liver between ob/ob mice treated with L-NIL and PBS (online appendix Fig. 4).
Insulin-stimulated tyrosine phosphorylation of IRS-1 and -2 in the liver was upregulated 1.5- and 3-fold by iNOS inhibitor, respectively, compared with PBS (Fig. 4). However, iNOS inhibitor did not alter insulin-stimulated tyrosine phosphorylation of IR. L-NIL treatment did not significantly alter basal (insulin-unstimulated) tyrosine phosphorylation of IR and IRS-1 and -2. The ratio of insulin-stimulated tyrosine phosphorylation to protein abundance for IR and IRS-1 and -2 was not significantly altered by L-NIL treatment (online appendix Fig. 5). These results suggest that the improvement by iNOS inhibitor in insulin-stimulated tyrosine phosphorylation of IRS-1 and -2 may be mainly attributable to the increased expression of IRS-1 and -2. Insulin-stimulated IRS-1eCand IRS-2eCassociated PI3-kinase activity was also enhanced by L-NIL up to 1.5- and 2.5-fold, respectively (Fig. 4D and E). L-NIL treatment also increased insulin-stimulated phosphorylation of Akt/protein kinase B, a downstream signaling molecule, while the expression of Akt/protein kinase B was not altered (Fig. 4F and G).
Glycogen content in the liver of fasted animals was significantly increased by L-NIL treatment (L-NIL: 14.9 ± 1.8; PBS: 7.3 ± 0.6 mg/g liver wt, P < 0.01). Triglyceride content in the liver of fasted animals appeared to be lower in L-NILeCtreated animals than in PBS-treated animals (L-NIL: 52.9 ± 5.6; PBS: 65.7 ± 4.0 e/g protein, P < 0.10), although there was no statistical difference. In skeletal muscle of ob/ob mice, L-NIL treatment increased IRS-1 protein expression, while IRS-2 abundance was unaltered. In adipose tissue, neither IRS-1 nor -2 expression was affected by L-NIL treatment (online appendix Fig. 6). We did not find a significant difference in circulating adiponectin (L-NIL: 6.32 ± 0.6; PBS: 6.00 ± 0.45 ng/ml) and free fatty acid (L-NIL: 1.83 ± 0.1; PBS: 1.71 ± 0.12 mEq/l) concentration between L-NILeCand PBS-administered ob/ob mice.
NO donor and iNOS reduced the expression of IRS-1 and -2 in cultured hepatocytes.
Next, we asked whether iNOS can downregulate IRS-1 and -2 expression in cultured hepatocytes. Exposure to NO donor, GSNO (1 mmol/l), for 24 h decreased the protein expression of IRS-1 and -2 in Hepalc1c7 and HepG2 cells (Fig. 5 and online appendix Fig. 7), whereas the expression of IR and p85 PI3-kinase was not affected by NO donor. Treatment with another NO donor, 3-morpholino-sydnonimine-1 (0.5 mmol/l), for 24 h also decreased the expression of IRS-1 and -2 (online appendix Fig. 7). Ectopically expressed iNOS reduced the expression of IRS-1 and -2 without altering p85 expression. The inhibitory effects of GSNO on IRS-1 and -2 expression were dose and time dependent (Fig. 6). In accordance with the greater increase in IRS-2 expression than in IRS-1 by L-NIL treatment in the liver of ob/ob mice (Fig. 3), the inhibitory effects of NO donor or iNOS transfection appeared to be more prominent on IRS-2 than -1 (Figs. 5 and 6). Treatment with 1 mmol/l GSNO for 24 h did not affect viability of the cells, as judged by the trypan blue exclusion test. Consistent with the effects of iNOS inhibitor on IRS-1 and -2 mRNA in the liver of ob/ob mice, 1 mmol/l GSNO decreased IRS-2 mRNA level but did not affect IRS-1 mRNA level in cultured Hepa1c1c7 cells (Fig. 7).
To further characterize the effects of NO donor on IRS-1 and -2, we examined the effects of ODQ, a soluble guanylate cyclase inhibitor, and Br-cGMP, a cell-permeable cGMP analog in Hepa1c1c7 cells. ODQ (1 eol/l) failed to inhibit GSNO-induced reduction in IRS-1 and -2 expression. cGMP analog, Br-cGMP (100 eol/l), did not affect the expression of IRS-1 and -2, either. These observations suggest that the inhibitory effects of GSNO are cGMP independent.
Since a recent study (33) revealed that SREBPs suppress the transcription of IRS-2 in the liver, we examined the effects of L-NIL and GSNO on SREBP-1c expression. SREBP-1c mRNA level was increased by GSNO (1 mmol/l) in cultured Hepa1c1c7 cells (Fig. 7C) and was reduced by L-NIL treatment in the liver of ob/ob mice under both fasted and fed conditions (Fig. 7D and E).
DISCUSSION
We found that iNOS expression was significantly elevated in the liver as well as skeletal muscle and adipose tissue in ob/ob mice compared with wild-type mice (Fig. 1) and that iNOS inhibitor reversed fasting hyperglycemia and ameliorated whole-body insulin resistance in ob/ob mice (Fig. 2). The improved glycemic control was accompanied by increased protein expression of IRS-1 and -2 and enhanced IRS-1eCand IRS-2eCmediated insulin signaling in the liver of ob/ob mice (Figs. 3 and 4). Moreover, we found that exposure to an NO donor and ectopically expressed iNOS reduced the protein expression of IRS-1 and -2 in cultured hepatocytes (Figs. 5 and 6). These results indicate that increased expression of iNOS may play an important role in obesity-induced fasting hyperglycemia and suggest that iNOS may contribute to the defects in hepatic insulin signaling, in particular, depressed expression of IRS-1 and -2 in ob/ob mice.
Fasting blood glucose levels are determined primarily by glucose output from the liver, and hepatic glucose output is suppressed by insulin in insulin-sensitive animals (21,22,25). Therefore, fasting hyperglycemia is accounted for by the failure of insulin to inhibit the production and release of glucose by the liver. A predominant role for defects in hepatic insulin signaling in fasting hyperglycemia is also supported by previous studies in mice with tissue-specific gene targeting of IR. Tissue-specific gene disruption of IR in skeletal muscle (34) or adipose tissue (35) was not associated with hyperglycemia, whereas liver-specific knockout of IR did exhibit hyperglycemia (36,37). Taken together, our findings suggest that increased iNOS expression may contribute to fasting hyperglycemia, in part, by exacerbating deranged insulin signaling in the liver of ob/ob mice.
In the liver, IRS-2 rather than IRS-1 plays a prominent role in metabolic actions of insulin, including the inhibition of hepatic glucose output, whereas IRS-1 has a major role in skeletal muscle (38eC41). Previous studies (42eC44) showed a marked reduction in IRS-2 expression in the liver of ob/ob mice, whereas IRS-1 expression was unaltered or modestly decreased. These findings suggest that defective IRS-2eCmediated insulin signaling is a major component of obesity-related hepatic insulin resistance.
iNOS inhibitor improved the protein expression of both IRS-1 and -2 in the liver of ob/ob mice. However, the beneficial effect of iNOS inhibitor on IRS-2 seemed more prominent compared with that on IRS-1 in the liver. Similarly, the reduction in protein expression induced by NO donor or iNOS transfection was more profound in IRS-2 compared with IRS-1 in Hepa1c1c7 and HepG2 cells. These findings suggest that iNOS may downregulate preferentially the expression of IRS-2 and, to a lesser extent, that of IRS-1 in the liver.
To date, limited knowledge is available about the regulatory mechanisms of IRS-2 expression in normal and disease states such as insulin resistance and diabetes. Previous studies (25,45,46) showed that insulin downregulates IRS-2 expression. Therefore, one can hypothesize that the improved expression of IRS-2 might be due to amelioration of hyperinsulinemia by L-NIL via as yet determined mechanisms rather than direct effects of iNOS inhibitor in the liver. However, our data argue against this hypothesis. NO donor and iNOS transfection reduced IRS-2 expression in cultured hepatocytes, suggesting that increased iNOS expression without hyperinsulinemia can reduce IRS-2 expression in the liver. Moreover, L-NIL increased IRS-2 expression at both protein and mRNA levels under fed conditions as well as in fasted animals, although the effects of L-NIL on plasma insulin level appeared to differ between fasted and fed ob/ob mice. Fasting plasma insulin concentration was significantly lower in L-NILeCtreated ob/ob mice than in PBS-treated animals, while plasma insulin concentration under fed condition tended to be higher in L-NILeCtreated animals, albeit with no statistical significance.
Recently, SREBPs were shown to negatively regulate the transcription of IRS-2 in the liver (33). We found that SREBP-1c expression was reduced by L-NIL in the liver in vivo and was elevated by NO donor in cultured hepatocytes (Fig. 7). In aggregate, therefore, it is possible that increased expression of IRS-2 may be associated with reduced SREBP-1 expression by iNOS inhibitor in the liver of ob/ob mice.
Protein expression of IRS-1 was increased by iNOS inhibitor in the liver of ob/ob mice, while NO donor and ectopically expressed iNOS decreased IRS-1 in cultured hepatocytes. However, mRNA of IRS-1 was not altered by iNOS inhibitor or NO donor. This is consistent with our preliminary observation that iNOS and NO donor decreased IRS-1 expression via proteasome-dependent proteolysis in skeletal muscle cells (H. Sugita, M.K., unpublished observations).
The effects of L-NIL on the expression of IRS-1 and -2 in skeletal muscle and adipose tissue were distinct from those in the liver. In contrast to the preferential improvement in IRS-2 expression in the liver, L-NIL treatment was associated with increased expression of IRS-1, but not of IRS-2, in skeletal muscle. In adipose tissue, L-NIL failed to modulate the expression of either IRS-1 or -2. These results in skeletal muscle are in agreement with our aforementioned unpublished observation that NO donor and iNOS transfection reduced IRS-1 expression but did not affect IRS-2 expression in skeletal muscle cells. Based on the predominant role of hepatic insulin signaling in fasting hyperglycemia, one can reasonably speculate that ameliorated expression of IRS-2 in the liver may be the more important contributor to the reversal of fasting hyperglycemia by L-NIL rather than improved IRS-1 expression in skeletal muscle. It is conceivable, however, that the protective effects of iNOS inhibitor in the skeletal muscle as well as in the liver may contribute in concert to improve whole-body insulin sensitivity in ob/ob mice.
The study of Perreault and Marette (12) showed that high-fat dieteCinduced elevation in iNOS expression was restricted to skeletal muscle and adipose tissue, but not observed in the liver in mice, in contrast to our results in ob/ob mice (Fig. 1). This discrepancy might be attributable to the methodological difference to induce obesity, namely leptin mutation in ob/ob mice versus high-fat diet. It is also possible that the difference in mouse strains could influence the results. We used ob/ob mice that were backcrossed onto C57BL/6J background, while C57BL/6X129SvEv mice were used by Perreault and Marette. Moreover, in contrast to the glucose-lowering effects of iNOS inhibitor (Fig. 2), iNOS disruption did not significantly alter high-fat dieteCinduced fasting hyperglycemia (12). The lack of iNOS induction in the liver might account for the ineffectiveness of iNOS disruption to mitigate fasting hyperglycemia in high-fat dieteCfed mice, although iNOS inhibitor effectively reversed fasting hyperglycemia in ob/ob mice in which iNOS expression was elevated in the liver.
Notably, there is a marked difference in the severity of fasting hyperglycemia between ob/ob mice and high-fat dieteCfed mice (12). In ob/ob mice, overt fasting hyperglycemia was associated with increased iNOS expression in the liver. But in mice fed a high-fat diet, fasting hyperglycemia was mild, and iNOS expression did not increase in the liver (12). Thus, iNOS induction in the liver seems to correlate with the magnitude of fasting hyperglycemia. Collectively, one can speculate that increased expression of iNOS in the liver might be a culprit for overt fasting hyperglycemia via suppressing IRS-1 and -2 expression. Further studies will be required to clarify this point.
In summary, the present data show that iNOS inhibitor reversed fasting hyperglycemia in parallel with the concomitant improvement in hepatic IRS-1eCand IRS-2eCmediated insulin signaling in ob/ob mice. The present study sheds a new light on a therapeutic potential of iNOS inhibitor to improve glycemic control in obesity-related diabetes.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health (R01DK058127 to M.K. and R01GM031569, R01GM055082, and R01GM061411 to J.A.J.M.) and grants from the Shriners Hospital for Children to J.A.J.M.
FOOTNOTES
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
Br-cGMP, 8-bromoguanosine-3',5'-cyclic monophosphorothioate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSNO, S-nitrosoglutathione; IKK, inhibitor B kinase; iNOS, inducible NO synthase; IR, insulin receptor; IRS, IR substrate; LPS, lipopolysaccharide; NF-B, nuclear factor-B; PI3, phosphatidylinositol-3; SREBP, sterol regulatory elementeCbinding protein
REFERENCES
Moller DE: Potential role of TNF-alpha in the pathogenesis of insulin resistance and type 2 diabetes. Trends Endocrinol Metab11 :212 eC217,2000
Febbraio MA, Pedersen BK: Muscle-derived interleukin-6: mechanisms for activation and possible biological roles. FASEB J16 :1335 eC1347,2002
Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE: Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikk-. Science293 :1673 eC1677,2001
Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI: Prevention of fat-induced insulin resistance by salicylate. J Clin Invest108 :437 eC446,2001
Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI: Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest109 :1321 eC1326,2002
Xie QW, Kashiwabara Y, Nathan C: Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem269 :4705 eC4708,1994
Elizalde M, Rydeen M, van Harmelen V, Eneroth P, Gyllenhammar H, Holm C, Ramel S, lund A, Arner P, Andersson K: Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans. J Lipid Res41 :1244 eC1251,2000
Shimabukuro M, Higa M, Zhou YT, Wang MY, Newgard CB, Unger RH: Lipoapoptosis in beta-cells of obese prediabetic fa/fa rats: role of serine palmitoyltransferase overexpression. J Biol Chem273 :32487 eC32490,1998
Sharma K, Danoff TM, DePiero A, Ziyadeh FN: Enhanced expression of inducible nitric oxide synthase in murine macrophages and glomerular mesangial cells by elevated glucose levels: possible mediation via protein kinase C. Biochem Biophys Res Commun207 :80 eC88,1995
Ceriello A, Quagliaro L, D’Amico M, Di Filippo C, Marfella R, Nappo F, Berrino L, Rossi F, Giugliano D: Acute hyperglycemia induces nitrotyrosine formation and apoptosis in perfused heart from rat. Diabetes51 :1076 eC1082,2002
Sugita H, Kaneki M, Tokunaga E, Sugita M, Koike C, Yasuhara S, Tompkins RG, Martyn JA: Inducible nitric oxide synthase plays a role in LPS-induced hyperglycemia and insulin resistance. Am J Physiol Endocrinol Metab282 :E386 eCE394,2002
Perreault M, Marette A: Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Nat Med7 :1138 eC1143,2001
Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, Baetens D, Orci L, Unger RH: Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci U S A97 :1784 eC1789,2000
Torres SH, De Sanctis JB, de L Briceo M, Hernandez N, Finol HJ: Inflammation and nitric oxide production in skeletal muscle of type 2 diabetic patients. J Endocrinol181 :419 eC427,2004
Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B, Mazzanti L, Mutus B: Evidence for iNOS-dependent peroxynitrite production in diabetic platelets. Diabetologia42 :539 eC544,1999
Ceriello A, Mercuri F, Quagliaro L, Assaloni R, Motz E, Tonutti L, Taboga C: Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia44 :834 eC838,2001
Frisbee JC, Maier KG, Stepp DW: Oxidant stress-induced increase in myogenic activation of skeletal muscle resistance arteries in obese Zucker rats. Am J Physiol Heart Circ Physiol283 :H2160 eCH2168,2002
Szabe?C, Zanchi A, Komjei K, Pacher P, Krolewski AS, Quist WC, LoGerfo FW, Horton ES, Veves A: Poly(ADP-ribose) polymerase is activated in subjects at risk of developing type 2 diabetes and is associated with impaired vascular reactivity. Circulation106 :2680 eC2686,2002
Ellis EA, Guberski DL, Hutson B, Grant MB: Time course of NADH oxidase, inducible nitric oxide synthase and peroxynitrite in diabetic retinopathy in the BBZ/WOR rat. Nitric Oxide6 :295 eC304,2002
Beedard S, Marcotte B, Marette A: Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase. Biochem J325 :487 eC493,1997
Dobashi K, Asayama K, Nakane T, Kodera K, Hayashibe H, Nakazawa S: Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo: study in 3T3eCL1 cells and Otsuka Long-Evans Tokushima Fatty rats. Life Sci67 :2093 eC2101,2000
Maggi LB Jr, Sadeghi H, Weigand C, Scarim AL, Heitmeier MR, Corbett JA: Anti-inflammatory actions of 15-deoxy-12,14-prostaglandin J2 and troglitazone: evidence for heat shockeCdependent and eCindependent inhibition of cytokine-induced inducible nitric oxide synthase expression. Diabetes49 :346 eC355,2000
Da Ros R, Assaloni R, Ceriello A: The preventive anti-oxidant action of thiazolidinediones: a new therapeutic prospect in diabetes and insulin resistance. Diabet Med21 :1249 eC1252,2004
Pilon G, Dallaire P, Marette A: Inhibition of inducible nitric-oxide synthase by activators of AMP-activated protein kinase: a new mechanism of action of insulin- sensitizing drugs. J Biol Chem279 :20767 eC20774,2004
Saltiel AR: New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell104 :517 eC529,2001
Taylor SI: Deconstructing type 2 diabetes. Cell97 :9 eC12,1999
Ekberg K, Landau BR, Wajngot A, Chandramouli V, Efendic S, Brunengraber H, Wahren J: Contributions by kidney and liver to glucose production in the postabsorptive state and after 60 h of fasting. Diabetes48 :292 eC298,1999
Hirose M, Kaneki M, Sugita H, Yasuhara S, Ibebunjo C, Martyn JAJ: Long-term denervation impairs insulin receptor substrate-1-mediated insulin signaling in skeletal muscle. Metabolism50 :216 eC222,2001
Kaneki M, Kharbanda S, Pandey P, Yoshida K, Takekawa M, Liou JR, Stone R, Kufe D: Functional role for protein kinase C as a regulator of stress-activated protein kinase activation and monocytic differentiation of myeloid leukemia cells. Mol Cell Biol19 :461 eC470,1999
Hirose M, Kaneki M, Sugita H, Yasuhara S, Martyn JA: Immobilization depresses insulin signaling in skeletal muscle. Am J Physiol Endocrinol Metab279 :E1235 eCE1241,2000
Inoue Y, Inoue J, Lambert G, Yim SH, Gonzalez FJ: Disruption of hepatic C/EBPalpha results in impaired glucose tolerance and age-dependent hepatosteatosis. J Biol Chem279 :44740 eC44748,2004
Lan H, Rabaglia ME, Stoehr JP, Nadler ST, Schueler KL, Zou F, Yandell BS, Attie AD: Gene expression profiles of nondiabetic and diabetic obese mice suggest a role of hepatic lipogenic capacity in diabetes susceptibility. Diabetes52 :688 eC700,2003
Ide T, Shimano H, Yahagi N, Matsuzaka T, Nakakuki M, Yamamoto T, Nakagawa Y, Takahashi A, Suzuki H, Sone H, Toyoshima H, Fukamizu A, Yamada N: SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat Cell Biol6 :351 eC357,2004
Wojtaszewski JF, Higaki Y, Hirshman MF, Michael MD, Dufresne SD, Kahn CR, Goodyear LJ: Exercise modulates postreceptor insulin signaling and glucose transport in muscle-specific insulin receptor knockout mice. J Clin Invest104 :1257 eC1264,1999
Bluher M, Michael MD, Peroni OD, Ueki K, Carter N, Kahn BB, Kahn CR: Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev Cell3 :25 eC38,2002
Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR: Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell6 :87 eC97,2000
Cohen SE, Tseng YH, Michael MD, Kahn CR: Effects of insulin-sensitising agents in mice with hepatic insulin resistance. Diabetologia47 :407 eC411,2004
Kido Y, Burks DJ, Withers D, Bruning JC, Kahn CR, White MF, Accili D: Tissue-specific insulin resistance in mice with mutations in the insulin receptor, IRS-1, and IRS-2. J Clin Invest105 :199 eC205,2000
Kubota N, Tobe K, Terauchi Y, Eto K, Yamauchi T, Suzuki R, Tsubamoto Y, Komeda K, Nakano R, Miki H, Satoh S, Sekihara H, Sciacchitano S, Lesniak M, Aizawa S, Nagai R, Kimura S, Akanuma Y, Taylor SI, Kadowaki T: Disruption of insulin receptor substrate 2 causes type 2 diabetes because of liver insulin resistance and lack of compensatory -cell hyperplasia. Diabetes49 :1880 eC1889,2000
Valverde AM, Burks DJ, Fabregat I, Fisher TL, Carretero J, White MF, Benito M: Molecular mechanisms of insulin resistance in IRS-2eCdeficient hepatocytes. Diabetes52 :2239 eC2248,2003
Suzuki R, Tobe K, Aoyama M, Inoue A, Sakamoto K, Yamauchi T, Kamon J, Kubota N, Terauchi Y, Yoshimatsu H, Matsuhisa M, Nagasaka S, Ogata H, Tokuyama K, Nagai R, Kadowaki T: Both insulin signaling defects in the liver and obesity contribute to insulin resistance and cause diabetes in IRS2(-/-) mice. J Biol Chem279 :25039 eC25049,2004
Kerouz NJ, Hrsch D, Pons S, Kahn CR: Differential regulation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinase isoforms in liver and muscle of the obese diabetic (ob/ob) mouse. J Clin Invest100 :3164 eC3172,1997
Anai M, Funaki M, Ogihara T, Terasaki J, Inukai K, Katagiri H, Fukushima Y, Yazaki Y, Kikuchi M, Oka Y, Asano T: Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats. Diabetes47 :13 eC23,1998
Shimomura I, Matsuda M, Hammer RE, Bashmakov Y, Brown MS, Goldstein JL: Decreased IRS-2 and increased SREBP-1c lead to mixed insulin resistance and sensitivity in livers of lipodystrophic and ob/ob mice. Mol Cell6 :77 eC86,2000
Rui L, Fisher TL, Thomas J, White MF: Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2. J Biol Chem276 :40362 eC40367,2001
Hirashima Y, Tsuruzoe K, Kodama S, Igata M, Toyonaga T, Ueki K, Kahn CR, Araki E: Insulin downregulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway. J Endocrinol179 :253 eC266,2003(Masaki Fujimoto, Nobuyuki)