Naoto Nagata

Abstract: The SH2 domain-containing protein-tyrosine phosphatase Shp2 is a negative regulator of hepatic insulin action in mice fed regular chow. To investigate the role of hepatic Shp2 in lipid metabolism and energy balance we determined the metabolic effects of its deletion in mice challenged with a high fat diet (HFD). We analyzed body mass, lipid metabolism, insulin sensitivity, and glucose tolerance in liver-specific Shp2-deficient (referred to herein as LSHKO) and control mice fed HFD. Hepatic Shp2 protein expression is regulated by nutritional status, increasing in mice fed HFD and decreasing during fasting. LSHKO mice gained less weight and exhibited increased energy expenditure compared with control mice. In addition, hepatic Shp2 deficiency led to decreased liver steatosis, enhanced insulin-induced suppression of hepatic glucose production, and impeded the development of insulin resistance following high fat feeding. At the molecular level, LSHKO exhibited decreased hepatic endoplasmic reticulum stress and inflammation compared with control mice. In addition, tyrosine and serine phosphorylation of total and mitochondrial signal transducer and activator of transcription 3 were enhanced in LSHKO compared with control mice. In line with this observation and the increased energy expenditure of LSHKO, oxygen consumption rate was higher in liver mitochondria of LSHKO compared with controls. Collectively, these studies identify hepatic Shp2 as a novel regulator of systemic energy balance under conditions of high fat feeding.

Abstract: Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.

Abstract: The SH2 domain-containing protein-tyrosine phosphatase Src homology phosphatase 2 (Shp2) has been implicated in a variety of growth factor signaling pathways, but its metabolic role in some peripheral insulin-responsive tissues remains unknown. To address the metabolic function of Shp2 in adipose tissue, we generated mice with adipose-specific Shp2 deletion using adiponectin-Cre transgenic mice. We then analyzed insulin sensitivity, glucose tolerance, and body mass in adipose-specific Shp2-deficient and control mice on regular chow and high-fat diet (HFD). Control mice on HFD exhibited increased Shp2 expression in various adipose depots compared with those on regular chow. Adiponectin-Cre mice enabled efficient and specific deletion of Shp2 in adipose tissue. However, adipose Shp2 deletion did not significantly alter body mass in mice on chow or HFD. In addition, mice with adipose Shp2 deletion exhibited comparable insulin sensitivity and glucose tolerance compared with controls. Consistent with this, basal and insulin-stimulated Erk and Akt phosphorylations were comparable in adipose tissue of Shp2-deficient and control mice. Our findings indicate that adipose-specific Shp2 deletion does not significantly alter systemic insulin sensitivity and glucose homeostasis.

Abstract: Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of insulin signaling and energy balance, but its role in brown fat adipogenesis requires additional investigation.

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Abstract: The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser(612)) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.

Abstract: The present study examined the influence of cimetidine on the nephrotoxicity and antitumor effects of cisplatin in vitro and in vivo. When the serum concentration of cimetidine was maintained over 20 µg/ml for 4 h by bolus and continuous intravenous infusion, cimetidine prevented nephrotoxicity of cisplatin without influencing antitumor activity. Cimetidine and the antioxidant N-acetylcysteine (NAC) significantly inhibited the in vitro growth inhibition of cisplatin in cells originating from the kidney, but not in SOSN2 osteosarcoma cells. Cimetidine (1 mM) also did not influence platinum concentration in the cells, regardless of whether the organic cation transporter 2 (OCT2) was expressed. Cisplatin did induce reactive oxygen species (ROS) in the KN41 kidney cell line and cimetidine and NAC significantly reduced ROS production. However, cisplatin did not produce ROS in osteosarcoma cells. From these results, cimetidine clearly inhibits nephrotoxicity induced by cisplatin without any influence on the antitumor activity of cisplatin on osteosarcoma in vitro and in vivo.

Abstract: The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin, by way of the production of secretory proteins, termed hepatokines. Here, we demonstrate that selenoprotein P (SeP), a liver-derived secretory protein, causes insulin resistance. Using serial analysis of gene expression (SAGE) and DNA chip methods, we found that hepatic SeP mRNA levels correlated with insulin resistance in humans. Administration of purified SeP impaired insulin signaling and dysregulated glucose metabolism in both hepatocytes and myocytes. Conversely, both genetic deletion and RNA interference-mediated knockdown of SeP improved systemic insulin sensitivity and glucose tolerance in mice. The metabolic actions of SeP were mediated, at least partly, by inactivation of adenosine monophosphate-activated protein kinase (AMPK). In summary, these results demonstrate a role of SeP in the regulation of glucose metabolism and insulin sensitivity and suggest that SeP may be a therapeutic target for type 2 diabetes.

Abstract: Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH.

Abstract: AIM/HYPOTHESIS: Recent studies have demonstrated relationships between circadian clock function and the development of metabolic diseases such as type 2 diabetes. We investigated whether the peripheral circadian clock is impaired in patients with type 2 diabetes. METHODS: Peripheral leucocytes were obtained from eight patients with diabetes and six comparatively young non-diabetic volunteers at 09:00, 15:00, 21:00 and 03:00 hours (study 1) and from 12 male patients with diabetes and 14 age-matched men at 09:00 hours (study 2). Transcript levels of clock genes (CLOCK, BMAL1 [also known as ARNTL], PER1, PER2, PER3 and CRY1) were determined by real-time quantitative PCR. RESULTS: In study 1, mRNA expression patterns of BMAL1, PER1, PER2 and PER3 exhibited 24 h rhythmicity in the leucocytes of all 14 individuals. The expression levels of these mRNAs were significantly (p < 0.05) lower in patients with diabetes than in non-diabetic individuals at one or more time points. Moreover, the amplitudes of mRNA expression rhythms of PER1 and PER3 genes tended to diminish in patients with diabetes. In study 2, leucocytes obtained from patients with diabetes expressed significantly (p < 0.05) lower transcript levels of BMAL1, PER1 and PER3 compared with leucocytes from control individuals, and transcript expression was inversely correlated with HbA(1c) levels (rho = -0.47 to -0.55, p < 0.05). CONCLUSIONS/INTERPRETATION: These results suggest that rhythmic mRNA expression of clock genes is dampened in peripheral leucocytes of patients with type 2 diabetes. The impairment of the circadian clock appears to be closely associated with the pathophysiology of type 2 diabetes in humans.

Abstract: Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH(2)-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid beta-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated beta-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance.

Abstract: Obesity is a major cause of insulin resistance and contributes to the development of type 2 diabetes. The altered expression of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) has been regarded as a key change in insulin-sensitive organs of patients with type 2 diabetes. This study explores possible molecular signatures of obesity and examines the clinical significance of OXPHOS gene expression in the livers of patients with type 2 diabetes. We analyzed gene expression in the livers of 21 patients with type 2 diabetes (10 obese and 11 nonobese patients; age, 53.0 +/- 2.1 years; BMI, 24.4 +/- 0.9 kg/m(2); fasting plasma glucose, 143.0 +/- 10.6 mg/dl) using a DNA chip. We screened 535 human pathways and extracted those metabolic pathways significantly altered by obesity. Genes involved in the OXPHOS pathway, together with glucose and lipid metabolism pathways, were coordinately upregulated in the liver in association with obesity. The mean centroid of OXPHOS gene expression was significantly correlated with insulin resistance indices and the hepatic expression of genes involved in gluconeogenesis, reactive oxygen species (ROS) generation, and transcriptional factors and nuclear co-activators associated with energy homeostasis. In conclusion, obesity may affect the pathophysiology of type 2 diabetes by upregulating genes involved in OXPHOS in association with insulin resistance markers and the expression of genes involved in hepatic gluconeogenesis and ROS generation.

Abstract: Insulin resistance is a major pathological condition associated with obesity and metabolic syndrome. Insulin resistance and the renin-angiotensin system are intimately linked. We evaluated the role of the renin-angiotensin system in the pathogenesis of insulin resistance-associated, non-alcoholic steatohepatitis by using the angiotensin II type 1 receptor blocker olmesartan medoxomil in a diabetic rat model. The effects of olmesartan on methionine- and choline-deficient (MCD) diet-induced steatohepatitis were investigated in obese, diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and control Long-Evans Tokushima Otsuka (LETO) rats. Components of the renin-angiotensin system were up-regulated in the livers of OLETF rats, compared with LETO rats. In OLETF, but not LETO, rats, oral administration of olmesartan for 8 weeks ameliorated insulin resistance. Moreover, olmesartan suppressed MCD diet-induced hepatic steatosis and the hepatic expression of lipogenic genes (sterol regulatory element-binding protein-1c and fatty acid synthase) in OLETF, but not LETO, rats. In both OLETF and LETO rats, olmesartan inhibited hepatic oxidative stress (4-hydroxy-2-nonenal-modified protein) and expression of NADPH oxidase. Olmesartan also inhibited hepatic fibrosis, stellate cell activation, and expression of fibrogenic genes (transforming growth factor-beta, alpha 1 [I] procollagen, plasminogen activator inhibitor-1) in both OLETF and LETO rats. In conclusion, pharmacological blockade of the angiotensin II type 1 receptor slows the development of steatohepatitis in the OLETF rat model. This angiotensin II type 1 receptor blocker may exert insulin resistance-associated effects against hepatic steatosis and inflammation as well as direct effects against the generation of reactive oxygen species and fibrogenesis.

Abstract: Conscious young adult male rats were given total parenteral nutrition (TPN) with or without soybean fat for 4 days. Those given fat-free TPN developed severe fatty liver, with hyperglycaemia, hyperinsulinaemia, and hypotriglyceridaemia. These disorders were clearly improved by supplementing TPN with soybean fat, in an amount equivalent to 20% of total calories, and correspondingly reducing glucose. Insulin resistance also developed over a 4-day infusion of fat-free TPN in mature rats. Even after over-night fasting after stopping the TPN infusion, the levels of serum glucose and insulin were higher in the fat-free TPN group than in the control group, and intravenous glucose tolerance test results indicated insulin resistance in the fat-free TPN group. The HOMA-IR index of insulin resistance was significantly improved by supplementation with soybean fat. In conclusion, fat-free TPN infusion induced hyperglycaemia and hyperinsulinaemia, leading to fatty liver and insulin resistance. TPN with glucose should be supplemented with soybean fat emulsion as replacement for part of the glucose calories.

Abstract: Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease and is one of the most common liver diseases in the developed world. The histological findings of NASH are characterized by hepatic steatosis, inflammation, and fibrosis. However, an optimal treatment for NASH has not been established. Tranilast, N-(3',4'-dimethoxycinnamoyl)-anthranilic acid, is an antifibrogenic agent that inhibits the action of transforming growth factor beta (TGF-beta). This drug is used clinically for fibrogenesis-associated skin disorders including hypertrophic scars and scleroderma. TGF-beta plays a central role in the development of hepatic fibrosis, and tranilast may thus ameliorate the pathogenesis of NASH. We investigated the effects of tranilast using an established dietary animal model of NASH, obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and nondiabetic control Long-Evans Tokushima Otsuka (LETO) rats fed a methionine-deficient and choline-deficient diet. Treatment with 2% tranilast (420 mg/kg/day) for 8 weeks prevented the development of hepatic fibrosis and the activation of stellate cells, and down-regulated the expression of genes for TGF-beta and TGF-beta-target molecules, including alpha1 procollagen and plasminogen activator-1. In addition, tranilast attenuated hepatic inflammation and Kupffer cell recruitment, and down-regulated the expression of tumor necrosis factor alpha. Unexpectedly, tranilast ameliorated hepatic steatosis and up-regulated the expression of genes involved in beta-oxidation, such as peroxisome proliferator-activated receptor alpha and carnitine O-palmitoyltransferase-1. Most of these effects were observed in LETO rats and OLETF rats, which suggest that the action of tranilast is mediated through the insulin resistance-independent pathway. CONCLUSION: Our findings suggest that targeting TGF-beta with tranilast represents a new mode of therapy for NASH.

Abstract: Tumor necrosis factor (TNF)-alpha and local activation of the renin-angiotensin system may contribute to insulin resistance and atherosclerosis. In this study, we investigated the involvement of these mediators in the liver. We found that the gene expression of renin-angiotensin system components, together with that of plasminogen activator inhibitor (PAI)-1, is upregulated in the liver of patients with obesity and type 2 diabetes. We next examined the role of the renin-angiotensin system on TNF-alpha-induced PAI-1 production in the nonmalignant human hepatocyte cell line THLE-5b. THLE-5b cells expressed genes encoding renin-angiotensin system components including angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin type 1 (AT(1)) receptor. ACE, angiotensinogen, and angiotensin AT(1) receptor mRNA expression were upregulated time-dependently by TNF-alpha. Moreover, angiotensin AT(1) receptor antagonist dose-dependently inhibited TNF-alpha-induced PAI-1 production. Interestingly, high-dose olmesartan, but not candesartan, reduced the increased expression of the angiotensin AT(1) receptor. These results suggest that TNF-alpha and the local renin-angiotensin system coordinately stimulate PAI-1 production in hepatocytes. Selective angiotensin AT(1) receptor antagonists inhibit both TNF-alpha- and angiotensin II-induced PAI-1 production in hepatocytes, suggesting a cross talk between both systems.

Abstract: We assessed the effects of different classes of flavonoids on insulin-stimulated 2-deoxy-D-[1-(3)H]glucose uptake by mouse MC3T3-G2/PA6 cells differentiated into mature adipose cells. Among the flavonoids examined, the flavones, apigenin and luteolin, the flavonols, kaempferol, quercetin and fisetin, an isoflavone, genistein, a flavanonol, silybin, and the flavanols, (-)-epigallocatechin gallate (EGCG) and theaflavins, significantly inhibited insulin-stimulated glucose uptake. Key structural features of flavonoids for inhibition of insulin-stimulated glucose uptake are the B-ring 4'- or 3',4'-OH group and the C-ring C2-C3 double bond of the flavones and flavonols, the A-ring 5-OH of isoflavones, and the galloyl group of EGCG and theaflavins. Luteolin significantly inhibits insulin-stimulated phosphorylation of insulin receptor-beta subunit (IR-beta), and apigenin, kaempferol, quercetin and fisetin, also tended to inhibit the IR-beta phosphorylation. On the other hand, isoflavones, flavanols or flavanonols did not affect insulin-stimulated IR-beta phosphorylation. Apigenin, luteolin, kaempferol, quercetin and fisetin also appeared to inhibit insulin-stimulated activation of Akt, a pivotal downstream effector of phosphatidylinositol 3-kinase (PI3K), and suppressed insulin-dependent translocation of a glucose transporter, (GLUT)4, into the plasma membrane. Although genistein, silybin, EGCG and theaflavins had no effect on the insulin-stimulated activation of Akt, they blocked insulin-dependent GLUT4 translocation. These results provide novel insights into the modulation by flavonoids of insulin's actions, including glucose uptake in adipocytes.

Abstract: Insulin resistance is a key pathophysiological feature of metabolic syndrome. However, the initial events triggering the development of insulin resistance and its causal relations with dysregulation of glucose and fatty acids metabolism remain unclear. We investigated biological pathways that have the potential to induce insulin resistance in mice fed a high-fat diet (HFD). We demonstrate that the pathways for reactive oxygen species (ROS) production and oxidative stress are coordinately up-regulated in both the liver and adipose tissue of mice fed an HFD before the onset of insulin resistance through discrete mechanism. In the liver, an HFD up-regulated genes involved in sterol regulatory element binding protein 1c-related fatty acid synthesis and peroxisome proliferator-activated receptor alpha-related fatty acid oxidation. In the adipose tissue, however, the HFD down-regulated genes involved in fatty acid synthesis and up-regulated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Furthermore, increased ROS production preceded the elevation of tumor necrosis factor-alpha and free fatty acids in the plasma and liver. The ROS may be an initial key event triggering HFD-induced insulin resistance.

Abstract: Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity. An adipocyte-derived hormone, adiponectin, may play a role in the pathophysiology of NAFLD through insulin-sensitizing and antifibrotic effects. We found that hepatic expression of adiponectin receptor AdipoR2, but not AdipoR1, was down-regulated in 14 patients with NAFLD compared with 7 patients with a normal liver (P < .05). To investigate the significance of the adiponectin system in obesity and NAFLD, we examined the regulation of AdipoR2 expression in a nonmalignant human hepatocyte cell line, the THLE-5b cells. Insulin down-regulated the levels of AdipoR2 messenger RNA (mRNA) and protein, whereas an adipocytokine, tumor necrosis factor alpha, up-regulated them. A thiazolidinedione, pioglitazone, up-regulated the expression of AdipoR2 mRNA and protein in THLE-5b cells. The AdipoR2 mRNA level was decreased in fatty THLE-5b cells induced by coincubating with fatty acids. These findings suggest that down-regulation of AdipoR2 in the liver caused by hyperinsulinemia and steatosis may play a role in the development of NAFLD.

Abstract: BACKGROUND & AIMS: The increasing prevalence of nonalcoholic steatohepatitis (NASH) is due to the epidemic of obesity and type 2 diabetes, both of which are associated with insulin resistance. METHODS: To clarify the causal relationship between insulin resistance and the development of NASH, steatohepatitis was induced in obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and nondiabetic control Long-Evans Tokushima Otsuka (LETO) rats by feeding them a methionine and choline-deficient (MCD) diet. Insulin sensitivity of the rats was altered by adding a high-fat (HF) diet or the peroxisomal-proliferator activated receptor-gamma agonist pioglitazone to the MCD diet. RESULTS: The MCD diet-induced steatohepatitis was accelerated in OLETF rats after 8 weeks. Steatosis preceded inflammation, which led to fibrosis and the development of steatohepatitis. The hepatic gene expression for transforming growth factor-beta, alpha1 procollagen and plasminogen activator inhibitor-1 was up-regulated in OLETF rats compared with LETO rats. The MCD + HF diet further enhanced insulin resistance and led to rapid development of pre-cirrhosis in OLETF rats by increasing the triglyceride pool, activating stellate cells, and up-regulating gene expression for sterol regulatory element-binding protein-1c and fatty acid synthase in the liver. In contrast, pioglitazone attenuated the MCD diet-induced steatohepatitis in OLETF rats but not in LETO rats by reversing the underlying pathogenesis involved in this model through improvement of insulin resistance. These results confirm a link between insulin resistance and the development/progression of steatohepatitis, at least partly via up-regulation of genes for lipogenesis, inflammation, and fibrogenesis, in animal models. CONCLUSIONS: Insulin resistance and/or diabetes may accelerate the entire pathologic spectrum of NASH.

Abstract: We hypothesized that systemically circulating peripheral blood mononuclear cells (PBMCs) reflect the pathophysiology of type 2 diabetes. PBMCs were obtained from 18 patients with type 2 diabetes and 16 non-diabetic subjects. The expression of genes in the PBMCs was analyzed by using a DNA chip followed by statistical analysis for specific gene sets for biological categories. The only gene set coordinately up-regulated by the existence of diabetes and down-regulated by glycemic control consisted of 48 genes involved in the c-Jun N-terminal kinase (JNK) pathway. In contrast, the only gene set coordinately down-regulated by the existence of diabetes, but not altered by glycemic control consisted of 92 genes involved in the mitochondrial oxidative phosphorylation (OXPHOS) pathway. Our findings suggest that genes involved in the JNK and OXPHOS pathways of PBMCs may be surrogate transcriptional markers for hyperglycemia-induced oxidative stress and morbidity of type 2 diabetes, respectively.

Abstract: Recently, nonalcoholic steatohepatitis (NASH) was found to be correlated with cardiovascular disease events independently of the metabolic syndrome. The aim of this study was to investigate whether an atherogenic (Ath) diet induces the pathology of steatohepatitis necessary for the diagnosis of human NASH and how cholesterol and triglyceride alter the hepatic gene expression profiles responsible for oxidative stress. We investigated the liver pathology and plasma and hepatic lipids of mice fed the Ath diet. The hepatic gene expression profile was examined with microarrays and real-time polymerase chain reactions. The Ath diet induced dyslipidemia, lipid peroxidation, and stellate cell activation in the liver and finally caused precirrhotic steatohepatitis after 24 weeks. Cellular ballooning, a necessary histological feature defining human NASH, was observed in contrast to existing animal models. The addition of a high-fat component to the Ath diet caused hepatic insulin resistance and further accelerated the pathology of steatohepatitis. A global gene expression analysis revealed that the Ath diet up-regulated the hepatic expression levels of genes for fatty acid synthesis, oxidative stress, inflammation, and fibrogenesis, which were further accelerated by the addition of a high-fat component. Conversely, the high-fat component down-regulated the hepatic gene expression of antioxidant enzymes and might have increased oxidative stress. CONCLUSION: The Ath diet induces oxidative stress and steatohepatitis with cellular ballooning. The high-fat component induces insulin resistance, down-regulates genes for antioxidant enzymes, and further aggravates the steatohepatitis. This model suggests the critical role of lipids in causing oxidative stress and insulin resistance leading to steatohepatitis.

Abstract: AIMS/HYPOTHESIS: Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in the pathophysiology of type 2 diabetes. Genes involved in OXPHOS have been reported to be down-regulated in skeletal muscle from patients with type 2 diabetes; however, hepatic regulation is unknown. MATERIALS AND METHODS: We analysed expression of genes involved in OXPHOS from the livers of 14 patients with type 2 diabetes and 14 subjects with NGT using serial analysis of gene expression (SAGE) and DNA chip analysis. We evaluated the correlation between expression levels of genes involved in OXPHOS and the clinical parameters of individuals with type 2 diabetes and NGT. RESULTS: Both gene analyses showed that genes involved in OXPHOS were significantly upregulated in the type 2 diabetic liver. In the SAGE analysis, tag count comparisons of mitochondrial transcripts showed that ribosomal RNAs (rRNA) were 3.5-fold over-expressed, and mRNAs were 1.2-fold over-expressed in the type 2 diabetes library. DNA chip analysis revealed that expression of genes involved in OXPHOS, which correlated with several nuclear factors, including estrogen-related receptor-alpha or peroxisome proliferator-activated receptor-gamma, was a predictor of fasting plasma glucose levels, independently of age, BMI, insulin resistance and fasting insulin levels (p = 0.04). Surprisingly, genes involved in OXPHOS did not correlate with peroxisome proliferator-activated receptor-gamma coactivator-1alpha or nuclear respiratory factor 1. CONCLUSIONS/INTERPRETATION: Our results indicate that upregulation of genes involved in OXPHOS in the liver, which are regulated by different mechanisms from genes in the skeletal muscle, is associated with fasting hyperglycaemia in patients with type 2 diabetes.