Metformin prevents liver tumorigenesis induced by high-fat diet in C57Bl/6 mice.

The prevalence of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) is increasing with the growing epidemics of obesity and diabetes. NAFLD encompasses a clinicopathologic spectrum of disease ranging from isolated hepatic steatosis to NASH, which is a more aggressive form of fatty liver disease, to cirrhosis and, finally, hepatocellular carcinoma (HCC). The exact mechanism behind the development of HCC in NASH remains unclear; however, it has been established that hepatic steatosis is the important risk factor in the development of HCC. Metformin has recently drawn attention because of its potential antitumor effect. Here, we investigated the effects of metformin on high-fat diet (HFD)-induced liver tumorigenesis, using a mouse model of NASH and liver tumor. Metformin prevented long-term HFD-induced liver tumorigenesis in C57Bl/6 mice. Of note, metformin failed to protect against liver tumorigenesis in mice that had already begun to develop NAFLD. Metformin improved short-term HFD-induced fat accumulation in the liver, associated with the suppression of adipose tissue inflammation. Collectively, these results suggest that metformin may prevent liver tumorigenesis via suppression of liver fat accumulation in the early stage, before the onset of NAFLD, which seems to be associated with a delay in the development of inflammation of the adipose tissue.

Protection from non-alcoholic steatohepatitis and liver tumourigenesis in high fat-fed insulin receptor substrate-1-knockout mice despite insulin resistance.

AIMS/HYPOTHESIS: Epidemiological studies have revealed that obesity and diabetes mellitus are independent risk factors for the development of non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma. However, the debate continues on whether insulin resistance as such is directly associated with NASH and liver tumourigenesis. Here, we investigated the incidence of NASH and liver tumourigenesis in Irs1 ( -/- ) mice subjected to a long-term high-fat (HF) diet. Our hypothesis was that hepatic steatosis, rather than insulin resistance may be related to the pathophysiology of these conditions. METHODS: Mice (8 weeks old, C57Bl/6J) were given free access to standard chow (SC) or an HF diet. The development of NASH and liver tumourigenesis was evaluated after mice had been on the above-mentioned diets for 60 weeks. Similarly, Irs1 ( -/- ) mice were also subjected to an HF diet for 60 weeks. RESULTS: Long-term HF diet loading, which causes obesity and insulin resistance, was sufficient to induce NASH and liver tumourigenesis in the C57Bl/6J mice. Obesity and insulin resistance were reduced by switching mice from the HF diet to SC, which also protected these mice against the development of NASH and liver tumourigenesis. However, compared with wild-type mice fed the HF diet, Irs1 ( -/- ) mice fed the HF diet were dramatically protected against NASH and liver tumourigenesis despite the presence of severe insulin resistance and marked postprandial hyperglycaemia. CONCLUSIONS/INTERPRETATION: IRS-1 inhibition might protect against HF diet-induced NASH and liver tumourigenesis, despite the presence of insulin resistance.

Hepatitis C virus directly associates with insulin resistance independent of the visceral fat area in nonobese and nondiabetic patients.

Insulin resistance (IR) is known to be associated with the visceral adipose tissue area. Elucidation of the relationship between hepatitis C virus (HCV) and IR is of great clinical relevance, because IR promotes liver fibrosis. In this study, we tested the hypothesis that HCV infection by itself may promote IR. We prospectively evaluated 47 patients with chronic HCV infection who underwent liver biopsy. Patients with obesity, type 2 diabetes mellitus (DM), or a history of alcohol consumption were excluded. IR was estimated by calculation of the modified homeostasis model of insulin resistance (HOMA-IR) index. Abdominal fat distribution was determined by computed tomography. Fasting blood glucose levels were within normal range in all the patients. The results of univariate analysis revealed a significant correlation between the quantity of HCV-RNA and the HOMA-IR (r = 0.368, P = 0.0291). While a significant correlation between the visceral adipose tissue area and the HOMA-IR was also observed in the 97 control, nondiabetic, non-HCV-infected patients (r = 0.398, P < 0.0001), no such significant correlation between the visceral adipose tissue area and the HOMA-IR (r = 0.124, P = 0.496) was observed in the patients with HCV infection. Multiple regression analysis with adjustment for age, gender and visceral adipose tissue area revealed a significant correlation between the HCV-RNA and the HOMA-IR (P = 0.0446). HCV is directly associated with IR in a dose-dependent manner, independent of the visceral adipose tissue area. This is the first report to demonstrate the direct involvement of HCV and IR in patients with chronic HCV infection.

The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance.

Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARgamma and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARgamma by PPARgamma agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARgamma activity by heterozygous PPARgamma deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARgamma deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARgamma deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor alpha, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARgamma deficiency and PPARgamma agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy.

Endogenous PPAR gamma mediates anti-inflammatory activity in murine ischemia-reperfusion injury.

BACKGROUND & AIMS: Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor whose activation has been linked to several physiologic pathways including those related to the regulation of intestinal inflammation. We sought to determine whether PPAR gamma could function as an endogenous anti-inflammatory pathway in a murine model of intestinal ischemia-reperfusion (I/R) injury. METHODS: PPAR gamma-deficient and wild-type mice were examined for their response to I/R procedure. Treatment with a PPAR gamma-specific ligand was also performed. RESULTS: In a murine model of intestinal I/R injury, we observed more severe injury in PPAR gamma-deficient mice and protection against local and remote tissue injury in mice treated with a PPAR gamma-activating ligand, BRL-49653. Activation of PPAR gamma resulted in down-regulation of intercellular adhesion molecule 1 expression by intestinal endothelium and tissue tumor necrosis factor alpha messenger RNA levels most likely by inhibition of the NF-kappa B pathway. CONCLUSIONS: These data strongly suggest that an endogenous PPAR gamma pathway exists in tissues that may be amenable to therapeutic manipulation in I/R-related injuries.

beta-cell glucokinase deficiency and hyperglycemia are associated with reduced islet amyloid deposition in a mouse model of type 2 diabetes.

Type 2 diabetes is characterized by impaired beta-cell function, hyperglycemia, and islet amyloid deposition. The primary constituent of islet amyloid is the 37-amino acid beta-cell product called islet amyloid polypeptide (IAPP) or amylin. To study mechanisms of islet amyloid formation, we developed a transgenic mouse model that produces and secretes the amyloidogenic human IAPP (hIAPP) molecule and have shown that 81% of male transgenic mice develop islet amyloid after 14 months on a high-fat diet. To test whether impaired beta-cell function and hyperglycemia could enhance islet amyloid formation, we cross-bred our hIAPP transgenic mice with beta-cell glucokinase-knockout mice (GKKO) that have impaired glucose-mediated insulin secretion and fasting hyperglycemia. The resulting new (hIAPPxGKKO) line of mice had higher basal plasma glucose concentrations than the hIAPP transgenic mice at 3, 6, and 12 months of age (P < 0.05), as did GKKO mice compared with hIAPP transgenic mice at 6 and 12 months of age (P < 0.05). Basal plasma immunoreactive insulin (IRI) levels were lower in hIAPP x GKKO mice than in hIAPP transgenic mice at 6 months of age (P < 0.05). The area under the glucose curve in response to an intraperitoneal glucose challenge (1 g/kg body weight) was larger in hIAPPxGKKO mice than in hIAPP transgenic mice at 3, 6, and 12 months of age (P < 0.005) and in GKKO mice compared with hIAPP transgenic mice at 6 and 12 months of age (P < 0.005). The area under the IRI curve was lower in hIAPPxGKKO mice at 6 and 12 months of age (P < 0.05) than in hIAPP transgenic mice and in GKKO mice compared with hIAPP transgenic mice at 12 months of age (P < 0.05). Despite the presence of hyperglycemia, hIAPPxGKKO mice had a lower incidence (4 of 17 vs. 12 of 19, P < 0.05) and amount (0.40 +/- 0.24 vs. 1.2 +/- 0.3 arbitrary units, P < 0.05) of islet amyloid than hIAPP transgenic mice had. As expected, no islet amyloid was observed in GKKO mice lacking the hIAPP transgene (0 of 13). There was no difference in pancreatic content of IRI and hIAPP among the three groups of mice. Thus, despite the presence of impaired islet function and hyperglycemia, hIAPPxGKKO mice had a decreased incidence and quantity of islet amyloid. Therefore, our data suggest that impaired beta-cell glucose metabolism or hyperglycemia are not likely to contribute to islet amyloid formation in diabetes. Furthermore, this finding may explain the lack of progression of glycemia in patients with maturity-onset diabetes of the young.

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion.

Glucose metabolism in glycolysis and in mitochondria is pivotal to glucose-induced insulin secretion from pancreatic beta cells. One or more factors derived from glycolysis other than pyruvate appear to be required for the generation of mitochondrial signals that lead to insulin secretion. The electrons of the glycolysis-derived reduced form of nicotinamide adenine dinucleotide (NADH) are transferred to mitochondria through the NADH shuttle system. By abolishing the NADH shuttle function, glucose-induced increases in NADH autofluorescence, mitochondrial membrane potential, and adenosine triphosphate content were reduced and glucose-induced insulin secretion was abrogated. The NADH shuttle evidently couples glycolysis with activation of mitochondrial energy metabolism to trigger insulin secretion.

Xid-like immunodeficiency in mice with disruption of the p85alpha subunit of phosphoinositide 3-kinase.

Mice with a targeted gene disruption of p85alpha, a regulatory subunit of phosphoinositide 3-kinase, had impaired B cell development at the pro-B cell stage, reduced numbers of mature B cells and peritoneal CD5+ Ly-1 B cells, reduced B cell proliferative responses, and no T cell-independent antibody production. These phenotypes are nearly identical to those of Btk-/- or xid (X-linked immunodeficiency) mice. These results provide evidence that p85alpha is functionally linked to the Btk pathway in antigen receptor-mediated signal transduction and is pivotal in B cell development and functions.

Glucose modulation of ATP-sensitive K-currents in wild-type, homozygous and heterozygous glucokinase knock-out mice.

One type of maturity-onset diabetes of the young (MODY2) is caused by mutations in the glucokinase gene, a key glycolytic enzyme in the beta cell and liver. Glucose fails to stimulate insulin secretion in mice in which the glucokinase gene has been selectively knocked out in the beta cell. We tested the hypothesis that this effect results from defective metabolic regulation of beta cell ATP-sensitive potassium (K(ATP)) channels. Glucose had little effect on K(ATP) currents in homozygous (-/-) mice but inhibited K(ATP) currents in wild-type (+/+) and heterozygous (+/-) mice with EC50 of 3.2 mM and 5.5 mM, respectively, in newborn animals, and of 4.7 mM and 9.9 mM, respectively, in 1.5-year-old mice. Glucose (20 mmol/l) did not affect the resting membrane potential of -/- beta cells but depolarised wild-type and + /- beta cells and induced electrical activity. In contrast, 20 mmol/l ketoisocaproic acid or 0.5 mmol/ l tolbutamide depolarised all three types of beta-cell. These results support the idea that defective glycolytic metabolism, produced by a loss (-/- mice) or reduction (+/- mice) of glucokinase activity, leads to defective K(ATP) channel regulation and thereby to the selective loss, or reduction, of glucose-induced insulin secretion.

Involvement of p85 in p53-dependent apoptotic response to oxidative stress.

Reactive oxygen species have damaging effects on cellular components and so trigger defensive responses by the cell and even programmed cell death, although the mechanisms by which mammalian cells transmit signals in response to oxidative damage are unknown. We report here that the protein p85, a regulator of the signalling protein phosphatidyl-3-OH kinase (PI(3)K), participates in the cell death process that is induced in response to oxidative stress and that this role of p85 in apoptosis does not involve PI(3)K. We show that disruption of p85 by homologous recombination impairs the cellular apoptotic response to oxidative stress. Because the protein p53 is required for cell death induced by oxidative damage, we examined the relation between p85 and p53. Using a chimaeric p53 fusion protein with the oestrogen receptor (p53ER) to supply p53 (p53 is induced upon binding of p53ER to oestradiol) in a p53-deficient cell line, we found that p85 is upregulated by p53 and that its involvement in p53-mediated apoptosis is independent of PI(3)K. We propose that p85 acts as a signal transducer in the cellular response to oxidative stress, mediating cell death regulated by p53.

Germ-line contribution of embryonic stem cells in chimeric mice: influence of karyotype and in vitro differentiation ability.

The effect of the karyotype and the ability to differentiate in vitro upon germ-line transmission by A3-1 embryonic stem (ES) cells in chimeric mice were examined. Germ-line transmission was confirmed in ES cells exhibiting 38% and more of the normal karyotype, but no chimeric mice and/or germ-line transmitters were observed regardless of the karyotype when the cystic embryoid body (CEB) was formed on day 8 and later in the suspension culture. Germ-line transmission of the ES cells was not significantly influenced by formation of the simple embryoid body (SEB). Germ-line transmitters were preferentially observed in chimeras when the ES cell contribution to coat color was markedly increased, but this contribution to coat color varied regardless of the karyotype or in vitro differentiation ability. These results suggest that A3-1 ES cells which exhibit CEB at 7 days after suspension culture and approximately 40% of normal karyotype are capable of germ-line transmission in chimeric mice.

[Mono-radiculopathy multiplex--multiple infarction of the cauda equina caused by intravascular lymphomatosis].

A 55-year-old man had felt numbness of the bilateral peroneal sides of legs for 6 months. Then hepatosplenomegaly, anemia, body weight loss and fever developed, and a diagnosis of malignant histiocytosis (MH) was made by revealing the presence of innumerable atypical histiocytes with hemophagocytosis in the bone marrow. Soon later, sensory disturbance of bilateral peroneal sides of legs (right side dominant) developed and aggravated with painful dysesthesia and weakness of the legs for the last 2 weeks before death. Electrophysiologically, sensory conduction velocity of the sural nerve was normal and somatosensory evoked potentials from tibial nerve were normal before P15 but were not evoked at all after the lumbar potential, suggesting lumbosacral radiculopathy. Autopsy showed multifocal ischemic lesions and secondary degeneration of the lumbosacral nerve roots associated with necrosis and fibrosis of the radicular vessels and intravascular infiltration of atypical mononuclear cells which were positive for B cell markers. The neurological manifestations and the distribution of ischemic lesions, which were similar to those of vasculopathic mononeuropathy multiplex, would deserve the name of "monoradiculopathy multiplex".