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Hepatitis C Leads to Insulin Resistance in Mice & Perhaps Diabetes in HCV+
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"Hepatitis C virus infection and diabetes: Direct involvement of the virus in the development of insulin resistance"
Yoshizumi Shintani
Gastroenterology
March 2004, Volume 126, Number 3
SUMMARY
Epidemiological studies have suggested a linkage between type 2 diabetes and chronic hepatitis C virus (HCV) infection. However, the presence of additional factors such as obesity, aging, or cirrhosis prevents the establishment of a definite relationship between these 2 conditions.
A mouse model transgenic for the HCV core gene was used.
In the glucose tolerance test, plasma glucose levels were higher at all time points including in the fasting state in the core gene transgenic mice than in control mice, although the difference was not statistically significant. In contrast, the transgenic mice exhibited a marked insulin resistance as revealed by the insulin tolerance test, as well as significantly higher basal serum insulin levels. Feeding with a high-fat diet led to the development of overt diabetes in the transgenic mice but not in control mice. A high level of tumor necrosis factor-, which has been also observed in human chronic hepatitis C patients, was considered to be one of the bases of insulin resistance in the transgenic mice, which acts by disturbing tyrosine phosphorylation of insulin receptor substrate-1. Moreover, administration of an anti-tumor necrosis factor- antibody restored insulin sensitivity.
The ability of insulin to lower the plasma glucose level in the HCV transgenic mice was impaired, as observed in chronic hepatitis C patients. These results provide a direct experimental evidence for the contribution of HCV in the development of insulin resistance in human HCV infection, which finally leads to the development of type 2 diabetes.
Approximately 200 million people are chronically infected with hepatitis C virus (HCV) in the world. Chronic HCV infection may lead to cirrhosis and hepatocellular carcinoma, thereby being a worldwide problem both in medical and socioeconomical aspects. In addition, chronic HCV infection is a multifaceted disease, which is associated with numerous clinical manifestations, such as essential mixed cryoglobulinemia, porphyria cutanea tarda, and membranoproliferative glomerulonephritis. Recent epidemiological studies have added another clinical condition, type 2 diabetes, to a spectrum of HCV-associated diseases. However, the establishment of a definite causative relationship between HCV infection and diabetes is hampered by the presence of other factors such as obesity, aging, or liver injury in patients with chronic HCV infection.
Type 2 diabetes is a complex, multisystem disease with a pathophysiology that includes a defect in insulin secretion, increased hepatic glucose production, and resistance to the action of insulin, all of which contribute to the development of overt hyperglycemia. Although the precise mechanisms whereby these factors interact to produce glucose intolerance and diabetes are uncertain, it has been suggested that the final common pathway responsible for the development of type 2 diabetes is the failure of the pancreatic -cells to compensate for the insulin resistance. Hyperinsulinemia in the fasting state is observed relatively early in type 2 diabetes, but it is considered to be a secondary response that compensates for the insulin resistance. Overt diabetes occurs over time when pancreatic -cells bearing the burden of increased insulin secretion fail to compensate for the insulin resistance.
In this study, to elucidate the role of HCV in a possible association between diabetes and HCV infection, transgenic mice that carry the core gene of HCV were analyzed. We found that these mice developed insulin resistance. An addition of a high-calorie diet led to the development of type 2 diabetes by disrupting the balance between insulin resistance and secretion.
DISCUSSION
Since Allison et al. reported an association between HCV infection and diabetes, evidence has been accumulating connecting these 2 conditions. In such studies, HCV infection has a significantly stronger association with diabetes than hepatitis B viral infection. The variables other than HCV infection that are associated with diabetes are cirrhosis, male sex, and aging. In addition to these clinic-based, case-control studies, Mehta et al. have reported the result of investigation at population level. In this cross-sectional national survey, persons 40 years or older with HCV infection were more than 3 times more likely to have type 2 diabetes than those without HCV infection. Thus, the association of HCV infection with diabetes has become closer as shown by epidemiological studies. However, there are some difficulties in establishing a definite relationship between HCV infection and diabetes on the basis of epidemiological studies; in patients, there are other numerous factors perturbing the verification of the definite relationship, such as obesity, aging, or particularly advanced liver injuries. Moreover, the biological mechanism underlying diabetes or insulin resistance in HCV infection is unknown. In vitro or cultured cell studies have a very limited utility for the study of insulin resistance or diabetes because insulin resistance is a condition that involves multiple organs, such as the skeletal muscles and liver. Thus, the use of good experimental animal model systems may be useful both in establishing a definite relationship between diabetes and HCV infection and in elucidating the role of HCV in the development of insulin resistance.
In the current study, the HCV core gene transgenic mice exhibited insulin resistance as early as 1-month old, despite an apparent absence of glucose intolerance. Development of insulin resistance without any liver injury or excessive body weight gain, as shown in the current study, clearly indicates that infection of HCV per se is a cause of the development of insulin resistance. Although only the core protein is expressed in these mice instead of HCV replication in humans, the fact that the intrahepatic core protein levels are similar between the core gene transgenic mice and chronic hepatitis C patients warrants extrapolating the result into hepatitis C patients. Certainly, dispersion in the intrahepatic core protein levels in human chronic hepatitis C patients compared with the constant amount of the core protein must be taken into account. The occurrence of insulin resistance in the core gene transgenic mice as early as 1-month old also excluded the possibility that aging is a cause of insulin resistance. Nonetheless, aging could be an aggravating factor for insulin resistance. Thus, the current analysis shows a definite causal relationship between HCV infection and the development of insulin resistance. Our earlier studies have shown the development of hepatic steatosis in these HCV core gene transgenic mice after the age of 3 months. However, insulin resistance invariably preceded the occurrence of hepatic steatosis, indicating that insulin resistance is not a consequence of hepatic steatosis in these mice. Certainly, it is possible that insulin resistance in the core gene transgenic mice may be affected or aggravated after the occurrence of hepatic steatosis. On the other hand, insulin resistance may be one of the factors that cause hepatic steatosis, whereas the impairment of very-low-density lipoprotein (VLDL) secretion from the liver and hypo--oxidation of fatty acids are considered to be the bases of development of hepatic steatosis in the core gene transgenic mice.
The general mechanism underlying insulin resistance is not precisely understood and is considered to be multifactorial. Chiefly, it involves glucose consumption by the skeletal muscle and glucose production in the liver. Our current analysis revealed a failure of insulin in the suppression of HPG in the liver and an absence of suppression of glucose uptake by the muscles in the core gene transgenic mice. Combined, these results indicate the insulin resistance in the core gene transgenic mice is chiefly due to hepatic insulin resistance. An elevated intrahepatic TNF- level plays one of the roles in causing insulin resistance through suppressing insulin-induced tyrosine phosphorylation of IRS-1. It should be noted that TNF- levels are invariably elevated in the sera of patients with HCV infection. Moreover, restoration of insulin sensitivity after anti-TNF- antibody administration strongly supports the notion that TNF- is, at least in this animal model, a major factor for the development of insulin resistance in HCV infection. Taken together, insulin resistance in the core gene transgenic mice mainly depends on suppression of the inhibitory effect of insulin on hepatic glucose production. This is consistent with the observation that the core protein is present only in the liver but absent in the skeletal muscle of the core gene transgenic mice (Tsutsumi T., unpublished data, December 2002). Impairment in other undetermined pathways may also be responsible for the development of insulin resistance in HCV infection.
Insulin resistance alone does not always lead to the development of overt diabetes in humans or murine models. Particularly, in the models with the C57/BL6 strain, hyperplasia of the islets of Langerhans in the pancreas compensates for insulin resistance by secreting higher amounts of insulin. Along with a gain in body weight by being fed a high-calorie diet, the core gene transgenic mice but no control mice developed overt diabetes, showing that obesity is a risk factor for diabetes as observed in patients or as shown in animal models for diabetes unrelated to HCV infection. This observation would suggest that HCV infection confers insulin resistance and additional factors such as obesity, aging, or possibly inflammation may contribute to the complete development of overt diabetes. The effect of high-fat diet on control C57BL/6 mice may be milder in the current study compared with a previous study. However, there was a substantial increase in FPG levels in high-fat-diet-fed control mice compared with normal-diet-fed control mice (Figures 1B and 4B). In addition, at fed-state, serum insulin levels in high-fat-diet-fed control mice were significantly increased compared with those in normal-diet-fed control mice (Figures 1B and 4B). It is unclear why plasma glucose levels were not very high at fed-state in control mice, but one possible explanation is the lower calorie content in the current study than those in the previous report: 4.70 kcal/g for our high-fat diet vs. 5.55 kcal/g for high-calorie diet in the previous study. A shorter duration of high-fat diet than the previous study (2 months vs. 6 months) may be another possible explanation. Such a mild elevation in plasma glucose levels in high-fat-diet-fed C57BL/6 mice as the one observed in our study has also been described in previous studies.
In conclusion, the HCV core protein induces insulin resistance in transgenic mice without gain in body weight at young age. These results indicate a direct involvement of HCV per se in the pathogenesis of diabetes in patients with HCV infection and provide a molecular basis for insulin resistance in such a condition.
Editorial
Hepatitis C: A metabolic liver disease
Gastroenterology
March 2004, Volume 126, Number 3
Steven A. Weinman
Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA
It is now 15 years since the hepatitis C virus (HCV) was first identified and tremendous progress has been made in understanding and treating chronic hepatitis C. The disease has a surprising degree of complexity in its manifestations in the liver, but it is primarily characterized by inflammation, slowly progressive fibrosis, and development of hepatocellular carcinoma. Current treatment achieves viral clearance in less than half of patients and is poorly tolerated. As a result, the treatment of chronically infected individuals remains a major clinical challenge. The mechanisms of liver pathogenesis in hepatitis C are incompletely understood. It is well established that specific and innate immune responses to the presence of the replicating virus play a central role in liver pathogenesis, but considerable evidence suggests additional direct effects of the viral proteins on cellular processes involved in lipid metabolism, oxidative stress, mitochondrial function, gene expression, and signaling pathways.
Alterations in hepatic lipid and carbohydrate metabolism are commonly observed in chronic hepatitis C. Early after discovery of the virus, it was noted that chronic hepatitis C was associated with hepatic steatosis to a greater extent than other inflammatory liver diseases. At least in the case of genotype 3 infection, this is clearly a cytopathic effect of the virus because it is present in approximately two thirds of infected individuals and resolves completely in patients who achieve sustained virologic response to therapy. In addition, there is a highly reproducible occurrence of lipid accumulation in transgenic mouse and cellular models of hepatitis C, which further shows that metabolic alterations resulting in fat accumulation in hepatocytes can be a direct effect of HCV. A second and possibly related metabolic effect of hepatitis C is its association with type 2 diabetes. In 1994, Allison et al. reported that, in patients with cirrhosis of various causes, infection with HCV was independently associated with type 2 diabetes mellitus. Multiple reports have subsequently confirmed this observation in large retrospective, case-control, and population-based epidemiologic studies. The association of hepatitis C infection and diabetes is present, in fact, even before the onset of cirrhosis and diabetes does not associate with other viral conditions such as hepatitis B. This combination of steatosis and insulin resistance, seen frequently in patients with hepatitis C, shares many features with nonalcoholic steatohepatitis and raises the possibility that these metabolic abnormalities contribute to pathogenesis. How then can HCV lead to insulin resistance and predispose to the onset of type 2 diabetes? The challenges are to understand the mechanism of this association and its implications for the progression of liver disease and to exploit this knowledge for new therapeutic approaches. The underlying mechanisms explaining the connection between HCV and the onset of type 2 diabetes are just beginning to emerge. Several possibilities have been suggested. First, insulin resistance may just be a consequence of steatosis. Hepatic steatosis is recognized as a component of the metabolic syndrome, a condition that arises from insulin resistance and that precedes the onset of type 2 diabetes. Intracellular fat accumulation by itself causes insulin resistance. Experimental interventions that increase hepatic triglyceride content in mice result in defects in insulin signaling and impairment of the ability of insulin to suppress hepatic glucose production. Likewise, interventions reducing intracellular triglyceride content improve insulin sensitivity. These data suggest that the connection between hepatitis C and diabetes could be secondary to the ability of HCV to induce hepatic steatosis.
Another possible explanation is a direct effect of HCV proteins on insulin-signaling pathways. Insulin action is mediated by binding to the cell-surface insulin receptor, a ligand-activated tyrosine kinase. After insulin binding, the receptor undergoes autophosphorylation with activation of kinase activity. The signal is transmitted by subsequent tyrosine phosphorylation of a family of insulin-receptor substrates (IRS) (IRS-1, IRS-2, IRS-3, and IRS-4). After tyrosine phosphorylation, the IRS proteins serve as docking sites for src homology (SH) domain proteins, which transmit the signal to the downstream molecules that control glucose metabolism, lipid metabolism, and cell growth and differentiation. In muscle, IRS-1 plays the primary role in increasing glucose uptake, whereas inhibition of hepatic glucose production is primarily the result of IRS-2 activation. IRS-1 is also a major site of regulation of insulin responsiveness because serine phosphorylation of IRS-1 reduces its tyrosine phosphorylation by the insulin receptor. Tumor necrosis factor (TNF)- induces serine phosphorylation of IRS-1 and thus inhibits its tyrosine phosphorylation and signaling activity. Defects in insulin receptor and IRS-1 are present in insulin resistance and type 2 diabetes.
It is in this context that the article by Shintani et al. in this issue of GASTROENTEROLOGY makes important contributions to our understanding in this field. The authors used a well-characterized transgenic mouse model that specifically expresses the HCV core protein at high levels in hepatocytes. These animals develop hepatic steatosis and hepatocellular carcinoma at increasing ages but lack the liver inflammation characteristic of the human disease. In this study, the authors evaluated insulin response in young mice, before the onset of steatosis. They noted that transgenic mice have normal plasma glucose concentrations but have elevated circulating insulin levels and islet cell hyperplasia. When challenged by a high-fat diet, the transgenic mice, but not their nontransgenic littermates, had a loss of glucose tolerance, as manifested by elevated plasma glucose levels in the fed state. The authors performed euglycemic insulin clamp and muscle glucose uptake studies and found defective insulin inhibition of hepatic glucose production and normal muscle glucose uptake. A clue to the mechanism of insulin resistance in this model comes from the author's previous findings that intrahepatic cytokine levels, particularly TNF-, are elevated in the transgenic mice. Because TNF- is known to inhibit IRS-1 phosphorylation, they proceeded to investigate the link between hepatic TNF- activity and insulin signaling. Insulin-induced tyrosine phosphorylation of IRS-1 was inhibited in the transgenic mice and this effect was prevented by administration of an anti-TNF- antibody to the hepatitis C transgenic mice.
The study makes a major contribution by showing that hepatic insulin resistance can be induced solely by expression of the HCV core protein, and that signaling abnormalities in the insulin receptor-IRS-1 pathway are present before the development of steatosis. The observations of Shintani et al. in the mouse model are given further significance by the important recent studies of Aytug et al. who observed a defect in IRS-1 tyrosine phosphorylation in liver biopsy specimens derived from patients with hepatitis C but not in those from noninfected controls.
It is important, as always, to interpret this new information cautiously and with a healthy respect for what is still to be learned. Although these findings are provocative in suggesting that TNF-a mediated alterations in IRS-1 signaling are relevant to hepatitis C-induced insulin resistance, several issues need further understanding. The first relates to TNF- itself. The data showing TNF- elevation in this mouse model is convincing, but the mechanism by which HCV core protein increases TNF-a in the absence of inflammation is not clear. Is this a property of this mouse model only, or is it a universal aspect of HCV infection? In patients with hepatitis C, TNF- is clearly elevated. However, other inflammatory liver diseases, such as hepatitis B, also have elevated circulating and intrahepatic TNF-a yet these are not associated with diabetes. Clearly, other factors must be involved as well, and perhaps TNF-a is necessary but not sufficient to produce the insulin resistance. Another unanswered question is whether the IRS-1-related changes are actually the cause of the hepatic insulin resistance. Studies in IRS-1 and IRS-2 knockout mice have shown that the regulation of hepatic gluconeogenesis is almost exclusively a consequence of IRS-2-mediated signaling, whereas the hepatic effects of IRS-1 are more involved in cell growth regulation. Shintani et al. did not observe an effect on IRS-2 in their model. Further investigation is needed to understand how insulin resistance is generated and whether IRS-1 is or is not causally related.
Although insulin resistance was present in the young mice, it was not until the mice were challenged with a high-fat diet that diabetes occurred. This is analogous to the clinical situation in which environmental stressors, such as those related to obesity, must be added to an underlying genetic susceptibility for type 2 diabetes to occur. In this study, the mice that developed diabetes after high-fat feeding were 4 months old and it is likely that, by that time, there was an increase in hepatic triglyceride content. Thus, it is very possible that the increased fatty deposits in the liver of the transgenic mice, together with the impairment in insulin signaling caused by the HCV core protein, led to the onset of diabetes.
A body of epidemiologic work suggests that the presence of diabetes and hepatic steatosis are risk factors of more rapid fibrosis progression in chronic hepatitis C. Studies evaluating the relationship of steatosis with the onset of diabetes in hepatitis C are eagerly awaited, because interventions that prevent the development of steatosis would have the potential to prevent HCV-associated type 2 diabetes and to slow the progression of the disease itself. The article by Shintani et al. makes an important contribution to putting the HCV-diabetes association on a mechanistic footing, thus elevating it from a curious association to an important disease process.
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