Potentiation of carbon tetrachloride hepatotoxicity and lethality in type 2 diabetic rats

ABSTRACT There is a need for well characterized and economical type 2 diabetic model that mimics the human disease. We have developed a type 2 diabetes rat model that closely resembles the diabetic patients and takes only 24 days to develop robust diabetes. Nonlethal doses of allyl alcohol (35 mg/kg i.p.), CCl 4 (2 ml/kg i.p.), or thioacetamide (300 mg/kg i.p.) yielded 80 to 100% mortality in diabetic rats. The objective of the present study was to investigate two hypotheses: higher CCl 4 bioactivation and/or inhibited compensatory tissue repair were the underlying mechanisms for increased CCl 4 hepatotoxicity in diabetic rats. Diabetes was induced by feeding high fat diet followed by a single dose of streptozotocin on day 14 (45 mg/kg i.p.) and was confirmed on day 24 by hyperglycemia, normoinsulinemia, and oral glucose intolerance. Time course studies (0 -96 h) of CCl 4 (2 ml/kg i.p.) indicated that although initial liver injury was the same in nondiabetic and diabetic rats, it progressed only in the latter, culminating in hepatic failure, and death. Hepatomicrosomal CYP2E1 protein and activity, lipid peroxidation, glutathione, and 14 CCl 4 covalent binding to liver tissue were the same in both groups, suggesting that higher bioactivation-based injury is not the mechanism. Inhibited tissue repair resulted in progression of injury and death in diabetic rats, whereas in the nondiabetic rats robust tissue repair resulted in regression of injury and survival after CCl 4 administration. These studies show high sensitivity of type 2 diabetes to model hepatotoxicants and suggest that CCl 4 hepatotoxicity is potentiated due to inhibited tissue repair. Several animal models resembling type 2 diabetes either occur spontaneously or can be induced experimentally. Most of the commonly used models of type 2 diabetes are genetic and have the disadvantage of prohibitive costs, unavailability, and failure to represent etiology of human disease. Consumption of high fat diet leads to insulin resistance and is considered to be a major predisposing factor for type 2 diabetes To address this need, we have refined and characterized an existing model based on high fat diet and a single dose of streptozotocin (STZ, 45 mg/kg i.p.). The principle behind the development of type 2 diabetes is simple. High fat diet elicits insulin resistance, and the rats maintain normoglycemia due to compensatory hyperinsulinemia. Administration of STZ (45 mg/kg i.p.) decreases insulin levels, destroying a population of pancreatic ␤-cells such that the insulin-resistant rats are now unable to maintain normal glucose levels and develop hyperglycemia, even though insulin levels in these rats are comparable with normal diet-fed normoglycemic rats. This is exactly what is seen in human diabetes where insulin resistance precedes hyperglycemia, thereby making this model a good representative of human type 2 diabetic (DB) condition. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.058834. ABBREVIATIONS: STZ, streptozotocin; DB, diabetic; NDB, nondiabetic; ND ϩ STZ, normal diet-fed rats injected streptozotocin; TA, thioacetamide; AA, allyl alcohol; SD, Sprague-Dawley; 3 H-T, tritiated thymidine; HFD, high fat diet-fed rats injected citrate buffer; HFD ϩ STZ, high fat diet-fed rats injected streptozotocin; ND, normal diet fed rats injected citrate buffer; ALT, alanine aminotransferase; AST, aspartate aminotransferase; PCNA, proliferating cell nuclear antigen; MES, 2-(N-morpholino)ethanesulfonic acid

Upregulation of calpastatin in regenerating and developing rat liver: Role in resistance against hepatotoxicity

Acute liver failure induced by hepatotoxic drugs results from rapid progression of injury. Substantial research has shown that timely liver regeneration can prevent progression of injury leading to a favorable prognosis. However, the mechanism by which compensatory regeneration prevents progression of injury is not known. We have recently reported that calpain released from necrotic hepatocytes mediates progression of liver injury even after the hepatotoxic drug is cleared from the body. By examining expression of calpastatin (CAST), an endogenous inhibitor of calpain in three liver cell division models known to be resistant to hepatotoxicity, we tested the hypothesis that increased CAST in the dividing hepatocytes affords resistance against progression of injury. Liver regeneration that follows CCL 4-induced liver injury, 70% partial hepatectomy, and postnatal liver development were used. In all three models, CAST was upregulated in the dividing/newly divided hepatocytes and declined to normal levels with the cessation of cell proliferation. To test whether CAST overexpression confers resistance against hepatotoxicity, CAST was overexpressed in the livers of normal SW mice using adenovirus before challenging them with acetaminophen (APAP) overdose. These mice exhibited markedly attenuated progression of liver injury and 57% survival. Whereas APAP-bioactivating enzymes and covalent binding of the APAP-derived reactive metabolites remained unaffected, degradation of calpain specific target substrates such as fodrin was significantly reduced in these mice. In conclusion, CAST overexpression could be used as a therapeutic strategy to prevent progression of liver injury where liver regeneration is severely hampered. Copyright © 2006 by the American Association for the Study of Liver Diseases