Liver Of Streptozotocin-diabetic Rats Research Articles

Evaluation of Antidiabetic, Antioxidant and Anti-Hyperlipidemic Effects of Solanum indicum Fruit Extract in Streptozotocin-Induced Diabetic Rats.

Globally, diabetes mellitus is the most common cause of premature mortality after cardiovascular diseases and tobacco chewing. It is a heterogeneous metabolic disorder characterised by the faulty metabolism of carbohydrates, fats and proteins as a result of defects in insulin secretion or resistance. It was estimated that approximately 463 million of the adult population are suffering from diabetes mellitus, which may grow up to 700 million by 2045. Solanum indicum is distributed all over India and all of the tropical and subtropical regions of the world. The different parts of the plant such as the roots, leaves and fruits were used traditionally in the treatment of cough, asthma and rhinitis. However, the hypoglycaemic activity of the plant is not scientifically validated. The present study aimed to evaluate the antioxidant, antidiabetic and anti-hyperlipidaemic activity of methanolic fruit extract of Solanum indicum (SIE) in streptozotocin (STZ) induced diabetic rats. Experimentally, type II diabetes was induced in rats by an i.p. injection of STZ at a dose of 60 mg/kg. The effect of the fruit extract was evaluated at doses of 100 and 200 mg/kg body weight in STZ-induced diabetic rats for 30 days. The oral administration of fruit extract caused a significant (p < 0.05) reduction in the blood glucose level with a more prominent effect at 200 mg/kg. The fruit extract showed dose-dependent α-amylase and α-glycosidase inhibitory activity. It reduced the serum cholesterol and triglyceride levels remarkably in diabetic rats compared to normal. The extract showed the reduced activity of endogenous antioxidants, superoxide dismutase, glutathione peroxidase and catalase in the liver of STZ diabetic rats. The result confirmed that the fruit extract of Solanum indicum showed a dose-dependent blood glucose lowering effect and significantly reduced elevated blood cholesterol and triglycerides. It prevented oxidative stress associated with type II diabetes in STZ rats.

EXPRESSION AND ACTIVITY OF AMINOTRANSFERASES IN THE LIVER OF STREPTOZOTOCIN-DIABETIC RATS: THE EFFECT OF MIFEPRISTONE.

Interventions that suppress hepatic gluconeogenesis from amino acids may be useful for improving glycemic control in diabetic patients. It was shown that administration of glucocorticoid receptor antagonist Mifepristone (MIF) leads to variously pronounced changes in the alanine-, aspartate-, tyrosine- aminotransferases (ALT, AST, TAT) activity in the liver of experimental animals. It has been suggested that this selective effect of MIF may be related to differences in the expression of the corresponding genes. The aim of the study was to investigate the gene expression and activity of ALT, AST and TAT in the liver of rats with streptozotocin-related diabetes (StD) under the long-term oral MIF administration. Male Wistar rats (n=48) with StD under the 10-days oral MIF administration were used. It was measured the activity of ALT, AST, TAT enzymes and relative expression of this genes in the liver of experimental animals. In rats with StD the gene expression of all three studied aminotransferases in the liver was statistically significantly increased and their activity was increased as well. MIF administration did not change the studied genes expression and enzymes activity to healthy rats and caused a decrease in expression of ALT and AST genes and activity of these enzymes to rats with StD. However, the expression of the TAT gene and the activity of this enzyme in the liver of rats with StD increased upon MIF administration in comparison with animals with StD. The introduction of MIF against the background of StD reduces the expression of genes and the activity of ALT and AST in the liver, what determine the transamination of amino acids to include them in gluconeogenesis, but increases the expression of genes and the activity of TAT, what determine the inclusion of tyrosine in the biogenic amines synthesis. The mechanisms of such selectivity require further study.

Hepatocellular Neoplasms Induced by Low-Number Pancreatic Islet Transplants in Autoimmune Diabetic BB/Pfd Rats

It has been shown that combined high local hyperinsulinism and hyperglycemia after low-number islet transplantation into the livers of streptozotocin-diabetic rats lead to the development of hepatocellular neoplasms but a substantial cocarcinogenic effect of genotoxic streptozotocin could not be ruled out completely. Thus, we herein investigated this model in BB/Pfd rats (n = 805; nine experimental groups), which develop spontaneous autoimmune diabetes similar to human type 1 diabetes. After low-number islet transplantation (n = 450), the liver acini downstream of the islets show insulin-induced alterations: massive glycogen and/or fat accumulation, translocation of the insulin receptor, decrease in glucose-6-phosphatase activity, increase in expression of insulin-like growth factor (IGF)-I, IGF-II/mannose-6-phosphate receptor, insulin receptor substrate-1, Raf-1, and Mek-1, corresponding to clear cell preneoplastic foci of altered hepatocytes known from chemical hepatocarcinogenesis and identical to that in streptozotocin-diabetic Lewis rats. After 6 months, many altered liver acini progressed to other types of preneoplasias often accompanied by an overexpression of the glutathione-S transferase (placental form), IGF-I receptor, and transforming growth factor (TGF)-alpha. After 12 to 15 and 15 to 18 months, 52% and 100% of the animals showed one or multiple hepatocellular adenomas or hepatocellular carcinomas (HCCs), respectively. Conclusively, this study identifies combined hyperinsulinism and hyperglycemia as a carcinogenic mechanism for the development of HCCs in diabetic rats. Hepatocarcinogenesis is independent from additional genotoxic compounds (i.e., streptozotocin), but is primarily triggered by increased intracellular insulin signaling via pathways associated with cell growth and proliferation, such as the Ras-Raf-mitogen-activated protein kinase pathway and the IGF system, and secondarily involves other growth factors, such as TGF-alpha.

Hepatocellular neoplasms induced by low-number pancreatic islet transplants in autoimmune diabetic BB/Pfd rats

It has been shown that combined high local hyperinsulinism and hyperglycemia after low-number islet transplantation into the livers of streptozotocin-diabetic rats lead to the development of hepatocellular neoplasms but a substantial cocarcinogenic effect of genotoxic streptozotocin could not be ruled out completely. Thus, we herein investigated this model in BB/Pfd rats ( n = 805; nine experimental groups), which develop spontaneous autoimmune diabetes similar to human type 1 diabetes. After low-number islet transplantation ( n = 450), the liver acini downstream of the islets show insulin-induced alterations: massive glycogen and/or fat accumulation, translocation of the insulin receptor, decrease in glucose-6-phosphatase activity, increase in expression of insulin-like growth factor (IGF)-I, IGF-II/mannose-6-phosphate receptor, insulin receptor substrate-1, Raf-1, and Mek-1, corresponding to clear cell preneoplastic foci of altered hepatocytes known from chemical hepatocarcinogenesis and identical to that in streptozotocin-diabetic Lewis rats. After 6 months, many altered liver acini progressed to other types of preneoplasias often accompanied by an overexpression of the glutathione- S transferase (placental form), IGF-I receptor, and transforming growth factor (TGF)-α. After 12 to 15 and 15 to 18 months, 52% and 100% of the animals showed one or multiple hepatocellular adenomas or hepatocellular carcinomas (HCCs), respectively. Conclusively, this study identifies combined hyperinsulinism and hyperglycemia as a carcinogenic mechanism for the development of HCCs in diabetic rats. Hepatocarcinogenesis is independent from additional genotoxic compounds (i.e., streptozotocin), but is primarily triggered by increased intracellular insulin signaling via pathways associated with cell growth and proliferation, such as the Ras-Raf-mitogen-activated protein kinase pathway and the IGF system, and secondarily involves other growth factors, such as TGF-α. (Cancer Res 2006; 66(3): 1833-43)

Apolipoprotein A-IV mRNA Overexpression in Early Preneoplastic Hepatic Foci Induced by Low-number Pancreatic Islet Transplants in Streptozotocin-diabetic Rats

After low-number transplantation of islets of Langerhans into the liver of streptozotocin-diabetic rats, the hepatocytes in the acini, draining the blood from the islets, are exposed to a local hyperinsulinemia, whereas the remaining tissue is affected by hypoinsulinemia. In this model, insulin induces alterations that resemble preneoplastic foci of altered hepatocytes (FAH) and develop into hepatocellular tumors in later stages of carcinogenesis. In rodents, apolipoprotein A-IV (A-IV) is synthesized in the small intestine and the liver. Whereas intestinal production is mainly influenced by lipid intake and chylomicrone formation, little is known about mechanisms regulating hepatic A-IV synthesis. As it is known that insulin modulates lipoprotein metabolism in different ways, we investigated the effect of insulin on hepatocytic A-IV mRNA expression in this model. After Laser microdissection of FAH and quantitative RT-PCR (LightCycler), we found a 3.2 to 7.4-fold increase of A-IV mRNA in the FAH. To the best of our knowledge, these results represent the first data of insulin-stimulated A-IV mRNA overexpression in rat hepatocytes in vivo, and are in line with previously reported results of experiments with cultured hepatocytes. It remains to be elucidated whether A-IV mRNA overexpression is only an epiphenomenon of insulin action or is relevant for hepatocarcinogenesis in this model.

In vivo treatment with stobadine prevents lipid peroxidation, protein glycation and calcium overload but does not ameliorate Ca 2+-ATPase activity in heart and liver of streptozotocin-diabetic rats: comparison with vitamin E

Hyperglycemia leads to excess production of reactive oxygen species (ROS), lipid peroxidation and protein glycation that may impair cellular calcium homeostasis and results in calcium sequestration and dysfunction in diabetic tissues. Stobadine (ST) is a pyridoindole antioxidant has been postulated as a new cardio- and neuroprotectant. This study was undertaken to test the hypothesis that the treatment with ST inhibits calcium accumulation, reduces lipid peroxidation and protein glycation and can change Ca 2+,Mg 2+-ATPase activity in diabetic animals. The effects of vitamin E treatment were also evaluated and compared with the effects of combined treatment with ST. Diabetes was induced by streptozotocin (STZ, 55 mg/kg i.p.). Some of diabetic rats and their age-matched controls were treated orally with a low dose of ST (24.7 mg/kg/day), vitamin E (400–500 IU/kg/day) or ST plus vitamin E for 10 weeks. ST and vitamin E separately produced, in a similar degree, reduction in diabetes-induced hyperglycemia. Each antioxidant alone significantly lowered the levels of plasma lipid peroxidation, cardiac and hepatic protein glycation in diabetic rats but vitamin E treatment was found to be more effective than ST treatment alone. Diabetes-induced increase in plasma triacylglycerol levels was not significantly altered by vitamin E treatment but markedly reduced by ST alone. The treatment with each antioxidant completely prevented calcium accumulation in diabetic heart and liver. Microsomal Ca 2+,Mg 2+-ATPase activity significantly decreased in both tissues of untreated diabetic rats. ST alone significantly increased microsomal Ca 2+,Mg 2+-ATPase activity in the heart of normal rats. However, neither treatment with ST nor vitamin E alone, nor their combination did change cardiac Ca 2+,Mg 2+-ATPase activity in diabetic heart. In normal rats, neither antioxidant had a significant effect on hepatic Ca 2+,Mg 2+-ATPase activity. Hepatic Ca 2+,Mg 2+-ATPase activity of diabetic rats was not changed by single treatment with ST, while vitamin E alone completely prevented diabetes-induced inhibition in microsomal Ca 2+,Mg 2+-ATPase activity in liver. Combined treatment with ST and vitamin E provided more benefits in the reduction of hyperglycemia and lipid peroxidation in diabetic animals. This study describes potential mechanisms on cellular effects of ST in the presence of diabetes-induced hyperglycemia that may delay or inhibit the development of diabetic complications. The use of ST together with vitamin E can better control hyperglycemia-induced oxidative stress.

Analysis of the IGF Axis in Preneoplastic Hepatic Foci and Hepatocellular Neoplasms Developing after Low-Number Pancreatic Islet Transplantation into the Livers of Streptozotocin Diabetic Rats

Preneoplastic hepatic foci have been demonstrated in liver acini, which drain the blood from intraportally transplanted pancreatic islets in streptozotocin-induced diabetic rats with mild persisting diabetes. In long-term studies of this animal model, hepatocellular adenomas and carcinomas (HCC) developed after a sequence of characteristic preneoplastic hepatic foci. In this experimental model, the local hyperinsulinism is thought to have a causative role. Because insulin and the insulin-like growth factor (IGF) axis are closely linked, an altered gene expression of the IGF axis components is likely. Therefore, preneoplastic hepatic foci and HCC were studied for the expression of IGF axis components. Glycogen-storing “early” preneoplastic hepatic foci were detectable several days after pancreatic islet transplantation. Northern blot analysis, in-situ hybridization, and immunohistochemical studies of these “early” lesions demonstrated increased expressions of IGF-I and IGF binding protein-4 (IGFBP-4) in altered parenchymal cells, and a decreased expression of IGFBP-1. IGF-II was not detected in these preneoplastic foci. HCC arising in this model had decreased expressions of IGF-I and IGFBP-4 but IGFBP-1 expression was not significantly altered. Some HCC showed a more than 100-fold overexpression of IGF-II, whereas other tumors were completely negative for IGF-II expression. Low IGF-I receptor expression was detected in preneoplastic foci and adjacent nonaltered liver tissue. However, HCC tissue consistently showed an increased IGF-I receptor expression, rendering these tissues susceptible to the mitogenic effects of IGF. The altered gene expression in glycogen-storing preneoplastic hepatic foci, especially the up-regulation of IGF-I and IGFBP-4 with the down-regulation of IGFBP-1, resemble the insulin-dependent regulation of these components in normal rat hepatocytes. These data agree with previous studies demonstrating a correspondence of the focal character, morphology, and enzyme pattern of preneoplastic hepatic foci with insulin effects on hepatocytes. The development from preneoplastic foci to HCC may be driven by insulin itself and/or an altered IGF axis component or yet unidentified factors.

Nonparenchymal cells in chronically hyperinsulinemic liver acini of diabetic rats, with special regard to hepatic stellate cells

Background/Aims: An increase in proliferative activity and other distinct hepatocellular alterations — resembling preneoplastic foci and progressing to hepatocellular tumors — have been shown to develop in liver acini draining the blood from islets of Langerhans, transplanted through the portal vein into the liver of streptozotocin-diabetic rats. Methods: Altered and unaltered liver acini were investigated for possible changes in hepatic stellate cells 4–76 days after islet transplantation. Results: Corresponding to a significant increase in the hepatocellular volume, the volume density of total non-parenchymal cells was significantly reduced in altered compared to unaltered liver acini. With regard to the total nonparenchymal cell volume, the hepatic stellate cell fraction was not different, whereas the fraction of Kupffer cells was significantly reduced and the fraction of sinusoidal endothelial cells was significantly increased in altered compared to unaltered liver acini, respectively. The volume density as well as the single volume of the hepatic stellate cell mitochondria increased significantly in altered compared to unaltered liver acini. Hepatic stellate cell lipid droplets did not show significant differences between altered and unaltered liver acini. In situ hybridization for hepatocyte growth factor mRNA showed no differences in intensity of the specific signals in hepatic stellate cells of altered versus unaltered liver acini. The transplanted islets were negative for hepatic growth factor mRNA. Conclusions: The results suggest that hepatic growth factor production by hepatic stellate cells or by islet cells is not relevant to hepatocellular proliferative activity in altered liver acini.

Identification of an Insulin-responsive Element in the Rat Insulin-like Growth Factor-binding Protein-3 Gene

The hepatic expression and serum levels of insulin-like growth factor-binding protein-3 (IGFBP-3) are decreased in insulin-dependent and insulin-resistant diabetes. Insulin increases hepatic IGFBP-3 expression by enhancing gene transcription. This report identifies sequences within the IGFBP-3 promoter that are necessary and sufficient for the response to insulin in hepatic nonparenchymal cells. By transient transfection, we mapped the insulin response element to the -1150 to -1124 base pair (bp) region of the rat IGFBP-3 promoter. Three tandem repeats of the -1150 to -1117 bp region conferred insulin responses in a heterologous promoter. Gel shift analyses revealed a 3-fold increase in DNA-protein complex formation with nuclear extracts obtained from insulin-stimulated nonparenchymal cells compared with cells incubated without insulin and revealed 3-4-fold decrease in complex formation with nuclear extracts obtained from the livers of streptozotocin-diabetic rats compared with control rats. Mutational analysis of this 34-bp region showed a core sequence of 10 bp (-1148 to -1139) that is critical for interaction with insulin-induced trans-acting factors. Southwestern blotting revealed a approximately 90-kDa protein that was increased 2-3-fold by the addition of insulin. Thus, we have identified cis-acting DNA sequences that are responsible for regulation of IGFBP-3 transcription by insulin and essential for binding of insulin-responsive nuclear factors.

Post-transcriptional regulation of insulin-like growth factor binding protein-2 mRNA in diabetic rat liver

IGFBP-1 and IGFBP-2 mRNAs are increased in the livers of streptozotocin-diabetic rats. A corresponding increase is observed in transcription of the IGFBP-1 but not the IGFBP-2 gene, indicating that the increase in steady-state levels of IGFBP-2 mRNA is a post-transcriptional effect. IGFBP-1 and IGFBP-2 mRNAs also differ in the rapidity of their response to insulin treatment: hepatic IGFBP-1 mRNA is normalized within 1 h, IGFBP-2 mRNA decreases more slowly. These differences suggest that IGFBP-2 may provide more chronic adaptation to metabolic change than IGFBP-1.

Rat hepatic glutaminase: Purification and immunochemical characterization

A method for the purification of phosphate-activated glutaminase from the liver of streptozotocin-diabetic rats is described. The procedure involves solubilization of glutaminase activity from isolated mitochondria by sonication, followed by ammonium sulfate precipitation, polyethylene glycol precipitation, and sequential chromatography on DEAE, hydroxylapatite, and zinc-chelated resins. The enzyme was purified 600-fold to a specific activity of 31–57 U/mg protein. The purified enzyme has an apparent subunit molecular mass of 58,000-Da and is >80% pure by scanning densitometry of sodium dodecyl sulfate-polyacrylamide gels. The purified enzyme has an apparent K m for glutamine of 17 m m and a pH optimum between 7.8 and 8.2. The physical and kinetic properties of this enzyme are similar to those of the enzyme from normal rat liver. Polyclonal antibodies raised against the enzyme specifically inhibit hepatic glutaminase activity and react primarily with a 58,000-Da peptide in liver fractions on immunoblots. These antibodies were used in equivalence point titrations and immunoblots to provide evidence for increased concentration of glutaminase protein in the liver of diabetic rats with no change in specific activity of the enzyme. In addition, the antibodies cross-react, at low affinity, with kidney-type glutaminases. On immunoblots, the antibodies did not react with fetal liver, mammary gland, or lung. Antibodies to rat hepatic glutaminase should prove useful as tools to study the long-term regulation of the enzyme.

Islet transplantation in experimental diabetes of the rat. X. Induction of cytotoxic antibodies and islet-cell-surface-antibodies after homologous islet transplantation in rats.

The mechanisms of islet allograft rejection are still unknown. 350 allogeneic rat islets (BDE/Han) were transplanted into the liver of Streptozotocin-diabetic rats (Lewis/Han). Animals were killed either on day 4, 7, 10 or 14. Immunofluorescence analysis for islet-cell-surface-antibodies (ICSA) was performed on BDE-islet-single-cells yielded by Collagenase-Dispase digestion. In order to detect cytoplasmic islet cell antibodies, immunofluorescence technique was used on BDE-pancreas cryosections. Cytotoxicity assay was tried out using 51Cr-release-test. After successful intraportal transplantation, rejection took place between day 7 and 10. ICSA could be observed on day 10 and 14. Cytoplasmic antibodies were not detected at any time. A specific cytotoxic activity in the recipient serum revealed itself on day 10 (23.0%) and day 14 (27.7%). Transplantation of freshly isolated allogeneic islets induced ICSA and cytotoxic antibodies. These results indicate an involvement of humoral mechanisms in islet allograft rejection.

Activity of some enzymes involved in the metabolism of polyamines in the liver of streptozotocin-diabetic rats

1. 1. The effect of diabetes on some enzymes of polyamine metabolism was studied in male rats 1–12 days after administration of streptozotocin. 2. 2. Hepatic ornithine decarboxylase activity decreased in the first days after the administration, but increased thereafter. The decrease was not due to an alteration of the ODC-antizyme concentration, nor to a posttranslational modification catalyzed by transglutaminase. 3. 3. S-adenosylmethionine decarboxylase and ornithine transaminase were both increased. 4. 4. Spermicline acetyltransferase activity was practically unchanged, while its inactivating factor was markedly decreased.

Effect of 1,3-diaminopropan-2-o1, an inhibitor of ornithine decarboxylase, on the metabolic response of liver to insulin.

The effect of diaminopropanol, an inhibitor of polyamine synthesis, on the metabolic response of liver to insulin was studied in streptozotocin-diabetic rats. Insulin elicited a prompt and very marked increase in ornithine and S-adenosylmethionine decarboxylase activities and in putrescine concentration. Pretreatment of rats with diaminopropanol prevented the increase in the decarboxylases and resulted in decreased spermidine and spermine content of liver. The insulin-induced increase in glycogen content was depressed by 50% and the increase in the rate of lipogenesis in vivo was completely prevented by prior injection of diaminopropanol. These studies implicate altered polyamine metabolism in the metabolic response of liver of streptozotocin-diabetic rats to insulin.

Studies on niacin biosynthesis from 3-hydroxyanthranilic acid in streptozotocin diabetic rats in vivo and in vitro.

Biosynthesis of niacin from 3-hydroxyanthranilic acid (3-OHAA) in normal and streptozotocin diabetic rats was studied in vivo and in vitro. Streptozotocin (SZ) diabetic rats were found to excrete lesser quantities of 3-OHAA, quinolinic acid, niacin and N1-methylnicotinamide (nmn) in their urines following 3-OHAA administration than corresponding normal rats. In vitro studies indicated that SZ diabetic livers form less quinolinic acid from 3-OHAA than normal livers. It appears that this may be due to elevated picolinic carboxylase activity in the livers of SZ diabetic rats.

Glycogen synthesis in the perfused liver of streptozotocin-diabetic rats.

1. Net glycogen accumulation was measured in sequentially removed samples during perfusion of the liver of starved streptozotocin-diabetic rats, and shown to be significantly impaired, compared with rates in normal (starved) rats. 2. In perfusions of normal livers with glucose plus C3 substrates, there was an increase in the proportion of glycogen synthetase 'a', compared with that in the absence of substrates. This response to substrates, followed in sequential synthesis and enzymic sensitivity in the perfused liver of diabetic rats were reversed by pretreatment in vivo with glucose plus fructose, or insulin. Glucose alone did not produce this effect. 4. Glucose, fructose, insulin or cortisol added to e perfusion medium (in the absence of pretreatment in vivo) did not stimulate glycogen synthesis in diabetic rats. 5. In intact diabetic rats, there was a decline in rates of net hepatic glycogen accumulation, and the response of glycogen synthetase to substrates. The most rapid rates of synthesis were obtained after fructose administration. 6. These results demonstrate that there is a marked inherent impairment in hepatic glycogen synthesis in starved diabetic rats, which can be rapidly reversed in vivo but no in perfusion. Thus hepatic glycogen synthesis does not appear to be sensitive to either the short-term direct action of insulin (added alone to perfusions) of to long-term insulin deprivation in vivo. The regulatory roles of substrates, insulin and glycogen synthetase in hepatic glycogen accumulation are discussed.

Glucose, glucose-6-phosphate, lactate and pyruvate content of the retina, blood and liver of streptozotocin-diabetic rats fed sucrose- or starch-rich diets.

The feeding of starch- or sucrose-rich diets to normal rats for 20 days resulted in a significant increase in lactate level and L/P ratio in the retina in the sucrose-fed animals. There was no correlation between the glucose and lactate content of the blood and retina and it was concluded that the increased lactate content of the retina was derived from the fructose moiety of the sucrose. Liver hypertrophy occurred in both normal and diabetic animals fed the sucrose diet. When streptozotocin-diabetic rats were maintained for 20 days on the same diets significant increases in the lactic acid content of the blood and liver occurred, but the high level of lactate in the retina of the sucrose-fed normal rats was not further elevated. The feeding of sucrose to diabetic rats resulted in better metabolic control of both blood glucose and lactate as compared with the maintenance of these animals on a starch diet.