Reduction in Obesity-Related Hepatic Fibrosis by SR1664.

Peroxisome-proliferator-activated receptor gamma (PPARγ) is a transcription factor with adipogenic, insulin-sensitizing, and antifibrotic properties. Strong PPARγ activators, such as the thiazolidinediones, can induce unwanted effects such as edema, weight gain, and bone loss, and therefore selective modulators of PPARγ are in development. We previously reported that one selective PPARγ modulator, SR1664, reduced toxin-induced hepatic fibrosis and the activation of hepatic stellate cells (HSCs), the main collagen-producing liver cell in fibrosis. In this study, we used a high fat and high carbohydrate (HFHC) model of hepatic steatosis and fibrosis to determine the effect of SR1664. Mice were placed on a standard chow or HFHC diet for 16 weeks, with SR1664 or control treatment for the final 4 weeks. SR1664 did not alter weight gain or fasting insulin or glucose levels. The size of lipid droplets in the HFHC group was reduced by SR1664, but there was no effect on total liver triglyceride levels. The degree of fibrosis was significantly reduced by SR1664 in mice on the HFHC diet, and this was accompanied by a decrease in activated HSC. In summary, SR1664 improved insulin sensitivity and reduced fibrosis in the HFHC diet, suggesting selective PPARγ modulation is effective in obesity-related liver fibrosis.

A Selective PPARγ Modulator Reduces Hepatic Fibrosis.

Hepatic fibrosis is the accumulation of excess collagen as a result of chronic liver injury. If left unabated, hepatic fibrosis can lead to the disruption of the liver architecture, portal hypertension, and increased risk of progression to cirrhosis and hepatocellular carcinoma. The thiazolidinedione class of antidiabetic drugs, through their target peroxisome proliferator-activated receptor γ (PPARγ), have protective effects against liver fibrosis, and can inhibit the profibrotic activity of hepatic stellate cells, the major collagen-producing liver cells. However, these drugs have been ineffective in the treatment of established fibrosis, possibly due to side effects such as increased weight and adiposity. Recently, selective PPARγ modulators that lack these side effects have been identified, but their role in treating fibrosis has not been studied. In this study, we tested the effectiveness of one of these selective modulators, SR1664, in the mouse carbon tetrachloride model of established hepatic fibrosis. Treatment with SR1664 reduced the total and type 1 collagen content without increasing body weight. The abundance of activated hepatic stellate cells was also significantly decreased. Finally, SR1664 inhibited the profibrotic phenotype of hepatic stellate cells. In summary, a selective PPARγ modulator was effective in the reduction of established hepatic fibrosis and the activated phenotype of hepatic stellate cells. This may represent a new treatment approach for hepatic fibrosis.

Serelaxin increases the antifibrotic action of rosiglitazone in a model of hepatic fibrosis.

AIM: To determine the effect of combined serelaxin and rosiglitazone treatment on established hepatic fibrosis. METHODS: Hepatic fibrosis was induced in mice by carbon tetrachloride administration for 6 wk, or vehicle alone (nonfibrotic mice). For the final 2 wk, mice were treated with rosiglitazone, serelaxin, or both rosiglitazone and serelaxin. Serum liver enzymes and relaxin levels were determined by standard methods. The degree of liver collagen content was determined by histology and immunohistochemistry. Expression of type I collagen was determined by quantitative PCR. Activation of hepatic stellate cells was assessed by alpha-smooth muscle actin (SMA) levels. Liver peroxisome proliferator activated receptor-gamma coactivator 1 alpha (PGC1α) was determined by Western blotting. RESULTS: Treatment of mice with CCl4 resulted in hepatic fibrosis as evidenced by increased liver enzyme levels (ALT and AST), and increased liver collagen and SMA. Monotherapy with either serelaxin or rosiglitazone for 2 wk was generally without effect. In contrast, the combination of serelaxin and rosiglitazone resulted in significantly improved ALT levels (P < 0.05). Total liver collagen content as determined by Sirius red staining revealed that only combination treatment was effective in reducing total liver collagen (P < 0.05). These results were supported by immunohistochemistry for type I collagen, in which only combination treatment reduced fibrillar collagen levels (P < 0.05). The level of hepatic stellate cell activation was modestly, but significantly, reduced by serelaxin treatment alone, but combination treatment resulted in significantly lower SMA levels. Finally, while hepatic fibrosis reduced liver PGC1α levels, the combination of serelaxin and rosiglitazone resulted in restoration of PGC1α protein levels. CONCLUSION: The combination of serelaxin and rosiglitazone treatment for 2 wk was effective in significantly reducing established hepatic fibrosis, providing a potential new treatment strategy.

Tacrolimus and sirolimus have distinct effects on insulin signaling in male and female rats.

Although the contribution of the immunosuppressants tacrolimus (TAC) and sirolimus (SIR) to the development of posttransplant diabetes mellitus (PTDM) are being increasingly recognized, the mechanisms of immunosuppressant-induced hyperglycemia are unclear. SIR induces insulin resistance predominantly, but is associated with ß-cell dysfunction in rodents. TAC affects islet function but is associated with worsening insulin sensitivity in a few, and improvement in some, clinical studies. We sought to clarify the contributions of TAC and SIR to insulin resistance and islet function. Four groups of male and female Sprague-Dawley rats received TAC, SIR, TAC and SIR, or control for 2 weeks. All rats were administered an oral glucose challenge at the end of treatment. Half the groups were sacrificed 10 minutes after administration of regular insulin whereas the other half did not receive insulin before sacrifice. Liver, pancreas, fat, and muscle were harvested subsequently. Quantification of Western blots revealed that SIR and TAC plus SIR suppressed the phospho-Akt (pAkt)-to-Akt ratios in liver, muscle, and fat compared with control, regardless of sex. TAC alone did not impair the pAkt-to-Akt ratios in any of the tissues in male and female rats. ß-Cell mass was reduced significantly after TAC treatment in male rats. SIR did not affect ß-cell mass, regardless of sex. Our study demonstrated very clearly that SIR impairs insulin signaling, without any effect on ß-cell mass, and TAC does not impair insulin signaling but reduces ß-cell mass. Our efforts are key to understanding the mechanisms of immunosuppressant-induced hyperglycemia and to tailoring treatments for PTDM.

Metformin improves immunosuppressant induced hyperglycemia and exocrine apoptosis in rats.

BACKGROUND: Immunosuppressants are an important cause of posttransplantation diabetes mellitus. We have shown that tacrolimus and sirolimus induce hyperglycemia and hyperinsulinemia in normal rats. We hypothesized that metformin, given concurrently with tacrolimus and/or sirolimus, prevents disturbances in glucose and insulin metabolism. METHODS: Eight groups (n=6) of normal Sprague-Dawley rats were studied: four groups received tacrolimus, sirolimus, tacrolimus/sirolimus, or control for 14 days, and four more groups received similar treatments along with metformin. Daily glucoses were measured. All rats were administered an oral glucose challenge before sacrifice. Pancreata were analyzed by terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling staining and immunohistochemistry. RESULTS: Tacrolimus, sirolimus, and tacrolimus/sirolimus impaired glucose tolerance compared to control. Sirolimus and tacrolimus/sirolimus also increased random blood glucose levels. Sirolimus alone resulted in hyperinsulinemia after oral glucose challenge compared to control. In the sirolimus/metformin and tacrolimus/sirolimus/metformin groups, mean daily random glucose was no longer increased, although the response to glucose challenge was still impaired. Metformin decreased pancreatic exocrine and trended to decrease endocrine apoptosis in tacrolimus/sirolimus group and reduced islet insulin content in sirolimus group. CONCLUSIONS: This is the first study to show that metformin can improve immunosuppressant-induced hyperglycemia, when administered concurrently, and reduces exocrine apoptosis (reducing the impact on potential islet progenitor cells).

Gene expression of vitamin D metabolic enzymes at baseline and in response to vitamin D treatment in thyroid cancer cell lines.

The association between vitamin D and thyroid cancer is unclear. It is unknown if CYP27A1 or CYP2R1 are present in normal thyroid or cancer cells and there is limited information regarding response to treatment with vitamin D. SV40 immortalized follicular cells (N-thy) and six thyroid cancer cell lines were treated with 10 µM vitamin D(3), 0.1 µM 1,25(OH)(2)D(3) or vehicle × 24 h. CYP27A1, CYP2R1, CYP27B1 and CYP24A1 mRNA were measured using quantitative real-time-PCR before and after treatment. Cell proliferation was also evaluated in TPC1 and C643 cells after treatment with D(3), 25(OH)D(3) and 1,25(OH)(2)D(3). Baseline CYP27A1 and CYP27B1 mRNA were present in all cells, CYP2R1 was higher and CYP24A1 mRNA was lower in cancer cell lines versus N-thy. TPC1 cells had increased CYP24A1 mRNA levels when treated with both D(3) (3.49, p < 0.001) and 1,25(OH)(2)D(3) (5.05, p < 0.001). C643 cells showed increased CYP24A1 mRNA expression when treated with 1,25(OH)(2)D(3) (5.36, p < 0.001). D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) all significantly decreased cell proliferation in TPC1 and C643 cells. Overall, both cancerous and N-thy cell lines express CYP27A1 and CYP2R1 in addition to CYP27B1, establishing the potential to metabolize D(3) to 1,25(OH)(2)D(3). Additionally, vitamin D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) all had an antiproliferative effect on two thyroid cancer cell lines.

Tacrolimus and sirolimus induce reproductive abnormalities in female rats.

BACKGROUND: Immunosuppression medications contribute to posttransplant diabetes mellitus in patients and can cause insulin resistance in male rats. Tacrolimus (TAC)-sirolimus (SIR) immunosuppression is also associated with appearance of ovarian cysts in transplant patients. Because insulin resistance is observed in patients with polycystic ovary syndrome, we hypothesized that TAC or SIR may induce reproductive abnormalities. METHODS: We monitored estrus cycles of adult female rats treated daily with TAC, SIR, and combination of TAC-SIR, or diluent (control) for 4 weeks. Animals were then challenged with oral glucose to determine their glucose and insulin responses, killed, and their blood and tissues, including ovaries and uteri harvested. RESULTS: TAC and TAC-SIR treatments increased mean random glucose concentrations (P<0.05). TAC, SIR, and TAC-SIR treatments also increased the glucose response to oral glucose challenge (P<0.05). The insulin response to glucose was significantly higher in rats treated with SIR compared with TAC (P<0.05). TAC, SIR and TAC-SIR treatments reduced number of estrus cycles (P<0.05). The ovaries were smaller after SIR and TAC-SIR treatment compared with controls. The TAC and TAC-SIR treatment groups had fewer preovulatory follicles. Corpora lutea were present in all groups. Ovarian aromatase expression was reduced in the SIR and TAC-SIR treatment groups. A significant (P<0.05) reduction in uterine size was observed in all treatment groups when compared with controls. CONCLUSION: In a model of immunosuppressant-induced hyperglycemia, both TAC and SIR induced reproductive abnormalities in adult female rats, likely through different mechanisms.

Redox regulation of insulin degradation by insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) is a thiol sensitive peptidase that degrades insulin and amyloid ß, and has been linked to type 2 diabetes mellitus and Alzheimer's disease. We examined the thiol sensitivity of IDE using S-nitrosoglutathione, reduced glutathione, and oxidized glutathione to distinguish the effects of nitric oxide from that of the redox state. The in vitro activity of IDE was studied using either partially purified cytosolic enzyme from male Sprague-Dawley rats, or purified rat recombinant enzyme. We confirm that nitric oxide inhibits the degrading activity of IDE, and that it affects proteasome activity through this interaction with IDE, but does not affect the proteasome directly. Oxidized glutathione inhibits IDE through glutathionylation, which was reversible by dithiothreitol but not by ascorbic acid. Reduced glutathione had no effect on IDE, but reacted with partially degraded insulin to disrupt its disulfide bonds and accelerate its breakdown to trichloroacetic acid soluble fragments. Our results demonstrate the sensitivity of insulin degradation by IDE to the redox environment and suggest another mechanism by which the cell's oxidation state may contribute to the development of, and the link between, type 2 diabetes and Alzheimer's disease.

Role of inflammation and insulin resistance in endothelial progenitor cell dysfunction.

OBJECTIVE: Endothelial progenitor cells (EPCs) are decreased in number and function in type 2 diabetes. Mechanisms by which this dysfunction occurs are largely unknown. We tested the hypothesis that a chronic inflammatory environment leads to insulin signaling defects in EPCs and thereby reduces their survival. Modifying EPCs by a knockdown of nuclear factor-κB (NF-κB) can reverse the insulin signaling defects, improve EPC survival, and decrease neointimal hyperplasia in Zucker fatty rats postangioplasty. RESEARCH DESIGN AND METHODS: EPCs from Zucker fatty insulin-resistant rats were cultured and exposed to tumor necrosis factor-α (TNF-α). Insulin signaling defects and apoptosis were measured in the presence and absence of an NF-κB inhibitor, BAY11. Then, EPCs were modified by a knockdown of NF-κB (RelA) and exposed to TNF-α. For in vivo experiments, Zucker fatty rats were given modified EPCs post-carotid angioplasty. Tracking of EPCs was done at various time points, and neointimal hyperplasia was measured 3 weeks later. RESULTS: Insulin signaling as measured by the phosphorylated-to-total AKT ratio was reduced by 56% in EPCs exposed to TNF-α. Apoptosis was increased by 71%. These defects were reversed by pretreatment with an NF-κB inhibitor, BAY11. Modified EPCs exposed to TNF-α showed a lesser reduction (RelA 20%) in insulin-stimulated AKT phosphorylation versus a 55% reduction in unmodified EPCs. Apoptosis was 41% decreased for RelA knockdown EPCs. Noeintimal hyperplasia postangioplasty was significantly less in rats receiving modified EPCs than in controls (intima-to-media ratio 0.58 vs. 1.62). CONCLUSIONS: In conclusion, we have shown that insulin signaling and EPC survival is impaired in Zucker fatty insulin resistant rats. For the first time, we have shown that this defect can be significantly ameliorated by a knockdown of NF-κB and that these EPCs given to Zucker fatty rats decrease neointimal hyperplasia post-carotid angioplasty.

Hyperglycemia induced by tacrolimus and sirolimus is reversible in normal sprague-dawley rats.

Post-transplant diabetes mellitus (PTDM) worsens outcomes after kidney transplantation, and immunosuppression agents contribute to PTDM. We have previously shown that tacrolimus (TAC) and sirolimus (SIR) cause hyperglycemia in normal rats. While there is little data on the mechanism for immunosuppressant-induced hyperglycemia, we hypothesized that the TAC and SIR-induced changes are reversible. To study this possibility, we compared normal rats treated for 2 weeks with either TAC, SIR, or a combination of TAC and SIR prior to evaluating their response to glucose challenge, with parallel groups also treated for 2 weeks after which treatment was stopped for 4 weeks, prior to studying their response to glucose challenge. Mean daily glucose and growth velocity was decreased in SIR, and TAC+SIR-treated animals compared to controls (P < 0.05). TAC, SIR, and TAC+SIR treatment also resulted in increased glucose response to glucose challenge, compared to controls (P < 0.05). SIR-treated animals also had elevated insulin concentrations in response to glucose challenge, compared to controls (P < 0.05). Insulin content was decreased in TAC and TAC+SIR, and islet apoptosis was also increased after TAC+SIR treatment (P < 0.05). Four weeks after treatments were stopped, all differences resolved between groups. In conclusion, TAC, SIR, and the combination of TAC+SIR-induced changes in glucose and insulin responses to glucose challenge that were accompanied by changes in islet apoptosis and insulin content. These changes were no longer present 4 weeks after cessation of therapy suggesting immunosuppressant-induced changes in glucose metabolism are likely reversible.

Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.

Effects of a PPAR-gamma agonist, on growth factor and insulin stimulated endothelial cells.

OBJECTIVE: PPAR-gamma agonists such as thiazolidinediones, used in patients with insulin resistance have been shown to reduce neointimal hyperplasia in the short term. However recent studies suggest increased cardiovascular risk for some thiazolidinediones. Longer-term animal studies show inhibition of endothelial regrowth post endothelial injury which may account for some of the increased risk. We studied the effect of pioglitazone on VEGF, FGF and insulin stimulated endothelial cells to determine if this was a mechanism of inhibition of endothelial regrowth. METHODS AND RESULTS: FGF/VEGF stimulated human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis was measured, in vitro, in the presence and absence of hyperinsulinemia, with and without treatment with the PPAR-gamma agonist pioglitazone. Activation of ERK 1/2 and p38MAPK was measured under the same conditions. There was 40% decrease in proliferation with pioglitazone in VEGF stimulated cells, which was reversed by insulin. ERK 1/2 activation was decreased by pioglitazone in VEGF stimulated cells and was partially reversed by insulin. p38MAPK activation was increased by pioglitazone and was unaffected by insulin or VEGF. Pioglitazone also increased endothelial cell apoptosis. CONCLUSION: PPAR-gamma agonists may have detrimental cardiovascular effects post angioplasty especially in patients with insulin resistance. We have shown that one of the mechanisms may be inhibition of endothelial regrowth and re-endothelialization by inhibition of VEGF/FGF stimulation of the ERK 1/2 pathways in endothelial cells.

Preliminary report: inhibition of cellular proteasome activity by free fatty acids.

There is evidence in animal studies that free fatty acids (FFA) can decrease protein degradation, but the exact mechanism is not known. We have shown that FFA can inhibit proteasome activity in vitro by interacting with insulin-degrading enzyme. Here we show that FFA can also inhibit the proteasome in whole cells. HepG2 cells were treated with various FFA, and proteasome activity was measured using a cell-permeable substrate for the chymotrypsin-like activity. Octanoic acid, a medium-chain fatty acid, did not affect proteasome activity. However, oleic and linoleic acids inhibited the chymotrypsin-like activity up to 80%, with approximate IC50s of 80 and 40 micromol/L, respectively. Insulin also inhibited but was not additive with the FFA, suggesting that they work through the same mechanism. These results show that the proteasome can be inhibited by FFA in whole cells and suggest that insulin-degrading enzyme may mediate this effect. This mechanism may be applicable to whole animals and represents a means to integrate hormonal and nutrient signals on the control of protein degradation.

Degradation of relaxin family peptides by insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) is a ubiquitously expressed metalloproteinase responsible for the intracellular degradation of insulin. IDE also interacts with other members of the insulin superfamily, including relaxin, but no studies have been reported regarding the interaction of other relaxin-like peptides with IDE. In this study, we determined that relaxin, relaxin-3, and InsL3 all competitively inhibited the degradation of insulin by IDE to different degrees, and all inhibited covalent cross-linking of insulin to IDE. Each of the peptides was degraded by IDE to various degrees (insulin > relaxin > InsL3 = relaxin-3). In summary, relaxin, InsL3, and relaxin-3 all bound to IDE, competed for the binding and degradation of insulin, and were all substrates for the proteolytic activity of IDE. Therefore, it is possible that in addition to insulin, IDE may be important for the cellular proteolysis of relaxin, InsL3, and relaxin-3.

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation.

Insulin-degrading enzyme (IDE) is responsible for the degradation of a number of hormones and peptides, including insulin and amyloid beta (Abeta). Genetic studies have linked IDE to both type 2 diabetes and Alzheimer's disease. Despite its potential importance in these diseases, relatively little is known about the factors that regulate the activity and function of IDE. Protein S-nitrosylation is now recognized as a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe a mechanism of inactivation of IDE by NO. NO donors decreased both insulin and Abeta degrading activities of IDE. Insulin-degrading activity appeared more sensitive to NO inhibition than Abeta degrading activity. IDE-mediated regulation of proteasome activity was affected similarly to insulin-degrading activity. We found IDE to be nitrosylated in the presence of NO donors compared to that of untreated enzyme and the control compound. S-nitrosylation of IDE enzyme did not affect the insulin degradation products produced by the enzyme, nor did NO affect insulin binding to IDE as determined by cross-linking studies. Kinetic analysis of NO inhibition of IDE confirmed that the inhibition was noncompetitive. These data suggest a possible reversible mechanism by which inhibition of IDE under conditions of nitrosative stress could contribute to pathological disease conditions such as Alzheimer's disease and type 2 diabetes.

Relaxin receptors in hepatic stellate cells and cirrhotic liver.

The polypeptide hormone relaxin has antifibrotic effects on a number of tissues, including the liver. Central to the progression of hepatic fibrosis is the transdifferentiation of hepatic stellate cells (HSC) from a quiescent state to an activated, myofibroblastic phenotype that secretes fibrillar collagen. Relaxin inhibits markers of HSC activation, but relaxin receptor expression in the liver is unclear. The purpose of this study was to determine the expression of the relaxin receptors LGR7 and LGR8 in activated HSC. Production of cAMP was induced by treatment of HSC with relaxin, or the relaxin-related peptides InsL3 or relaxin-3, selective activators of LGR8 and LGR7, respectively. Quiescent HSC expressed low levels of LGR7 but not LGR8. During progression to the activated phenotype, expression of both receptors increased markedly. Immunocytochemistry confirmed the presence of both receptors in activated HSC. In normal rat liver, LGR7, but not LGR8, was expressed at low levels. In cirrhotic liver, expression of both receptors significantly increased. Neither receptor was detectable in normal liver by immunohistochemistry, but both LGR7 and LGR8 were readily detectable in cirrhosis. These results were confirmed in human cirrhotic tissue, with the additional finding of occasional perisinusoidal LGR7 immunoreactivity in non-cirrhotic tissue. In conclusion, the expression of LGR7 and LGR8 is increased with activation of HSC in culture. Cirrhosis also caused increased expression of both receptors. Therefore, agents that stimulate LGR8 and LGR7 may be therapeutically useful to limit the activation of hepatic stellate cells in liver injury.

Long-term effects of a PPAR-gamma agonist, pioglitazone, on neointimal hyperplasia and endothelial regrowth in insulin resistant rats.

OBJECTIVE: Insulin resistance is an independent risk factor for cardiovascular disease. PPAR-gamma agonists like pioglitazone decrease insulin resistance and have been shown to reduce neointimal hyperplasia in the short-term. However long-term studies on endothelial regrowth and neointimal hyperplasia have not been done. METHODS AND RESULTS: We used hyperinsulinemic, normoglycemic Zucker fatty rats. Rats were treated with either 10 mg/kg body wt. pioglitazone or placebo till the end of the experiment. Rats underwent carotid angioplasty at age 12-14 weeks, 1 week after treatment was begun. In one set of experiments rats were sacrificed at 6 months and neointimal hyperplasia and VEGF expression was assessed. In another set of experiments rats were sacrificed at 3 and 6 months. Endothelial regrowth was determined. The rats were all normoglycemic and hyperinsulinemic. Pioglitazone treated rats had a significantly lesser degree of neointimal hyperplasia than control rats. Treated rats also had decreased VEGF expression. Endothelial regrowth was decreased in the treated rats at 6 months. CONCLUSION: We conclude that although pioglitazone decreases neointimal hyperplasia even at 6 months, it retards endothelial regrowth, which could predispose the denuded vessel to thrombotic events. This may be modulated by a suppression of VEGF expression.

Tacrolimus and sirolimus cause insulin resistance in normal sprague dawley rats.

BACKGROUND: Tacrolimus-sirolimus immunosuppression has improved islet graft survival but may affect islet function. METHODS: We studied the effects of tacrolimus, sirolimus, or both in normal adult male Sprague Dawley rats. Glucose and insulin response to oral glucose load and pancreas pathology were evaluated after two weeks of daily tacrolimus (1-8 mg/kg/day), sirolimus (0.08-8 mg/kg/day), or low-dose sirolimus (0.08 mg/kg/day) plus tacrolimus (1 mg/kg/day) treatment compared to controls. RESULTS: Tacrolimus and sirolimus each caused dose-dependent hyperglycemia with hyperinsulinemia in response to oral glucose compared to controls, suggesting insulin resistance. At the highest doses of sirolimus, fasting insulin concentrations were high and did not increase with oral glucose suggesting loss of first phase insulin release. The combination of low doses of tacrolimus and sirolimus, at concentrations used in clinical transplantation, resulted in hyperglycemia without hyperinsulinemia after oral glucose administration. The combination of tacrolimus and sirolimus decreased islet size, and increased islet apoptosis more than either medication alone, or controls. CONCLUSIONS: In summary, short-term therapy with either tacrolimus or sirolimus causes insulin resistance in normal rats. Combination tacrolimus-sirolimus causes greater islet changes suggesting early islet failure.

Neointimal hyperplasia and vascular endothelial growth factor expression are increased in normoglycemic, insulin resistant, obese fatty rats.

OBJECTIVE: Insulin resistance is associated with a constellation of factors that enhance the artherosclerotic process. Vessel injury results in the formation of a markedly increased neointima in type 2 diabetes. Increased neointimal hyperplasia (NH) and vascular endothelial growth factor (VEGF) expression may lead to restenosis post angioplasty. We studied NH and VEGF expression in an obese, insulin resistant, but normoglycemic rat model, after carotid balloon injury. METHODS AND RESULTS: Diabetic rats (ZDF, n=10), normoglycemic, insulin-resistant rats (ZDF-normoglycemic, n=6) as well as Zucker fatty rats (FZ, n=6), and lean Zucker rats (LZ, n=6), all 13-16 weeks old, were subjected to right carotid injury by an angioplasty catheter introduced via the femoral artery. Three weeks later the rats were sacrificed and serum and carotids obtained. The intima-media ratio (I/M) was then calculated. ZDF-normoglycemic, FZ and ZDF-diabetic rats were all hyperinsulinemic and hypertriglyceridemic when compared to LZ rats. ZDF diabetic rats were hyperglycemic while FZ, ZDF-normoglycemic and LZ rats were normoglycemic. The I/M ratio for ZDF and FZ rats were significantly greater than for LZ rats. The VEGF expression was significantly greater in ZDF and FZ rats than LZ rats. CONCLUSIONS: In conclusion, insulin resistance increases neointimal hyperplasia and VEGF expression even with normoglycemia, after carotid angioplasty in rats.