The Tellurium compound, AS101, increases SIRT1 level and activity and prevents type 2 diabetes

Abstract

The histone deacetylase, SIRT1, plays a major role in glucose regulation and lipid metabolism. Ammonium Trichloro (dioxoethylene-o,o') Tellurate, AS101, is a potent in vitro and in vivo immunomodulator, with several potential therapeutic applications. AS101 administration resulted in upregulation of SIRT1 protein expression and activity. These effects were associated with decreased levels of serum insulin like growth factor-1 (IGF-1) and of insulin. The properties of AS101 prompted us to investigate its potential therapeutic role in rats with type 2 diabetes (T2D). T2D was induced by a high fat diet combined with a low dose of Streptozotocin (STZ). Treatment with AS101 before manifestation of hyperglycemia, resulted in increased insulin sensitivity, and decreased blood glucose levels, and prevented symptoms of diabetes including defective glucose clearance, fatty liver, and abnormal distribution of insulin-producing beta cells in the pancreas. Treatment after disease emergence resulted in partial restoration of normal glucose homeostasis. Diabetic rats showed a reduction in liver SIRT1 levels. In both treatment regimens the reduction in SIRT1 levels in the liver were blocked by AS101 consumption. Together, these findings demonstrate the therapeutic potential of AS101 for treating T2D, and for reversing impaired fat and glucose metabolism.

Figure 1. AS101 increases SIRT1 levels in vitro and in vivo

(A-C) AS101 increases SIRT1 expression in several cell lines. For in vitro studies, AS101 and PBS were added to cultures of HEK293 (A), HL-60 (B) and Rin-5f (C) cells at a concentration of 0.1-2.5μg/ml, for 48 hours. Each experiment was done in three independent replicates and representative blots are shown. (D-E) AS101 increases SIRT1 expression in healthy rat livers. For the in vivo assay, healthy rats were injected daily i.p with AS101 or PBS at different concentrations for 14 days (0.25-1mg/kg AS101(D)) and for different periods of time (0.5 mg/kg) (E) (n=4 for each group.) SIRT1 levels in protein extracts from cell lines or rat liver, were measured by western blot analysis with anti- SIRT1 antibodies; actin was used as a loading control.

Figure 2. AS101 increases SIRT1 protein activity in vitro and in vivo

(A) AS101 induces activity of recombinant SIRT1. To determine in vitro SIRT1 activity, recombinant SIRT1 was incubated with AS101 at different concentration (0.1-2.5 μg/ml) or with PBS for 1 hour. Activity was examined based on SIRT1 de-acetylation of a fluorogenic acetylated peptide substrate. Results shown are average ±SEM of three independent experiments (#p=0.05,*p<0.05 versus PBS). (B-C) AS101 treatment reduces the acetylation levels of SIRT1 substrates. In vivo SIRT1 activity was measured by de-acetylation of its substrates PGC1α (B), and p53 (C) in rat liver extracts from treated for 14 days with AS101 or PBS at different concentrations. To detect acetylation levels, immunoprecipitated PGC1α and p53 were immunoblotted with antibodies against themselves and against pan acetyl antibodies. IgG was used as a control for the immunoprecipitation. Results shown are representative of the experiments from three independent replicates that gave similar results.

Figure 3. AS101 treatment increases SIRT1 expression through a possible mechanism of serum IGF-1 reduction

(A) Sera from AS101 treated rats contained significantly reduced IGF-1 levels. AS101 (0.5mg/kg) or PBS was injected daily i.p into healthy rats for 14 days and IGF-1 levels were measured by ELISA (*p<0.01 versus PBS). Error bars represent the ±SEM of all animals (AS101 n=5, PBS n-4). (B) SIRT1 expression increased in HEK293 cells incubated with AS101-treated rat serum. Supplementing with rIGF-1 back to normal levels (500 ng/ml) restored SIRT1 levels as in control mice. Cell extracts, were used for western blot analysis with anti- SIRT1 antibodies; Actin was used as a loading control. (C) Sera from AS101 treated rats contained significantly reduced insulin levels. AS101 (0.25-1mg/kg) and PBS were injected daily i.p to healthy rats for 14 days. Serum insulin levels were measured by ELISA kit after 4 hours starvation (*p<0.001 versus PBS) (n=3 in each group). (D-E) AS101 decreases PPARγ levels in tissue culture in vitro. AS101 and PBS were added to tissue cultures of HEK293 (D) and HL-60 (E) cells at the indicated concentrations for 48 hours. Western blots were probed with PPARγ antibody; actin was used as a loading control. (F) AS101 maintains normal insulin levels in HFD fed rats. AS101 (0.5-1mg/kg) and PBS was injected daily i.p into HFD/ND rats for 14 days. Serum insulin was measured by ELISA after 4 hours of fasting (*p<0.001 versus HFD+PBS, n=3 in each group). Error bars represent the SEM. Results shown in B, D and E are representative of three independent replicates that gave similar results (using different batches of rat sera).

Figure 4. AS101 reverses HFD+STZ induced T2D

(A) Course of experiment. (B) AS101 treatment prevents glucose increase in HFD+STZ induced T2D rat model. (*p<0.01 all groups vs. HFD+PBS group, n=6 in each group). (C) AS101 treatment maintains glucose tolerance. On day 17 (following the onset of diabetes) rats were fasted for 12 hours before i.p injection of 2mg/kg glucose. (*p<0.001 versus HFD+PBS+STZ, n=6 in each group). The histogram represents the incremental area under the glucose curve. (D) AS101 treatment maintains proper insulin levels in the serum of rats after 4 hours starvation (*p<0.001 all groups versus HFD+PBS+STZ, n=6 for each group). (E) AS101 preserves insulin production in the pancreas. Immunohistological staining for insulin (brown) and glucagon (red) of the rat pancreas. Pictures represent two out of the six animals in each group. Error bars represent the ±SEM.

Figure 5. AS101 treatment protects rats from HFD+STZ induced hepatosteatosis

(A) Oil red staining of lipid droplets in frozen liver sections. Pictures represent two out of the six animals in each group. (B) AS101 treatment reduces serum ALP levels. (*p<0.05 decrease vs. diabetic group, n=6 in each group).

Figure 6. AS101 treatment after disease induction results in partial beneficial effects in the HFD+STZ T2D rat model

(A) Course of experiment. (B) AS101 reduced glucose levels in HFD+STZ T2D rat model. Glucose levels were measured in peripheral blood at different times (*p<0.05 all groups vs. HFD+PBS group, n=6 in each HFD group, n=3 in ND group). (C) AS101 treatment restores proper glucose tolerance. Rats fasted for 12 hours before i.p injection of 2mg/kg glucose. (*p<0.01 versus HFD+PBS+STZ, n=6 in each group). The histogram represents the incremental area under the glucose curve. (D) AS101 (1 mg/kg) treated rats show higher SIRT1 protein levels in liver extract prepared at the end of the experiment. Western blot analysis was done with anti- SIRT1 antibodies; actin was used as a loading control. Error bars represent the ±SEM.