FXR agonist INT-747 upregulates DDAH expression and enhances insulin sensitivity in high-salt fed Dahl rats

Abstract

Aims: Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.

Methods and results: In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.

Conclusion: Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.

Figure 1. Schematic showing the time frame and experimental protocol followed to induce hypertension by high-salt feeding.

Measurements period of BP, glucose challenge, urinary collection and blood draw are also shown. BP = blood pressure.

Figure 2. High-salt diet progressively increases systemic blood pressure.

Weekly measurement of: a) systolic blood pressure (SBP) and b) heart rate (HR) by tail-cuff in Dahl salt-sensitive rats fed diet containing low salt (n = 9); high-salt and treated with vehicle (n = 15); high-salt and treated with 10 mg/kg/day INT-747 (n = 15) or high-salt and treated with high dose of INT-747 at 30 mg/kg/day (n = 15). Data is expressed as Mean±SEM. (*p<0.05 versus high-salt diet data. ANOVA followed by Bonferroni post-test).

Figure 3. Assessing renal morphology and fibrosis.

a: H&E staining to assess the renal morphology of Dahl rats following: LS-diet with nearly normal morphology (A) or HS-diet and administration of vehicle, showing fibrinoid arteriolar necrosis with extravasation of erythrocytes and thrombosis of glomerular capillaries (B); INT-747 at 10 mg/kg/day shows similar findings (C) as does INT-747 at 30 mg/kg/day (D) for 6 weeks. Representative images are shown. (400X mag). b: Masson's Trichrome staining to evaluate renal fibrosis (seen as blue-colored expansion of the interstitium between the tubules) in Dahl rat kidneys following: LS-diet (A) or HS-diet and administration of vehicle (B); INT-747 at 10 mg/kg/day (C) or INT-747 at 30 mg/kg/day (D) for 6 weeks. Representative images are shown. (100X mag).

Figure 4. The effect of INT-747 treatment on DDAH1 expression in Liver.

Animals were fed low (control)- or high-salt diet and treated with vehicle or INT-747 at 10 or 30 mg/kg/day for 6 weeks. Liver lysates were compared for DDAH1 expression by Western blot. rDDAH1: purified recombinant human DDAH1 described previously . The DDAH1 expression was normalized to β-Actin (ACTB). DDAH = dimethylarginine dimethylaminohydrolase. Data is expressed as Mean±SEM. (*p<0.05 versus control value. ANOVA followed by Bonferroni post-test).

Figure 5. Measurements of serum ADMA and NO in Dahl rats at baseline and 6-weeks after feeding high-salt diet in the presence of vehicle or INT-747 treatment.

NO and ADMA were measured as described in the text. Data is expressed as Mean±SEM. NO = nitric oxide; ADMA = asymmetric dimethylarginine.

Figure 6. Blood glucose and insulin measurements to assess insulin sensitivity: Measurement of a) blood glucose \and b) plasma insulin concentration over time during GTT in Dahl rats fed with low or high-salt diet for 5-weeks prior to the glucose challenge test.

Values are Mean ± SEM for: Control (n  = 6); Vehicle (n  = 7); INT-10 mg/kg/day (n  = 5) and INT-30 mg/kg/day (n = 9). *p<0.05 versus high-salt diet data. ANOVA followed by Bonferroni post-test. GTT = glucose tolerance test. In c), the effect of INT-747 treatment on insulin sensitivity is shown. Insulin Resistance (IR) index was calculated as described in the text. Data is expressed as Mean±SEM. (*p<0.05 versus low-salt diet data. ANOVA followed by Bonferroni post-test). LS = low salt; HS = high salt; V = vehicle; INT-10 = INT-747 at 10 mg/kg/day and INT-30 = INT-747 at 30 mg/kg/day.