The antianginal ranolazine mitigates obesity-induced nonalcoholic fatty liver disease and increases hepatic pyruvate dehydrogenase activity

Abstract

Obese individuals are often at risk for nonalcoholic fatty liver disease (NAFLD), insulin resistance, type 2 diabetes (T2D), and cardiovascular diseases such as angina, thereby requiring combination therapies for their comorbidities. Ranolazine is a second-line antianginal agent that also improves glycemia, and our aim was to determine whether ranolazine modifies the progression of obesity-induced NAFLD. Twelve-week-old C57BL/6J male mice were fed a low-fat or high-fat diet for 10 weeks and then treated for 30 days with either vehicle control or ranolazine (50 mg/kg via daily s.c. injection). Glycemia was monitored via glucose/pyruvate/insulin tolerance testing, whereas in vivo metabolism was assessed via indirect calorimetry. Hepatic triacylglycerol content was quantified via the Bligh and Dyer method. Consistent with previous reports, ranolazine treatment reversed obesity-induced glucose intolerance, which was associated with reduced body weight and hepatic steatosis, as well as increased hepatic pyruvate dehydrogenase (PDH) activity. Ranolazine's actions on hepatic PDH activity may be directly mediated, as ranolazine treatment reduced PDH phosphorylation (indicative of increased PDH activity) in HepG2 cells. Therefore, in addition to mitigating angina, ranolazine also reverses NAFLD, which may contribute to its documented glucose-lowering actions, situating ranolazine as an ideal antianginal therapy for obese patients comorbid for NAFLD and T2D.

Keywords: Glucose metabolism; Hepatology; Metabolism; Obesity; Pharmacology.

Figure 1. Ranolazine treatment improves glucose homeostasis.

(A) Circulating glucose levels measured in lean and obese mice during the random-fed state over 30 days of vehicle control (VC) or ranolazine (Ran) treatment (n = 11–16). (B and C) Glucose tolerance in lean and obese mice treated with either VC or Ran; and AUC during the glucose tolerance test (n = 12–15). (D) Plasma insulin levels during the glucose tolerance test at 0 and 15 minutes after glucose administration (n = 5). Values represent means ± SEM. Differences were determined using a 2-way ANOVA, followed by a Bonferroni post hoc analysis. *P < 0.05, significantly different from VC-treated counterpart.

Figure 2. Ranolazine treatment does not influence insulin sensitivity.

(A and B) Insulin tolerance in lean and obese mice treated with either vehicle control (VC) or ranolazine (Ran) (n = 12–15). (C) Insulin signaling (Akt and GSK3β phosphorylation) in soleus from obese mice treated with either VC or Ran (n = 4). Values represent means ± SEM.

Figure 3. Ranolazine treatment reduces adiposity and decreases body weight gain.

(A) Body weight measured over 30 days following vehicle control (VC) or ranolazine (Ran) treatment (Tx) in lean and obese mice (n = 11–16). (B and C) Body composition analysis in lean and obese mice treated with either VC or Ran (n = 6–8). Values represent means ± SEM. Differences were determined using a 2-way ANOVA, followed by a Bonferroni post hoc analysis. *P < 0.05, significantly different from VC-treated counterpart.

Figure 4. Ranolazine treatment improves whole-body oxygen consumption rates in obese mice.

(A and B) Twenty-four–hour (light cycle from 0–12 hours; dark cycle from 12–24 hours) and average whole-body oxygen consumption rates in lean and obese mice treated with either vehicle control (VC) or ranolazine (Ran) (n = 5). (C) Average whole-body carbon dioxide production rates in lean and obese mice treated with either VC or Ran (n = 5). (D and E) Twenty-four–hour (light cycle from 0–12 hours; dark cycle from 12–24 hours) and average respiratory exchange ratios (RER) in lean and obese mice treated with either VC or Ran (n = 5–6). (F) Twenty-four–hour ambulatory activity in lean and obese mice treated with either VC or Ran (n = 5–6). Values represent means ± SEM. Differences were determined using a 2-way ANOVA, followed by a Bonferroni post hoc analysis. *P < 0.05, significantly different from VC-treated counterpart.

Figure 5. Ranolazine treatment reverses obesity-induced hepatic steatosis.

(A) Liver weight/body weight ratios in lean and obese mice treated with either vehicle control (VC) or ranolazine (Ran) (n = 5–8). (B) Liver TAG content in lean and obese mice treated with either VC or Ran (n = 4). (C and D) Plasma TAG and NEFA levels in lean and obese mice treated with either VC or Ran (n = 4). (E and F) Pyruvate tolerance in lean and obese mice treated with either VC or Ran, and the associated AUC (n = 10–13). Values represent means ± SEM. Differences were determined with a 2-way ANOVA, followed by a Bonferroni post hoc analysis. *P < 0.05, significantly different from VC-treated counterpart.

Figure 6. Ranolazine-mediated reductions in body weight are not required for ranolazine’s salutary actions on NAFLD and dysglycemia.

(A) Pyruvate tolerance and associated AUC. (B) Liver TAG content and (C) body weights in obese mice treated acutely with either vehicle control (VC) or ranolazine (Ran) (n = 4). (D) Pyruvate tolerance and associated AUC. (E) Liver TAG content and (F) body weights in obese mice treated with either VC or Ran for 1 week (n = 5–6). Values represent means ± SEM. Differences were determined with an unpaired, 2-tailed Student’s t test or a 2-way ANOVA, followed by a Bonferroni post hoc analysis. *P < 0.05, significantly different from VC-treated counterpart.

Figure 7. Ranolazine decreases hepatic PDH phosphorylation and Pdk4/PDK4 mRNA expression.

(A) PDH phosphorylation (serine 293) in livers from obese mice treated with either vehicle control (VC) or ranolazine (Ran) for 1 week (n = 4). (B) Pdk mRNA expression in liver RNA extracts from obese mice treated with either VC or Ran for 1 week (n = 4–5). (C) PDH enzymatic activity in liver protein extracts from obese mice treated with either VC or Ran for 1 week (n = 5–6). (D) PDH phosphorylation (serine 293) in HepG2 cell treated with either VC or Ran (10 μM) (n = 4). (E) Pdk mRNA expression in HepG2 cell RNA extracts treated with either VC or Ran (10 μM) (n = 4–5). Values represent means ± SEM. Differences were determined with an unpaired, 2-tailed Student’s t test. *P < 0.05, significantly different from VC-treated counterpart.