Long-term Fenretinide treatment prevents high-fat diet-induced obesity, insulin resistance, and hepatic steatosis

Abstract

The synthetic retinoid Fenretinide (FEN) increases insulin sensitivity in obese rodents and is in early clinical trials for treatment of insulin resistance in obese humans with hepatic steatosis (46). We aimed to determine the physiological mechanisms for the insulin-sensitizing effects of FEN. Wild-type mice were fed a high-fat diet (HFD) with or without FEN from 4-5 wk to 36-37 wk of age (preventive study) or following 22 wk of HF diet-induced obesity (12 wk intervention study). Retinol-binding protein-4 (RBP4) knockout mice were also fed the HFD with or without FEN in a preventive study. FEN had minimal effects on HFD-induced body weight gain but markedly reduced HFD-induced adiposity and hyperleptinemia in both studies. FEN-HFD mice gained epididymal fat but not subcutaneous or visceral fat mass in contrast to HFD mice without FEN. FEN did not have a measurable effect on energy expenditure, food intake, physical activity, or stool lipid content. Glucose infusion rate during hyperinsulinemic-euglycemic clamp was reduced 86% in HFD mice compared with controls and was improved 3.6-fold in FEN-HFD compared with HFD mice. FEN improved insulin action on glucose uptake and glycogen levels in muscle, insulin-stimulated suppression of hepatic glucose production, and suppression of serum FFA levels in HFD mice. Remarkably, FEN also reduced hepatic steatosis. In RBP4 knockout mice, FEN reduced the HFD-induced increase in adiposity and hyperleptinemia. In conclusion, long-term therapy with FEN partially prevents or reverses obesity, insulin resistance, and hepatic steatosis in mice on HFD. The anti-adiposity effects are independent of the RBP4 lowering effect.

Fig. 1.

Fenretinide partially prevents high-fat diet (HFD)-induced obesity. A: body weight curves of male FVB mice on standard CHOW (○), HFD (▴), or HFD plus 0.1% wt/wt Fenretinide (FEN-HFD, ▵) from 4–5 wk of age. B: body fat content at 9 and 19 wk on diet (CHOW, open bar; HFD, filled bar, or FEN-HFD, gray bar). C: serum leptin levels at 8 and 22 wk on diet. D: visceral (VIS), subcutaneous (SC), and epididymal (EPI) white adipose tissue (WAT) depot weights dissected at 34 wk diet. E: association of dissected WAT depot weights with body weight at 34 wk diet. F: average size of adipocytes in SC-WAT expressed as amount of lipid per adipocyte (left), number of adipocytes in SC-WAT depot per mouse (right). Results are means ± SE from 12–14 mice per dietary group. *P < 0.05 vs. CHOW, #P < 0.05 vs. HFD.

Fig. 2.

Fenretinide does not affect food intake or energy expenditure. A: cumulative food intake during first 3 wk (left) and 9–11 wk (right) of diet in mice put on HFD (▴) or FEN-HFD (▵) diet from 4–5 wk of age. B: stool lipid content at 30 wk of diet. C: energy expenditure expressed as O₂ consumption of individual mice (left) and respiratory exchange ratio (RER; right) at 17 wk of diet. Results are means ± SE from 12–14 mice per dietary group. *P < 0.05 vs. CHOW, #P < 0.05 vs. HFD.

Fig. 3.

Fenretinide partially prevents HFD-induced insulin resistance in skeletal muscle and liver. A: insulin tolerance test at 14 wk of diet (CHOW, ○; HFD, ▴; FEN-HFD, ▵) in male FVB mice put on diet from 4–5 wk of age. B–F: euglycemic-hyperinsulinemic (10 mU·kg⁻¹·min⁻¹ insulin) clamp at 16–18 wk diet (CHOW, open bar; HFD, filled bar; FEN-HFD, gray bar). B: insulin-stimulated glucose infusion rate (G_inf) and basal and insulin-stimulated whole body glucose uptake (R_d). C: insulin-stimulated glucose uptake into skeletal muscle (gastrocnemius and soleus) and WAT. D: glycogen content in tibialis anterior muscle at the end of the clamp. E: insulin-stimulated suppression of hepatic glucose production. F: liver mass (left) and liver triglyceride levels (right) at 16–18 wk of diet post-euglycemic-hyperinsulinemic clamp. G: insulin-stimulated suppression of serum FFA levels (%suppression relative to basal) at 16–18 wk of diet during euglycemic-hyperinsulinemic clamp. Results are means ± SE from 11–14 mice per dietary group. *P < 0.05 vs. CHOW, #P < 0.05 vs. HFD.

Fig. 4.

Fenretinide treatment ameliorates obesity and insulin resistance in diet-induced obese mice. A: body weight curves of HFD-induced obese male FVB mice treated with Fenretinide (0.1% wt/wt HFD, FEN-HFD, ▵), untreated HFD-induced obese mice (HFD, ▴), and CHOW-fed controls (○) from 26–27 wk of age. B: fat mass during the course of Fenretinide treatment. (HFD, ▴; FEN-HFD, ▵). C: total fat mass gained and lean mass gained at 9 wk of Fenretinide treatment (HFD, filled bar; FEN-HFD, gray bar). D: serum leptin levels before and at 12 wk of Fenretinide treatment (CHOW, open bar; HFD, filled bar; FEN-HFD, gray bar). E: VIS-, SC-, and EPI-WAT weights at 12 wk of Fenretinide treatment. F: average size of adipocytes in SC-WAT expressed as amount of lipid per adipocyte (left) and number of adipocytes in SC-WAT depot per mouse (right). G: cumulative food intake during first 9 wk of Fenretinide treatment. H: serum insulin levels before and at 12 wk of Fenretinide treatment (5-h-fasted levels). I: insulin tolerance test pretreatment and at 12 wk of Fenretinide treatment (CHOW, ○; HFD, ▴; FEN-HFD, ▵). Results are means ± SE from 8–10 mice per dietary group. *P < 0.05 vs. CHOW, #P < 0.05 vs. HFD. Statistical symbols indicate a difference for the curve.

Fig. 5.

Fenretinide partially prevents HFD-induced obesity in mice lacking serum RBP4. A: serum RBP4 levels in WT and retinol-binding protein-4 knockout (RBP4KO) male mice on C57BL/6J × 129Sv mixed background after 22 wk of either standard CHOW (open bar), HFD (filled bar), or HFD + Fenretinide (0.1% wt/wt; FEN-HFD, gray bar) from 4–5 wk of age. Right: representative Western blot. B: body weight progression of WT and RBP4KO mice on either CHOW (○), HFD (▴), or FEN-HFD (▵) from 4–5 wk of age. In both WT and RBP4KO genotypes, body weight progression was significantly different among all 3 treatment groups (repeated-measures ANOVA, P < 2 × 10e⁻⁷). Body weights in WT FEN-HFD were significantly different from WT HFD at weeks 21 and 22 of diet treatment (P < 0.05, ANOVA with Bonferroni post hoc test). C: body fat content at 20 wk on diet. (CHOW, open bar; HFD, filled bar; FEN-HFD, gray bar). D: ad libitum-fed serum leptin levels at 22 wk on diet. E: cumulative food intake during 17–20 wk of diet. Results are means ± SE from 10–15 mice per dietary group. *P < 0.05 vs. CHOW, #P < 0.05 vs. HFD.