Fenretinide prevents lipid-induced insulin resistance by blocking ceramide biosynthesis

Abstract

Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.

FIGURE 1.

Ceramide inhibition with fenretinide protects insulin signaling. A, murine C2C12 myotubes were treated with 0.75 mm BSA-conjugated PA for 16 h in the presence or absence of FEN (5 μm) followed by 10 min of insulin treatment (100 nm). pAkt, phosphorylated Akt; pGSK3β, phospho-glycogen synthase kinase 3β; GSK3β, glycogen synthase kinase 3β. B and C, the readdition of C2-ceramide (100 μm) bypassed the protection offered by FEN (B), an effect seen with as little as 20 μm ceramide (C). D, the improvement in insulin signaling with FEN does not require RBP4 as C2C12 cells appear to lack the protein. E and F, although PA induces ceramide and dihydroceramide accumulation, FEN prevents ceramide synthesis (E), whereas accumulating dihydroceramides (F). *, p < 0.05 for treatment versus BSA. +, p < 0.05 for PA+FEN versus PA (n = 3–6).

FIGURE 2.

Resveratrol inhibits Des1 and protects insulin signaling. A, similar to fenretinide treatment, the addition of 20 μm RSV improved insulin signaling (100 nm, 10 min) in C2C12 myotubes exposed to 0.75 mm PA for 16 h. pAkt, phosphorylated Akt; Sirt1 KO, Sirt1 knock-out. C and D, RSV significantly reduced ceramides and increased dihydroceramides when added to PA-containing medium in comparison with PA alone. A, B, and E, to confirm that these effects occurred independently of Sirt1, similar to Sirt1 inhibition with nicotinamide (NAM) (A), Sirt1 ablation with shRNA (B) had no effect on RSV-mediated improvements in insulin signaling (E). *, p < 0.05 for treatment versus BSA. +, p < 0.05 for PA+RSV versus PA (n = 3–6).

FIGURE 3.

Ablation of Des1 inhibits ceramide accumulation and protects insulin signaling. A, Akt Ser-473 phosphorylation (pAkt) was determined in C2C12 myotubes 48 h after transfection with Des1 siRNA or control. Cells were treated for 16 h with 0.75 mm PA followed by insulin stimulation (100 nm; 10 min). Des1 knockdown protected insulin signaling. pGSK3β, phospho-glycogen synthase kinase 3β; GSK3β, glycogen synthase kinase 3β. A and B, Des1 knockdown was confirmed by Western blot (A) and quantitative PCR (B). To confirm the quality of Des1 knockdown, levels of ceramides and dihydroceramides were determined and found to vary significantly between treatments. C and D, Des1 knockdown robustly inhibited ceramide accumulation (C) and induced a significant increase in dihydroceramides in response to 0.75 mm PA (D) when compared with control conditions. *, p < 0.05 for treatment versus BSA. +, p < 0.05 for dihydroceramides versus ceramides in BSA with Des1 siRNA (n = 4–5).

FIGURE 4.

Fenretinide prevents lipid-induced reductions in insulin-stimulated glucose uptake. Following excision, rat soleus was incubated in gassed medium for 6 h with 1 mm PA in the presence or absence of FEN (10 μm) followed by 1 h of insulin incubation (300 microunits). A, 2-[³H]DOG uptake was found to be significantly improved with the addition of FEN to PA when compared with PA alone. B, to confirm that the effect occurs independently of RBP4, we measured RBP4 protein in whole muscle and found extremely low levels. *, p < 0.05 for insulin stimulation versus basal. +, p < 0.05 for PA+FEN versus PA alone (n = 5). Skm, skeletal muscle.

FIGURE 5.

Acute fenretinide injection selectively inhibits ceramide accumulation. HFD-fed male C57Bl/6 mice received 10 mg/kg of FEN via intraperitoneal injection 12 h prior to sacrifice. A and C, ceramides tended to decrease in both the soleus (Sol.) and the liver (Hep.) (A and C), but did not reach significance. B and D, however, FEN injection induced a roughly 6-fold increase in dihydroceramides in the soleus (B) and a 2-fold increase in the liver (D). *, p < 0.05 for treatment versus PBS injection (n = 5).

FIGURE 6.

Chronic fenretinide treatment improves glucose tolerance and insulin sensitivity in diet-induced obese mice. Male mice (C57Bl/6) were made obese by HFD for 12 weeks prior to 4-week FEN treatment administered in drinking water (10 μg/ml). Following the treatment period, intraperitoneal glucose (1g/kg of body weight) and insulin (0.75 units/kg of body weight) tolerance tests were conducted. A and B, FEN treatment resulted in improved glucose (A) and insulin (B) tolerance when compared with HFD alone (area under curve (AUC) also shown). *, p < 0.05 for HFD+FEN versus HFD. Fasting insulin and glucose were used to determine the HOMA-IR. C, HFD-fed mice had a significantly elevated HOMA-IR value when compared with animals fed SD, and this was reduced in the HFD+FEN group. D, body weight increased on HFD, but was unaffected by FEN. *, p < 0.05 for treatment versus SD. +, p < 0.05 for HFD+FEN versus HFD (n = 6).

FIGURE 7.

Fenretinide reduces muscle and liver ceramides and increases dihydroceramides. Lipids from soleus (Sol.) and liver (Hep.) were extracted from tissues of mice receiving SD, HFD, or HFD with fenretinide (HFD+FEN) after 4 weeks of treatment. A–D, in both soleus (A and B) and liver (C and D), the HFD-induced increase in ceramides was prevented with FEN treatment (A and C). Moreover, FEN increased dihydroceramides in both tissues (B and D). *, p < 0.05 for treatment versus SD. +, p < 0.05 for HFD+FEN versus HFD (n = 6).

FIGURE 8.

Fenretinide reduces soleus and liver neutral lipid content. A, DAG increased significantly with HFD versus SD, and FEN had no effect. B and C, in contrast, the increased TAG content in liver with HFD was significantly inhibited with FEN, suggesting a reversal of hepatic steatosis with FEN. *, p < 0.05 for treatment versus SD. +, p < 0.05 for HFD+FEN versus HFD (n = 6).

FIGURE 9.

Fenretinide inhibits high-fat diet-induced increase in Des1 transcription. Liver and soleus were extracted from mice fed SD, HFD, or HFD with fenretinide (HFD+FEN) for 4 weeks. A–C, hepatic Des1 mRNA and protein levels were markedly elevated with HFD (A and C), but less so in soleus (B). However, inclusion of FEN to HFD resulted in a significant reduction in Des1 transcript in soleus when compared with HFD alone. *, p < 0.05 for treatment versus SD. +, p < 0.05 for HFD+FEN versus HFD (n = 6).