Targeting a ceramide double bond improves insulin resistance and hepatic steatosis

Abstract

Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.

Fig. 1.. Deletion of Degs1 from leptin-deficient ob/ob mice improves glucose homeostasis and resolves hepatic steatosis.

(A) Schematic depicting the DES1 reaction. Dihydroceramides are produced through a biosynthetic pathway fueled by palmitoyl-CoA and serine, which produces the sphingosine backbone that subsequently incorporates additional fatty acids. DES1 then inserts the highly conserved 4,5-trans double bond into the sphingoid backbone. Both ceramides and dihydroceramides serve as precursors for the more predominant, complex sphingolipids. (B to L) Adult ob/ob Degs1^fl/fl and ob/ob Degs1^{Rosa26/ERT2-Cre} mice (20 weeks old) were injected with tamoxifen (3 mg) once daily over a 5-day time period. Animals were euthanized and tissues collected at 31 weeks of age. (B) Degs1 mRNA expression and (C) ceramide/dihydroceramide ratios in tissues and serum; (D) body mass; (E) fat and lean mass when the animals were 30 weeks of age; (F) tissue mass; (G and H) glucose tolerance tests and area under the curve (AUC) when the mice were 23 weeks of age; (I and J) insulin tolerance tests and AUC when the mice were 24 weeks of age; (K) adipocyte size and liver lipid droplet area; and (L) plasma ALT and AST levels. For each experiment, n = 14 to 16 mice per group were used, except for the hematoxylin and eosin staining used to calculate adipocyte and liver droplet size (n = 3 to 8 mice) or the AST/ALT determinations (n = 7 to 13 mice). Values are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 versus control. The “ns” denotes that the comparison was not significantly different. dhCer, dihydroceramide; Cer, ceramide.

Fig. 2.. Tissue-specific deletion of Degs1 improves glucose homeostasis and resolves hepatic steatosis in mice.

(A to J) Degs1^δAdipo or Degs1^fl/fl littermate (control) mice were placed on a HFD at 4 weeks of age. At 12 weeks of age, Degs1^fl/fl mice were injected with AAV-Tbg-Cre to produce Degs1^δLiver or Degs1^{δLiver/Adipo} mice (i.e., where Degs1 is specifically deleted in the liver or liver plus adipose tissues, respectively). The other mice received control empty viruses (AAV-Tbg-Null). Euthanasia and tissue harvesting were performed when mice were 16 weeks of age. (A) Degs1 transcript levels from indicated tissues; (B) ceramide/dihydroceramide ratios in tissues and serum; (C) body mass; (D) fat versus lean mass; (E) tissue mass; (F) fed and fasted blood glucose; (G) glucose tolerance tests were performed in animals when they were 14 weeks of age; (H) insulin tolerance tests were performed when they were 15 weeks of age; (I) serum insulin; (J) serum FFAs (n = 3 to 7 mice per group; replicate cohorts of animals were phenotyped separately, with the data included in figs. S3 to S7). Values are expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 versus control. eWAT, epididymal white adipose tissue; sWAT, subcutaneous white adipose tissue; BAT, brown adipose tissue.

Fig. 3.. Hepatic Degs1 inhibition with AAV-Degs1-shRNA improves glucose homeostasis.

(A to J) Intervention study: 26-week-old C57BL/6 mice were placed on a HFD for 12 weeks. They were then injected with a single injection (1 × 10¹¹ or 3 × 10¹¹ genome copies/mouse, low dose or high dose, respectively) of AAV-Degs1 expressing shRNA8 (AAV-Degs1) or a scrambled control (AAV-control). Mice were analyzed 12 weeks later. (A) Quantification of Degs1 mRNA in the liver, eWAT, and muscle 12 weeks after administration of AAV-Degs1-shRNA or control; (B) liver and plasma ceramide/dihydroceramide ratios; (C) body mass; (D) fasting blood glucose; (E) glucose tolerance; (F) insulin tolerance; (G) fasting plasma insulin; (H) HOMA-IR; (I) glucose infusion rate (GIR), glucose disposal rate (GDR), insulin-stimulated GDR, and (J) hepatic glucose production. (K to O) Prevention study: 20-week old C57BL/6 mice were given a single injection (1 × 10¹¹ or 3 × 10¹¹ genome copies/mouse, low dose or high dose, respectively) of AAV-Degs1 expressing shRNA8 (AAV-Degs1) or a scrambled control (AAV-control). They were then placed on a HFD for 12 weeks. (K) Ceramide/dihydroceramide ratios in liver, adipose tissue, and plasma; (L) body mass; (M) food intake; (N) percent blood glucose change; and (O) insulin tolerance. n = 10 to 12 mice for both the intervention and prevention studies. Values are expressed as mean ± SEM, *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001 versus control.

Fig. 4.. Ceramides, but not dihydroceramides, induce selective insulin resistance.

(A) Hepa cells were treated with vehicle, C₂-ceramide (Cer), or C₂-dihydroceramide (dhCer) for 2 hours before stimulation with insulin (1 μM,10 min). Lysates were resolved by SDS-polyacrylamide gel electrophoresis, and Western blots were performed using antibodies recognizing phosphorylated Akt (p-Akt) or total Akt. (B) HFD-control and HFD-Degs1^δLiver mice (as described in Fig. 1) were fasted for 5 hours before receiving a single dose of insulin (1 U/kg). The animals were euthanized 30 min later and tissues were flash frozen. p-Akt and Akt levels were determined by Western blotting as in (A). (C) C57BL/6 mice underwent the dietary and phenotyping regimen described for the intervention study (Fig. 3). Insulin stimulation of Akt/PKB phosphorylationwas determined by multiplex analysis (as described in the supplementary materials). (D) RNA-seq experiments were performed on livers from control and Degs1^δLiver animals under chow-fed conditions. Similar RNA-seq data are presented in the supplementary materials (fig. S11B) for animals maintained on the HFD. (E) Quantitative PCR determination of transcripts for SREBP-target genes in livers isolated from HFD-fed control, HFD-Degs1^δLiver, HFD-Degs1^δAdipo, and HFD-Degs1^{δLiver/Adipo} mice (n = 3 to 5 per group, treatment regimen described in Fig. 2). (F) FFA uptake in primary hepatocytes isolated from control and Degs1^{Rosa26/ERT2-Cre} mouse. (G) CD36 immunofluorescence in Hepa cells after treatment with either vehicle, Cer, or dhCer, as described in the supplementary materials. Scale bars, 10 μm. (H) Mitochondrial complex activity in WAT obtained from the control and Degs1^{Rosa26/ERT2-Cre} mice maintained on a chow diet (2 weeks after receiving the five doses of tamoxifen). (I) Western blots depicting phosphorylated [pHSL(S563)] or total HSL in primary adipocytes treated with vehicle, Cer, C₆-dihydroceramide (dhCer), and/or isoproterenol (Iso), as described in the supplementary materials. (J) Quantification of pHSL(S563) from (I) (n = 3 independent experiments). (K) Western blots depicting Isoinduced phosphorylation of pHSL (S563 and S660 phosphorylation sites) after treatment with Cer for 4 hours and/or the PP2A inhibitor microcystin-LR (MLR) for 1 hour in primary adipocytes. (L) Quantification of pHSL (S563) and pHSL (S660) from (K) (n = 4 independent experiments). (M) Sphingolipid flux was determined by measuring the incorporation of L-serine-¹³C_3,¹⁵N into ceramide in primary adipocytes treated with Iso (n = 4). Values are expressed as mean ± SEM, *P < 0.05, **P < 0.001, ***P < 0.0001 versus control.