Drug‐like sphingolipid SH‐BC‐893 opposes ceramide‐induced mitochondrial fission and corrects diet‐induced obesity

Abstract Ceramide‐induced mitochondrial fission drives high‐fat diet (HFD)‐induced obesity. However, molecules targeting mitochondrial dynamics have shown limited benefits in murine obesity models. Here, we reveal that these compounds are either unable to block ceramide‐induced mitochondrial fission or require extended incubation periods to be effective. In contrast, targeting endolysosomal trafficking events important for mitochondrial fission rapidly and robustly prevented ceramide‐induced disruptions in mitochondrial form and function. By simultaneously inhibiting ARF6‐ and PIKfyve‐dependent trafficking events, the synthetic sphingolipid SH‐BC‐893 blocked palmitate‐ and ceramide‐induced mitochondrial fission, preserved mitochondrial function, and prevented ER stress in vitro. Similar benefits were observed in the tissues of HFD‐fed mice. Within 4 h of oral administration, SH‐BC‐893 normalized mitochondrial morphology in the livers and brains of HFD‐fed mice, improved mitochondrial function in white adipose tissue, and corrected aberrant plasma leptin and adiponectin levels. As an interventional agent, SH‐BC‐893 restored normal body weight, glucose disposal, and hepatic lipid levels in mice consuming a HFD. In sum, the sphingolipid analog SH‐BC‐893 robustly and acutely blocks ceramide‐induced mitochondrial dysfunction, correcting diet‐induced obesity and its metabolic sequelae.

.  Figure EV2. Existing agents do not protect from ceramide-induced mitochondrial fission.
A After a 1-h pre-treatment with vehicle (DMSO) or mdivi-1 and M1 (50 lM, 5 lM) for 1 h or 24 h, MEFs were treated for 3 h with vehicle (ethanol) or C16-CER (100 lM) and stained for citrate synthase. B-D Aspect ratio (B), branch length (C), or roundness (D) of mitochondria in the cells in (A). E-H As in (A-D), but in MEFs pre-treated with vehicle (methanol) or leflunomide (50 lM) for 1 h or 24 h. I-L As in (A-D), but in MEFs pre-treated with vehicle (water) or P110 (10 lM) for 1 h or P110 (1 lM) for 12 h. M MEFs treated for 3 h with BSA or palmitate (250 lM) after a 3-h pre-treatment with vehicle (DMSO), celastrol (500 nM), or withaferin A (WFA, 500 nM). N-P Aspect ratio (N), branch length (O), or roundness (P) of mitochondria in the cells in (M). Q MEFs were pre-treated with vehicle (water) or SH-BC-893 for 3 h and then treated with vehicle (DMSO) or FCCP (1 lM) for 1 h. R-T Aspect ratio (R), branch length (S), or roundness (T) of mitochondria in the cells in (Q).

EMBO Molecular Medicine
Vaishali Jayashankar et al A Mitochondrial networks in freshly resected livers from mice fed a SD (left panels) or a HFD (right panels) imaged by NAD(P)H autofluorescence. Images are maximum intensity Z-projections derived from 6 Z-slices. Binarized mitochondrial networks were segmented to tag individual objects. Aspect ratio (tubule width/length) and roundness ((4 × area)/(p × width)) were measured on a per field basis. In violin plots (left), the center line is the median and the quartiles define the 25 th to 75 th percentile; data from the representative field of view shown. The scatterplots show the mean AE SD from 8 to 12 fields of view taken from each of 4 mice per group. The same strategy was applied to quantify hypothalamic and cortical mitochondria visualized with a citrate synthase antibody except that 6 fields of view were evaluated from each of 3 mice per group. Scale bar, 20 µm. B NAD(P)H and MitoTracker Green (200 nM) co-localization in MEFs. Scale bar, 20 µm.

EMBO Molecular Medicine
Vaishali Jayashankar et al  Figure EV4. SH-BC-893 reduces food intake and triggers weight loss without toxicity.
A, B Average energy expenditure using the Weir formula (A) or activity (B) from ZT12-ZT24 in the mice from Fig 6A-D that were fed a HFD for 10 weeks and then treated with either vehicle (n = 8) or 120 mg/kg 893 (n = 6-8) by gavage on days 1 and 3. C-E Average daily distance run each day (C) or time spent on the wheel each day (D) or average daily time on wheels per mouse over the entire treatment period (E) in HFD-fed mice gavaged with vehicle (n = 8) or 120 mg/kg 893 (n = 6) Mon/Wed/Fri for 4 weeks. F-I Relative (F,H) or absolute (G,I) fat (F,G) or lean (H,I) mass as measured by EchoMRI after 25 days of treatment in the mice shown in (C-E) and Fig 6L-N; n = 6-10. J, K Daily food intake for the mice in Fig 6O-R. Mice were fed a chow diet or the HFD for 9-10 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 Mon/Wed/Fri for 3 weeks; (n = 9). Average food intake per mouse in the 24 h after each of 8 different treatments (indicated with arrows) is shown; mice were euthanized 4 h after the ninth treatment. On day 6 in (J), the University Lab Animal Resources technician unexpectedly added food to the hopper and intake could not be accurately quantified (break in line).