Disulfiram Treatment Normalizes Body Weight in Obese Mice

Abstract

Obesity is a top public health concern, and a molecule that safely treats obesity is urgently needed. Disulfiram (known commercially as Antabuse), an FDA-approved treatment for chronic alcohol addiction, exhibits anti-inflammatory properties and helps protect against certain types of cancer. Here, we show that in mice disulfiram treatment prevented body weight gain and abrogated the adverse impact of an obesogenic diet on insulin responsiveness while mitigating liver steatosis and pancreatic islet hypertrophy. Additionally, disulfiram treatment reversed established diet-induced obesity and metabolic dysfunctions in middle-aged mice. Reductions in feeding efficiency and increases in energy expenditure were associated with body weight regulation in response to long-term disulfiram treatment. Loss of fat tissue and an increase in liver fenestrations were also observed in rats on disulfiram. Given the potent anti-obesogenic effects in rodents, repurposing disulfiram in the clinic could represent a new strategy to treat obesity and its metabolic comorbidities.

Keywords: beta-cell hyperplasia; fibrosis; hepatic steatosis; inflammation; insulin resistance; obesity; weight gain.

Figure 1.

DSF improves health and insulin responsiveness in mice fed a high-fat diet (HFD). (A) Average body weight trajectory in HFD-fed mice supplemented without or with low (HFDL) and high (HFDH) doses of DSF, n =14–22 per group. (B) Timetable for phenotypic, metabolic and biochemical analyses. (C) Average daily food consumption across 42 weeks. (D) Feeding efficiency expressed as change in BW over average number of calories consumed per day. (E) Lean-to-fat ratio measured by nuclear magnetic resonance spectroscopy. (F) Metabolic cage analyses for the determination of the Respiratory Exchange Ratio (RER) and average heat production across 60 h. D, dark cycle; L, light cycle. (G) Average signals associated with D1-D3 (dark) and L1-L2 (light) represented as scatter plots. *** and ****, P < 0.001 and 0.0001 vs. HFD using one-way ANOVA coupled with Dunnett’s post-hoc test. (H) Relationship between heat production and body weight in HFD, HFDL, HFDH mice. Dotted line represents the transposition of the HFD slope to that of HFDH; 48 weeks of age, 32 weeks on diet, n = 7 per group. (I-J) Insulin tolerance test (I) with area under the curve (AUC) (J); 39 weeks of age, 23 weeks on diet, n = 6 per group. (K) Insulin levels after a 16-h fast. (L) The homeostatic model assessment calculation of insulin resistance (HOMA-IR); 45 weeks of age, 23 weeks on diet, n =4–6 per group. *, P < 0.05 vs. HFD. (M-O) Response to DSF supplementation in Aldh2-KO mice fed HFD. (M) Body weight gain trajectories over the course of 13-weeks expressed as % change over baseline with (N) AUC; mean ± SEM. (O) Fasting blood glucose collected at the time of sacrifice. WT mice fed HFD +/− DSF, n = 7/group; Aldh2-KO mice on HFD +/− DSF, n = 4–5/group. Statistics for the overall effects of treatment, genotype, and the interaction (treatment x genotype) represent the P value from a two-way ANOVA for each measurement; ** and ****, P < 0.01 and 0.0001, Tukey post-hoc test. See also Figure S1 and Table S1.

Figure 2.

Disulfiram confers protection of liver and pancreas in HFD-fed mice. (A) Representative photomicrographs of fixed liver sections after staining with Oil red O, Sirius Red, periodic acid-Schiff (PAS), and H&E (x400). (n =5 per group. This was performed on at least 10–15 fields per slide, 3 slides/liver). (B) Representative SEM images of fixed liver sections (x1000). Scale bar = 10 μm. Arrows denote lipid droplets and * represent hepatic stellate cells. (n = 5 per group, multiple fields per micrograph/animal). (C) Hepatic triglyceride (TG) levels after 40 weeks on diet, n = 3–4 per group. Data are shown as mean ± SEM (n = 5 per group). * P ≤0.05 compared to diet without DSF; #, P ≤0.05 compared to low DSF. (D) Representative images of immunostaining for insulin (red), glucagon (green), and TOPRO-3 (blue) in mouse islets after 40 weeks on the indicated dietary intervention. Scale bar = 100 μm. (n =5 per group. This was performed on at least 10 to 15 sections separated by at least 200 μm from each other section, were assessed for signal intensity per animal). (E) Quantification of total islet size. (F) Percentage of β-cells (insulin positive) and α-cells (glucagon positive). (G) Number of glucagon-producing α-cells per islet. See also Figure S2.

Figure 3.

DSF Treatment Suppresses Body Weight Gain and Improves Adiposity and Glucose Intolerance in Mice at Mid-life Fed a High-fat Diet for the Previous 3 Months. (A) Treatment protocol. (B) Feeding efficiency expressed as change in BW over average number of calories consumed per day for 12 weeks (week 12–24). The green shading indicates the results obtained in females, and it applies to all panels throughout the figure. (C) Body weight trajectories in male (top panel) and female (bottom panel) mice. Red arrow indicates the start of the DSF supplementation or SD feeding. (D) Body weight at baseline and after HFD for 12 weeks (hatched bars), and at the study completion after an additional 12 weeks of treatment. (E-F) Percent fat mass (E) and lean body mass (F) measured by NMR. (G) Lean-to-fat ratio calculated from panels E and F. (H) Representative MRI images from a male mouse. (n =53 per sex, 12 weeks on HFD) and following the completion of the intervention period (n =12 per sex and diet). (I-J) Quantitative measures of visceral fat volume (I) and calculated ratio of subcutaneous/visceral fat volumes (J) from abdominal MRI scans. Panels B-I: n = 8–12 for males and n = 9–12 for females in each experimental group unless indicated otherwise. (K) Blood glucose after a 6-h fast, n = 6–8 for males and n = 5–7 for females. (L) Relative degree of steatosis in fixed liver sections from male (n =3–6) and female (n =4–6) mice. The total number of images per diet group ranged from 60–120. Representative stained liver sections can be found in Figure S3H. (M-N) Serum liver enzymes (ALT and AST) at the conclusion of the 12-week treatment in both male (n =5–11) and female (n =5–11) mice. Dotted line indicates the upper normal limit in mice. All data are means ± SEM, * P ≤ 0.05. The treatment groups included: HFD, HFD-fed without DSF supplementation; HFDL and HFDH, diet supplementation with low and high doses of DSF, respectively; SD, standard diet. See also Figure S3 and Table S2.

Figure 4.

Diet switching reveals reversible actions of DSF against diet-induced metabolic dysfunction. (A) Trajectories of body weight as percent change from baseline (upper panel) and average weekly food consumption per mouse (lower panel) fed HFD. (B) Trajectories of body weight as percent change from baseline (upper panel) and average weekly food consumption per mouse (lower panel) fed either SD or HFD+DSF. Black arrow, diet switch from HFD to the indicated diet at the start of the experiment (t= 0); orange arrow, second diet switch; purple arrow, sac. (C) Feeding efficiency shown by the ratio of body weight change per the number of calories ingested (n = 8, mean ± SEM). (D) Organ weights at sac as percent of body weight (HFD-> HFD+DSF: n = 13; SD-> HFD+DSF: n = 4; HFD+DSF-> HFD: n = 8, mean ± SEM). ***, P < 0.001.

Figure 5.

Effects of a 12-week treatment with DSF in Sprague-Dawley rats. (A) Upper panel, trajectories of daily food consumption by rats on the indicated diets; lower panel, trajectories of change in body weight gain from baseline of the same rats. (B) Changes in fat mass after normalization for BW at the indicated time points. Baseline values were set at ‘0’. Data are represented in box and whisker plot format (n =8 per group). Statistics for the overall effects of treatment length, DSF treatment (T), and the interaction represent the P value from a two-way ANOVA with Tukey’s post-hoc tests. (C) Organ weights at the time of killing (sac) as percent of total body weight. (D) Frequency of fenestrations in liver (left) and average diameter-gaps (right). Most of the data are represented in box and whisker plot format (n =8 per group). *, *** = P < 0.05 and 0.001 vs. control chow diet. See also Figure S4.