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. 2018 Mar 22;8(1):5057.
doi: 10.1038/s41598-018-22988-1.

Crotamine induces browning of adipose tissue and increases energy expenditure in mice

Marcelo P Marinovic  1 , Joana D Campeiro  1 , Sunamita C Lima  1 , Andrea L Rocha  2 , Marcela B Nering  1 , Eduardo B Oliveira  3 , Marcelo A Mori  2   4 , Mirian A F Hayashi  5
Affiliations

Crotamine induces browning of adipose tissue and increases energy expenditure in mice

Marcelo P Marinovic et al. Sci Rep. .

Abstract

Crotamine, originally isolated from rattlesnake venom, has been extensively studied due to its pleiotropic biological properties, and special attention has been paid to its antitumor activity. However, long-term treatment with crotamine was accompanied by a reduction in animal body weight gain and by increases in glucose tolerance. As cancer is commonly associated with cachexia, to preclude the possible cancer cachexia-like effect of crotamine, herein this polypeptide was administered in healthy wild-type C57/BL6 mice by the oral route daily, for 21 days. Reduced body weight gain, in addition to decreased white adipose tissue (WAT) and increased brown adipose tissue (BAT) mass were observed in healthy animals in the absence of tumor. In addition, we observed improved glucose tolerance and increased insulin sensitivity, accompanied by a reduction of plasma lipid levels and decreased levels of biomarkers of liver damage and kidney disfunctions. Importantly, long-term treatment with crotamine increased the basal metabolic rate in vivo, which was consistent with the increased expression of thermogenic markers in BAT and WAT. Interestingly, cultured brown adipocyte cells induced to differentiation in the presence of crotamine also showed increases in some of these markers and in lipid droplets number and size, indicating increased brown adipocyte maturation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Body weight gain, food intake rate, fat depot weights and fat index of animals after oral treatment. (a) Mean body weight (g), (b) body weight gain (g), (c) food intake (g), (d) weight of brown adipose tissue (BAT, g), (e) weight of subcutaneous white adipose tissue (WAT, g), (f) weight of the epididymal WAT (EAT, (g), and (g) fat index. The results are presented as the mean ± SEM of three independent experiments (in which cohorts were composed of 5 animals in each group). *p < 0.05 for comparison between the treated and untreated control groups.
Figure 2
Figure 2
Evaluation of glucose tolerance and insulin sensitivity. (a) Glucose tolerance test (GTT) and (b) insulin tolerance test (ITT), at 0, 5, 15, 30, 60 and 90 min after stimulus. The respective area under the curve (AUC) values for each curve are presented in the insets. (c) The constant rate of glucose disappearance calculated from (b), the glucose decay index (kITT). The results are presented as the mean ± SEM of seven independent experiments. *p < 0.05 for comparison between the treated and untreated control groups.
Figure 3
Figure 3
Biochemical evaluation of treated and untreated mice serum. (a) Triglycerides, (b) cholesterol, (c) LDL, (d) HDL, (e) ALT, (f) AST, (g) gamma-GT, (h) creatinine, and (i) uric acid. The results are presented as the mean ± SEM of six independent experiments. *p < 0.05 for comparison between the treated and untreated control groups.
Figure 4
Figure 4
Basal metabolic profile assessed by calorimetry of treated and untreated animals. (a) Energy expenditure (HEAT). (b) oxygen consumption (VO2), (c) carbon dioxide consumption (VCO2), (d) respiratory exchange ratio (RER), and (e) ambulatorial displacement. The results are presented as the mean ± SEM of two independent experiments (in which cohorts were composed of 4 animals in each group). Statistical analysis was performed using one-way ANOVA test with Bonferroni post hoc analysis to compare the quantitative results among samples from treated and untreated groups at light and dark cycles (#p < 0.05). Student T-test (*p < 0.05) was also used to show significant differences between the light and dark cycles.
Figure 5
Figure 5
Evaluation of thermogenic marker expression in BAT and WAT of treated and untreated animals. Gene expression of (a) β-3 adrenergic receptor (Adrb3), (b) Pparα, (c) Pgc1α, and (d) Ucp1 in BAT, and gene expression of transcription factor (e) Prdm16 and (f) Ucp1 in WAT, all normalized against 18S. Data are the mean ± SEM of three independent experiments. *p < 0.05 for comparison between the treated and untreated control groups.
Figure 6
Figure 6
Evaluation of expression of markers for differentiation and activation of brown adipocytes (9B). Gene expression of the transcription factors (a) Prdm16 and (b) Pparγ, (c) Pgc1α, and (d) Ucp1 normalized against 18S is shown, and the results are presented as the mean ± SEM of five independent experiments. *p < 0.05 for comparison between the treated and untreated control groups.
Figure 7
Figure 7
Confocal imaging of differentiated adipocyte cells. Immortalized brown preadipocytes were induced to adipocyte differentiation in the absence (a and b) or presence (c and d) of crotamine (5 μM). Differential interference contrast (DIC) imaging (a and c) allows the morphological visualization of cells and of lipid vesicles, whereas the nucleus of the cells and lipid vesicles are shown by DAPI (λEx 405 nm/λEm 420–470 nm) and red O oil (λEx 543 nm/λEm 555–625 nm) staining, respectively (b and d). Scale bars = 10 μm. The average number of lipid droplets per cell (e) and the mean value of diameter of lipid droplets (f) were determined by using ImageJ software (N = 50 cells). *p < 0.05 for comparison between the treated and untreated control groups.
Figure 8
Figure 8
Schematic representation of the cell differentiation process. Immortalized brown preadipocytes cell line 9B were induced to adipocyte differentiation in the absence or presence of crotamine (5 μM). Crotamine was added in each day of medium change for replacement of the different drugs of the cocktail for differentiation, as indicated by the vertical arrows.
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