Deletion of inducible nitric-oxide synthase in leptin-deficient mice improves brown adipose tissue function

Abstract

Background: Leptin and nitric oxide (NO) on their own participate in the control of non-shivering thermogenesis. However, the functional interplay between both factors in this process has not been explored so far. Therefore, the aim of the present study was to analyze the impact of the absence of the inducible NO synthase (iNOS) gene in the regulation of energy balance in ob/ob mice.

Methods and findings: Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated, and the expression of molecules involved in the control of brown fat cell function was analyzed by real-time PCR, western-blot and immunohistochemistry. Twelve week-old DBKO mice exhibited reduced body weight (p<0.05), decreased amounts of total fat pads (p<0.05), lower food efficiency rates (p<0.05) and higher rectal temperature (p<0.05) than ob/ob mice. Ablation of iNOS also improved the carbohydrate and lipid metabolism of ob/ob mice. DBKO showed a marked reduction in the size of brown adipocytes compared to ob/ob mutants. In this sense, in comparison to ob/ob mice, DBKO rodents showed an increase in the expression of PR domain containing 16 (Prdm16), a transcriptional regulator of brown adipogenesis. Moreover, iNOS deletion enhanced the expression of mitochondria-related proteins, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1alpha), sirtuin-1 (Sirt-1) and sirtuin-3 (Sirt-3). Accordingly, mitochondrial uncoupling proteins 1 and 3 (Ucp-1 and Ucp-3) were upregulated in brown adipose tissue (BAT) of DBKO mice as compared to ob/ob rodents.

Conclusion: Ablation of iNOS improved the energy balance of ob/ob mice by decreasing food efficiency through an increase in thermogenesis. These effects may be mediated, in part, through the recovery of the BAT phenotype and brown fat cell function improvement.

Figure 1. Growth and metabolic variables of mice of the four experimental groups.

Growth curves of 4–12 week-old-mice (A) together with epididymal (EWAT), subcutaneous (SCWAT) and whole-body fat content (B) of the experimental animals. Cumulative food intake (C), food efficiency (D) and rectal temperature (E) are also shown. Representative images illustrating the differences in size between 12-week-old ob/ob and DBKO mice (F). Values are the mean ± SEM (n = 10 per group). Differences between groups were analyzed by two-way ANOVA. ***p<0.001, effect of the absence of the ob gene. +p<0.05, ++p<0.01, effect of the absence of the iNOS gene. One-way ANOVA followed by Tukey's post hoc test was applied for variables with interaction between factors. ¶¶¶p<0.001 vs wild type, #p<0.05 vs ob/ob mice. EWAT, epididymal white adipose tissue; SCWAT, subcutaneous white adipose tissue; WAT, white adipose tissue; bw, body weight.

Figure 2. Phenotype of BAT of the experimental groups.

(A) Representative histological sections of BAT stained with hematoxylin-eosin. Magnification X100 (scale bar = 50 µm). BAT weight, general cell surface area (B), and mean values (C) of the cell surface area in relation to the percentage of brown adipocytes contributing to the final cell size in each of the experimental groups. Values are the mean ± SEM (n = 6 per group). (D) MicroPET scans depicting interscapular BAT uptake of experimental animals using ¹⁸F-FDG as a probe; signals are shown in %ID/g at the region of interest over the background. Differences between groups were analyzed by one-way ANOVA followed by Tukey's post hoc test. ¶¶¶p<0.001 vs wild type, ###p<0.001 vs ob/ob mice.

Figure 3. Gene and protein expression levels of genes involved in thermogenesis.

Expression levels of UCP-1 (A) and UCP-3 (B) in BAT. mRNA and protein data were normalized for the expression of 18S rRNA and β-actin, respectively. The expression in wild type mice was assumed to be 1. Representative blots are shown on top of the histograms. Immunohistochemistry of UCP-1 and UCP-3 in BAT corresponding to each experimental groups is shown at the bottom of the histograms. Magnification X100 (scale bar = 50 µm). Values are the mean±SEM (n = 6 per group). Differences between groups were analyzed by two-way ANOVA. *p<0.05, **p<0.01, effect of the absence of the ob gene. +p<0.05, ++p<0.01, effect of the absence of the iNOS gene.

Figure 4. Expression of genes involved in brown fat differentiation.

Gene expression levels of Prdm16 (A), Bmp7 (B) and Rip140 (C). Data were normalized for the expression of 18S rRNA and gene expression levels in wild type mice were assumed to be 1. Values are the mean ± SEM (n = 6 per group). Protein levels of Bmp7 are also shown (B). Protein data were normalized for the expression of β-actin. Differences between groups were analyzed by two-way ANOVA. *p<0.05, effect of the absence of the ob gene. Ø p = 0.056, effect of the absence of the iNOS gene. One way ANOVA followed by Tukey's post hoc test was applied for variables with interaction between factors. ¶¶p<0.01 vs wild type.

Figure 5. Effect of the lack of both genes on molecules involved in the regulation of thermogenesis.

Bar graphs show the transcript and protein levels of peroxisome proliferator-activated γ coactivator-1 α (PGC-1α) (A), sirtuin-1 (SIRT1) (B), and sirtuin-3 (SIRT3) (C) in BAT of experimental animals. mRNA and protein data were normalized for the expression of 18S rRNA and β-actin, respectively. The expression in wild type mice was assumed to be 1. Representative blots are shown on the top of the histograms. Values are the mean±SEM (n = 6 per group). Differences between groups were analyzed by two-way ANOVA. *p<0.05, effect of the absence of the ob gene. +p<0.05, ++p<0.01, effect of the absence of the iNOS gene. Ø p = 0.089, effect of the absence of the iNOS gene; X p = 0.084, effect of the absence of the iNOS gene.