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

<p>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 <i>ob/ob</i> and DBKO mice (F). Values are the mean ± SEM (n = 10 per group). Differences between groups were analyzed by two-way ANOVA. ***<i>p<</i>0.001, effect of the absence of the <i>ob</i> gene. +<i>p<</i>0.05, ++<i>p<</i>0.01, effect of the absence of the <i>iNOS</i> gene. One-way ANOVA followed by Tukey's <i>post hoc</i> test was applied for variables with interaction between factors. ¶¶¶<i>p<</i>0.001 vs wild type, #<i>p<</i>0.05 vs <i>ob/ob</i> mice. EWAT, epididymal white adipose tissue; SCWAT, subcutaneous white adipose tissue; WAT, white adipose tissue; bw, body weight.</p

Metabolic characteristics of 12-week-old experimental animals.

<p>Data are means ± SEM of 8–10 animals. <i>P</i> values obtained by two-way ANOVA are shown. One way ANOVA followed by Tukey's <i>post hoc</i> tests were applied for variables with interaction between factors.</p>a<p>effect of the absence of the <i>ob</i> gene (<i>p<</i>0.01);</p>b<p>effect of the absence of the <i>iNOS</i> gene (<i>p<</i>0.01);</p>c<p>interaction between factors (<i>p<</i>0.05);</p>d<p><i>p<</i>0.05 vs wild type;</p>e<p><i>p<</i>0.001 vs wild type;</p>f<p><i>p = </i>0.071 vs <i>ob/ob</i>. FFA: free fatty acids, HOMA: homeostasis model assessment, TG: triglycerides. DBKO: double knockout mice simultaneously lacking the <i>ob</i> and <i>iNOS</i> genes.</p

Phenotype of BAT of the experimental groups.

<p>(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 <i>post hoc</i> test. ¶¶¶<i>p<</i>0.001 vs wild type, ###<i>p<</i>0.001 vs <i>ob/ob</i> mice.</p

Sequences of the primers and TaqMan® probes.

<p><i>Prdm16</i>, PR domain containing 16; <i>Rip140</i>, receptor-interacting protein 140; <i>Bmp7</i>, bone morphogenetic protein 7; <i>Sirt-1</i>, sirtuin-1, <i>Sirt-3</i>, sirtuin-3; <i>Pgc-1α</i>, peroxisome proliferative activated receptor γ coactivator 1 α; <i>Ucp-1</i>, uncoupling protein 1, <i>Ucp-3</i>, uncoupling protein 3.</p

Genes involved in vesicle-mediated transport altered by leptin in the gastrocnemius muscle of <i>ob/ob</i> mice.

<p>Differential expression of genes is indicated as fold changes with respect to the wild type group presenting only the genes which were significantly different (<i>P</i><0.05) between the leptin-treated and the control <i>ob/ob</i> groups. Ratio: fold change value for leptin-treated <i>vs</i> the <i>ob/ob</i> group.</p

Expression of genes involved in brown fat differentiation.

<p>Gene expression levels of <i>Prdm16</i> (A), <i>Bmp7</i> (B) and <i>Rip140</i> (C). Data were normalized for the expression of <i>18S</i> 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. *<i>p<</i>0.05, effect of the absence of the <i>ob</i> gene. Ø <i>p = </i>0.056, effect of the absence of the <i>iNOS</i> gene. One way ANOVA followed by Tukey's <i>post hoc</i> test was applied for variables with interaction between factors. ¶¶<i>p<</i>0.01 vs wild type.</p

Effect of leptin on the expression in skeletal muscle of the negative regulators of GLUT4 translocation, TBC1D1 and TBC1D4.

<p>Real-Time PCR analysis of <i>Tbc1d1</i> (A) and <i>Tbc1d4</i> (B) mRNA in the gastrocnemius, extensor digitorum longus (EDL) and soleus muscles of wild type, control <i>ob/ob</i>, pair-fed <i>ob/ob</i> and leptin-treated <i>ob/ob</i> mice (n = 6 per group). Values are presented as means±SEM. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 by one-way ANOVA followed by LSD tests.</p

Biological processes according to Gene Ontology (GO) altered by leptin deficiency and leptin administration in the gastrocnemius muscle.

<p><i>P</i> values (FDR adjusted <i>P</i> values) reflect the significance of change in prevalence of genes in each category under the leptin deficiency (<i>ob/ob</i>) and leptin administration (leptin) conditions in <i>ob/ob</i> mice to the expected prevalence of genes in each category. Statistical significant <i>P</i> values are highlighted in bold.</p

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

<p>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 <i>18S</i> 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. *<i>p<</i>0.05, effect of the absence of the <i>ob</i> gene. +<i>p<</i>0.05, ++<i>p<</i>0.01, effect of the absence of the <i>iNOS</i> gene. Ø <i>p = </i>0.089, effect of the absence of the <i>iNOS</i> gene; X <i>p = </i>0.084, effect of the absence of the <i>iNOS</i> gene.</p

Sequences of the primers and Taqman® probes used in the Real-Time PCR analysis.

<p>Sequences of the primers and Taqman® probes used in the Real-Time PCR analysis.</p