Perinatal Treatment with Leptin, but Not Celastrol, Protects from Metabolically Obese, Normal-Weight Phenotype in Rats

Figure 1. Body weight evolution during lactation in pups treated with vehicle (Control), leptin, celastrol, and with leptin and celastrol (Lep+Cel). Data represent means ± SEMs (n = 8–16). Statistics: The # symbol shows the significance vs. Control animals (U Mann–Whitney, p < 0.05).

Figure 2. Liver analysis. Liver fat content (A) (triglycerides and total lipids) and (B) liver sections stained with hematoxylin and eosin in adult control rats fed a normal-fat diet (NF-Control) and in adult rats that were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel), and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. In (A), data represent means ± SEMs (n = 8–10). Statistics: The # symbol shows the significance of all the HF-groups vs. the NF-Control group (U Mann–Whitney, p < 0.05). In (B), representative sections are shown.

Figure 3. Liver gene expression analysis in adult control rats fed a normal-fat diet (NF-Control) and in adult rats which were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel) and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. mRNA expression was measured by RT-qPCR. Data represent means ± SEMs (n = 8–10) of ratios of specific mRNA levels relative to Gdi (used as reference gene), expressed as a percentage of the value of the NF-Control group that was set to 100%. Statistics: One-way ANOVA was only performed in the HF-fed animals. Data not sharing a common letter (a, b) are significantly different. LSD post hoc was used after ANOVA analysis. The # symbol shows the significance of all the HF-fed groups vs. the NF-Control group (U Mann–Whitney, p < 0.05); the * symbol shows the significance of the HF-treated groups vs. the HF-Control group (U Mann–Whitney, p < 0.05).

Figure 4. Retroperitoneal WAT (rWAT) gene expression analysis in adult control rats fed a normal-fat diet (NF-Control) and in adult rats that were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel) and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. mRNA expression was measured by RT-qPCR. Data represent means ± SEMs (n= 8–10) of ratios of specific mRNA levels relative to Gdi (used reference gene), expressed as a percentage of the value of the NF-Control group that was set to 100%. Statistics: One-way ANOVA was performed only in the HF-fed animals. Data not sharing a common letter (a, b) are significantly different. LSD post hoc was used after ANOVA analysis. The # symbol shows the significance of all the HF-fed groups vs. the NF-Control group (U Mann–Whitney, p < 0.05); the * symbol shows the significance of the HF-treated groups vs. the HF-Control group (U Mann–Whitney, p < 0.05).

Figure 5. Epididymal WAT (eWAT) gene expression analysis in adult control rats fed a normal-fat diet (NF-Control) and in adult rats that were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel) and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. mRNA expression was measured by RT-qPCR. Data represent means ± SEMs (n= 8–10) of ratios of specific mRNA levels relative to Gdi (used reference gene), expressed as a percentage of the value of the NF-Control group that was set to 100%. Statistics: The # symbol shows the significance of all the HF-fed groups vs. the NF-Control group (U Mann–Whitney, p < 0.05).

Figure 6. Retroperitoneal WAT (rWAT) analysis. (A) UCP1 immunostaining; (B) Ucp1 gene expression analyzed by RT-qPCR; and (C) number of adipocytes per area (mm2). Groups: adult control rats fed a normal-fat diet (NF-Control) and adult rats that were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel), and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. In (B), data represent threshold cycle (Ct) values. The number of animals in each group in which Ucp1 gene expression was detected (+animals) is indicated. In (C), data represent means ± SEMs (n = 6–10). The # symbol shows the significance of all the HF-fed groups vs. the NF-Control group (U Mann–Whitney, p < 0.05); the * symbol shows the significance of the HF-treated groups vs. the HF-Control group (U Mann–Whitney, p < 0.05).

Figure 7. Brown adipose tissue (BAT) gene expression analysis in adult control rats fed a normal-fat diet (NF-Control) and in adult rats that were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel) and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. mRNA expression was measured by RT-qPCR. Data represent means ± SEMs (n = 8–10) of ratios of specific mRNA levels relative to Gdi (used reference gene), expressed as a percentage of the value of the NF-Control group that was set to 100%. Statistics: The # symbol shows the significance of all the HF-fed groups vs. the NF-Control group (U Mann–Whitney, p < 0.05); the * symbol shows the significance of the HF-treated groups vs. the HF-Control group (U Mann–Whitney, p < 0.05).

Figure 8. Hypothalamic gene expression analysis in adult control rats fed a normal-fat diet (NF-Control) and in adult rats who were treated during lactation with vehicle (Control), leptin, celastrol, or leptin and celastrol (Lep+Cel) and subsequently pair-fed for 11 weeks with high-fat diet (HF) administered under isocaloric conditions to the NF-Control group. mRNA expression was measured by RT-qPCR. Data represent means ± SEMs (n = 8–10) of ratios of specific mRNA levels relative to Gdi (used reference gene), expressed as a percentage of the value of the NF-Control group that was set to 100%. Statistics: One-way ANOVA was performed only in the HF-fed animals. Data not sharing a common letter (a, b) are significantly different. LSD post hoc was used after ANOVA analysis. The # symbol shows the significance of all the HF-fed groups vs. the NF-Control group (U Mann–Whitney, p < 0.05); the * symbol shows the significance of the HF-treated groups vs. the HF-Control group (U Mann–Whitney, p < 0.05).