Human CIDEC transgene improves lipid metabolism and protects against high-fat diet–induced glucose intolerance in mice

Figures

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  Figure 1. CIDEC expression negatively associates with lipolysis in the adipose tissue of patients with obesity.A, basal lipolysis, assessed as glycerol release, was measured in 12 SAT and 15 VAT depots of human patients and normalized to total protein. Data are presented as mean ± SEM. B, basal ATGL mRNA in paired SAT and VAT depots of 27 human study participants. Data are presented as mean ± SEM. C, ATGL protein expression in SAT and VAT paired samples from eight randomly selected patients, with obesity among the 27 samples. Data are presented as mean ± SEM. D, basal HSL mRNA in paired SAT and VAT depots of 27 human participants. Data are presented as mean ± SEM. E, HSL protein expression in SAT and VAT paired samples from eight randomly selected patients with obesity. F, CIDEC mRNA in 27 paired SAT and VAT depots of patients with obesity. Data are presented as mean ± SEM. G, CIDEC protein level measured in paired SAT and VAT depots of eight randomly selected patients. Data are presented as mean ± SEM. For statistical significance, unpaired t test was used to analyze the significance between two groups. SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue.

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  Figure 2. Generation and characterization of Ad-CIDECtg transgenic mice expressing human CIDEC specifically in adipose tissue.A, schematic representation of the generation of Ad-CIDECtg mice expressing CIDEC in adipose tissue. B, genotyping of mice to assess the presence of human CIDEC and/or Cre recombinase. C, relative gene expression of human CIDEC in different organs of Ad-CIDECtg mice (n = 3 per group). D, protein expression of CIDEC in different adipose tissue depots including perigonadal white adipose tissue, inguinal adipose tissue (subcutaneous adipose tissue), and interscapular adipose tissue (brown adipose tissue) of male floxed and male Ad-CIDECtg mice (n = 3 per group).

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  Figure 3. Ad-CIDECtg mice exhibit protection against high fat diet (HFD)–induced glucose intolerance.A, i.p. GTT in 4.5 month old (3 months + 1.5 months HFD) male floxed or Ad-CIDECtg mice either on a regular diet (RD) or HFD for 10 weeks. The plot on the right-hand side represents area under the curve (AUC). ∗ signifies statistical significance between the HFD-fed groups, and $ signifies statistical significance between the RD-fed groups. B, ITT of floxed or Ad-CIDECtg mice either on a RD or HFD. The plot on the right-hand side represents the AUC. ∗ signifies statistical significance between the HFD-fed groups, and $ signifies statistical significance between the RD-fed groups. C, fasting blood glucose levels in mice. D, serum leptin levels, (E) serum adiponectin levels, and (F) leptin/adiponectin ratio. In C–F, blue, green, black, and red bars represent floxed RD, Ad-CIDECtg RD, floxed HFD, and Ad-CIDECtg HFD, respectively. G, Western blot analysis representing relative insulin-stimulated AKT phosphorylation (Ser473) levels in insulin-sensitive tissues including white adipose tissue, liver, and muscle (gastrocnemius), and (H) densitometry analysis of Western blots. Data are represented…

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  Figure 4. RNA-seq analysis in white adipose tissue of floxed and Ad-CIDECtg mice.A, significantly enriched (q value <0.05) biological pathways among upregulated DEGs, and (B) downregulated pathways in adipose tissue of Ad-CIDECtg mice. The rich factor is the ratio of the number of DEGs to the total gene number in a pathway. Here, the q-value is a corrected p-value. The color and size of the dots represent the range of q-value and the number of DEGs mapped to the indicated pathways, respectively. C, volcano plot representing differently expressed genes in floxed versus Ad-CIDECtg mice. D, heat map showing altered genes associated with lipid metabolism in perigonadal adipose tissue of Ad-hCIDECtg mice. DEG, differentially expressed gene.

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  Figure 5. CIDEC interacts with CGI-58. Fasting serum lipid parameters were analyzed in floxed and Ad-CIDECtg mice fed regular chow or HFD. A, serum triglycerides, (B) serum total cholesterol, (C) low-density lipoprotein, (D) high-density lipoprotein, and (E) nonesterified fatty acids. F, Western blot analysis to confirm the overexpression of human ATGL, human CGI-58, and human CIDEC. G, immunoprecipitation of human CIDEC from cell lysates of human CIDEC and human CGI-58 overexpressing HEK-293 cells and immunoblotting against CGI-58 antibody (top panel). Immunoprecipitation of CGI-58 from cell lysates of human CIDEC and human CGI-58 overexpressing HEK-293 cells and immunoblotting against CIDEC (lower panel). H, a Z-slice of 0.2 μm showing localization of CIDEC (green) and CGI-58 (red) in culture human adipocytes. Yellow color shows colocalization of CIDEC and CGI-58. I, semiquantitative analysis of CIDEC colocalized to CGI-58 (green to red), and CGI-58 colocalized to CIDEC (red to green) (n = 10). HFD, high-fat diet.

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  Figure 6. CIDEC limits CGI-58-induced ATGL activation to release FFAs.A, measurement of glyceryl trilinoleate lipolysis upon addition of cell lysates overexpressing either ATGL, CGI-58, or CIDEC alone or in different combinations to assess ATGL-induced lipolytic activity. ∗ signifies statistical significance compared to controls, $ signifies statistical significance compared to the lipolysis measured in column 6 (ATGL+CGI-58). One-way ANOVA was performed to analyze the significance between multiple groups (n = 3 per group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and $p < 0.05, $$p < 0.01, $$$p < 0.001. B, glycerol release was assessed in ex vivo explants of white adipose tissue of mice either unstimulated or stimulation with CL-316243 (2 μM) for 2 h. Data are represented as mean ± SEM. For statistical analyses, a student’s t test was applied to compare two groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. C, glycerol release was assessed in the adipose tissue from SAT depot (n = 10) after siRNA-mediated CIDEC knockdown. Scrambled siRNA was used as a control. Top right panel is a representative Western blot showing siRNA-mediated CIDEC knockdown in SAT. D, glycerol release from the VAT depot (n =…

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  Figure 7. Generation, characterization, and metabolic studies in Ad-CIDECmut mice.A, DNA sequence of human CIDEC transgene expressed in Ad-CIDECtg mice compared to mutant CIDEC transgene expressed in Ad-CIDECmut mice. B, genotyping of Ad-CIDECmut mice for the presence of mutant CIDEC and/or Cre recombinase. M Cidec represents mouse Cidec gene. C, body weight gain of floxed and Ad-CIDECmut mice fed either regular chow or HFD. D, organ weight of floxed and Ad-CIDECmut mice. Blue, green, black, and red bars represent floxed RD, Ad-CIDECmut RD, floxed HFD, and Ad-CIDECmut HFD, respectively. E, food intake and (F) energy intake of floxed and Ad-CIDECmut mice fed either regular chow or HFD. G, GTT and (H) ITT of mice. I–L, fasting serum total cholesterol (I), nonesterified fatty acids (J), LDL cholesterol (K), and HDL cholesterol (L) of floxed and Ad-CIDECmut mice fed either regular chow or HFD. Data are represented as mean ± SEM. For statistical analysis, two-way ANOVA followed by Bonferroni post hoc analysis was performed for (G) and (H). For other graphs, a student’s t test was applied to compare the significance between the two groups. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (n=6–8 per group).…