Sex-Dependent Role of Adipose Tissue HDAC9 in Diet-Induced Obesity and Metabolic Dysfunction

Figures

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  Figure 1. HDAC9 expression is increased in adipose tissue under obese-conditions. (A) HDAC9 mRNA expression in SQ and VF from CD- and HFD-fed female WT mice. HDAC9 mRNA expression in SVF and MAF from SQ (B) and VF (C) of female CD- and HFD-fed WT mice (n = 5–6). (D) HDAC9 protein expression in human SQ correlated with body mass index (n = 12). Data represent mean ± SEM. * p < 0.05, ** p < 0.01, ns, not significant. SQ, subcutaneous adipose tissue; VF, visceral adipose tissue; CD, chow diet; HFD, high fat diet; SVF, stromovascular fraction; MAF, mature adipocyte fraction.

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  Figure 2. Generation of adipocyte-specific HDAC9 KO mice. HDAC9 mRNA expression in SQ (A) and VF (B) from male and female HFD-fed mice (n = 5–9). (C) HDAC9 mRNA expression level in tissues from female WT and A-KO fed a HFD (n = 4). (D) Western blot validation of HDAC9 protein expression in VF from HFD-fed female mice (n = 6). Data represent mean ± SEM. * p < 0.05, ** p < 0.01, ns, not significant. A-KO, adipocyte-specific HDAC9 KO; SQ, subcutaneous adipose tissue; VF, visceral adipose tissue.

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  Figure 3. Female A-KO mice have reduced weight gain and fat mass following HFD-feeding. (A) Growth curves of female WT and A-KO mice fed a HFD (n = 11–15). (B) Cumulative locomotor activity over a 24 h period (n = 8). (C) Cumulative food consumption over a 24 h period (n = 7). (D) Fat mass and lean mass measured by whole body composition nuclear magnetic resonance (n = 11–16). (E) Tissue weight measurements of SQ, VF and liver at time of sacrifice (n = 10–15). (F) Hepatic triglyceride measured per gram liver (n = 10–16). Data represent mean ± SEM. * p < 0.05, ** p < 0.01, ns, not significant. SQ, subcutaneous adipose tissue; VF, visceral adipose tissue.

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  Figure 4. Adipocyte-specific HDAC9 gene deletion increases energy expenditure and metabolic health in HFD-fed female mice. Comprehensive lab animal monitoring system (CLAMS) measurement of energy expenditure (A), VO2 consumption (B), and respiratory exchange ratio (RER) (C) from HFD-fed female mice (n = 8). Representative H&E images of SQ (D) and VF (E) from HFD-fed female WT and A-KO mice with quantification of adipocyte size (n = 3). Glucose tolerance (F) and insulin tolerance test (G) quantified by area under the curve (n = 5–7). Data represent mean ± SEM. * p < 0.05; ** p < 0.01, ns, not significant. SQ, subcutaneous adipose tissue; VF, visceral adipose tissue.

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  Figure 5. Adipocyte-specific HDAC9 gene deletion increases expression of adipogenic differentiation and metabolism-associated genes and enhances adaptive thermogenesis in female mice. mRNA expression level of (A) adipogenic and (B) metabolism genes in SQ of HFD-fed female mice. (C) Hourly body temperature measurements during 4 °C acute cold challenge (n = 4). mRNA expression level of (D) classical beiging and (E) UCP-1 independent thermogenesis associated genes in SQ of HFD-fed female mice. Data represent mean ± SEM. * p < 0.05; ** p < 0.01, # p < 0.08. SQ, subcutaneous adipose tissue; PPARɣ, peroxisome proliferator-activated receptor gamma; Pgc1ɑ, PPARɣ coactivator 1-alpha; C/ebpɑ, CCAAT/enhancer-binding protein alpha; Fabp4, fatty acid binding protein 4; Pfkfb3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; Ndfus1, NADH-ubiquinone oxidoreductase; Ndufv2, NADH-ubiquinone oxidoreductase flavoprotein 2; Atp5a1, ATP synthase F1 subunit alpha; Cpt1a, carnitine palmitoyltransferase 1a; Ldha, lactate dehydrogenase A; Prdm16, PR/SET domain 16; Fgf21, fibroblast growth factor 21; Ucp1, uncoupling protein 1; Serca2b, sarco/endoplasmic reticulum Ca2+ ATPase 2b; Ryr2, ryanodine receptor 2;…

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  Figure 6. Adipocyte-specific HDAC9 gene deletion does not improve metabolic health in HFD-fed male mice. (A) Growth curves of HFD-fed male WT and A-KO mice (n = 11–15). (B) Fat mass and lean mass measured by whole body composition nuclear magnetic resonance (n = 10–12). (C) Tissue weight measurements of SQ, VF and liver at time of sacrifice (n = 10–12). Glucose tolerance (D) and insulin tolerance test (E) quantified by area under the curve (n = 4–5). mRNA expression of (F) adipogenic, (G) metabolic, (H) classic beiging and (I) UCP1-independent beiging genes in SQ from HFD-fed male mice (n = 8). Data represent mean ± SEM. * p < 0.05, ns, not significant. SQ, subcutaneous adipose tissue; VF, visceral adipose tissue; PPARɣ, peroxisome proliferator-activated receptor gamma; Pgc1ɑ, PPARɣ coactivator 1-alpha; C/ebpɑ, CCAAT/enhancer-binding protein alpha; Pfkfb3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; Ndfus1, NADH-ubiquinone oxidoreductase; Ndufv2, NADH-ubiquinone oxidoreductase flavoprotein 2; Atp5a1, ATP synthase F1 subunit alpha; Prdm16, PR/SET domain 16; Serca2b; sarco/endoplasmic reticulum Ca2+ ATPase 2b.

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  Figure 7. HDAC9 is differentially expressed in SVF of male compared to female mice. (A) HDAC9 mRNA expression in SQ and VF from HFD-fed female and male WT mice (n = 4–7). HDAC9 mRNA expression in SVF and MAF from SQ (B) and VF (C) of HFD-fed WT mice (n = 6–7). Data represent mean ± SEM. * p < 0.05, ns, not significant. SQ, subcutaneous adipose tissue; VF, visceral adipose tissue; SVF, stromovascular fraction; MAF, mature adipocyte fraction.