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. 2019 Apr 15;10(1):19.
doi: 10.1186/s13293-019-0233-y.

Siah2 modulates sex-dependent metabolic and inflammatory responses in adipose tissue to a high-fat diet challenge

Sujoy Ghosh  1   2 , Jessica L Taylor  1 , Tamra M Mendoza  1 , Thanh Dang  1 , David H Burk  1 , Yongmei Yu  1 , Gail Kilroy  1 , Z Elizabeth Floyd  3
Affiliations

Siah2 modulates sex-dependent metabolic and inflammatory responses in adipose tissue to a high-fat diet challenge

Sujoy Ghosh et al. Biol Sex Differ. .

Erratum in

Abstract

Background: The obesity-related risk of developing metabolic syndrome is higher in males than in females of reproductive age, likely due to estrogen-mediated reduced adipose tissue inflammation and fibrosis with hypertrophied adipocytes. Depletion of the ubiquitin ligase Siah2 reduced white adipose tissue inflammation and improved glucose metabolism in obese male mice. Siah2 is a transcriptional target of estrogen, but data is lacking about the effect of Siah2 on adipose tissue of females. We therefore evaluated the impact of Siah2 deficiency on white and brown adipose tissue in females of reproductive age.

Methods: Body composition, adipose tissue morphology, brown adipose tissue gene, and protein expression and adipocyte sizing were evaluated in wild-type and Siah2KO female and male mice fed a low-fat or high-fat diet. Glucose and insulin tolerance, fasting glucose, insulin, fatty acids and triglycerides, and gene expression of inflammation markers in perigonadal fat were evaluated in wild-type and Siah2KO female mice. Microarray analysis of brown fat gene expression was carried out in both sexes. Statistical analysis was assessed by unpaired two-tailed t test and repeated measures ANOVA.

Results: Siah2 deficiency improves glucose and insulin tolerance in the presence of hypertrophied white adipocytes in high-fat-fed female mice with percent fat comparable to male mice. While previous studies showed Siah2KO reduces the white adipose tissue inflammatory response in male mice, the response in females is biased toward the upregulation of M2-like markers in white adipose tissue. In contrast, loss of Siah2 leads to increased whitening of brown fat in males, but not in females. This corresponded to increased expression of markers of inflammation (F4/80, Ccl2) and thermogenic genes (Pgc1alpha, Dio2, Ucp-1) and proteins (PGC-1α, UCP-1) in females. Contrary to expectations, increased expression of thermogenic markers in females was coupled with a downregulation of ERalpha and ERRgamma protein levels.

Conclusions: The most striking sex-related effect of Siah2 deficiency is reduced whitening of brown fat in high-fat-fed females. Protection from accumulating unilocular adipocytes in the brown fat corresponds to increased expression of thermogenic genes and proteins in female, but not in male mice. These results raise the possibility that Siah2 contributes to the estrogen-related effects on brown fat function in males and females.

Keywords: Adipose tissue; Estrogen; Estrogen receptor; Estrogen-related receptor; Inflammation; Obesity; Sex; Siah2; Ubiquitin.

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Conflict of interest statement

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition) and approved by the Pennington Biomedical Research Center Animal Care and Use Committee (protocol #1030).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Wild-type and Siah2KO female mice fed a high-fat diet have percent fat mass comparable to male mice, but higher levels of brown fat relative to total fat mass. a Body weight, b percent fat mass, and c fat pad weight/total fat mass were measured in the wild-type (WT) and Siah2KO female and male mice fed a defined low (LFD)- or high (HFD)-fat diet over 16 weeks. Statistical significance was determined using repeated measures ANOVA in a and b and two-tailed, unpaired t test in c, a, p < 0.05; within sex comparison between genotypes. b, p < 0.05; between sex comparison of related genotype
Fig. 2
Fig. 2
Carbohydrate metabolism is improved in the lean or obese Siah2KO female mice. a Glucose tolerance and b insulin tolerance testing were carried out at 12 weeks on the LFD or HFD. c Fasting blood glucose, d insulin, e triglycerides, and f free fatty acid levels were assayed after 16 weeks on the LFD or HFD in the female mice. Statistical significance was determined using repeated measures ANOVA in a and b and two-tailed, unpaired t test in c–f
Fig. 3
Fig. 3
Female Siah2KO adipocytes increase in size with HFD in white adipose tissue, but accumulate fewer crown-like structures and less fibrosis compared to male white adipose tissue. a H&E and b trichrome staining of HFD male (M) and female (F) wild-type and Siah2KO inguinal and perigonadal fat
Fig. 4
Fig. 4
Siah2KO affect adipocyte size and number in male, but not female gonadal and inguinal fat. Adipocyte size (area) in the LFD and HFD-fed female and male wild-type (WT) or Siah2KO (KO) mice was determined by automated cell counting of H&E stained tissue using Image J software. Adipocyte number/fat pad was estimated by converting the adipocyte area to an adipocyte volume (pL) and converting fat pad weight to a volume using the density of lipids. a Adipocyte volume (pL) and b adipocyte number/fat pad for gonadal fat. c Adipocyte volume (pL) and d adipocyte number/fat pad for inguinal fat. Statistical significance was determined using two-tailed, unpaired t test
Fig. 5
Fig. 5
Siah2 regulates mRNA expression of markers of gonadal adipose tissue inflammation in female mice. Gene expression of markers of inflammation, cytokines, and chemokines was assayed in the perigonadal adipose tissue of wild-type (WT) and Siah2KO (KO) female mice after 16 weeks on the LFD or HFD using real-time qRT-PCR. Statistical significance of Siah2KO compared to wild-type within diet, * p < 0.05, *** p < 0.001
Fig. 6
Fig. 6
Female brown adipose tissue accumulates less unilocular fat than males on a HFD. a H&E and b trichrome staining of brown fat in wild-type and Siah2KO male (M) and female (F) mice fed a HFD for 16 weeks. c Adipocyte volume (pL) in the LFD and HFD-fed female wild-type (WT) or Siah2KO (KO) mice was based on adipocyte area determined by automated cell counting using Image J software of laminin stained brown adipose tissue. d Adipocyte number/fat pad was estimated by converting adipocyte area to an adipocyte volume (pL) and fat pad weight to a volume using the density of lipids. Statistical significance was determined using two-tailed, unpaired t test
Fig. 7
Fig. 7
Microarray analysis of brown fat transcriptomics in HFD-fed male and female mice. a–d Analysis of gene expression in male and female BAT samples. a Overlap of differentially expressed genes (nominal P < 0.001, absolute fold-change > 1.5) in male and female samples. b–d Mean-average plots for gene targets of transcription factors Nfe2l2 and Ppargc1a (male BAT samples) and Srebf1 (female BAT samples), based on over-representation analysis in Ingenuity Pathway Analysis tool. Transcription factor target genes are shown as solid circles, whereas the remaining genes on the microarray are shown as open circles
Fig. 8
Fig. 8
Siah2 regulates expression of markers of thermogenesis in female, but not male brown adipose tissue. Gene expression of markers of a lipid metabolism, b mitochondrial function, c thermogenesis, and d inflammation were assayed in brown adipose tissue of wild-type (WT) and Siah2KO (KO) male (M) and female(F) mice after 16 weeks on the HFD using real-time qRT-PCR. e Serum leptin levels were assayed in male and female wild-type (WT) and Siah2KO (KO) at 16 weeks on the HFD. Statistical significance was determined using a two-tailed, unpaired t test, a, p < 0.05, b, p < 0.01, c, p < 0.001; Siah2KO compared to wild-type within the same sex
Fig. 9
Fig. 9
Loss of Siah2 upregulates thermogenic proteins, but downregulates ERα and ERRγ in brown adipose tissue independent of changes in gene expression in female, not male mice fed a HFD. a PGC1α and UCP1 and c PPARγ, ERRγ, ERα, AMPK1/2, and phosphorylated AMPK levels in brown adipose tissue were assayed via western blot analysis in male and female wild-type (WT) and Siah2KO (KO) mice after 16 weeks on the HFD and b, d quantified using Un-Scan-It software. e Gene expression of Eralpha and Errgamma was analyzed via real-time qRT-PCR. β-actin is included as a loading control in a, c. Statistical significance was determined using a two-tailed, unpaired t test
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