Estrogen Impairs Adipose Tissue Expansion and Cardiometabolic Profile in Obese-Diabetic Female Rats

Figures

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  Figure 1. Ovariectomy and E2 treatment effects on body weight, food intake and adiposity in female ZDF rats. (A) Body weight and (B) food intake over the time (left panels) and at termination of the study (right panels). In A and B the arrow indicates the moment of ovariectomy and the start of E2 or vehicle treatment. (C) The Adiposity index corresponds to the sum of all adipose depots weight referred to 100 g of body weight. (D) WAT depot weight. WAT, white adipose tissue; gWAT, gonadal WAT; mWAT, mesenteric WAT; rpWAT, retroperitoneal WAT; prWAT, perirenal WAT; iWAT, inguinal WAT; E2, 17β-estradiol. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test. Student’s t-test: a, SHAM vs. LEAN. One-way ANOVA: b, OVA or OVA + E2 vs. SHAM; c, OVA + E2 vs. OVA.

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  Figure 2. Ovariectomy and E2 treatment effects on serum metabolic parameters. (A) Glycemia. (B) Insulin levels. (C) QUICKI. (D) HbA1c. (E) TG. (F) NEFA. (G) LDL-c. (H) HDL-c. (I) Atherogenic index of plasma. QUICKI, Quantitative insulin sensitivity check; HbA1c, glycated hemoglobin; TG, triglycerides; NEFA, non-esterified fatty acids; LDL-c, low-density lipoprotein cholesterol; HDL-c, high-density lipoprotein cholesterol. LEAN, control rats (fa/+); SHAM, obese-diabetic (fa/fa) rats undergoing sham surgery; OVA, ovariectomized obese-diabetic (fa/fa) rats; OVA + E2, OVA rats treated with E2 (3 µg/day) for 5 weeks. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.

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  Figure 3. Ovariectomy and E2 treatment effects on oral glucose and insulin tolerance tests in female ZDF rats. (A) Oral Glucose Tolerance Test (OGTT). (B) Insulin Tolerant Test (ITT). In A and B, left panels represent blood glucose curves and right panels represent AUC. Blood samples were obtained from the tail vein at indicated time points. (C) Ratio of pAKT/AKT and protein levels of GLUT4 in WAT. (D) Cd36 and Ap2 mRNA expression levels in WAT. AUC, area under the curve; AKT, protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLUT4, glucose transporter type 4; Cd36, cluster of differentiation 36; Ap2, adaptor protein 2. In C pAKT protein bands were normalized to total AKT protein intensity, and GLUT4 protein bands were normalized to GAPDH loading control. mRNA levels were normalized to housekeeping Gapdh expression. In C and D, data are expressed in arbitrary units (au) related to LEAN group. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc…

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  Figure 4. Ovariectomy and E2 treatment effects on WAT composition and expandability in female ZDF rats. (A) Adipose tissue histology. Representative slides of WAT hematoxylin-eosin stained sections (scale bar, 100 μm). (B) Adipocyte area. (C) Protein/DNA ratio and (D) lipid content. (E,F) mRNA expression of ECM remodeling markers. ECM, extracellular matrix; Col, collagen; Mmp, matrix metalloproteinase. mRNA levels were normalized to housekeeping Gapdh expression. In E and F, data are expressed in arbitrary units (au) related to LEAN group. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.

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  Figure 5. Ovariectomy and E2 treatment effects on (A) inflammatory adipokine expression profile and (B) markers of apoptosis in WAT of female ZDF rats. Il, interleukin; Tnf, tumor necrosis factor alpha; Serpine1, plasminogen activator inhibitor-1; Ccl3, macrophage inflammatory protein-1 alpha; Cd68, cluster of differentiation 68; Bad, Bcl-2-associated death promoter; Bcl2, B-cell lymphoma 2. mRNA levels were normalized to housekeeping Gapdh mRNA expression. Values are expressed as the mean ± SEM in arbitrary units (au) related to LEAN group. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.

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  Figure 6. Ovariectomy and E2 treatment effects on adiponectin and mediators of adiponectin signaling pathway in female ZDF rats. (A) Serum adiponectin levels. WAT protein levels of (B) adiponectin, (C) ADIPOR1 and ADIPOR2, (D) APPL1, (E) PPARG. ADIPOR, adiponectin receptor protein; APPL1, adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1; PPARG, peroxisome proliferator activated receptor gamma. Protein bands were normalized to loading control GAPDH protein intensity and expressed in arbitrary units (au) related to LEAN group. Values are expressed as the mean ± SEM. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.

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  Figure 7. Ovariectomy and E2 treatment effects on cardiac fat content, metabolic and fibrosis markers. (A) Heart weight and (B) TG content. (C) Insulin pathway and expression of Cd36. mRNA expression of (D) fibrosis markers and (E) Serpine1. Fn1, fibronectin 1; Tgfb1, transforming growth factor beta. pAKT protein bands were normalized to total AKT protein intensity. mRNA levels were normalized to housekeeping Gapdh expression. Values are expressed as the mean ± SEM. Values in C, D and E are expressed in arbitrary units (au) related to LEAN group. Differences between obese-diabetic (SHAM) and control (LEAN) animals were analyzed by Student’s t-test. Differences between obese-diabetic groups with different hormonal manipulation were analyzed by One-way ANOVA with Fisher’s LSD post-hoc test.