Heme oxygenase-1 induction remodels adipose tissue and improves insulin sensitivity in obesity-induced diabetic rats

Abstract

Obesity-associated inflammation causes insulin resistance. Obese adipose tissue displays hypertrophied adipocytes and increased expression of the cannabinoid-1 receptor. Cobalt protoporphyrin (CoPP) increases heme oxygenase-1 (HO-1) activity, increasing adiponectin and reducing inflammatory cytokines. We hypothesize that CoPP administration to Zucker diabetic fat (ZDF) rats would improve insulin sensitivity and remodel adipose tissue. Twelve-week-old Zucker lean and ZDF rats were divided into 4 groups: Zucker lean, Zucker lean-CoPP, ZDF, and ZDF-CoPP. Control groups received vehicle and treatment groups received CoPP (2 mg/kg body weight) once weekly for 6 weeks. Serum insulin levels and glucose response to insulin injection were measured. At 18 weeks of age, rats were euthanized, and aorta, kidney, and subcutaneous and visceral adipose tissues were harvested. HO-1 expression was measured by Western blot analysis and HO-1 activity by serum carbon monoxide content. Adipocyte size and cannabinoid-1 expression were measured. Adipose tissue volumes were determined using MRI. CoPP significantly increased HO-1 activity, phosphorylated AKT and phosphorylated AMP kinase, and serum adiponectin in ZDF rats. HO-1 induction improved hyperinsulinemia and insulin sensitivity in ZDF rats. Subcutaneous and visceral adipose tissue volumes were significantly decreased in ZDF rats. Adipocyte size and cannabinoid-1 expression were both significantly reduced in ZDF-CoPP rats in subcutaneous and visceral adipose tissues. This study demonstrates that HO-1 induction improves insulin sensitivity, downregulates the peripheral endocannabinoid system, reduces adipose tissue volume, and causes adipose tissue remodeling in a model of obesity-induced insulin resistance. These findings suggest HO-1 as a potential therapeutic target for obesity and its associated health risks.

Figure 1

A, Aortic tissue homogenates were subjected to Western blotting for HO-1 and HO-2 protein determination and densitometry analyses of HO-1 and HO-2/actin ratio. *P<0.05; **P<0.05 vs ZDF rats; #P<0.001. CoPP was administered once per week for 6 weeks. B, CO levels were lower in ZDF control rats. Results are mean±SE; n=4. CO release was determined as in Methods and expressed as pmol/mg per 30 minutes. Significance levels were *P<0.05 lean vs ZDF rats and **P<0.05 vs ZDF rats. C, Effect of CoPP on serum adiponectin levels in lean, ZDF, and ZDF–CoPP rats. CoPP was administered weekly for 6 weeks, and serum samples were obtained immediately before euthanization. Results are expressed as μg/mL serum; mean±SE; n=8 to 12. Significance levels were *P<0.05 lean vs ZDF rats and **P<0.005 ZDF vs ZDF–CoPP rats.

Figure 2

Effect of HO-1 expression on insulin sensitivity. Intra-peritoneal insulin sensitivity tests were performed on ZDF controls and ZDF–CoPP rats as described in Methods. Results are expressed as mean±SEM; n=3. *P<0.05; **P<0.01 ZDF vs ZDF–CoPP rats.

Figure 3

Effect of CoPP on body weight appearance and visceral fat content in lean, lean–CoPP, ZDF, and ZDF–CoPP rats. A, Effect of CoPP administration on body appearance. B, Examination of SAT and examples of SAT and VAT. C, Dissected fat from lean and ZDF rats. Representative photographs show 1 rat from each group after 6 weeks of treatment (n=8) and untreated ZDF, ZDF–CoPP, and obese–CoPP rats after 6 weeks of treatment.

Figure 4

MRI sagital image with representative axial cross sections of a ZDF control and ZDF–CoPP rat. VAT is highlighted in red (top). Percent decrease in adipose tissue volumes is represented graphically. HO-1 induction significantly reduced SAT and VAT volumes. *P<0.05; n=4.

Figure 5

A and B, Immunostaining for HO-1 expression (arrows) in SAT (Aa and Ab) and VAT (Ac and Ad) from ZDF (Aa and Ac) and ZDF–CoPP (Ab and Ad) rats. B, CoPP treatment increases HO-1 expression in SAT and VAT. C, HO-1 induction resulted in significantly smaller adipocytes in the SAT and VAT of ZDF rats and (D) IOD after immunostaining for the CB-1 receptor in subcutaneous and visceral aortic adipose tissues from ZDF and ZDF–CoPP rats. *P<0.05 control vs diabetic animals; mean±SE; n=40.

Figure 6

Western blot and densitometry analysis of effect of HO-1 expression on pAKT and total AKT in proteins of aortas from lean, lean–CoPP, ZDF, and ZDF–CoPP rats. Quantitative densitometry evaluation of pAMPK and pAKT in the aorta was determined. *P<0.05. Diabetic–obese vs control or diabetic–obese-CoPP. Each bar represents mean±SE of 4 experiments.