Novel fat depot-specific mechanisms underlie resistance to visceral obesity and inflammation in 11 β-hydroxysteroid dehydrogenase type 1-deficient mice

Abstract

Objective: The study objective was to determine the key early mechanisms underlying the beneficial redistribution, function, and inflammatory profile of adipose tissue in 11β-hydroxysteroid dehydrogenase type 1 knockout (11β-HSD1(-/-)) mice fed a high-fat (HF) diet.

Research design and methods: By focusing on the earliest divergence in visceral adiposity, subcutaneous and visceral fat depots from 11β-HSD1(-/-) and C57Bl/6J control mice fed an HF diet for 4 weeks were used for comparative microarray analysis of gene expression, and differences were validated with real-time PCR. Key changes in metabolic signaling pathways were confirmed using Western blotting/immunoprecipitation, and fat cell size was compared with the respective chow-fed control groups. Altered adipose inflammatory cell content and function after 4 weeks (early) and 18 weeks (chronic) of HF feeding was investigated using fluorescence (and magnetic)-activated cell sorting analysis, immunohistochemistry, and in situ hybridization.

Results: In subcutaneous fat, HF-fed 11β-HSD1(-/-) mice showed evidence of enhanced insulin and β-adrenergic signaling associated with accretion of smaller metabolically active adipocytes. In contrast, reduced 11β-HSD1(-/-) visceral fat accumulation was characterized by maintained AMP kinase activation, not insulin sensitization, and higher adipocyte interleukin-6 release. Intracellular glucocorticoid deficiency was unexpectedly associated with suppressed inflammatory signaling and lower adipocyte monocyte chemoattractant protein-1 secretion with strikingly reduced cytotoxic T-cell and macrophage infiltration, predominantly in visceral fat.

Conclusions: Our data define for the first time the novel and distinct depot-specific mechanisms driving healthier fat patterning and function as a result of reduced intra-adipose glucocorticoid levels.

FIG. 1.

Quantitative RT-PCR validation of microarray gene expression differences between HF-fed C57BL/6J and 11β-HSD1^−/− adipose. C57BL/6J (▨) and 11β-HSD1^−/− (▤) mice were fed an HF diet for 4 weeks. Levels of mRNA were measured in two fat depots: subcutaneous (sc) and mesenteric (mes). A: Representative genes involved in lipid and glucose metabolism altered in subcutaneous fat. B: Representative genes involved in inflammatory cell signaling and function altered in mesenteric fat. Data are presented as a mean ± SEM of two independent HF diet studies and are expressed as a ratio of the gene of interest to the TATA-binding protein internal control. n = 7–10. Analyses were by two-way ANOVA. Significant effects of genotype are shown: *P < 0.05. Significant genotype-by-depot interaction is shown: †P < 0.05.

FIG. 2.

Phosphorylation of proteins in the insulin and AMPK signaling pathways in adipose tissues of C57BL/6J and 11β-HSD1^−/− mice. A: Immunoprecipitation using an anti-IRS1 antibody followed by Western blotting for IRS1-phosphotyrosine and p-85 PI3K and Western blot for phospho-AKTser473 (AKT^p) and pan-AKT (AKT) in insulin-treated C57BL/6J mice fed control (B6, ■) or HF (B6HF, ▨) diet and 11β-HSD1^−/− mice on control (KO, □) or HF (KOHF, ▤) diet. B: Western blot for phospho/pan-AKT in mesenteric fat and for phospho-AMPKthr172 (AMPK^p) and pan-AMPK in mesenteric fat. n = 6–8. Effects of diet are shown as significant: †P < 0.05. Effects of genotype are shown as significant: *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

T-cell levels in adipose tissues of C57BL/6J and 11β-HSD1^−/− mice fed HF diet for 4 weeks. A: Anti-CD3 staining in mesenteric adipose sections from C57BL/6J (B6) and 11β-HSD1^−/− mice (KO) fed control or HF diet (B6HF, KOHF) (representative of n = 5). Note fat cell expansion causes the appearance of lower CD3⁺ cells/area, but there is actually an increase per depot as shown in B. FACS quantification of T-cell numbers in mesenteric (B) adipose SVC, and (C) adipose lymph nodes from C57BL/6J mice fed control (■) or HF (▨) diet and 11β-HSD1^−/− mice fed control (□) or HF (▤) diet. CD8+ cytotoxic T-cells are shown on the left, and CD3⁺CD8⁻ (a surrogate for CD4⁺ T-helper cells) FACS data are shown on the right; n = 4, with adipose pooled from two mice per condition. Effects of diet are shown as significant: †P < 0.05. Effects of genotype are shown as significant: *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Macrophage numbers in adipose tissues from C57BL/6J and 11β-HSD1^−/− mice after 18-week HF diet. A: FACS quantification of total macrophages (Mϕ) content (Cd11b⁺) in subcutaneous and mesenteric fat, as a percentage of the total SVC number, in C57BL/6J (■) or 11β-HSD1^−/− mice (□) on control diet. B: FACS quantification of macrophage number (CD11b⁺) as cells per gram of adipose tissue in subcutaneous and mesenteric fat after 18-week HF diet in C57BL/6J (▨) and 11β-HSD1^−/− mice (▤); n = 6 with adipose pooled from two mice per condition. C: Quantitative results from in situ hybridization with an antisense riboprobe (top) hybridized against the macrophage marker F4/80 in mesenteric fat of C57BL/6J mice fed control (B6: ■) or HF (B6HF: ▨) diet and in genetically obese Lepob mice (Ob: ) and Lepob mice that are 11β-HSD1 deficient (Ob HSD1^−/−: ); n = 6, effects of genotype are shown as significant: *P < 0.05. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

11β-HSD1 mRNA levels in MACS-enriched adipose macrophages from 18-week HF diet–induced and genetically obese mice. Adipose stromal macrophages (Mϕ) (CD11b⁺, left) were enriched with magnetic-bead cell sorting using the anti-CD11b antibody from other SVCs (CD11b⁻, right) in A the subcutaneous and mesenteric adipose tissues of C57BL/6J mice fed control (B6: ■) or HF (B6HF: ▨) diet or in B genetically obese Lepob mice (Ob: ). Effects of genotype (†) and diet (*) are shown as significant: P < 0.05.

FIG. 6.

11β-HSD1 activity suppresses IL-6–mediated activation of AMP kinase in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were exposed to increasing concentrations of IL-6 (□) alone or in the presence of the 11β-HSD1 substrate 11-DHC (200 nM, ■) for 24 h. Cells were homogenized, and levels of phosphorylated (activated) AMPK were determined by Western blot. *P < 0.05 for effects of 100 ng/mL IL-6 compared with basal and †P < 0.05 for effects of 11-DHC on IL-6–stimulated AMPK activation. Data are mean ± SEM, n = 4.

FIG. 7.

Summary of the effects of 11β-HSD1 deficiency (low intracellular glucocorticoid action) on subcutaneous and visceral fat in obesity after early (4-week) and chronic (18-week) HF diet exposure. HF feeding causes a differential expansion of adipose mass (pink double-sided arrow) in the genotypes. After an initial period of generally attenuated fat mass accumulation (4-week HF diet), fat becomes favorably redistributed toward safer peripheral (subcutaneous) fat stores and away from detrimental visceral (mesenteric) fat stores in 11β-HSD1^−/− mice (11β-KO) with chronic (18-week) HF diet (9). In subcutaneous fat, higher PPARγ (and β-adrenergic) remodeling drives increased numbers of small metabolically competent adipocytes that maintain insulin sensitivity, increased glucose uptake, and potentially oxidative drive, despite overall greater fat mass with chronic HF diet (9). In visceral fat, higher PPARγ and increased adipocyte IL-6 secretion drives maintained AMPK-mediated fat oxidation, independently of insulin sensitization. Reduced visceral fat inflammatory responses in 11β-HSD1^−/− mice become accentuated with HF diet, particularly an early (4-week) reduction in CD8+ T-cells and a later reduction in macrophage content due, in part, to reduced adipocyte MCP1 secretion from 11β-HSD1^−/− adipocytes. Visceral fat of 11β-HSD-1^−/− mice also exhibits reduced adipogenesis (30). WT: C57BL/6J mice, 11β-KO: 11β-HSD1^−/− mice.