Enhanced hexose-6-phosphate dehydrogenase expression in adipose tissue may contribute to diet-induced visceral adiposity

Abstract

Background: Visceral fat accumulation increases the risk of developing type 2 diabetes and metabolic syndrome, and is associated with excessive glucocorticoids (GCs). Fat depot-specific GC action is tightly controlled by 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) coupled with the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Mice with inactivation or activation of H6PDH genes show altered adipose 11ß-HSD1 activity and lipid storage. We hypothesized that adipose tissue H6PDH activation is a leading cause for the visceral obesity and insulin resistance. Here, we explored the role and possible mechanism of enhancing adipose H6PDH in the development of visceral adiposity in vivo.

Methods: We investigated the potential contribution of adipose H6PDH activation to the accumulation of visceral fat by characterization of visceral fat obese gene expression profiles, fat distribution, adipocyte metabolic molecules, and abdominal fat-specific GC signaling mechanisms underlying the diet-induced visceral obesity and insulin resistance in H6PDH transgenic mice fed a standard of high-fat diet (HFD).

Results: Transgenic H6PDH mice display increased abdominal fat accumulation, which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPα and PPARγ mRNA levels within adipose tissue. Transgenic H6PDH mice fed a high-fat diet (HFD) gained more abdominal visceral fat mass coupled with activation of GSK3β and induction of XBP1/IRE1α, but reduced pThr³⁰⁸ Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls. HFD-fed H6PDH transgenic mice also had impaired insulin sensitivity and exhibited elevated levels of intra-adipose GCs with induction of adipose 11ß-HSD1.

Conclusion: These data provide the first in vivo mechanistic evidence for the adverse metabolic effects of adipose H6PDH activation on visceral fat distribution, fat metabolism, and adipocyte function through enhancing 11ß-HSD1-driven intra-adipose GC action.

Figure 1

Lipogenic gene ACC and ACL mRNA and protein expression in epididymal fat (Ep fat), mesenteric fat (Mes fat) and subcutaneous fat (Sub fat) of aP2-H6PDH (■) or WT (□) mice (a, d, g) Relative expression of ACC and ACL mRNA levels were measured by real-time RT-PCR and normalized to 18S (n =8). (b, c, e, f, h, i) Relative expression levels of t-ACC, t-ACL, p-Ser⁷⁹ ACC and p-Ser⁴⁵⁵ ACL proteins were normalized to GAPDH. Data are means ± SE of 7–8 mice/group. *P<0.01 vs. WT controls; **P < 0.001 vs. WT controls.

Figure 2

Alterations of obese gene expression in epididymal fat (Ep fat), mesenteric fat (Mes fat) and subcutaneous fat (Sub fat) between aP2-H6PDH (■) or WT (□) mice. (a, c, e). Relative expression levels of lipid metabolic gene FAS and PEPCK, and lipogenic gene transcriptor C/EBPα, PPARγ, and SREBP mRNA levels in epididymal fat (a), mesenteric fat (c), and subcutaneous fat (e) were normalized to 18S. (b, d, f) Relative expression of leptin, FOXO1, and SIRT1 mRNA levels in epididymal fat (b), mesenteric fat (d) and subcutaneous fat (f) of aP2-H6PDH Tg (■) or WT (□) mice. Data are mean ± SE from six to eight mice per group. *P<0.01 vs. WT controls; **P < 0.005 vs. WT controls.

Figure 3

ER stress response and the relative alterations of Akt/PKB and GSKβ gene expression in epididymal fat (Ep fat) of aP2-H6PDH Tg (■) and their WT controls (□). (a, b) Relative expression of IREα, XBP1, AKT, and GSK3β mRNA levels measured by real-time RT-PCR and normalized to 18S. (c and e) Western blotting analyses were performed to compare the expression levels of t-REα, XBP1 and p-Ser⁷²⁴ IRE. (d and f) Relative adipose p-Th³⁰⁸ Akt and p-Ser⁹ GSK3β protein was standardized to GAPDH. *P < 0.01 vs. WT controls; **P < 0.02 vs. WT controls.

Figure 4

Body weight and visceral fat mass of aP2-H6PDH transgenic and WT mice. (a) Body weight gain of aP2-H6PDH and WT mice fed chow diet (C) or high-fat diet (HFD) (○, non-Tg mice fed chow diet; ■, Tg mice fed chow diet; △, non-Tg mice fed HFD; ● Tg mice fed HFD, n = 8–10 mice/group). (b) Ep fat and Mes fat mass of H6PDH mice fed control chow diet or HFD. (c) Lipid droplet accumulation was monitored by fluorescent microscopy using Oil Red O staining with DAPI. (d, e) Relative mesenteric fat obese gene mRNA levels in aP2-H6PDH Tg (▨) and their WT controls (■) and WT mice fed HFD. (f) Relative alterations of Akt, p-Th³⁰⁸Akt and p-Ser⁹ GSK3β content in Mes fat of mice fed HFD. *P < 0.01 and #P < 0.02 vs. WT controls; **P < 0.02 vs. HFD-fed controls; †P < 0.02 and ‡P < 0.01 vs. HFD-fed WT controls. (g) Schematic diagram representing the consequences of enhancing adipose H6PDH expression. H6PDH activation provides the fuel for the production of 11β-HSD1-driven intra-adipose CORT and results in fat accumulation through: (1) CORT stimulation of IREα/XBP1 pathway activates C/EBPα and PPARγ expression, facilitating lipogenesis and TG synthesis, (2) C/EBPα can activate 11β-HSD1, further enhancing CORT availability and (3) CORT-mediated suppression of p-Akt induces insulin resistance and activation of GSK3β signaling that is required for adipogenesis. Additionally, GSK3β activation can promote CORT to bind/activate GR and further amplifying GC action in adipose tissue.

Figure 5

Glucose and insulin tolerance tests in aP2-H6PDH Tg mice and WT controls. (a) Glucose tolerance test (GTT; □, non-Tg mice fed chow diet (C); ○, non-Tg mice fed high-fat diet (HFD); ■, Tg mice fed chow diet; ● Tg mice fed HFD). (b) Insulin tolerance test (ITT; △, non-Tg mice fed chow diet; ○, Tg mice fed chow diet; ▲, non-Tg mice fed HFD; ● Tg mice fed HFD). (c) Serum FFA levels. (d) Adipose glucose uptake in visceral fat tissue from aP2-H6PDH mice and WT control. Data are means ± SE of 7–8 mice/group. †P < 0.02 vs. HFD-fed WT controls; *P < 0.001 and #P < 0.01 vs. WT controls; **P < 0.01 vs. HFD-fed WT controls.

Figure 6

Adipose target gene expression and corticosterone levels in epididymal fat (Ep fat) and mesenteric fat (Mes fat) of aP2-H6PDH (white bar) and WT mice fed HFD (black bar). (a, b) Quantitative real-time RT-PCR analysis demonstrating the relative alterations of H6PDH, G6PT, 11β-HSD1, and GR mRNA levels in Ep fat (a) and Mes fat (b) of aP2-H6PDH and WT mice fed HFD. (c) Western blot analysis of expression of 11β-HSD1 in epididymal fat (Ep fat) and mesenteric fat (Mes fat) of aP2-H6PDH and WT mice fed HFD. (d) Adipose 11β-HSD1 reductase activity was measured in adipose microsomes using 11-dehydrocorticosterone (DHC) as the substrate in the presence of NADPH. Enzyme activity was expressed as % DHC converted to corticosterone (B). (e) Tissue corticosterone concentrations in epididymal fat and mesenteric visceral fat of aP2-H6PDH and WT mice fed HFD. (f) Plasma corticosterone levels in aP2-H6PDH and WT mice fed HFD. Data are means ± SE of 7–8 mice/group. *P < 0.001 vs. HFD-fed WT controls; †P < 0.01 vs. HFD-fed WT controls.