Large increases in adipose triacylglycerol flux in Cushingoid CRH-Tg mice are explained by futile cycling

Abstract

Glucocorticoids are extremely effective anti-inflammatory therapies, but their clinical use is limited due to severe side effects, including osteoporosis, muscle wasting, fat redistribution, and skin thinning. Here we use heavy water labeling and mass spectrometry to measure fluxes through metabolic pathways impacted by glucocorticoids. We combine these methods with measurements of body composition in corticotropin-releasing hormone (CRH)-transgenic (Tg)(+) mice that have chronically elevated, endogenously produced corticosterone and a phenotype that closely mimics Cushing's disease in humans. CRH-Tg(+) mice had increased adipose mass, adipose triglyceride synthesis, and greatly increased triglyceride/fatty acid cycling in subcutaneous and abdominal fat depots and increased de novo lipogenesis in the abdominal depot. In bone, CRH-Tg(+) mice had decreased bone mass, absolute collagen synthesis rates, and collagen breakdown rate. In skin, CRH-Tg(+) mice had decreased skin thickness and absolute collagen synthesis rates but no decrease in the collagen breakdown rate. In muscle, CRH-Tg(+) mice had decreased muscle mass and absolute protein synthesis but no decrease in the protein breakdown rate. We conclude that chronic exposure to endogenous glucocorticoid excess in mice is associated with ongoing decreases in bone collagen, skin collagen, and muscle protein synthesis without compensatory reduction (coupling) of breakdown rates in skin and muscle. Both of these actions contribute to reduced protein pool sizes. We also conclude that increased cycling between triglycerides and free fatty acids occurs in both abdominal and subcutaneous fat depots in CRH-Tg(+) mice. CRH-Tg mice have both increased lipolysis and increased triglyceride synthesis in adipose tissue.

Fig. 1.

Body mass and body composition in wild-type (WT) and corticotropin-releasing hormone (CRH)-transgenic (Tg)⁺ mice. A: average group body mass. B: body composition by dual energy X-ray absorptiometry (DEXA), including lean mass, fat mass, and percent fat; n = 6 for the WT group and n = 8 for the CRH-Tg⁺ group. C: weights of individual adipose depots. BAT, brown adipose tissue. *P < 0.05 compared with WT. ***P < 0.001 compared with WT by t-test.

Fig. 2.

Bone parameters in WT and CRH-Tg⁺ mice. A: bone mineral content (BMC) in grams. B: bone area in cm². C: bone mineral density (BMD) in g/cm². D: fraction new collagen (f) in bone. E and F: micro-computed tomography (CT) scan of WT femur (E) and CRH-Tg⁺ femur (F). G: estimated absolute rate of bone collagen synthesis in arbitrary units/wk; n = 6 for WT and n = 8 for CRH-Tg⁺ for A, B, C, and D. *P < 0.05 and ***P < 0.001 compared with WT by t-test.

Fig. 3.

Skin parameters in WT and CRH-Tg⁺ mice. A: ear skin thickness in μm, n = 10 for WT and n = 11 for CRH-Tg⁺. B: fraction of new collagen in skin, n = 6 for WT and n = 8 for CRH-Tg⁺. C: estimated absolute rate of skin collagen synthesis in arbitrary units/wk. *P < 0.05 and ***P < 0.001 compared with WT by t-test.

Fig. 4.

Skeletal muscle parameters in WT and CRH-Tg⁺ mice. A: mass of quadriceps muscles. B: fraction new protein (alanine) in quadriceps skeletal muscle. C: estimated absolute rate of muscle protein synthesis in quadriceps skeletal muscle in mg/wk; n = 6 for WT and n = 8 for CRH-Tg⁺. ***P < 0.001 compared with WT by t-test.

Fig. 5.

Absolute, retained triglyceride (TG) synthesis and de novo lipogenesis (DNL) over the week-long labeling period in inguinal and epididymal fat pads in WT and CRH-Tg⁺ mice. A: absolute TG synthesis in the inguinal depot. B: absolute TG synthesis in the epididymal depot. C: absolute DNL in the inguinal depot. D: absolute DNL in the epididymal depot; n = 6 for WT and n = 8 for CRH-Tg⁺. **P < 0.01 and ***P < 0.001 compared with WT by t-test.

Fig. 6.

Percent glyceroneogenesis in inguinal and epididymal fat depots and liver in WT and CRH-Tg⁺ mice. Percent glyceroneogenesis indicates what percent of the TG-glycerol came from glyceroneogenic, rather than glycolytic, pathways (i.e., 100% glyceroneogenesis means 100% of TG-glycerol came from glyceroneogenic pathways and 0% came from glycolytic pathways). A: inguinal depot. B: epididymal depot. C: liver; n = 6 for WT and n = 8 for CRH-Tg⁺. No significant difference between genotypes in any tissue was found, but %glyceroneogenesis in liver was significantly different from %glyceroneogenesis in both fat depots (P < 0.001) by one-way ANOVA followed by Tukey's posttests.