Abstract
Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor, is induced during adipocyte differentiation; however, its precise contribution to this process is unknown. Using both in vitro and in vivo systems, we show that p21 is crucial for maintaining adipocyte hypertrophy and obesity-induced insulin resistance. The absence of p21 in 3T3-L1 fibroblasts by RNA-mediated interference knockdown or in embryonic fibroblasts from p21(-/-) mice impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet, p21(-/-) mice fed high energy diets had reduced adipose tissue mass and adipocyte size accompanied by a marked improvement in insulin sensitivity. Knockdown of p21 in enlarged epididymal fat of diet-induced obese mice and also in fully differentiated 3T3-L1 adipocytes caused vigorous apoptosis by activating p53. Thus, p21 is involved in both adipocyte differentiation and in protecting hypertrophied adipocytes against apoptosis. Via both of these mechanisms, p21 promotes adipose tissue expansion during high fat diet feeding, leading to increased downstream pathophysiological consequences such as insulin resistance.
Highlights
The process of adipocyte differentiation has been extensively characterized in cultures of preadipocyte clonal cell lines such as mouse 3T3-L1 and 3T3-F442A [1, 2]
It was previously suggested that expression of p21 and p27 is changed during the adipogenesis [3]. p21 has been reported to be induced by Foxo1 and has been implicated in entry of adipocytes into the clonal expansion phase of adipogenesis; p27 is critical for adipocyte hyperplasia [3,4,5]
Role of p21 in Adipocyte Differentiation and Apoptosis— Our current study clearly demonstrates that p21 plays a crucial role in later stages of differentiation and hypertrophy of adipocytes. p21 is induced during adipogenesis and sustained during terminal differentiation. p21 knockdown experiments demonstrated that p21 contributes to but is not absolutely required for adipocyte differentiation
Summary
Cell Culture and Adipocyte Differentiation—3T3-L1 cells (ATCC) were maintained in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal calf serum in 5% CO2. We performed weekly collections of blood for metabolic analysis (measurement of blood glucose, insulin, triglyceride, total cholesterol, and free fatty acid) and measured body weight. They were subjected to intravenous glucose tolerance tests 4 weeks after the HFHS diet started, insulin tolerance tests 5 weeks after the diet started, dual energy x-ray absorptiometry (DEXA) analysis, and sacrifice at 6 weeks. Determination of Adipocyte Number and Size—Epididymal fat pads from normal mice (ϳ50 mg) and HFHS-fed mice (ϳ150 mg) were fixed in osmium tetroxide-collidine buffer for 3 days. For Northern blot analysis, equal aliquots of total RNA from 5– 6 mice and culture cells were pooled (5–10 g of total RNA), denatured with formaldehyde and formamide, subjected to electrophoresis in a 1% agarose gel, and transferred to Hybond N membranes (Amersham Biosciences) for hybridization.
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