Biochemical and Biophysical Research Communications
Loss of FKBP5 impedes adipocyte differentiation under both normoxia and hypoxic stress
Introduction
Obesity is a global health problem and major risk factor for metabolic syndrome, type 2 diabetes, nonalcoholic fatty liver disease, cardiovascular disease, and certain types of cancer [1], [2], [3]. Many factors (e.g. dietary pattern, genetic predisposition, psychological status, and environment) have been linked to abnormal lipid metabolism and subsequent obesity [1], [2], [3].
Multiple pathways and adipogenic-associated genes, including PPARγ2, RXRα, SEBP1c, HIF-1 and C/EBPs, have been associated with adipogenesis. Emerging new research found that the glucocorticoid receptor (GR) and its binding proteins, (e.g. The FK506-binding protein 51, FKBP5) are implicated in fat synthesis and metabolism as well [4], [5], [6]. Deleting the GR gene delays adipogenesis in cultured preadipocytes, by directly targeting the adipogenic transcription factors C/EBPα, C/EBPβ, and PPARγ [7]. Activation of GRα by glucocorticoid (GC) promotes lipolysis and lipid secretion [4], while an increase in GRβ leads to adipocyte hypertrophy and lipogenesis [8]. These studies imply that dysregulation of GR function could result in an imbalance of lipid homeostasis, leading to obesity and metabolic disorders.
FKBP51, a co-chaperone of HSP90, is best known for its essential regulation of GRα activity [9], [10], [11]. FKBP5 binding to the GRα receptor complex can diminish the binding affinity of GC to the receptor, dampening GRα nuclear translocation [12]. GR activation upregulates the mRNA and protein level of FKBP51, resulting in a negative feed-back loop for GR sensitivity [13]. Additionally, FKBP5 is a crucial chaperone to the Akt-specific phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) [14], [15]. The loss of FKBP5 enhances Akt/p38 pathway activity, enhancing the phosphorylation of both GRα and PPARγ [16]. This suggests an important function of FKBP5 in GRα- and PPARγ-controlled metabolic events. Recent studies have shown that FKBP5 deficiency lowers lipid accumulation, suppresses differentiation of 3T3-L1 cells, slows maturation of MEFs to adipocytes [5], and renders mice resistant to diet-induced obesity [6]. These studies suggest that FKBP5 could be a promising therapeutic target for obesity.
Physiological hypoxia is critical for normal embryogenesis, organ formation, and functional maintenance [17]. For example, the tissue oxygen tension is remarkably low in developing embryos [18], and the proliferation, differentiation, and self-renewal of stem cells requires low oxygen tension in niches where stem cells reside [19]. Adipose tissues, particularly the enlarged adipocytes observed in obese individuals, are thought to experience severe hypoxia, as their expanded size exceeds the normal oxygen diffusion distance from the vessels [20]. This hypoxic state can promote angiogenesis, erythropoiesis, vasodilation, and glycolysis [21]. Many studies of adipogenesis and obesity development have been conducted under normoxic conditions, however physiological oxygen tension in organs and tissues is about 2–9%, much lower than the oxygen level in the atmosphere (21%) or in standard in vitro culture systems (∼18%) [17]. Studies of adipogenesis in hypoxic environments have revealed distinct results. For instance, preadipocyte adipogenesis is blocked under hypoxia (0.01% or 1–2% O2) through inhibition of PPARγ2 gene expression [22]. Low oxygen tension (less than 2% O2) has also been shown to suppress the differentiation of cultured human MSCs into adipocytes [23]. Additionally, other studies found that extreme hypoxia (0.2% O2) enhances the adipogenic differentiation of MSCs [24]. These results emphasize the importance of hypoxic stress on adipogenesis, and imply that the degree of hypoxia is critical in this process. Understanding the underlying mechanism by which oxygen tension affects adipogenesis is crucial for establishing a complete conception of obesity.
Given the importance of the hypoxic microenvironment on adipogenesis and lipid metabolism, and the critical role of FKBP5 in the GR-mediated stress response, it is necessary to investigate the potential role of FKBP5 plays in regulating adipogenesis and lipid metabolism in response to hypoxic stress. Particularly interested to us is if adipocyte machinery including genes affect adipogenesis and lipolysis were altered in KO, such as myelin P2 homolog (αP2), glucose transporter 4 (Glut4), adipocyte membrane glycoprotein CD36 and others [25]. The aP2 is a cytosolic fatty acid binding protein and Glut-4 promotes adipokine secretion, both of their transcriptions are mediated by members of the PPAR family and C/EBPα [26]. The adipocyte membrane glycoprotein CD36 binds and promotes the transport of long-chain fatty acids into cells, its transcription is directed by the lipogenic transcription factors PPARγ, LXR, and PXR [27], [28]. Adiponectin is also important due to it functions as a sensitizer to insulin and is reduced in obese individuals. Glycerophosphodiester phosphodiesterase (Gdpd1) metabolizes glycerol by catalyzing the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. It may also function as a lysophospholipase D, generating N-acylethanolamines and lysophosphatidic acid [29], [30]. The mitochondrial uncoupling protein UCP1 controls mitochondrial proton leak and stimulates energy dissipation. When overexpressed in white fat, it can induce obesity resistance, decrease mitochondrial membrane potential, and elevate oxygen consumption [31], [32].
To this end, we isolated mouse embryonic fibroblasts (MEFs) from WT and FKBP5 KO embryos, and compared their differentiation capacity towards adipocytes under both normoxic and hypoxic conditions. We have studied the adipogenesis differences between FKBP5 KO and WT MEFs at normoxic and hypoxic conditions, and identified FKBP5 gene plays an important role in adipogenesis.
Section snippets
Animals
The FKBP5 knockout (KO) mouse line was generated previously [6], and maintained with heterozygous animals. Heterozygous FKBP5 (Fkbp5+/−) mice were intercrossed to obtain homozygous FKBP5 KO (FKBP5-/-) and WT littermates. All animal experiments were conducted in accordance with “Guide for the Care and Use of Laboratory Animals” and were approved by Animal Care and Research Advisory Committee in the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences.
Cell culture and induction of adipocyte differentiation under normoxia and hypoxia
MEFs were isolated
Hypoxia promotes the differentiation of wild type MEFs towards adipocytes
To further elucidate the effect of hypoxia on adipogenesis, we first cultured WT-MEFs with differentiation medium under normoxia (21% O2) or hypoxia (5% O2). As shown in Fig. 1A–B, the remarkable amount of lipid droplets formed in the cells on Day 6 of differentiation, indicates that MEFs were successfully induced into adipocytes under normoxic conditions. Importantly, after 6 days of culture with differentiation medium, the lipid droplets were strikingly increased in the cells under hypoxia,
Discussion
As a stress response gene, FKBP5 has been shown to be involved in lipid accumulation and obesity development both in vitro and in vivo [5], [6], [36]. However, the exact role of FKBP5 in adipogenic and metabolic dysregulation in the hypoxic microenvironment observed in obese individuals' adipocytes is still unclear. In this study, we have confirmed that adipogenesis is enhanced under hypoxic conditions, and for the first time revealed that the absence of FKBP5 modifies adipogenesis-related gene
Acknowledgments
This research was supported by grants from National Basic Research Program (No. 2013CB945001); Beijing Natural Science Foundation (NO. 7164278); National Mega-projects for Infectious Diseases (No. 2014ZX10004002-003-001); PUMC Youth Fund (NO. 33320140086); the scientific research fund of the Institute of Laboratory Animal Science, CAMS&PUMC (NO. DWS201508, 2016ZX310043); and the National Institutes of Health (NO. NIAAA P60AA007611, U01AA013522 and R24AA015512).
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