Elsevier

Experimental Cell Research

Volume 317, Issue 16, 1 October 2011, Pages 2299-2307
Experimental Cell Research

Research Article
Impaired response of mature adipocytes of diabetic mice to hypoxia

https://doi.org/10.1016/j.yexcr.2011.07.004Get rights and content

Abstract

Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.

Introduction

Mammals have two functionally distinct adipose tissues; white and brown. While brown adipose tissues dissipate stored energy by producing heat, white adipose tissues store energy as triglycerides [1], [2]. Excessive storage of triglycerides in adipose tissue can result in obesity, which is a complex metabolic disorder related with hypertension, stroke, cardiovascular diseases, and type 2 diabetes [3], [4], [5]. Adipose tissues contain various cells such as infiltrated monocytes/macrophages and endothelial cells in addition to preadipocytes and adipocytes. Adipocytes have an endocrine function by secreting many cytokines known as adipokines such as monocyte chemoattractant protein 1 (MCP1), IL-6, and TNF-α. In addition adipocytes express adipose specific cytokines such as leptin, adiponectin, and resistin [2], [6], [7], [8], [9], [10]. Adipocytes communicate with other organs – such as brain, liver, and β-islet cells – through adipokines. Dysregulation of adipokines in adipocyte tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes [6], [10], [11], [12].

Wound repair is a complex and dynamic process which consists of inflammation, angiogenesis, and tissue formation and remodeling [13], [14], [15]. Impediment of normal wound repair often results in chronic non-healing wounds. Specifically, impaired wound healing related with diabetes is a major medical problem and often leads to lower extremity amputation [16], [17]. Although the pathogenesis of diabetic wounds is not clearly understood, several underling mechanisms have been suggested [18]. Decreased angiogenesis has been postulated in impaired diabetic wounds and when topical treatment of VEGF was applied in a diabetic wounds, accelerated healing was found by increased angiogenesis [19]. In peripheral vascular complications of type 2 diabetes decreased number of circulating endothelial progenitor cells (EPCs), which are essential for vasculogenesis, was found [20]. Treatment of hypoxia and stromal cell-derived factor (SDF)-1α enhanced EPC mobilization and wound healing in diabetes [21]. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds [22]. Thus, growth factors and cytokines play important roles for the proper wound repair.

Subcutaneous adipose tissue is located underneath the skin, which consists of epidermis and dermis. Adipocytes can contribute to wound repair since they secrete many cytokines which are important in skin wound repair. Adipose tissues have enormous plasticity since their volume can be dramatically increased or decreased by environmental signals such as nutrient intake [23], [24], [25]. In type 2 diabetes, subcutaneous fat enormously increased in amount clinically, and thus its behavior to hypoxia is of interest. Adipocytes are important parts of injured tissue subject to hypoxia which is insufficient to cause full necrosis. Therefore, understanding the proliferation and differentiation of adipocytes is crucial for the mechanism study of wound repair as well as obesity and diabetes. Established preadipocyte cell lines, such as 3T3-L1, and primary preadipose cells isolated from fat pads have been used for previous adipogenesis studies [26], [27]. These cells are efficiently differentiated to adipocytes, and have characteristics of mature adipocytes, by adipogenic cocktails containing dexamethasone, isobutylmethylxanthine (IBMX), and insulin. A complex transcriptional cascade regulating adipogenesis has been elucidated using those cells [27], [28], [29], [30], [31]. However, those differentiated adipocytes in these models are imperfect. Cells are still attached to the culture plates and form multilocular lipid droplets. In contrast, mature adipocytes, called unilocular cells, in white adipose tissues in vivo have unique structure where nucleus is located peripherally and a large lipid droplet occupying the entire cytoplasm. Adipocytes in vivo exist in a three dimensional matrix environment including HA and collagen, and so studying cells in this environment is more relevant than floating cells.

Difficulty of using conventional cell culture methods for mature adipocytes, due to their buoyancy, has limited their functional study in vitro. We established a novel in vitro culture method for the mature adipocytes by enclosing them in a HA based hydrogel in this report. We hypothesize that mature adipocytes play a critical role in response to stress such as hypoxia. We show that mature adipocytes of wild type mice, C57BL/6, responded to hypoxia. In comparison, mature adipocytes of db/db and TallyHo mice in a monogenic and polygenic animal model for human type 2 diabetes, respectively, did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells and their abnormal function can be one of the factors causing morbidity in type 2 diabetes.

Section snippets

Isolation and culture of mature adipocytes

Subcutaneous inguinal fat pads from 6 to 8 week old male mice were used to isolate mature adipocytes. C57BL/6, leptin receptor knockout (db/db) mice (strain B6.Cg-m +/+ Leprdb/J), TallyHo (TallyHo/JNJ, #005314), and green fluorescence protein (GFP) expressing transgenic mice (strain C57BL/6-Tg(ACTB-EGFP)1Osb/J) were purchased from the Jackson laboratory. Mature adipocytes were isolated as previously described with some modifications [32], [33]. Briefly, the fat pads were minced with scissors,

Mature adipocyte culture in hydrogel

Mature adipocytes cannot be cultured in conventional culture methods because they float on the surface of culture media due to their high lipid content in the cytoplasm. To overcome this problem and to provide a microenvironment which reflects in vivo conditions, we used a three dimensional culture. We chose a HA-derived hydrogel, Extracel-HP, because HA is a major constituent of the extracellular matrix which mediates cell proliferation, differentiation, and adhesion [36], [37]. Hydrogel is

Discussion

Invaluable information has been discovered in adipogenesis of which dysregulation is involved in obesity using primary preadipocytes and preadipocyte cell lines. The major function of white mature adipocytes has been thought as energy storage. However recently progress has suggested they are metabolically active and secrete adipokines which functions as autocrine and paracrine signaling [6], [7], [8]. However, molecular studies of mature adipocytes have not been well addressed compared to

Acknowledgments

We would like to thank Matthew Geringer for his critical review of the manuscript. This work was supported by internal funding from the Division of Plastic and Reconstructive Surgery.

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