A new paradigm for energy metabolism research
Yuan-Di C. Halvorsen
Peter Pingerelli Matt C. Petre
Michael R. Briggs
SmithKline Beecham Pharmaceuticals
Stratagene introduces new tools to facilitate the study of human adipocyte biology: Human Predifferentiated Adipocytes and Human Differentiated Adipocytes. Adipose tissue is a differentiated endocrine organ that primarily functions to manage energy homeostasis. Human primary adipocytes represent a model system to study these complex processes. The stromal-vascular fraction from adipose tissue can be processed to isolate preadipocytes and induced with hormones to differentiate into adipocytes. The resulting in vitro differentiated cells are functionally similar to primary human adipocytes and can be cultured for weeks without loss of adipocyte-specific markers, unlike the fragile primary adipocytes that die within 24 hours.
The study of adipocyte biology is important not only to understand metabolic diseases (including obesity, diabetes, dyslipidemia, and atherosclerosis), but to develop pharmacological agents to treat such maladies. Adipose tissue is critical for maintaining energy balance, because it serves both as a storage depot and an endocrine organ.1 It is actively involved in sensing the nutritional state of the organism through several different signaling pathways. It responds by altering the expression of genetic pathways to mobilize or store energy and activating signals to other tissues to regulate energy balance.
Previously, all research in adipocyte biology was conducted using rodent cells, mainly transformed cell lines.2 However, because humans and rodents have distinct genetic backgrounds and metabolic pathways, potential experimental pitfalls were inevitable. For example, compounds effective in activating the b3-adrenergic pathway in rodents failed to stimulate human adipocytes to burn more fat.3 Stratagene now offers Human Predifferentiated Adipocytes and Human Differentiated Adipocytes, so rodent systems can be bypassed for human systems in experiments involving adipocyte signal transduction pathways or gene regulation.
When excess preadipocytes are recruited into the differentiated adipocyte, obesity results. Since from adolescence through adulthood humans continue to recruit cells to become adipocytes, it is of interest to study the environmental and humoral factors that initiate and maintain the differentiated phenotype of adipocytes. Human preadipocytes provide the tools to explore the most relevant factors that influence human obesity.
Stratagene recently developed the Adipocyte Differentiation Kit, which uses a simple and efficient method to differentiate human primary preadipocytes into mature, fully functional adipocytes. Undifferentiated preadipocytes are cultured in DMEM/F-10 (1:1, v/v) with 10% fetal bovine serum. The cells can be passaged for expansion, frozen for later reuse, or differentiated. The absence of staining with antisera to factor VIII and lack of vascular endothelial growth factor receptor mRNA, shown by Northern analysis, 4 demonstrated that no endothelial cells are present in the preadipocyte culture.
Earlier passage cells are more robust in both growth and differentiation potential; passages one to three are ideal. Stratagene uses only passage-two human preadipocytes for the Adipocyte Differentiation Kit. Typically, a production lot of cells is isogenic, and lots vary within the 10% to 20% range for total differentiation potential, as measured by lipid accumulation.
Up to 90% of the Human Predifferentiated Adipocytes can be induced to differentiate after they have been treated with a hormone and chemical cocktail. This treatment promotes a cascade of transcription factors activation and adipocyte-specific gene expression that leads to the mature adipocyte phenotype (Figure 1, panel A and B). Predifferentiated Adipocyte Differentiation Medium is also available from Stratagene as a separate reagent.
During the differentiation process5, key adipocyte- specific genes, such as PPARg, fatty acid binding protein, and obese gene, are expressed.6,7,8 Expression of signaling molecules, such as leptin, the obese gene product is induced during differentiation (Figure 2).
By using Stratagenes Human Predifferentiated Adipocytes, various questions can be addressed: What makes these cells differentiate from fibroblast-like cells to adipocytes? What are adipocytes similarities and differences? What chemical and endocrine effectors transform preadipocytes? What gene expression changes are necessary and/or sufficient for preadipocyte transformation?
Human Predifferentiated Adipocytes are available in a 24- or 96-well format and are shipped ready to use. Besides providing a unique tool to study the processes that trigger and promote differentiation, these cells can be used to identify agents that block or slow the differentiation process.
By characterizing gene expression and regulation, we may unravel the mysteries of the adipocytefrom its role as an endocrine receptor to its role as a mediator of energy homeostasis. Transfection experiments, which lead to overexpression of wild type or mutated protein forms, are used to characterize many gene functions. The phenotype of the altered expression state allows the effects of single gene changes to be examined, which may model metabolic derangement associated with particular diseases.9
To date we have successfully transfected Human Differentiated Adipocytes with the human adipose- specific obese gene and peroxisome proliferator activated receptor gamma (PPARg) gene promoter-driven reporters (data not shown). In experiments to improve the transfectability of Human Differentiated Adipocytes or Human Predifferentiated Adipocytes using Stratagenes Transfection MBS Mammalian Transfection Kit or LipoTAXI Transfection Reagent, respectively, we showed that including the Primary Transfection ENHANCER results in a higher transfection efficiency (Figure 3, panel A and panel B).
One of the key functions of adipocytes is to adjust lipid storage according to the physiological energy needs of the organism. When extra calories are required, adipocyte intracellular triglyceride stores are metabolized to glycerol and fatty acids for further oxidation, a process termed lipolysis.10 Our Human Differentiated Adipocytes respond to adrenergic hormone receptor agonists, such as norepinephrine or isoproterenol, to induce this metabolic pathway. The lipolysis response can be assayed by measuring the release of glycerol into the culture medium (Figure 4). Thus, these cells are useful for analyzing compounds or genes that induce, or are involved with, this important function of the mature adipocyte.
With Stratagenes new Human Predifferentiated Adipocytes and Human Differentiated Adipocytes, researchers have a new class of reagents with which to study human adipose biology. It is now possible to directly study the factors that affect human adipose differentiation, and to identify agents which may promote or interfere with the progression of obesity. Gene expression in both adipocytes and preadipocytes can be achieved with the aid of Primary Transfection ENHANCER, a new reagent developed by Stratagene to enhance primary cell transfection.
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