RESUMEN
Plant extracts continue to represent an untapped source of renewable therapeutic compounds for the treatment and prevention of illnesses including chronic metabolic disorders. With the increase in worldwide obesity and its related morbidities, the need for identifying safe and effective treatments is also rising. As such, use of primary human adipose-derived stem cells represents a physiologically relevant cell system to screen for bioactive agents in the prevention and treatment of obesity and its related complications. By using these cells in a primary screen, the risk and cost of identifying artifacts due to interspecies variation and immortalized cell lines is eliminated. We demonstrate that these cells can be formatted into 384-well high throughput screens to rapidly identify botanical extracts that affect lipogenesis and lipolysis. Additionally, counterscreening with human primary stem cells from distinct adipose depots can be routinely performed to identify tissue specific responses. In our study, over 500 botanical extracts were screened and 16 (2.7%) were found to affect lipogenesis and 4 (0.7%) affected lipolysis.
Asunto(s)
Grasa Intraabdominal/citología , Extractos Vegetales/farmacología , Células Madre/efectos de los fármacos , Grasa Subcutánea/citología , Adipocitos/efectos de los fármacos , Adulto , Células Cultivadas , Femenino , Humanos , Lipogénesis/efectos de los fármacos , Lipólisis/efectos de los fármacos , Síndrome Metabólico/fisiopatologíaRESUMEN
OBJECTIVE: Adipocyte infiltration of the musculoskeletal system is well recognized as a hallmark of aging, obesity, and type 2 diabetes. Intermuscular adipocytes might serve as a benign storage site for surplus lipid or play a role in disrupting energy homeostasis as a result of dysregulated lipolysis or secretion of proinflammatory cytokines. This investigation sought to understand the net impact of local adipocytes on skeletal myocyte metabolism. RESEARCH DESIGN AND METHODS: Interactions between these two tissues were modeled using a coculture system composed of primary human adipocytes and human skeletal myotubes derived from lean or obese donors. Metabolic analysis of myocytes was performed after coculture with lipolytically silent or activated adipocytes and included transcript and metabolite profiling along with assessment of substrate selection and insulin action. RESULTS: Cocultured adipocytes increased myotube mRNA expression of genes involved in oxidative metabolism, regardless of the donor and degree of lipolytic activity. Adipocytes in the basal state sequestered free fatty acids, thereby forcing neighboring myotubes to rely more heavily on glucose fuel. Under this condition, insulin action was enhanced in myotubes from lean but not obese donors. In contrast, when exposed to lipolytically active adipocytes, cocultured myotubes shifted substrate use in favor of fatty acids, which was accompanied by intracellular accumulation of triacylglycerol and even-chain acylcarnitines, decreased glucose oxidation, and modest attenuation of insulin signaling. CONCLUSIONS: The effects of cocultured adipocytes on myocyte substrate selection and insulin action depended on the metabolic state of the system. These findings are relevant to understanding the metabolic consequences of intermuscular adipogenesis.
Asunto(s)
Adipocitos/metabolismo , Lipólisis , Fibras Musculares Esqueléticas/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Técnicas de Cocultivo , Ácidos Grasos no Esterificados/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Insulina , Resistencia a la Insulina , Fibras Musculares Esqueléticas/efectos de los fármacos , Obesidad/metabolismo , Delgadez/metabolismo , Triglicéridos/metabolismoRESUMEN
Drug discovery efforts have an increasing focus on functional cell-based screening to identify compounds that modulate targets presented in a relevant format. Historically, immortalized cell lines have been used in primary and secondary screens due to their ease of manipulation, transformation, and propagation. However, more researchers are using primary cells that present their drug targets in their natural context. Human primary cell isolation and propagation procedures have become efficient enough to provide these cells in the necessary scale for early stage drug discovery. Adult human stem cells provide an opportunity for investigating multiple pathways of differentiation, development, regeneration, and toxicity using a single cell source and type. Adipose-derived stem cells (ASCs) are an attractive adult human primary stem cell for drug discovery due their abundance in adipose tissue, ease of isolation, and propagation in culture. They can be expanded in high numbers and retain their unique properties to differentiate into multiple lineages. In this chapter, we describe a protocol to identify modulators of human ASC lipogenesis following partial differentiation to adipocytes.