(Cllick to Enlarge) Human Adipocytes (HAd) (differentiated) accumulate lipid droplets visualized by Oil Red O (A). Western blot analysis of undifferentiated human pre-adipocytes (lane 1) and differentiated mature adipocytes (lane 2) lysates (B, C) . Western blot analysis of the unstimulated (lane 1) and insulin-stimulated (lane 2) differentiated human adipocytes using anti-phospho-insulin receptor antibodies (left
...Human Preadipocytes: S-HPAd: Pre-Screened
Description
Human Preadipocytes (HPAd) Pre-Screened
HPAd are prescreened for Adipogenesis Signaling: Â Primary cultures of adipocytes are prepared by inducing differentiation of human primary preadipocytes and are prescreened for the expression of the following major adipocyte markers:
- PPARγ (Peroxisome proliferator-activated receptor gamma), an adipocyte-specific nuclear hormone receptor
- C/EBPα (CCAAT/enhancer binding protein alpha), a transcription factor involved in creating and maintaining the adipocyte phenotype
- IRÂ (Insulin receptor), which plays a critical role in induction of adipocyte differentiation, as well as functional regulation
- ACCα (Acetyl-CoA carboxylase alpha), a key enzyme for de novo fatty acid synthesis and lipogenic capability of adipocytes
- GSK-3β (Glycogen synthase kinase-3-beta), a ubiquitous kinase implicated in regulation of adipocyte gene expression, signaling, adipogenesis, insulin action and glycogen synthesis
- Adiponectin, a bioactive adipokine specifically secreted by differentiated adipocyte and involved in metabolic regulation
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Details
(Detail for Figure) Human Preadipocytes: HPAd: Pre-Screened. High level expression of both PPARg and C/EBPa proteins was detected in our prescreened human adipocytes but not in the undifferentiated preadipocytes (Fig. B). Additionally, IR expression is dramatically increased in the prescreened human adipocytes as compared to that in undifferentiated preadipocytes. There was also a slight increase in Akt expression in differentiated adipocytes. Our results further demonstrate higher expression levels for both metabolic enzymes, ACCa and GSK-3b, in the mature adipocytes compared to the undifferentiated preadipocytes. Adiponectin was also detected only in the mature adipocytes, but not in the undifferentiated preadipocytes (Fig. C). Insulin is not only critical to adipocyte differentiation, but also serves as a key metabolic regulator. Thus, insulin response is a characteristic assay for adipocyte function. We confirmed that the pre-screened adipocytes respond strongly to insulin stimulation (Fig. D). Strong phosphorilation of Tyr 1165/6 of insulin receptor was detected in the insulin-stimulated (lane 2), but not in untreated adipocytes (lane 1). Total insulin receptor levels did not change before and after insulin stimulation (right blot).
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