Prolonged efficiency of siRNA‐mediated gene silencing in primary cultures of human preadipocytes and adipocytes

Abstract

ObjectivePrimary human preadipocytes and differentiated adipocytes in culture are valuable cell culture systems to study adipogenesis and adipose function in relation to human adipose biology. To use these systems for mechanistic studies, we studied siRNA-mediated knockdown of genes for its effectiveness.Design and MethodsMethods were developed to effectively deliver siRNA to for gene silencing in primary preadipocytes isolated from human subcutaneous adipose tissue and newly-differentiated adipocytes. Expression level of genes and proteins was measured using quantitative RT-PCR and western blotting. Lipid droplet morphology was observed using microscopy and glycerol release was quantified as a measure of lipolysis.ResultssiRNA-mediated knockdown of genes in primary human preadipocytes resulted in prolonged silencing effects, suppressing genes throughout the process of their differentiation. In newly differentiated adipocytes, siRNA-mediated gene knockdown allowed proteins to stay depleted for at least 5 days. It was possible to re-express a protein after its siRNA-mediated depletion. Importantly, siRNA transfected human adipocytes remained metabolically active, responding to β-adrenergic stimulation to increase lipolysis.ConclusionsOur study describes the methods of gene silencing in primary cultures of human preadipocytes and adipocytes and their prolonged effectiveness.

Human White and Brite Adipogenesis is Supported by MSCA1 and is Impaired by Immune Cells

“…D). While our data showed that esiRNA treatment resulted in a modest reduction in the levels of MSCA1 transcript, this was in agreement with the range of esiRNA efficiency that can be obtained with native progenitor cells . We confirmed a functional role for MSCA1 in brite adipogenesis by treating human native progenitor cells with the pharmacological AP inhibitor, tetramisole .…”

Fat-specific Protein 27 (FSP27) Interacts with Adipose Triglyceride Lipase (ATGL) to Regulate Lipolysis and Insulin Sensitivity in Human Adipocytes

“…A 2-to 3-fold increase in overall FSP27 expression was observed with adenovirus (supplemental Fig. 2), similar to the effect shown previously with lentivirus (47). As expected, FSP27 expression decreased basal lipolysis by 65% and stimulated lipolysis by 35%, whereas ATGL overexpression increased basal lipolysis by 50% and stimulated lipolysis by 30% (Fig.…”

“…siRNA Transfection of Human Adipocytes-Human adipocytes were transfected with siRNA duplexes using Lipofectamine reagent and PLUS (Invitrogen) on day 9 of differentiation, as described previously (47).…”

Cholecystokinin is involved in triglyceride fatty acid uptake by rat adipose tissue

“…The medium was replaced by differentiation media after overnight incubation. Two days after transfection, the medium was changed to α-MEM containing 0.1% BSA (Lee et al 2014), then CCK-8 (10 −6 mol/L) was added and incubated for 2 h (37°C, 5% CO 2 ). Media was removed and mRNA (2 wells) was extracted as detailed below.…”