Dysregulated Alternative Splicing Pattern of PKC during Differentiation of Human Preadipocytes Represents Distinct Differences between Lean and Obese Adipocytes
Obesity and its comorbidities affect millions of people. Here, we demonstrate that human preadipocytes are susceptible to programmed cell death (apoptosis) while mature adipocytes are resistant to apoptosis. The molecular mechanisms underlying the phenotype of apoptosis-resistant adipocytes are lesser known. To study the role of apoptosis and define molecular differences in the developmental process of adipogenesis, human preadipocytes were differentiated in vitro to mature adipocytes. Many genes in the apoptosis pathway are alternatively spliced. Our data demonstrates that during differentiation PKCδ, Bclx, and Caspase9 switch to their prosurvival splice variants along with an increase in Bcl2 expression when the cells terminally differentiate into mature adipocytes. Next we determined the expression pattern of these genes in obesity. Our data indicated high expression of PKCδVIII in adipose tissue of obese patient in different depots. We demonstrate a shift in the in vitro expression of these splice variants in differentiating preadipocytes derived from obese patients along with a decrease in adipogenesis markers. Hence, the programmed splicing of antiapoptotic proteins is a pivotal switch in differentiation that commits adipocytes to a prosurvival pathway. The expression pattern of these genes is dysregulated in obesity and may contribute to adipose tissue dysfunction. 1. Introduction The human proteome is changing constantly in response to hormones, age, and developmental stage or disease. The genetic makeup of the body indicates about 25,000 genes responsible for close to 100,000 proteins in a given proteome. Alternative splicing is a quintessential mechanism to generate proteins with distinct functions from the same gene. Alternative splicing occurs in more than 90% of genes and is a powerful step in gene expression to diversify the genomic repertoire. Genetic, environmental, and cultural factors contribute to the onset of obesity. In order to develop a therapeutic agent to combat obesity, it is essential to understand the molecular mechanisms underlying adipogenesis. Differentiation of preadipocytes to mature adipocytes is usually studied in 3T3L1 and 3T3F442A murine preadipocyte cell lines as they reproduce adipogenesis in vitro including expression of adipogenic genes and morphological changes. However, beyond the obvious species differences, preadipocytes from mouse and humans show differences as shown by gene-centric analysis of adipogenesis marker genes such as PPARγ and C/EBP , β and δ [1]. It is also known that, unlike murine adipocytes,
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