Date of Award

5-2008

Degree Type

Thesis

Degree Name

Master of Science

Major

Comparative and Experimental Medicine

Major Professor

Madhu S. Dhar

Committee Members

Patricia K. Tithof, Roger C. Carroll

Abstract

A recently identified phospholipid translocase, Atp10c, may be involved in the modulation of glucose and lipid metabolism in mouse adipocytes. Aminophospholipid translocases have previously been associated with cell signaling and intracellular protein trafficking. In this study, the role of Atp10c in the insulin signaling pathway and adipogenesis was investigated using the murine 3T3-L1 cell line.

Atp10c mRNA is highly expressed in undifferentiated cells and 2-fold down-regulated during adipogenesis. Interestingly, Western blotting showed a band for ATP10C protein at 70 kDa, a lower molecular weight than expected; ATP10C expression increased during differentiation. Possible reasons for these discrepancies will be discussed.

Since PPARgamma is considered to be the master regulator of adipogenesis and controls the expression of adipocyte genes, the effect of PPARgamma agonists on Atp10c expression was examined. The PPARgamma agonists and anti-diabetic drugs MCC555 and troglitazone both promoted adipogenesis and consequently reduced Atp10c expression by 5-fold and 3-fold, respectively. When used in combination with the PPARgamma antagonist GW9662, treatment rescued some of the decrease in Atp10c due to MCC555 or troglitazone alone. The pattern of Atp10c expression is shown to be opposite to that of PPARgamma.

Additionally, the correlation between effectors of glucose and lipid metabolism and Atp10c was investigated. Treatment of 3T3-L1 adipocytes with insulin and dexamethasone showed significantly increased expression of Atp10c; treatment with the β-adrenergic factor isoproterenol slightly decreased expression. This suggests transcriptional control of Atp10c by hormonal factors, but not β-adrenergic factors, during differentiation.

To further explore the function of Atp10c in lipid metabolism, ATP10C silencing through transient transfection of 3T3-L1 cells was first optimized to ultimately allow the assessment of changes in proteins related to obesity and diabetes. An 84% and 77% knockdown of Atp10c gene expression in preadipocytes and adipocytes, respectively, was achieved. To elucidate a potential role for Atp10c/ATP10C in the insulin signaling pathway, glucose uptake was investigated. Following Atp10c silencing in 3T3-L1 adipocytes, glucose uptake was significantly increased at both 24 and 48 hours.

Based on the results presented herein, the Atp10c gene and ATP10C protein have an important role in the insulin signaling pathway and may be involved in adipogenesis.,/p>

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