Anne Schaar

Date of Award

January 2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Brij Singh

Abstract

A multitude of organs play a critical role in managing metabolism including the liver, pancreas, gut, brain, muscle, and adipose tissue. Abnormal function in any one of these can offset the metabolic balance and negatively affect whole body function thereby contributing to the development of metabolic complications and obesity. Although no single metabolic abnormality is responsible for obesity, understanding how normal cellular processes are regulated provides us with knowledge to reverse them during times of dysfunction. The main focus of this dissertation is on the regulation of adipose tissue due to its dual role of storing excess lipids and endocrine organ capabilities. Adipocytes, along with a majority of other cell types, are highly dependent on calcium (Ca2+), thus the role of Ca2+ and the involvement of the Ca2+ channel Transient Receptor Potential Canonical 1 (TRPC1) were studied. The evidence provided in this dissertation shows that Ca2+ entry in adipocytes, especially upon store-depletion, plays an important role in adipocyte differentiation, autophagy, and adipokine secretion and subsequently metabolic regulation. The endogenous Ca2+ entry channel in both subcutaneous and visceral adipocytes was found to be dependent on TRPC1-STIM1 complexes and altering or blocking TRPC1 resulted in dysfunctional adipocytes.

Adipocyte differentiation, the process of pre-adipocytes converting to adipocytes, is a tightly regulated process with Ca2+ dependency. Blockage of TRPC1-mediated Ca2+ entry with SKF-96365 inhibited differentiation which was indicated by decreased lipid accumulation and expression of PPAR, FAPB4, and perilipin in both subcutaneous adipose tissue (Subc AT) and visceral adipose tissue (VAT). Loss of TRPC1 in either adipose tissue type resulted in a reduced ability to differentiate which occurred prior to PPAR expression indicating TRPC1-mediated Ca2+ entry is necessary for the initial stages of differentiation.

Diets high in fat induce unhealthy expansion of adipose tissue, while exercise reduces adipocyte size and lipid content. Mice deficient of TRPC1 function and challenged with both a high-fat diet and exercise had lower fat mass and fasting glucose concentrations along with decreased adipocyte numbers. Further investigation indicated a decrease in autophagy with a concurrent increase in apoptosis. Together, this data shows that TRPC1 inhibits the positive effects of exercise under a high-fat diet-induced obesity environment.

Adipose tissue is an important endocrine organ responsible for secreting a number of cytokines, including adiponectin and leptin, which have a functional role in modulating metabolism. Secretion of adiponectin from adipose tissue has been found to be Ca2+ dependent, but the identity of the responsible Ca2+ channel is unknown. This study provides evidence that TRPC1 deficient mice have reduced serum adiponectin concentration which is believed to be due to an inability of adipose tissue to properly secrete adiponectin. Further, it was shown that loss of TRPC1-mediated Ca2+ influx is involved in SNARE complex formation necessary for proper exocytosis of adiponectin loaded vesicles.

Serum adiponectin concentrations have been shown to be correlated to adiponectin receptor expression, thus it was investigated whether reduces in serum adiponectin concentrations observed in TRPC1 deficient mice was due to dysfunctional adiponectin signaling in muscle. Within this study, it was observed that skeletal muscle of TRPC1 deficient mice have reduced adiponectin targets including PGC1α, lipid metabolism, and mitochondrial biogenesis mRNA expression. Though data suggests AdipoRon initiated adiponectin signaling in muscle is Ca2+ and SOCE dependent, TRPC1 is not a contributing member as loss of TRPC1 did not inhibit Ca2+ influx or PGC1α expression indicating decreased adiponectin targets is likely due to reduce serum adiponectin concentrations.

Overall, evidence from the combined studies suggests TRPC1-mediated Ca2+ influx is an important regulator of adipocyte processes necessary for maintaining a healthy body.

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