Date of Award

Spring 1-1-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Bradley B. Olwin

Second Advisor

James Orth

Third Advisor

Jingshi Shen

Fourth Advisor

James DeGregori

Fifth Advisor

Robert Garcea

Abstract

Excessive circulating triglycerides due to reduction or loss of lipoprotein lipase (LPL) activity contribute to hypertriglyceridemia and increased risk for pancreatitis. The only gene therapy treatment for LPL deficiency decreases pancreatitis but minimally reduces hypertriglyceridemia. Synthesized in striated muscle and adipose tissue, LPL is then trafficked to blood vessel endothelial cells where it hydrolyzes triglycerides into free fatty acids. We conditionally knocked out LPL in differentiated striated muscle tissue lowering striated muscle LPL activity and causing hypertriglyceridemia. We crossed these LPL knockout mice with mice possessing a conditional avian retroviral receptor gene and infected mice with human LPL or mCherry retrovirus. Plasma LPL activity increased for three weeks following human LPL infection. Human LPL infected mice also had significantly lower blood triglyceride levels. Thus, targeted delivery of human LPL into striated muscle tissue identifies a potential therapeutic target for LPL deficiency.

To repair skeletal muscle, the resident stem cells, termed satellite cells, exit reversible quiescence, proliferate, and differentiate to repair damaged muscle. The transition from quiescence to cycling is poorly understood, particularly with regard to the molecular clock. Pharmacological and genetic perturbations of the molecular clock in satellite cells disrupts activation blunting muscle regeneration. Bmal1 deficient satellite cells have an altered citric acid cycle metabolism increasing succinate levels and altering the alpha-ketoglutarate/succinate ratio. Succinate promotes satellite cell G-alert and premature activation via inhibition of prolyl-hydroxylase 2 (PHD2) increasing the levels of PHD2 substrates. Exogenous succinate and PHD2 inhibitors mimic Bmal1cKO satellite cells, while α-ketoglutarate rescues Bmal1cKO cells. Thus, the molecular clock is intimately coupled to myogenesis, providing a link between metabolism and cell transitions in satellite cells.

The satellite cell transition from quiescence to cycling also relies upon post-transcriptional regulation. Tristetraprolin, an RNA decay protein, degrades mRNAs during quiescence including MyoD mRNA which is involved in activation. Upon activation, tristetraprolin is inhibited and target mRNAs are transcribed. Inhibition of tristeraprolin in vivo leads to aberrant activation and fusion into muscle, suggesting additional target mRNAs are involved. We identified additional tristetraprolin mRNA targets in satellite cells involved in activation and environmental sensing, providing a mechanism for rapid cell fate decisions.

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