California State University, Northridge

CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Effects of Silencing Lipoprotein Lipase on Metabolic Enzymes in Rat Muscle Cells A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biochemistry By Adam Scott Mogul May 2019 The thesis of Adam Mogul is approved: ______________________________________ _____________________ Dr. Simon Garrett Date ______________________________________ _____________________ Dr. Daniel Tamae Date ______________________________________ _____________________ Dr. Jheem Medh, Chair Date California State University, Northridge ii Acknowledgements I would like to thank everyone in the CSUN Chemistry and Biochemistry department for helping me along this journey, especially those of you that helped supply me with helpful tips, reagents, or buffer solutions in my times of dire need. All of my friends in the department, the graduate and undergraduate students that have made my time at CSUN so enjoyable. The great group of people that have been a part of Dr. Medh’s lab, both past and present. Finally, a heartfelt thank you to Dr. Medh, for all of the help, understanding, and support through the time that I have spent in this program. Your guidance and patience have provided the foundation for my successful navigation of the world of biochemistry, and I truly appreciate it. iii Dedication I would like to dedicate this thesis to my Mom and Dad, who have believed in me and supported me through my academic career and beyond. To my grandparents, who have genuinely tried to be interested when I talk about my research, and provided me with love, food, and a place to sleep near school on late nights. To my sister, for being my life-long companion. And to my friends, new and old, who understood when I was too busy to see them but still made the effort to see me when I was available. iv Table of Contents Signature Page ii Acknowledgements iii Dedication iv List of Tables viii List of Figures ix List of Abbreviations xi Abstract xiii Chapter 1 – Introduction Section 1 – Prevalence and mechanism of diabetes 1 Section 2 – Muscle cells and metabolism 1 Section 3 – Lipoprotein Lipase (LPL) 2 Section 4 – Physiological role of LPL products 6 Section 5 – Glycolysis, glycogen synthesis, and LPL 6 Section 6 – Previous study of LPL and its impact on glucose utilization 10 Section 7 – Research goals 10 v Chapter 2 – Materials and Methods Section 1 – L6 rat muscle cell culture 12 Section 2 – L6 rat muscle cell differentiation 13 Section 3 – The LPL-knock-down L6 cell line 14 Section 4 – RNA isolation 15 Section 5 – RNA quantification 16 Section 6 – RT-PCR to make cDNA 17 Section 7 – End-point PCR 18 Section 8 – Quantitative PCR 20 Section 9 – L6 cell lysis for protein isolation 22 Section 10 – Protein quantification assay 22 Section 11 – Western blot 24 Section 12 – Hexokinase activity assay 27 Section 13 – Statistical analysis 32 Chapter 3 – Results Section 1 – Morphology of LPL-KD cells 33 Section 2 – Effects of LPL-KD on RNA expression levels 34 Section 3 – Effects of LPL-KD on protein expression levels 36 vi Section 4 – Effects of LPL-KD on hexokinase activity 37 Chapter 4 – Discussion Section 1 – Effect of LPL on gene expression 45 Section 2 – Effect of LPL on hexokinase II protein levels 47 Section 3 – Effect of LPL on the activity of hexokinase 49 Section 4 – Literature review of altered LPL expression and metabolism 55 Chapter 5 – Conclusion 58 References 60 vii List of Tables Table 1. Primers used for PCR reactions. 19 Table 2. Antibodies used in western blotting 26 Table 3. Baseline NADH formation in HK assay samples. 41 Table 4. Raw absorbance value comparison of concentrated lysates. 42 viii List of Figures Figure 1. Overview of the processing and transport of dietary lipids. 3 Figure 2. LPL catalyzes the hydrolysis of triglyceride into glycerol and free fatty acids 3 Figure 3. LPL dimer bound to GPIHBP1. 4 Figure 4. The catalytic triad of the active site of LPL. 5 Figure 5. Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate 7 Figure 6. Phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate by the enzyme phosphofructokinase-1. 9 Figure 7. Parallel differentiation of wild-type cells for experimentation. 14 Figure 8. Theory behind the hexokinase activity assay. 28 Figure 9. Comparison of the morphology of the L6 WT vs LPL-KD cells. 33 Figure 10. End-point PCR products separated via 2% agarose gel electrophoresis. 34 Figure 11. Regulation of gene transcription in LPL-KD cells as determined using qPCR. 35 Figure 12. Western blot analysis of HK II protein concentration in L6 cells. 37 Figure 13. Standard curve used for the hexokinase assay data analysis. 38 ix Figure 14. Hexokinase activity in WT and LPL-KD L6 cells. 39 Figure 15. Comparison of the nmol of NADH formed per mg of protein per minute for each sample. 40 Figure 16. Comparison of NADH formation in the concentrated WT and KD samples after background-subtraction. 43 Figure 17. Summary of the main findings of this research. 44 Figure 18. Proposed explanation for the regulation of hexokinase II activity in LPL-KD cells. 58 x List of abbreviations 6PG 6-phospho-D-glucono-1,5-lactone APS Ammonium persulfate BCA Bicinchoninic acid Bis-acrylamide N’N’-bis-methylene-acrylamide BG sample Background sample BSA Bovine serum albumin cDNA Complementary DNA CT value Cycles required to reach fluorescence threshold DMSO Dimethyl sulfoxide dsDNA Double-stranded DNA F16-BP Fructose 1,6-bisphosphate F6-P Fructose 6-phosphate faCoAs Fatty acyl-CoAs FBS Fetal bovine serum FFA Free fatty acid G6-P Glucose 6-phosphate G6PD Glucose 6-phosphate dehydrogenase GPIHBP1 Glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 HK Hexokinase hLPL0 Heart-specific LPL knock-out mice HRP Horseradish peroxidase IDL Intermediate-density lipoprotein IRS-1 Insulin receptor substrate-1 KD Knock-down (of LPL unless otherwise noted) LCACoA Long-chain acyl CoA LPL Lipoprotein lipase xi LPL-KD Lipoprotein lipase-knock-down M-MLV Moloney Murine Leukemia Virus M-MLV MM Moloney Murine Leukemia Virus master mix M-MLV RT Moloney Murine Leukemia Virus Reverse Transcriptase mRNA Messenger RNA PBS Phosphate buffered saline PCR Polymerase chain reaction PFK Phosphofructokinase PI Phosphatidylinositol PMSF Phenylmethanesulfonyl fluoride PPP Pentose phosphate pathway qPCR Quantitative PCR rcf Relative centrifugal force RNAi RNA interference RT-PCR Reverse transcriptase polymerase chain reaction SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulfate – polyacrylamide gel electrophoresis shRNA Short hairpin RNA T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus TEMED N,N,N’,N’-tetramethylethylenediamine TG Triglyceride VLDL Very-low-density lipoprotein WT Wild-type xii Abstract Effects of Silencing Lipoprotein Lipase on Metabolic Enzymes in Rat Muscle Cells By Adam Scott Mogul Master of Science in Biochemistry Lipoprotein lipase (LPL) is an enzyme required for the hydrolysis of triglycerides to free fatty acids and glycerol. In earlier reports, we demonstrated that in a muscle cell line, LPL levels are directly correlated with insulin resistance, and reducing LPL expression increased insulin-stimulated glucose uptake. Additionally, LPL-knock-down (LPL-KD) L6 rat muscle cells showed increased glucose oxidation compared to wild- type (WT) L6 cells. Glycolysis is the first pathway in glucose oxidation, and two important enzymes for regulating glycolysis in skeletal muscle are hexokinase II (HK II) and phosphofructokinase-1 (PFK-1). Our goal was to compare the expression and activity of the glycolytic enzymes hexokinase and phosphofructokinase in LPL-KD and WT L6 muscle cells. The cells used in this project were prepared in a previous study. For LPL-KD cells, the LPL gene was silenced in rat skeletal muscle cells (L6 cells) by lentiviral- mediated RNA interference. Total RNA was isolated and specific primer pairs were used xiii for end-point and real-time PCR amplification of LPL, Hexokinase II, and PFKM. A western blot analysis was carried out for HK II protein mass. β-actin was used as a housekeeping gene during all PCR and western blot analyses. Additionally, HK activity assays were performed to determine the HK enzyme activity in the cell lysates. Quantitative PCR showed that LPL-KD cells have <1% of the LPL expression as their WT counterparts due to the shRNA silencing. Silencing of the LPL gene resulted in a dramatic increase in the gene transcription of HKII (253% of WT levels) and a decrease in that of PFKM (72.1% of WT levels). These findings were supported by data from end- point PCR and western blot analysis of hexokinase II. The hexokinase activity assay suggested a decrease in HK activity in LPL-KD cells compared to WT cells. We hypothesize that increased HK II expression and translation facilitates glucose uptake into the cell. The finding that the glycolytic rate-limiting enzyme PFK-1 is repressed suggests that glucose 6-phosphate may be diverted to glycogen synthesis, the pentose phosphate pathway, or generally build up to a high enough concentration to inhibit the overall HK II activity, in spite of the increased amount of HK II protein in the LPL-KD cells. xiv Chapter 1 – Introduction Section 1 – Prevalence and mechanism of diabetes Diabetes mellitus is a widespread and chronic metabolic disease that afflicts an estimated 8.3% of the world’s adult population.1 The disease can be categorized into two main types and is characterized by hyperglycemia, which leads to complications that include cardiovascular diseases, neuropathy, retinopathy, and nephropathy. Type 1 diabetes mellitus (T1DM) is caused by an autoimmune response against the β (beta) cells of the pancreas, which are responsible for the secretion of insulin. Destruction of the β cells leads to the inability of a patient with T1DM to produce enough insulin to regulate metabolic needs, which results in hyperglycemia.

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