Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

Skeletal muscle is one of the largest tissues in humans and provides many pivotal functions to support life. Abnormality in skeletal muscle functions can lead to disease. For example, insulin resistance in skeletal muscle leads to type II diabetes. The underlying mechanisms that control energy balance in skeletal muscle remain largely elusive, especially at the genetic level. Here in the second chapter, I showed that MyoD mediated the transcriptional regulation of ACSL5, a mitochondrial protein, in C2C12 myoblasts via two E-box elements. A SNP rs2419621 (T) created a de novo E-box that together with the two pre-existing proximal E-boxes strongly enhances ACSL5 expression in both CV1 and C2C12 cells. In the third chapter, I identified a novel VGLL4-interacting protein IRF2BP2 and verified the interaction with co-immunoprecipitation and mammalian two-hybrid assays. Functionally, overexpression of IRF2BP2 and transcription factor TEAD1 activates mouse VEGF-A promoter in CV1 cells and enhances the biosynthesis of VEGF-A in C2C12 myoblasts. In vivo studies showed that ischemia induced the expression of IRF2BP2 by more than three fold, suggesting that IRF2BP2 could play a pivotal role during tissue ischemia. IRF2BP2 is a nuclear protein in both mouse cardiac myocytes and C2C12 myoblasts as demonstrated by immunohistochemistry and immunocytochemistry, respectively. Therefore, I sought to delineate the mechanism for the nuclear shuttling of IRF2BP2 in the fourth chapter. With various DNA alternations, I mapped the NLS to an evolutionarily conserved sequence 354ARKRKPSP361 in IRF2BP2. Deletion of the positively charged amino acids resulted in the abolishment of the NLS signal. Next, I showed that phosphorylation of serine 360 (S360) mediates the nuclear import of the protein. Whereas an alanine substitution (S360A) at the site resulted in perinuclear accumulation of the protein, an aspartic acid substitution (S360D) forced the nuclear accumulation. Nevertheless, the forced accumulation of the S360D mutant did not enhance the activation of VEGF-A promoter in CV1 cells as did the wild-type protein. My studies revealed two novel mechanisms by which skeletal muscle could harvest energy, thus providing new insight into the energy metabolism in skeletal muscle

It is estimated by the World Health Organization (WHO) that 422 million people had diabetes worldwide in 2014, including 30.3 million people in the US. The cost of treating the disease is has tripled from 2003-2013 due to the increased number of patients. One of the genes strongly associated with type 2 diabetes (T2D) is the transcription factor 7 like 2 (TCF7L2). A single nucleotide polymorphism (SNP) of the TCF7L2 results in increased expression of long chain acyl-CoA synthetase 5 (ACSL5) while deletion of this part of the TCF7L2 gene reduces ACSL5 mRNA level. The regulation of ACSL5 gene expression by the high risk TCF7L2 allele highlights the importance of investigating the role of ACSL5 in T2D. ACSL5 is one of a family of enzymes that activates FA to its CoA ester and is required for FA metabolism within cells. Mice lacking this protein have reduced fat mass and are more insulin sensitive. Chronic exposure of clonal pancreatic ß-cells to excess nutrients has been shown to result in increased intrinsic lipid droplets, reduced insulin content, a left-shift in glucose dose-dependent insulin secretion curve characterized by basal insulin hypersecretion (IH) and blunted glucose stimulated insulin secretion (GSIS). We tested the hypothesis that the use of a putative ACSL5 inhibitor (Adipo C) can reduce accumulated lipid droplets, rescue insulin content and reverse the left-shift in glucose dose-dependent insulin secretion curve. INS-1 (823/13) cells were cultured in either 4 mM or 11 mM glucose media representing physiological and excess nutrients environment. Adipo C (10-25 µM) was added to cells to both acutely (2 hrs) and chronically (72 hrs) inhibit ACSL5 activity. Thin layer chromatography with C11 Bodipy fatty acid (BFA) was used to detect acute fatty acid incorporation into neutral lipids. Nile red was used to visualize intrinsic lipid droplets inside cells. Intracellular Ca2+ activity was detected using fura 2. Insulin assay was measured by HTRF. Acute fatty acid incorporation and lipid accumulation were reduced in cells exposed to Adipo C. An Adipo C concentration dependent right shift of glucose dose-dependent insulin release and increased insulin content were observed. 11 mM glucose cells cultured in 25 µM Adipo C showed decreased intracellular Ca2+ activity at 3 mM glucose and increased Ca2+ activity at 12 mM glucose, which are characteristic of cells cultured in 4 mM glucose having reduced lipid stores. These results all indicate possible protective effects on -cells exposed to excess nutrients. Islets of T2D patients who have a physiologically elevated blood glucose level are exposed to a similar excess nutrient environment. Therefore, the results illustrated here warrant further research on Adipo C compound to explore its therapeutic potential on T2D.

OBJECTIVE: The close relationship between obesity and type 2 diabetes (T2D) highlights the fact that most diabetes patients are overweight or obese. We propose that elevated glucose and free fatty acid levels in those patients cause beta-cell dysfunction. Chronic exposure to excess nutrients (glucose and free fatty acid) leads to glucolipotoxicity, characterized by basal insulin hypersecretion, a left-shift in the glucose dose-dependent insulin secretion curve, and blunted glucose-stimulated insulin secretion. One of the suggested reasons for this defect is elevated intracellular lipid. In this study, our objective was to investigate whether reducing beta-cell lipid levels can reverse basal insulin hypersecretion. METHODS: INS-1 (823/13) cells were cultured in 4 or 11 mM glucose media. Elevated glucose and KCl doses were added to cells in the insulin secretion experiments. In the KCl-induced insulin secretion experiment, cells were treated with a combination of 12 mM glucose and 250 μM diazoxide, then assigned to different test concentrations with elevated KCl doses. Insulin release and content were measured by the insulin ultra-sensitive homogenous time-resolved fluorescence (HTRF) kit (Cisbio). Following that, we monitored intracellular Ca2+ activity of KCl-induced insulin secretion on a fluorescence spectrophotometer F-2000 (Hitachi). Additionally, we acutely added Adipo C (20 µM) or fatty acid-free BSA to cells to reduce the lipids levels in the ß-cells. We also stained with Nile Red (Sigma) to examine the intrinsic lipid droplets in those cells. RESULTS: ß-cells cultured in excess nutrients (11 mM glucose) exhibited a left shift in the glucose dose-dependent response curve. The hypersecretion at low glucose could be blocked by the KATP channel activator, diazoxide, indicating that Ca2+ influx drives the increase in secretion at glucose concentrations normally considered basal. Here we extend this left shift to include KCl-induced insulin secretion, supporting a role for Ca2+ in the observed hypersensitivity. KCl-induced Ca2+ influx was also left-shifted. Interestingly, we found acute exposure to Adipo C or fatty acid-free BSA reversed the basal hypersecretion in cells cultured in excess nutrients. CONCLUSION: The work presented in this study provided supporting evidence that ß-cells cultured in excess nutrients were hypersensitive to glucose while extending these studies to KCl-induced insulin release. The excess nutrient-induced left shift in both glucose and KCl-stimulated insulin secretion was mediated by increased Ca2+. Thus, we postulate that excess nutrient exposure increases ß-cell plasma membrane lipids that alter Ca2+ handling to allow increased Ca2+ influx at inappropriate low glucose concentrations. Our results demonstrated that cells acutely exposed to the putative long-chain acyl-CoA synthetase inhibitor Adipo C or fatty acid-free BSA reversed basal insulin hypersecretion and supports a role for lipids mediating the adverse effect. T2D patients with obesity have a similar physiologically elevated fasting blood glucose and lipid. Thus, our findings suggest lowering lipid levels in ß-cells may have therapeutic potential in treating hyperinsulinemia leading to T2D.