SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION
Woodlief, Tracey L.
An oversupply of fatty acids and inadequacies in their partitioning toward oxidation and/or storage will lead to disruptions in cellular homeostasis. Reductions in mitochondrial content and lower rates of fatty acid oxidation have been associated with the accumulation of bioactive lipids [e.g., acyl-coenzyme-A and diacylgleycerol (DAG)] and superoxide derived free radicals. Decreased mitochondrial fatty acid partitioning towards oxidation as well as increased fatty acid partitioning towards synthesis are both associated with skeletal muscle in the obese condition. These fatty acid partitioning paradigms are believed to establish a state of insulin resistance and the development of type II diabetes, as both increased bioactive fatty acids and mitochondrial superoxides have been linked to down regulating the insulin signaling cascade. An accumulating body of evidence, derived mostly from studies in rodents, suggests that long-chain acyl-CoA synthetase (ACSL) and diglyceride acyltransferase-1 (DGAT-1) are significant metabolic regulators involved in lipid partitioning toward lipid oxidation and synthesis with alterations in ACSL and DGAT-1 activities likely to impact insulin signaling and cellular redox potential. To determine the function of these proteins in human skeletal muscle, ACSL-5 (localized on the liver mitochondrial membrane and postulated to be associated with oxidative lipid partitioning) and DGAT-1 (partitions fatty acids toward triacylglycerol synthesis) were overexpressed in primary human myotubes derived from muscle biopsies (HSKMC). We hypothesized that ACSL-5 overexpression would be associated with an upregulation in insulin signaling by virtue of increasing fatty acid flux into the mitochondria for oxidation and hence reducing cytosolic acyl-CoAs. Acyl-CoAs are known activators of kinases that impair the insulin signaling cascade. In addition, we hypothesized that DGAT-1 overexpression would also lead to increases in the insulin signaling cascade due to increased removal of bioactive DAGs. DAGs are a known activator of kinases established to decrease insulin signaling at the level of the insulin receptor tyrosine kinase activity and IRS-1 serine phosphorylation. Primary myoblasts isolated from vastus lateralis of obese women (N=6) were transfected with ACSL-5 and DGAT-1 plasmid DNA alone or concurrently, differentiated into myotubes, incubated on day 5-7 with 125µM:125µM palmitate:oleate lipid incubation ± 100nM insulin (15min- day 7) and then harvested on day 7. Increased ACSL-5 and DGAT-1 protein expression alone and concurrently were assessed by Western Blot. Mitochondrial complete and incomplete oxidation were assessed by radiolabeled [superscript]14C-palmitate oxidation, as well as synthesis of total and lipid specific subspecies monoacylglcerol, diacylglcerol and triacylglcerol (MAG, DAG, TAG) by [superscript]14C-palmitate incorporation. MitoSOX red was utilized to measure mitochondrial specific superoxide formation via flow cytometry, while manganese superoxide dismutase (MnSOD) expression was utilized to determine the cellular antioxidant response. MnSOD is one primary oxidant defense protein localized in the mitochondria exclusively. The effects of ACSL-5 or DGAT-1 overexpression or co-expression on insulin signaling was assessed at the levels of p-IRS-1[superscript]ser307, p-Akt[superscript]ser347 and p-AS160[superscript]thr642 by Western Blot procedures. Analyses confirmed significant increases in both ACSL-5 (2.2 fold change, P < 0.05) and DGAT-1 (2.1 fold change, P<0.05) protein expression following transfection. As hypothesized, ACSL-5 overexpression resulted in increased complete (2.7-fold, P < 0.05) and incomplete oxidation (2.6-fold, P < 0.05). Inconsistent with our hypothesis however, ACSL-5 overexpression resulted in increases in total (1.8-fold, P < 0.05) and lipid specie specific synthesis [TAG, (2-fold, P > 0.05) and DAG (1.5-fold, P < 0.05)]. ACSL-5 overexpression also resulted in a significant increase in superoxide production (20% and P < 0.05), but was also accompanied with a significant increase in MnSOD (3.2-fold, P <0.05) expression. Lastly, ACSL-5 overexpression resulted in no significant changes in insulin signaling. As expected, DGAT-1 overexpression was associated with significant increases in total lipid synthesis (2.6-fold, P <0.05) and TAG (3-fold, P <0.05) synthesis. However, DAG (1.8-fold, P <0.05) and MAG (2.2-fold and P <0.05) content were also increased. Unexpectedly, DGAT-1 overexpression resulted in elevations in complete (1.8-fold, P <0.05) fatty acid oxidation which was accompanied with a reduction in IRS-1[superscript]ser307 (1.6-fold and P <0.05) but elevations in pAkt[superscript]ser347 (1.4-fold P <0.05) (no change in pAS160[superscript]thr642). DGAT-1 overexpression was also associated with elevations in superoxide formation (20%, P <0.05). However, contrary to ACSL-5 overexpression, no change in MnSOD was noted. Lastly, when ACSL-5 and DGAT-1 were co-overexpressed, elevations in fatty acid oxidation (2.2-fold, P <0.05), total lipid synthesis (1.8-fold, P <0.05), mitochondrial superoxide production (30%, P <0.05) and MnSOD (4.8-fold, P <0.05) protein expression were noted. As with DGAT-1 overexpression, co-overexpression was associated with reduced IRS-1[superscript]ser307 (1.6-fold and P <0.05) and elevations in pAkt[superscript]ser347 (1.4-fold, P <0.05) (no change in pAS160[superscript]thr642). To date this is the first study to demonstrate the successful overexpression/co-expression of the lipid partitioning proteins ACSL-5 and DGAT-1 in human skeletal muscle. In addition, these results demonstrate a role for these protein isoforms in human skeletal muscle fatty acid partitioning towards oxidation and lipid synthetic pathways. It appears that ACSL-5 overexpression may provide positive effects on lipid partitioning in skeletal muscle by increasing mitochondrial oxidation (known to be reduced in the insulin resistant state) and by increasing lipid synthesis capacity by increasing lipid partitioning towards TAG (inert storage specie) synthesis. However, ACSL-5 overexpression is insufficient to upregulate insulin signaling. This may be due, in part, to ACSL-5's effects on increasing fatty acid flux and hence reducing equivalents to the electron transport chain resulting in increased mitochondrial superoxide formation. DGAT-1 overexpression facilitates mitochondrial fatty acid oxidation and increases lipid partitioning of potentially disruptive bioactive lipids toward storage as biologically inert triacylglycerols. DGAT-1's effects on fatty acid oxidation may be due to increases in fatty acid oxidative machinery in response to an increase in fatty acid turnover in the skeletal muscle cell, which would cause an increase in reducing equivalents to the electron transport chain and thereby increase superoxide production. However, DGAT-1 overexpression resulted in an upregualtion in insulin signaling despite increases in superoxide production, possibly due to an increase in the turnover of bioactive lipid species such as DAGs to TAGs. These effects are associated with an upregulation in insulin signaling independently or in conjunction with ACSL-5 overexpression. These and future studies will expand our understanding of the regulation of fatty acid trafficking in human skeletal muscle and its effects on glucose homeostasis and insulin signaling in the obese and/or diabetic condition.
Woodlief, Tracey L.. (January 2011). SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION (Doctoral Dissertation, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/3763.)
Woodlief, Tracey L.. SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION. Doctoral Dissertation. East Carolina University, January 2011. The Scholarship. http://hdl.handle.net/10342/3763. August 18, 2019.
Woodlief, Tracey L., “SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION” (Doctoral Dissertation., East Carolina University, January 2011).
Woodlief, Tracey L.. SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION [Doctoral Dissertation]. Greenville, NC: East Carolina University; January 2011.
East Carolina University