Overexpression of Long-Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Oxidation and Free Radical Formation While Attenuating Insulin Signaling in Primary Human Skeletal Myotubes

In rodent skeletal muscle, acyl-coenzyme A (CoA) synthetase 5 (ACSL-5) is suggested to localize to the mitochondria but its precise function in human skeletal muscle is unknown. The purpose of these studies was to define the role of ACSL-5 in mitochondrial fatty acid metabolism and the potential effects on insulin action in human skeletal muscle cells (HSKMC). Primary myoblasts isolated from vastus lateralis (obese women (body mass index (BMI) = 34.7 ± 3.1 kg/m2)) were transfected with ACSL-5 plasmid DNA or green fluorescent protein (GFP) vector (control), differentiated into myotubes, and harvested (7 days). HSKMC were assayed for complete and incomplete fatty acid oxidation ([1-14C] palmitate) or permeabilized to determine mitochondrial respiratory capacity (basal (non-ADP stimulated state 4), maximal uncoupled (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-linked) respiration, and free radical (superoxide) emitting potential). Protein levels of ACSL-5 were 2-fold higher in ACSL-5 overexpressed HSKMC. Both complete and incomplete fatty acid oxidation increased by 2-fold (p < 0.05). In permeabilized HSKMC, ACSL-5 overexpression significantly increased basal and maximal uncoupled respiration (p < 0.05). Unexpectedly, however, elevated ACSL-5 expression increased mitochondrial superoxide production (+30%), which was associated with a significant reduction (p < 0.05) in insulin-stimulated p-Akt and p-AS160 protein levels. We concluded that ACSL-5 in human skeletal muscle functions to increase mitochondrial fatty acid oxidation, but contrary to conventional wisdom, is associated with increased free radical production and reduced insulin signaling

Metformin Improves Insulin Signaling in Obese Rats via Reduced IKK Action in a Fiber-Type Specific Manner

Metformin is a widely used insulin-sensitizing drug, though its mechanisms are not fully understood. Metformin has been shown to activate AMPK in skeletal muscle; however, its effects on the inhibitor of κB kinaseβ (IKKβ) in this same tissue are unknown. The aim of this study was to (1) determine the ability of metformin to attenuate IKKβ action, (2) determine whether changes in AMPK activity are associated with changes in IKKβ action in skeletal muscle, and (3) examine whether changes in AMPK and IKKβ function are consistent with improved insulin signaling. Lean and obese male Zuckers received either vehicle or metformin by oral gavage daily for four weeks (four groups of eight). Proteins were measured in white gastrocnemius (WG), red gastrocnemius (RG), and soleus. AMPK phosphorylation increased (P < .05) in WG in both lean (57%) and obese (106%), and this was supported by an increase in phospho-ACC in WG. Further, metformin increased IκBα levels in both WG (150%) and RG (67%) of obese rats, indicative of reduced IKKβ activity (P < .05), and was associated with reduced IRS1-pSer307 (30%) in the WG of obese rats (P < .02). From these data we conclude that metformin treatment appears to exert an inhibitory influence on skeletal muscle IKKβ activity, as evidenced by elevated IκBα levels and reduced IRS1-Ser307 phosphorylation in a fiber-type specific manner

Mechanism of benefit of combination thrombolytic therapy for acute myocardial infarction: A quantitative angiographic and hematologic study

AbstractObjectives. The goal of this study was to lend insight into the mechanisms responsible for the beneficial effects of combination thrombolytic therapy.Background. Combination thrombolytic therapy for acute myocardial infarction bas been associated with less reocclusion and fewer in-hospital clinical events than has monotherapy.Methods. Infarct-related quantitative coronary dimensions and hemostatic protein levels were evaluated in 287 patients with acute myocardial infarction during the early (90-min) and convalescent (7-day) phases after administration of recombinant tissue-type plasminogen activator (rt-PA), urokinase or combination rt-PA and urokinase.Results. Minimal lumen diameter was similar in the 90-min and 7-day phases after treatment with rt-PA, urokinase and combination rt-PA and urokinase (0.72 ± 0.45 mm, 0.62 ± 0.53 mm and 0.75 ± 0.58 mm, respectively, at 90 min, p = 0.16; and 1.05 ± 0.56 mm, 1.12 ± 0.72 mm and 0.94 ± 0.54 mm, respectively, at 7 days, p = 0.22). In-hospital clinical event and reocclusion rates were less frequent in patients receiving combination therapy than in those receiving monotherapy (25% vs. 38% and 32% for rt-PA and urokinase, respectively, p = 0.084; and 3% vs. 13% and 9% for rt-PA and urokinase, respectively, p = 0.03), but these events were unrelated to early or late coronary dimensions. Patients receiving combination therapy or urokinase monotherapy had significantly higher peak fibrin degradation products (1,307 ± 860 and 1,285 ± 898 μg/ml vs. 435 ± 717 μg/ml, respectively, p < 0.0001) and lower nadir fibrinogen levels (0.85 ± 1.00 and 0.75 ± 0.53 g/liter vs. 1.90 ± 0.86 g/liter, respectively, p < 0.0001) than did those receiving rt-PA monotherapy. Peak fibrinogen degradation products indirectly correlated (p = 0.004) and baseline (p = 0.026) and nadir (p = 0.089) fibrinogen levels directly correlated with reocclusion.Conclusions. Lower in-hospital clinical event and reocclusion rates observed with combination thrombolytic therapy may relate to systemic hematologic factors rather than to the residual lumen obstruction after thrombolysis

Mitochondrial Hâ‚‚Oâ‚‚ emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans

High dietary fat intake leads to insulin resistance in skeletal muscle, and this represents a major risk factor for type 2 diabetes and cardiovascular disease. Mitochondrial dysfunction and oxidative stress have been implicated in the disease process, but the underlying mechanisms are still unknown. Here we show that in skeletal muscle of both rodents and humans, a diet high in fat increases the Hâ‚‚Oâ‚‚-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Furthermore, we show that attenuating mitochondrial Hâ‚‚Oâ‚‚ emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the overexpression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet. These findings place the etiology of insulin resistance in the context of mitochondrial bioenergetics by demonstrating that mitochondrial Hâ‚‚Oâ‚‚ emission serves as both a gauge of energy balance and a regulator of cellular redox environment, linking intracellular metabolic balance to the control of insulin sensitivity. Original version available at http://www.jci.org/articles/view/3704

Time From Symptom Onset to Treatment and Outcomes after Thrombolytic Therapy

OBJECTIVES: This study sought to examine the relations among patient characteristics, time to thrombolysis and outcomes in the international GUSTO-I trial. BACKGROUND: Studies have shown better left ventricular function and decreased infarct size as well as increased survival with earlier thrombolysis, but the relative benefits of various thrombolytic agents with earlier administration are uncertain. METHODS: We evaluated the relations of baseline characteristics to three prospectively defined time variables: symptom onset to treatment, symptom onset to hospital arrival (presentation delay) and hospital arrival to treatment (treatment delay). We also examined the relations of delays to clinical outcomes and to the relative 30-day mortality benefit with accelerated tissue-type plasminogen activator (t-PA) versus streptokinase. RESULTS: Female, elderly, diabetic and hypertensive patients had longer delays at all stages. Previous infarction or bypass surgery was an additional risk factor for treatment delay. Early thrombolysis was associated with lower overall mortality rate ( 4 h, 9.0%), but no additional relative benefit resulted from earlier treatment with accelerated t-PA versus streptokinase (p = 0.38). Longer presentation and treatment delays were both associated with increased mortality rate (presentation delay 4 h, 8.6%; treatment delay 90 min, 8.1%). As time to treatment increased, the incidence of recurrent ischemia or reinfarction decreased, but the rates of shock, heart failure and stroke increased. CONCLUSIONS: Earlier treatment resulted in better outcomes, regardless of thrombolytic strategy. Elderly, female and diabetic patients were treated later, adding to their already substantial risk

SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION

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 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.  Ph.D

SKELETAL MUSCLE LIPID PARTITIONING : CONSEQUENCES OF ALTERATIONS IN ACYL-COA SYNTHETASE-5 AND DIGLYCERIDE ACYLTRANFERASE-1 EXPRESSION

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 DAG (1.5-fold P &lt; 0.05)]. ACSL-5 overexpression also resulted in a significant increase in superoxide production (20% and P &lt; 0.05) but was also accompanied with a significant increase in MnSOD (3.2-fold P &lt;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 &lt;0.05) and TAG (3-fold P &lt;0.05) synthesis. However DAG (1.8-fold P &lt;0.05) and MAG (2.2-fold and P &lt;0.05) content were also increased. Unexpectedly DGAT-1 overexpression resulted in elevations in complete (1.8-fold P &lt;0.05) fatty acid oxidation which was accompanied with a reduction in IRS-1[superscript]ser307 (1.6-fold and P &lt;0.05) but elevations in pAkt[superscript]ser347 (1.4-fold P &lt;0.05) (no change in pAS160[superscript]thr642). DGAT-1 overexpression was also associated with elevations in superoxide formation (20% P &lt;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 &lt;0.05) total lipid synthesis (1.8-fold P &lt;0.05) mitochondrial superoxide production (30% P &lt;0.05) and MnSOD (4.8-fold P &lt;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 &lt;0.05) and elevations in pAkt[superscript]ser347 (1.4-fold P &lt;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.

Overexpression of Long-Chain Acyl-CoA Synthetase 5 Increases Fatty Acid Oxidation and Free Radical Formation While Attenuating Insulin Signaling in Primary Human Skeletal Myotubes

In rodent skeletal muscle, acyl-coenzyme A (CoA) synthetase 5 (ACSL-5) is suggested to localize to the mitochondria but its precise function in human skeletal muscle is unknown. The purpose of these studies was to define the role of ACSL-5 in mitochondrial fatty acid metabolism and the potential effects on insulin action in human skeletal muscle cells (HSKMC). Primary myoblasts isolated from vastus lateralis (obese women (body mass index (BMI) = 34.7 ± 3.1 kg/m2)) were transfected with ACSL-5 plasmid DNA or green fluorescent protein (GFP) vector (control), differentiated into myotubes, and harvested (7 days). HSKMC were assayed for complete and incomplete fatty acid oxidation ([1-14C] palmitate) or permeabilized to determine mitochondrial respiratory capacity (basal (non-ADP stimulated state 4), maximal uncoupled (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-linked) respiration, and free radical (superoxide) emitting potential). Protein levels of ACSL-5 were 2-fold higher in ACSL-5 overexpressed HSKMC. Both complete and incomplete fatty acid oxidation increased by 2-fold (p &lt; 0.05). In permeabilized HSKMC, ACSL-5 overexpression significantly increased basal and maximal uncoupled respiration (p &lt; 0.05). Unexpectedly, however, elevated ACSL-5 expression increased mitochondrial superoxide production (+30%), which was associated with a significant reduction (p &lt; 0.05) in insulin-stimulated p-Akt and p-AS160 protein levels. We concluded that ACSL-5 in human skeletal muscle functions to increase mitochondrial fatty acid oxidation, but contrary to conventional wisdom, is associated with increased free radical production and reduced insulin signaling