Chemistry and Physics of Lipids 187 (2015) 56–61
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Dietary docosahexaenoic acid supplementation reduces SERCA Ca2+ transport efficiency in rat skeletal muscle Val Andrew Fajardo a , Eric Bombardier a , Thomas Irvine a , Adam H. Metherel a , Ken D. Stark a , Todd Duhamel b,c, James W.E. Rush a , Howard J. Green a , A. Russell Tupling a, * a
Department of Kinesiology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada Health, Leisure, and Human Performance Research Institute, Faculty of Kinesiology and Recreation Management, University of Manitoba, Winnipeg, MB R3T 2N2 Canada c Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, 351 Tache Avenue, Winnipeg MB R2H 2A6, Canada b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2014 Received in revised form 13 February 2015 Accepted 11 March 2015 Available online 12 March 2015
Docosahexaenoic acid (DHA) can reduce the efficiency and increase the energy consumption of Na+/K+ATPase pump and mitochondrial electron transport chain by promoting Na+ and H+ membrane permeability, respectively. In skeletal muscle, the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) pumps are major contributors to resting metabolic rate. Whether DHA can affect SERCA efficiency remains unknown. Here, we examined the hypothesis that dietary supplementation with DHA would reduce Ca2+ transport efficiency of the SERCA pumps in skeletal muscle. Total lipids were extracted from enriched sarcoplasmic reticulum (SR) membranes that were isolated from red vastus lateralis skeletal muscles of rats that were either fed a standard chow diet supplemented with soybean oil or supplemented with DHA for 8 weeks. The fatty acid composition of total SR membrane lipids and the major phospholipid species were determined using electrospray ionization mass spectrometry (ESI-MS). After 8 weeks of DHA supplementation, total SR DHA content was significantly elevated (control, 4.1 1.0% vs. DHA, 9.9 1.7%; weight percent of total fatty acids) while total arachidonic acid was reduced (control, 13.5 0.4% vs. DHA-fed, 9.4 0.2). Similar changes in these fatty acids were observed in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, altogether indicating successful incorporation of DHA into the SR membranes post-diet. As hypothesized, DHA supplementation reduced SERCA Ca2+ transport efficiency (control, 0.018 0.0002 vs. DHA-fed, 0.014 0.0009) possibly through enhanced SR Ca2+ permeability (ionophore ratio: control, 2.8 0.2 vs. DHA-fed, 2.2 0.3). Collectively, our results suggest that DHA may promote skeletal muscle-based metabolism and thermogenesis through its influence on SERCA. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Docosahexaenoic acid SERCA Ca2+ transport efficiency Sarcoplasmic reticulum
1. Introduction Docosahexaenoic acid (DHA, 22:6n-3) is a highly unsaturated fatty acid (HUFA; 20 carbons, 3 double bonds) found in biological membranes thought to be important for the regulation of cellular activities, including thermogenesis and metabolic regulation (Hulbert and Else, 1989; Hulbert et al., 2005). Consequently, DHA has been viewed as an attractive nutritional supplement potentially useful for combating obesity (Delarue
* Corresponding author at: University of Waterloo, Department of Kinesiology, 200 University Ave. W., Waterloo, ON N2L 3G1, Canada. Tel.: +1 519 888 4567x33652; fax: +1 519 885 0470. E-mail address: [email protected] (A. R. Tupling). http://dx.doi.org/10.1016/j.chemphyslip.2015.03.001 0009-3084/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
et al., 2004; Li et al., 2008; Lorente-Cebrian et al., 2013; Rossmeisl et al., 2009; Ruzickova et al., 2004; Vasickova et al., 2011). At the cellular level, DHA has been positively correlated with mitochondrial proton leak (Hulbert, 2003), and increasing mitochondrial DHA content through lipid infusion or dietary treatment augments proton movement and state 4 respiration (Stillwell et al., 1997). In addition, DHA enhances Na+ membrane permeability (Hendriks et al., 1976; Stillwell and Wassall, 2003) and Na+/K+-ATPase activity (Else and Wu, 1999; Turner et al., 2003; Wu et al., 2004). Collectively, these studies have led to the notion that DHA may increase cellular energy expenditure by inducing a Na+ leak-pump cycle and/or by enhancing mitochondrial uncoupling (Hulbert and Else, 1999; Hulbert et al., 2005). We and others have shown that the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) pumps are significant contributors
V.A. Fajardo et al. / Chemistry and Physics of Lipids 187 (2015) 56–61
to resting muscle metabolic rate (Chinet et al., 1992; Smith et al., 2013). The SERCA pumps actively transport Ca2+ across the sarcoplasmic reticulum (SR) membrane from the cytosol and into the SR and play a primary role in the maintenance of a large cytosolic-SR Ca2+ gradient (1:10 000). (Gamu et al., 2014). Whether DHA can promote a Ca2+ leak-pump cycle and reduce SERCA’s Ca2+ transport efficiency in skeletal muscle is currently unknown and sets the basis for the present study. Indeed, incorporation of DHA in the SR membranes of mouse cardiac muscle resulted in reduced SERCA Ca2+ uptake, possibly through enhanced SR Ca2+ permeability; however, this theory had never been fully tested (Croset et al., 1989). Like cardiac muscle (Croset and Kinsella, 1989), DHA content in rat skeletal muscle can be influenced by diet (Stark et al., 2007). Thus, to more directly examine the influence of DHA on SERCA Ca2+ transport efficiency in skeletal muscle, we assessed the SERCA coupling ratio in red vastus lateralis (RV) homogenates obtained from rats fed a normal chow diet and those supplemented with DHA for 8 weeks. We hypothesized that dietary DHA supplementation would result in incorporation of this HUFA into the SR membrane and that this would lead to a reduction in SERCA Ca2+ transport efficiency. 2. Methods
57
Table 2 Fatty acid composition of diets administered. Fatty acid
14:0 16:0 18:0 20:0 22:0 24:0 Total saturates 18:1n-9 20:1n-9 22:1n-9 Total monounsaturates 18:2n-6 18:3n-6 20:2n-6 20:4n-6 18:3n-3 20:3n-3 20:5n-3 22:5n-3 22:6n-3 Total polyunsaturates Total fatty acids
Control
DHA
mg/g diet
Energy %
mg/g
Energy %
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Dietary docosahexaenoic acid supplementation reduces SERCA Ca2+ transport efficiency in rat skeletal muscle Val Andrew Fajardo a , Eric Bombardier a , Thomas Irvine a , Adam H. Metherel a , Ken D. Stark a , Todd Duhamel b,c, James W.E. Rush a , Howard J. Green a , A. Russell Tupling a, * a
Department of Kinesiology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada Health, Leisure, and Human Performance Research Institute, Faculty of Kinesiology and Recreation Management, University of Manitoba, Winnipeg, MB R3T 2N2 Canada c Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, 351 Tache Avenue, Winnipeg MB R2H 2A6, Canada b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2014 Received in revised form 13 February 2015 Accepted 11 March 2015 Available online 12 March 2015
Docosahexaenoic acid (DHA) can reduce the efficiency and increase the energy consumption of Na+/K+ATPase pump and mitochondrial electron transport chain by promoting Na+ and H+ membrane permeability, respectively. In skeletal muscle, the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) pumps are major contributors to resting metabolic rate. Whether DHA can affect SERCA efficiency remains unknown. Here, we examined the hypothesis that dietary supplementation with DHA would reduce Ca2+ transport efficiency of the SERCA pumps in skeletal muscle. Total lipids were extracted from enriched sarcoplasmic reticulum (SR) membranes that were isolated from red vastus lateralis skeletal muscles of rats that were either fed a standard chow diet supplemented with soybean oil or supplemented with DHA for 8 weeks. The fatty acid composition of total SR membrane lipids and the major phospholipid species were determined using electrospray ionization mass spectrometry (ESI-MS). After 8 weeks of DHA supplementation, total SR DHA content was significantly elevated (control, 4.1 1.0% vs. DHA, 9.9 1.7%; weight percent of total fatty acids) while total arachidonic acid was reduced (control, 13.5 0.4% vs. DHA-fed, 9.4 0.2). Similar changes in these fatty acids were observed in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, altogether indicating successful incorporation of DHA into the SR membranes post-diet. As hypothesized, DHA supplementation reduced SERCA Ca2+ transport efficiency (control, 0.018 0.0002 vs. DHA-fed, 0.014 0.0009) possibly through enhanced SR Ca2+ permeability (ionophore ratio: control, 2.8 0.2 vs. DHA-fed, 2.2 0.3). Collectively, our results suggest that DHA may promote skeletal muscle-based metabolism and thermogenesis through its influence on SERCA. ã 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Docosahexaenoic acid SERCA Ca2+ transport efficiency Sarcoplasmic reticulum
1. Introduction Docosahexaenoic acid (DHA, 22:6n-3) is a highly unsaturated fatty acid (HUFA; 20 carbons, 3 double bonds) found in biological membranes thought to be important for the regulation of cellular activities, including thermogenesis and metabolic regulation (Hulbert and Else, 1989; Hulbert et al., 2005). Consequently, DHA has been viewed as an attractive nutritional supplement potentially useful for combating obesity (Delarue
* Corresponding author at: University of Waterloo, Department of Kinesiology, 200 University Ave. W., Waterloo, ON N2L 3G1, Canada. Tel.: +1 519 888 4567x33652; fax: +1 519 885 0470. E-mail address: [email protected] (A. R. Tupling). http://dx.doi.org/10.1016/j.chemphyslip.2015.03.001 0009-3084/ ã 2015 Elsevier Ireland Ltd. All rights reserved.
et al., 2004; Li et al., 2008; Lorente-Cebrian et al., 2013; Rossmeisl et al., 2009; Ruzickova et al., 2004; Vasickova et al., 2011). At the cellular level, DHA has been positively correlated with mitochondrial proton leak (Hulbert, 2003), and increasing mitochondrial DHA content through lipid infusion or dietary treatment augments proton movement and state 4 respiration (Stillwell et al., 1997). In addition, DHA enhances Na+ membrane permeability (Hendriks et al., 1976; Stillwell and Wassall, 2003) and Na+/K+-ATPase activity (Else and Wu, 1999; Turner et al., 2003; Wu et al., 2004). Collectively, these studies have led to the notion that DHA may increase cellular energy expenditure by inducing a Na+ leak-pump cycle and/or by enhancing mitochondrial uncoupling (Hulbert and Else, 1999; Hulbert et al., 2005). We and others have shown that the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) pumps are significant contributors
V.A. Fajardo et al. / Chemistry and Physics of Lipids 187 (2015) 56–61
to resting muscle metabolic rate (Chinet et al., 1992; Smith et al., 2013). The SERCA pumps actively transport Ca2+ across the sarcoplasmic reticulum (SR) membrane from the cytosol and into the SR and play a primary role in the maintenance of a large cytosolic-SR Ca2+ gradient (1:10 000). (Gamu et al., 2014). Whether DHA can promote a Ca2+ leak-pump cycle and reduce SERCA’s Ca2+ transport efficiency in skeletal muscle is currently unknown and sets the basis for the present study. Indeed, incorporation of DHA in the SR membranes of mouse cardiac muscle resulted in reduced SERCA Ca2+ uptake, possibly through enhanced SR Ca2+ permeability; however, this theory had never been fully tested (Croset et al., 1989). Like cardiac muscle (Croset and Kinsella, 1989), DHA content in rat skeletal muscle can be influenced by diet (Stark et al., 2007). Thus, to more directly examine the influence of DHA on SERCA Ca2+ transport efficiency in skeletal muscle, we assessed the SERCA coupling ratio in red vastus lateralis (RV) homogenates obtained from rats fed a normal chow diet and those supplemented with DHA for 8 weeks. We hypothesized that dietary DHA supplementation would result in incorporation of this HUFA into the SR membrane and that this would lead to a reduction in SERCA Ca2+ transport efficiency. 2. Methods
57
Table 2 Fatty acid composition of diets administered. Fatty acid
14:0 16:0 18:0 20:0 22:0 24:0 Total saturates 18:1n-9 20:1n-9 22:1n-9 Total monounsaturates 18:2n-6 18:3n-6 20:2n-6 20:4n-6 18:3n-3 20:3n-3 20:5n-3 22:5n-3 22:6n-3 Total polyunsaturates Total fatty acids
Control
DHA
mg/g diet
Energy %
mg/g
Energy %
