Title: Relationship of glucose and oleate metabolism to cardiac function in lipin-1 deficient (fld) mice
Abstract: Lipin-1 is the major phosphatidate phosphatase (PAP) in the heart and a transcriptional coactivator that regulates fatty acid (FA) oxidation in the liver. As the control of FA metabolism is essential for maintaining cardiac function, we investigated whether lipin-1 deficiency affects cardiac metabolism and performance. Cardiac PAP activity in lipin-1 deficient [fatty liver dystrophy (fld)] mice was decreased by >80% compared with controls. Surprisingly, oleate oxidation and incorporation in triacylglycerol (TG), as well as glucose oxidation, were not significantly different in perfused working fld hearts. Despite this, [3H]oleate accumulation in phosphatidate and phosphatidylinositol was increased in fld hearts, reflecting the decreased PAP activity. Phosphatidate accumulation was linked to increased cardiac mammalian target of rapamycin complex 1 (mTORC1) signaling and endoplasmic reticulum (ER) stress. Transthoracic echocardiography showed decreased cardiac function in fld mice; however, cardiac dysfunction was not observed in ex vivo perfused working fld hearts. This showed that changes in systemic factors due to the global absence of lipin-1 could contribute to the decreased cardiac function in vivo. Collectively, this study shows that fld hearts exhibit unchanged oleate esterification, as well as oleate and glucose oxidation, despite the absence of lipin-1. However, lipin-1 deficiency increases the accumulation of newly synthesized phosphatidate and induces aberrant cell signaling. Lipin-1 is the major phosphatidate phosphatase (PAP) in the heart and a transcriptional coactivator that regulates fatty acid (FA) oxidation in the liver. As the control of FA metabolism is essential for maintaining cardiac function, we investigated whether lipin-1 deficiency affects cardiac metabolism and performance. Cardiac PAP activity in lipin-1 deficient [fatty liver dystrophy (fld)] mice was decreased by >80% compared with controls. Surprisingly, oleate oxidation and incorporation in triacylglycerol (TG), as well as glucose oxidation, were not significantly different in perfused working fld hearts. Despite this, [3H]oleate accumulation in phosphatidate and phosphatidylinositol was increased in fld hearts, reflecting the decreased PAP activity. Phosphatidate accumulation was linked to increased cardiac mammalian target of rapamycin complex 1 (mTORC1) signaling and endoplasmic reticulum (ER) stress. Transthoracic echocardiography showed decreased cardiac function in fld mice; however, cardiac dysfunction was not observed in ex vivo perfused working fld hearts. This showed that changes in systemic factors due to the global absence of lipin-1 could contribute to the decreased cardiac function in vivo. Collectively, this study shows that fld hearts exhibit unchanged oleate esterification, as well as oleate and glucose oxidation, despite the absence of lipin-1. However, lipin-1 deficiency increases the accumulation of newly synthesized phosphatidate and induces aberrant cell signaling. The constant requirement for ATP in the beating heart is satisfied by the utilization of diverse substrates, such as fatty acids (FA), glucose, lactate, ketones, and amino acids, with FA oxidation (FAO) providing the majority (50–75%) of cardiac ATP production (1.Stanley W.C. Recchia F.A. Lopaschuk G.D. Myocardial substrate metabolism in the normal and failing heart.Physiol. Rev. 2005; 85: 1093-1129Crossref PubMed Scopus (1459) Google Scholar). FAs are also incorporated into triacylglycerol (TG), which is stored in lipid droplets. Cardiac TG turnover is rapid (2.Lopaschuk G.D. Ussher J.R. Folmes C.D. Jaswal J.S. Stanley W.C. Myocardial fatty acid metabolism in health and disease.Physiol. Rev. 2010; 90: 207-258Crossref PubMed Scopus (1410) Google Scholar) and FAs released from TG can contribute up to 10–20% of ATP production (3.Saddik M. Lopaschuk G.D. Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts.J. Biol. Chem. 1991; 266: 8162-8170Abstract Full Text PDF PubMed Google Scholar). Plasma FA and TG levels rise in insulin resistance and diabetes (4.Lewis G.F. Carpentier A. Adeli K. Giacca A. Disordered fat storage and mobilization in the pathogenesis of insulin resistance and type 2 diabetes.Endocr. Rev. 2002; 23: 201-229Crossref PubMed Scopus (829) Google Scholar, 5.McArthur M.D. Graham S.E. Russell J.C. Brindley D.N. Exaggerated stress-induced release of nonesterified fatty acids in JCR:LA-corpulent rats.Metabolism. 1998; 47: 1383-1390Abstract Full Text PDF PubMed Scopus (17) Google Scholar), and cardiac FA utilization can increase to supply as much as 90% of ATP (6.Boudina S. Sena S. Theobald H. Sheng X. Wright J.J. Hu X.X. Aziz S. Johnson J.I. Bugger H. Zaha V.G. et al.Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins.Diabetes. 2007; 56: 2457-2466Crossref PubMed Scopus (464) Google Scholar, 7.Buchanan J. Mazumder P.K. Hu P. Chakrabarti G. Roberts M.W. Yun U.J. Cooksey R.C. Litwin S.E. Abel E.D. Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity.Endocrinology. 2005; 146: 5341-5349Crossref PubMed Scopus (419) Google Scholar). Despite this increase in FAO, FA uptake can exceed the ability of the heart to utilize FAs, leading to excess accumulation of TG in cardiomyocytes (6.Boudina S. Sena S. Theobald H. Sheng X. Wright J.J. Hu X.X. Aziz S. Johnson J.I. Bugger H. Zaha V.G. et al.Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins.Diabetes. 2007; 56: 2457-2466Crossref PubMed Scopus (464) Google Scholar, 7.Buchanan J. Mazumder P.K. Hu P. Chakrabarti G. Roberts M.W. Yun U.J. Cooksey R.C. Litwin S.E. Abel E.D. Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity.Endocrinology. 2005; 146: 5341-5349Crossref PubMed Scopus (419) Google Scholar, 8.Aasum E. Cooper M. Severson D.L. Larsen T.S. Effect of BM 17.0744, a PPARalpha ligand, on the metabolism of perfused hearts from control and diabetic mice.Can. J. Physiol. Pharmacol. 2005; 83: 183-190Crossref PubMed Scopus (34) Google Scholar, 9.Boudina S. Abel E.D. Mitochondrial uncoupling: a key contributor to reduced cardiac efficiency in diabetes.Physiology (Bethesda). 2006; 21: 250-258Crossref PubMed Scopus (145) Google Scholar). The compensatory changes that occur in response to excessive FA availability eventually become maladaptive, and the heart is locked in a pathological and inflexible metabolic state that contributes to cardiac dysfunction (6.Boudina S. Sena S. Theobald H. Sheng X. Wright J.J. Hu X.X. Aziz S. Johnson J.I. Bugger H. Zaha V.G. et al.Mitochondrial energetics in the heart in obesity-related diabetes: direct evidence for increased uncoupled respiration and activation of uncoupling proteins.Diabetes. 2007; 56: 2457-2466Crossref PubMed Scopus (464) Google Scholar, 7.Buchanan J. Mazumder P.K. Hu P. Chakrabarti G. Roberts M.W. Yun U.J. Cooksey R.C. Litwin S.E. Abel E.D. Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity.Endocrinology. 2005; 146: 5341-5349Crossref PubMed Scopus (419) Google Scholar, 10.Brindley D.N. Kok B.P. Kienesberger P.C. Lehner R. Dyck J.R. Shedding light on the enigma of myocardial lipotoxicity: the involvement of known and putative regulators of fatty acid storage and mobilization.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E897-E908Crossref PubMed Scopus (80) Google Scholar). As such, maintenance of metabolic flexibility is essential for normal cardiac function. Whereas many proteins have been identified to contribute to cardiac FA uptake and metabolism (1.Stanley W.C. Recchia F.A. Lopaschuk G.D. Myocardial substrate metabolism in the normal and failing heart.Physiol. Rev. 2005; 85: 1093-1129Crossref PubMed Scopus (1459) Google Scholar, 10.Brindley D.N. Kok B.P. Kienesberger P.C. Lehner R. Dyck J.R. Shedding light on the enigma of myocardial lipotoxicity: the involvement of known and putative regulators of fatty acid storage and mobilization.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E897-E908Crossref PubMed Scopus (80) Google Scholar), relatively little information is available about the role of lipin-1 in the heart. Lipin-1 is an important protein involved in regulating both TG synthesis and FAO in other organs (11.Reue K. Brindley D.N. Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism.J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Instead of being considered as mutually antagonistic processes, FAO and TG synthesis are now widely viewed as being companion pathways because augmenting cardiac TG synthesis increases FAO (12.Liu L. Shi X. Bharadwaj K.G. Ikeda S. Yamashita H. Yagyu H. Schaffer J.E. Yu Y.H. Goldberg I.J. DGAT1 expression increases heart triglyceride content but ameliorates lipotoxicity.J. Biol. Chem. 2009; 284: 36312-36323Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Moreover, FAs released through lipolysis of endogenous TG stores contribute significantly to FAO (3.Saddik M. Lopaschuk G.D. Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts.J. Biol. Chem. 1991; 266: 8162-8170Abstract Full Text PDF PubMed Google Scholar, 13.Banke N.H. Wende A.R. Leone T.C. O'Donnell J.M. Abel E.D. Kelly D.P. Lewandowski E.D. Preferential oxidation of triacylglyceride-derived fatty acids in heart is augmented by the nuclear receptor PPARalpha.Circ. Res. 2010; 107: 233-241Crossref PubMed Scopus (126) Google Scholar). Lipin-1 could play a prominent role in this regulation as it is a bifunctional protein involved in regulating both TG synthesis and FAO in the liver (11.Reue K. Brindley D.N. Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism.J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). First, lipin-1 functions as a phosphatidate phosphatase (PAP) through its catalytic DxDxT motif, which generates the diacylglycerol (DG) required for the synthesis of TG, phosphatidylethanolamine (PE), and phosphatidylcholine (PC) (14.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). There are three members in the lipin family, of which lipin-1 is the best characterized. All three lipins have PAP activity, but they are expressed in a tissue-specific manner (14.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Lipin-1 appears to be the predominant lipin in the heart because of the apparent absence of cardiac PAP activity in lipin-1-deficient mice (15.Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr, J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). These animals are known as fatty liver dystrophy (fld) mice because they develop transient fatty livers and hypertriglyceridemia, which resolve upon weaning (16.Reue K. Xu P. Wang X.P. Slavin B.G. Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene.J. Lipid Res. 2000; 41: 1067-1076Abstract Full Text Full Text PDF PubMed Google Scholar, 17.Péterfy M. Phan J. Xu P. Reue K. Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (472) Google Scholar, 18.Langner C.A. Birkenmeier E.H. Ben-Zeev O. Schotz M.C. Sweet H.O. Davisson M.T. Gordon J.I. The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities.J. Biol. Chem. 1989; 264: 7994-8003Abstract Full Text PDF PubMed Google Scholar). Fld mice are devoid of mature adipose tissue, which is attributed to the role of lipin-1 in inducing peroxisome proliferator activated receptor γ (PPARγ) expression during adipocyte differentiation (19.Phan J. Péterfy M. Reue K. Lipin expression preceding peroxisome proliferator-activated receptor-gamma is critical for adipogenesis in vivo and in vitro.J. Biol. Chem. 2004; 279: 29558-29564Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The fld mouse is also insulin resistant and is prone to developing atherosclerosis when fed a high cholesterol/cholate diet (16.Reue K. Xu P. Wang X.P. Slavin B.G. Adipose tissue deficiency, glucose intolerance, and increased atherosclerosis result from mutation in the mouse fatty liver dystrophy (fld) gene.J. Lipid Res. 2000; 41: 1067-1076Abstract Full Text Full Text PDF PubMed Google Scholar, 20.Gazit V. Weymann A. Hartman E. Finck B.N. Hruz P.W. Tzekov A. Rudnick D.A. Liver regeneration is impaired in lipodystrophic fatty liver dystrophy mice.Hepatology. 2010; 52: 2109-2117Crossref PubMed Scopus (49) Google Scholar). In addition, phosphatidate (PA) accumulation leads to demyelination in Schwann cells through aberrant ERK1/2 activation, which subsequently causes the development of peripheral neuropathy in fld mice (21.Langner C.A. Birkenmeier E.H. Roth K.A. Bronson R.T. Gordon J.I. Characterization of the peripheral neuropathy in neonatal and adult mice that are homozygous for the fatty liver dystrophy (fld) mutation.J. Biol. Chem. 1991; 266: 11955-11964Abstract Full Text PDF PubMed Google Scholar, 22.Nadra K. de Preux Charles A.S. Medard J.J. Hendriks W.T. Han G.S. Gres S. Carman G.M. Saulnier-Blache J.S. Verheijen M.H. Chrast R. Phosphatidic acid mediates demyelination in Lpin1 mutant mice.Genes Dev. 2008; 22: 1647-1661Crossref PubMed Scopus (105) Google Scholar). Lipin-1 also acts as a transcriptional coactivator with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and PPARα to upregulate the expression of proteins involved in FAO in liver (23.Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr, J.C. Kelly D.P. Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). Consequently, fld mice are defective in the fasting-induced hepatic expression of PPARα and its downstream targets (23.Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr, J.C. Kelly D.P. Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). We decided to determine whether lipin-1 has a similar function in the heart. Lipin-1 consists of full-length lipin-1B and the lipin-1A splice variant, which lacks a stretch of 20 amino acids (24.Péterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Lipin-1B is the predominant isoform in the heart, and it exhibits both PAP and transcriptional coactivator activities (23.Finck B.N. Gropler M.C. Chen Z. Leone T.C. Croce M.A. Harris T.E. Lawrence Jr, J.C. Kelly D.P. Lipin 1 is an inducible amplifier of the hepatic PGC-1alpha/PPARalpha regulatory pathway.Cell Metab. 2006; 4: 199-210Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 24.Péterfy M. Phan J. Reue K. Alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis.J. Biol. Chem. 2005; 280: 32883-32889Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Lipin-1 expression is dynamically regulated in the liver (25.Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.J. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26.Whiting P.H. Bowley M. Sturton R.G. Pritchard P.H. Brindley D.N. Hawthorne J.N. The effect of chronic diabetes, induced by streptozotocin, on the activities of some enzymes of glycerolipid synthesis in rat liver.Biochem. J. 1977; 168: 147-153Crossref PubMed Google Scholar), heart (27.Schoonderwoerd K. Broekhoven-Schokker S. Hülsmann W.C. Stam H. Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.Biochem. J. 1990; 268: 487-492Crossref PubMed Scopus (24) Google Scholar), and adipose tissue (28.Zhang P. O'Loughlin L. Brindley D.N. Reue K. Regulation of lipin-1 gene expression by glucocorticoids during adipogenesis.J. Lipid Res. 2008; 49: 1519-1528Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Lpin1 transcription and, thus, PAP activity are increased in the liver in fasting and diabetes due to the synergistic actions of glucagon (or epinephrine) through cAMP production and glucocorticoids (11.Reue K. Brindley D.N. Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism.J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Insulin antagonizes these actions in rat and mouse hepatocytes (25.Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.J. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Lpin1 gene expression is regulated in neonatal rat cardiomyocytes in a way similar to that in the liver (B. P. C. Kok and D. N. Brindley, unpublished results). Lpin2 and Lpin3 expression are not regulated significantly by glucocorticoids, cAMP, or insulin in hepatocytes (25.Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.J. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Increased lipin-1 expression is thought to provide a reservoir of PAP activity which, together with the FA-induced translocation of lipin-1 from the cytosol to the endoplasmic reticulum (ER) (15.Harris T.E. Huffman T.A. Chi A. Shabanowitz J. Hunt D.F. Kumar A. Lawrence Jr, J.C. Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1.J. Biol. Chem. 2007; 282: 277-286Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 29.Cascales C. Mangiapane E.H. Brindley D.N. Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes.Biochem. J. 1984; 219: 911-916Crossref PubMed Scopus (110) Google Scholar, 30.Swanton E.M. Saggerson E.D. Glycerolipid metabolizing enzymes in rat ventricle and in cardiac myocytes.Biochim. Biophys. Acta. 1997; 1346: 93-102Crossref PubMed Scopus (22) Google Scholar), enhances the capacity for FA sequestration into TG in the face of increased FA uptake (10.Brindley D.N. Kok B.P. Kienesberger P.C. Lehner R. Dyck J.R. Shedding light on the enigma of myocardial lipotoxicity: the involvement of known and putative regulators of fatty acid storage and mobilization.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E897-E908Crossref PubMed Scopus (80) Google Scholar, 11.Reue K. Brindley D.N. Thematic Review Series: Glycerolipids. Multiple roles for lipins/phosphatidate phosphatase enzymes in lipid metabolism.J. Lipid Res. 2008; 49: 2493-2503Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Furthermore, increased lipin-1 expression in fasting and diabetes could also promote FAO through its role as a transcriptional coactivator. In the fed state, lipin-1 expression is suppressed by insulin action (25.Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.J. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and this presumably helps to promote glucose utilization with a concurrent decline in FAO. Despite this, cardiac PAP activity has been shown to be decreased in insulin-resistant rat models as well as in Type 2 diabetic patients (31.Burgdorf C. Hansel L. Heidbreder M. Johren O. Schutte F. Schunkert H. Kurz T. Suppression of cardiac phosphatidate phosphohydrolase 1 activity and lipin mRNA expression in Zucker diabetic fatty rats and humans with type 2 diabetes mellitus.Biochem. Biophys. Res. Commun. 2009; 390: 165-170Crossref PubMed Scopus (23) Google Scholar, 32.Jamal Z. Martin A. Gómez-Muñoz A. Hales P. Chang E. Russell J.C. Brindley D.N. Phosphatidate phosphohydrolases in liver, heart and adipose tissue of the JCR:LA corpulent rat and the lean genotypes: implications for glycerolipid synthesis and signal transduction.Int. J. Obes. Relat. Metab. Disord. 1992; 16: 789-799PubMed Google Scholar). The importance of lipin-1 function in metabolism was highlighted by numerous studies demonstrating associations of lipin-1 polymorphisms with different metabolic disease states (33.Loos R.J. Rankinen T. Perusse L. Tremblay A. Despres J.P. Bouchard C. Association of lipin 1 gene polymorphisms with measures of energy and glucose metabolism.Obesity (Silver Spring). 2007; 15: 2723-2732Crossref PubMed Scopus (41) Google Scholar, 34.Wiedmann S. Fischer M. Koehler M. Neureuther K. Riegger G. Doering A. Schunkert H. Hengstenberg C. Baessler A. Genetic variants within the LPIN1 gene, encoding lipin, are influencing phenotypes of the metabolic syndrome in humans.Diabetes. 2008; 57: 209-217Crossref PubMed Scopus (62) Google Scholar, 35.Donkor J. Sparks L.M. Xie H. Smith S.R. Reue K. Adipose tissue lipin-1 expression is correlated with peroxisome proliferator-activated receptor alpha gene expression and insulin sensitivity in healthy young men.J. Clin. Endocrinol. Metab. 2008; 93: 233-239Crossref PubMed Scopus (57) Google Scholar). We, therefore, hypothesized that lipin-1 deficiency in the heart would severely inhibit TG synthesis and FAO and, thereby, cardiac function. We determined that there was cardiac dysfunction in the lipin-1-deficient mice in vivo. Surprisingly, lipin-1 deficiency did not produce major modifications in FA esterification or oxidation in isolated perfused working hearts. There was also no indication of impaired cardiac function in this ex vivo system, indicating that the dysfunction in vivo probably resulted from systemic influences on the heart due to global lipin-1 deficiency. However, there were differences in phosphatidate and phosphatidylinositol turnover and in cell signaling. We established a breeding colony of Balb/cByJ-Lpin1fld/J mice from the Jackson Laboratory, Bar Harbor, ME. We used two breeding triads, the first consisting of two female fld mice bred with one male heterozygous mouse, which produced only fld and heterozygous offspring. The other triad contained two female heterozygous mice and one male heterozygous mouse, resulting in fld, heterozygous, and wild-type offspring. The wild-type and heterozygous mice were both designated as the control mice. Mice were fed Lab Diet 5058 containing 9% fat by weight. Male mice were used in the experiments. The research was conducted in accordance with the policies of the Canadian Council on Animal Care, as approved by the University of Alberta Animal Policy and Welfare Committee. Transthoracic echocardiography was performed on mildly anesthetized (1.5% isoflurane and 95% O2) mice using a Vevo 770 Imaging System (VisualSonics, Toronto, ON) (36.Dolinsky V.W. Morton J.S. Oka T. Robillard-Frayne I. Bagdan M. Lopaschuk G.D. Des Rosiers C. Walsh K. Davidge S.T. Dyck J.R. Calorie restriction prevents hypertension and cardiac hypertrophy in the spontaneously hypertensive rat.Hypertension. 2010; 56: 412-421Crossref PubMed Scopus (96) Google Scholar). The Tei index was calculated as the sum of the isovolumic relaxation and contraction time divided by the ejection time (37.Tei C. Ling L.H. Hodge D.O. Bailey K.R. Oh J.K. Rodeheffer R.J. Tajik A.J. Seward J.B. New index of combined systolic and diastolic myocardial performance: a simple and reproducible measure of cardiac function--a study in normals and dilated cardiomyopathy.J. Cardiol. 1995; 26: 357-366PubMed Google Scholar). One week after echocardiographies, 11-week-old mice were fasted from 0900 to 1300 h (0600–1800 h light/dark cycle) before being euthanized by decapitation, after which cardiac tissues were collected for RT-PCR and Western blot analysis. Serum samples were also collected and assayed with the glucose-c kit (Wako Chemicals, Richmond, VA). Serum TG and unesterified FAs were measured using the TG GPO kit (Pointe Scientific, Canton, MI) and NEFA kit (Wako Chemicals), respectively. For serum lipid measurements, food was withheld from 19- to 23-week-old mice from 0900 to 1100 h. mRNA concentrations were measured by quantitative RT-PCR relative to that of TATA-binding protein (Tbp) (25.Manmontri B. Sariahmetoglu M. Donkor J. Khalil M.B. Sundaram M. Yao Z. Reue K. Lehner R. Brindley D.N. Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically.J. Lipid Res. 2008; 49: 1056-1067Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Similar results were obtained with hypoxanthine-guanine phosphoribosyltransferase and cyclophilin A as reference genes. Primer sequences are listed in supplementary Table I. SDS-PAGE and Western blots were performed using antibodies as described in supplementary Table II (38.Dyck J.R. Cheng J.F. Stanley W.C. Barr R. Chandler M.P. Brown S. Wallace D. Arrhenius T. Harmon C. Yang G. et al.Malonyl coenzyme a decarboxylase inhibition protects the ischemic heart by inhibiting fatty acid oxidation and stimulating glucose oxidation.Circ. Res. 2004; 94: e78-e84Crossref PubMed Google Scholar). The antibody against the C terminus of lipin-1 was a gift from Dr. Thurl Harris (University of Virginia, Charlottesville, VA). Quantitative densitometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD). Antibodies against lipin-2 were raised in rabbits using the peptide sequence N′-PKGELIQERTKGNK-C′ followed by affinity purification (Genscript, Piscataway, NJ). The lipin-2 antibody was verified by comparing endogenous lipin-2 in the heart to recombinant lipin-2 protein overexpressed in MCF-7 breast cancer cells (Fig. 1B, upper panel). PAP assays were performed at pH 6.5 and 7.4 using two methods for preparing the PA (14.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns.J. Biol. Chem. 2007; 282: 3450-3457Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The first method used a dispersion of PA with PC, which was designed to maximize the activity of PAP versus that of lipid phosphate phosphatase (LPP). Each sample was assayed in a total volume of 100 µl consisting of 100 mM Tris/maleate buffer (pH 6.5) or Tris/HCl buffer (pH 7.4) in addition to 0.6 mM dithiothreitol, 1.5 mM MgCl2 (pH 7.4) and 5 mM MgCl2 (pH 6.5), 2 mg/ml FA-poor BSA, protease inhibitor cocktail (Sigma-Aldrich), 30 nM microcystin-LR, 0.6 mM PA labeled with [3H]palmitate (approximately 6 × 104 dpm per assay), 1 mM EDTA/EGTA, 0.4 mM PC, and 200 µM tetrahydrolipstatin to inhibit the degradation of the DG product by lipase activity (39.Martin A. Gómez-Muñoz A. Jamal Z. Brindley D.N. Characterization and assay of phosphatidate phosphatase.Methods Enzymol. 1991; 197: 553-563Crossref PubMed Scopus (49) Google Scholar). In the second assay, 45 mM Triton X-100 was used to disperse 5 mM PA in micelles and PC was omitted. Each sample was assayed in a total volume of 100 µl consisting of 100 mM Tris/maleate (pH 6.5) or Tris/HCl (pH 7.4) in addition to 0.6 mM dithiothreitol, 1 or 6 mM MgCl2 (pH 7.4 and 6.5 respectively), protease inhibitor cocktail, 30 nM microcystin-LR, 1 mM PA labeled with [3H]palmitate (approximately 6 × 104 dpm per assay), 9 mM Triton X-100 (from the substrate preparation), and 200 µM tetrahydrolipstatin. Parallel measurements were performed in the absence of Mg2+ or in the presence of 8 mM N-ethylmaleimide to determine the contribution from LPP activity (14.Donkor J. Sariahmetoglu M. Dewald J. Brindley D.N. Reue K. Three mammalian lipins act as phosphatidate phos