Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Cardiac Expression of Microsomal Triglyceride Transfer Protein Is Increased in Obesity and Serves to Attenuate Cardiac Triglyceride Accumulation

  • Emil D. Bartels,

    Affiliation Department of Clinical Biochemistry, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark

  • Jan M. Nielsen,

    Affiliation Department of Cardiology, Aarhus University Hospital, Skejby, Denmark

  • Lars I. Hellgren,

    Affiliation Department of Systems Biology and Centre for Advanced Food Studies, Technical University of Denmark, Lyngby, Denmark

  • Thorkil Ploug,

    Affiliation Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

  • Lars B. Nielsen

    larsbo@rh.dk

    Affiliations Department of Clinical Biochemistry, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark, Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

Cardiac Expression of Microsomal Triglyceride Transfer Protein Is Increased in Obesity and Serves to Attenuate Cardiac Triglyceride Accumulation

  • Emil D. Bartels, 
  • Jan M. Nielsen, 
  • Lars I. Hellgren, 
  • Thorkil Ploug, 
  • Lars B. Nielsen
PLOS
x

Abstract

Obesity causes lipid accumulation in the heart and may lead to lipotoxic heart disease. Traditionally, the size of the cardiac triglyceride pool is thought to reflect the balance between uptake and β-oxidation of fatty acids. However, triglycerides can also be exported from cardiomyocytes via secretion of apolipoproteinB-containing (apoB) lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP); the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism remain unknown, we investigated how cardiac lipoprotein synthesis affects cardiac expression of triglyceride metabolism-controlling genes, insulin sensitivity, and function in obese mice. Heart-specific ablation of MTP-A in mice using Cre-loxP technology impaired upregulation of MTP expression in response to increased fatty acid availability during fasting and fat feeding. This resulted in cardiac triglyceride accumulation but unaffected cardiac insulin-stimulated glucose uptake. Long-term fat-feeding of male C57Bl/6 mice increased cardiac triglycerides, induced cardiac expression of triglyceride metabolism-controlling genes and attenuated heart function. Abolishing cardiac triglyceride accumulation in fat-fed mice by overexpression of an apoB transgene in the heart prevented the induction of triglyceride metabolism-controlling genes and improved heart function. The results suggest that in obesity, the physiological increase of cardiac MTP expression serves to attenuate cardiac triglyceride accumulation albeit without major effects on cardiac insulin sensitivity. Nevertheless, the data suggest that genetically increased lipoprotein secretion prevents development of obesity-induced lipotoxic heart disease.

Introduction

Obesity is associated with increased risk of cardiac failure and death [1], [2]. As a consequence of the increasing prevalence of obesity it is pertinent to improve understanding of the metabolic alterations causing cardiac dysfunction in overweight individuals. Obesity instigates intracellular accumulation of triglycerides in cardiac myocytes [3][5]. Lipid accumulation and altered metabolism of free fatty acids are associated with development of myocardial contractile dysfunction and may lead to cardiac myocyte apoptosis [5][8]. Although, the mechanisms involved in development of obesity-associated heart disease remain to be fully clarified, they appear to be independent from those leading to ischemia-induced heart dysfunction. Consequently, lipotoxic heart disease has been proposed as a distinct form of cardiomyopathy associated with obesity and type 2 diabetes [9][11].

Lipid accumulation in the heart results from an imbalance between uptake and utilization of free fatty acids. Under normal conditions, ∼70% of the energy production in the heart is derived from fatty acids [10]. Heart failure is accompanied by myocyte lipid accumulation, which has been explained by a switch in substrate utilization from fatty acids to glucose [12] leading to storage of the excess fatty acids as triglycerides. However, obesity is accompanied by both increased utilization of fatty acids for energy production and lipid accumulation both in humans and mice [4], [5], [13]. This apparent paradox may be explained by increased delivery of fatty acids to the heart in obesity [14], [15]. Increased plasma free fatty acids promote increased cardiac uptake reflecting the concentration gradient between plasma and the intracellular milieu in the cardiac myocytes [16], [17]. Moreover, obesity and cardiac lipid accumulation has been associated with increased heart expression of genes that stimulate local production (i.e. lipoprotein lipase (LPL)) and transport protein mediated uptake (i.e., fatty acid translocase (FAT/CD36), fatty acid transporter protein 1 (FATP1) and fatty acid transporter protein 4 (FATP4)) of free fatty acids [5]. The regulatory role of cardiac free fatty acid uptake in lipotoxic heart disease is underscored by seminal studies showing that overexpression of LPL and FATP1 causes cardiac triglyceride accumulation and cardiac dysfunction in lean mice [18], [19]. In the obese heart, the increased availability of free fatty acids is also associated with upregulation of genes involved with fatty acid metabolization and increased utilization of fat for energy production [8].

Divergence of the net metabolic flux of fatty acids from the oxidative pathway leads to myocardial triglyceride accumulation and development of lipotoxic heart disease in mouse models that overexpress either PPARα or long-chain acyl-CoA synthetase (ACSL) [20], [21], or are long-chain acyl-CoA dehydrogenase-deficient (LCAD) [22]. The obese rat heart displays increased expression of uncoupling protein 3 (UCP3) shunting free fatty acids out of the mitochondria and increasing cytosolic fatty acid levels [23]. The excess availability of fatty acids that are not used for energy production leads to accumulation of intracellular triglycerides. The turnover time of triglycerides in the heart is extremely rapid (∼5 hours) compared to adipose tissue (200–270 days) [10], [24], [25]. This implies that cardiac triglyceride accumulation is not inert but rather is associated with markedly increased intracellular fluxes of free fatty acids.

The human heart expresses the two genes that are mandatory for formation of apoB-containing lipoproteins, i.e. the microsomal triglyceride transfer protein (MTP) gene and the apoB gene [26]. MTP is an intracellular protein located in the endoplasmatic reticulum, which transfers neutral lipids onto the apoB polypeptide and participates in the subsequent lipidation of the nascent lipoproteins [27]. Two recent reports independently demonstrated that there are two isoforms of the MTP gene in the mouse MTP-A and MTP-B (also named MTP and MTPv1) [28], [29]. The MTP-B variant has a unique exon 1 located 2.7 kb 5′ to the canonical exon 1 resulting in translation of a different signal peptide and addition of three amino acids at the NH2-terminus of the mature MTP protein [28], [29]. The physiological role of apoB is to package and secrete lipids in the form of lipoproteins. Indeed, cardiac myocytes secrete apoB-containing lipoproteins [30] and cardiac triglyceride accumulation is attenuated in apoB-overexpressing transgenic mice with streptozotocin-induced type 1 diabetes [31], lipoprotein lipase overexpressing mice [32], and LCAD-deficient mice [33]. These findings indicate that export of triglycerides in the form of lipoproteins can reduce pathological triglyceride accumulation. Therefore, export of triglycerides in the form of lipoproteins may represent a novel pathway in cardiac lipid metabolism that deserves further exploration.

We have utilized mice overexpressing a full length human apoB transgene and mice lacking cardiac expression of MTP-A to explore the role of cardiac lipoprotein formation on lipid metabolism and heart function in mice with diet-induced obesity. The data suggest that local lipoprotein secretion is integrated with cardiac lipid metabolism and protects against obesity-induced lipotoxic heart disease.

Results

Induction of MTP-B in MTP-A-deficient mouse hearts

Ob/ob mice have increased cardiac triglyceride stores, which are associated with increased total MTP mRNA expression and MTP activity in the myocardium [5]. This may reflect that increased lipoprotein formation is a compensatory mechanism dampening cardiac lipid accumulation in the setting of excess supplies of fatty acids.

Real-time PCR studies with MTP-A and MTP-B specific assays showed that the expression of both isoforms is increased in mice with diet-induced obesity after fat-feeding (60% energy from fat) for ∼1 yr (Fig. S1). To explore the importance of the two MTP isoforms in the heart of obese mice, we bred mice with loxP sites flanking exon 1 of the MTP-A gene (Mttpflox/flox) [34] with transgenic mice that express Cre-recombinase in the heart and skeletal muscle, but not in liver (Mck-Cre+/o mice) (Fig. 1A). Predictably, deletion of exon-1 of MTP-A should not abolish MTP-B expression since exon 1 of the MTP-B isoform is located ∼2.7 kb upstream of exon 1 of MTP-A. Chow-fed (12% energy from fat) Mttpflox/floxMck-Cre+/o mice had a >95% reduction of MTP-A mRNA in the heart and unchanged MTP-A mRNA expression in the liver as compared with Mttpflox/flox mice indicating that ablation of MTP-A in cardiac myocytes was highly effective and tissue specific (Fig. 1B). Nevertheless, the cardiac total MTP (MTP-A+MTP-B) mRNA expression was increased 1.4 fold in Mttpflox/floxMck-Cre+/o mice compared with Mttpflox/flox littermates (Fig. 1C). This increase was attributable to a ∼3.6 fold elevation (P = 0.0006) of the MTP-B mRNA expression (Fig. 1D) and may reflect increased transcription of MTP-B from the Cre-loxP-modified MTP gene locus.

thumbnail
Figure 1. Effect of heart-specific MTP-A-deficiency in chow-fed mice.

Mttpflox/flox mice were bred with Mck-Cre+/o mice to generate mice with heart-specific MTP-A-deficiency. A) Cre mRNA expression in liver (n = 4) and heart (n = 4) of Mttpflox/floxMck-Cre+/o mice, B) MTP-A mRNA expression in the liver (n = 4 in each group) and heart (n = 8 in each group) of Mttpflox/flox and Mttpflox/floxMck-Cre+/o mice, C) Total MTP mRNA expression in the heart of Mttpflox/flox (n = 8) and Mttpflox/floxMck-Cre+/o mice (n = 8), D) Cardiac MTP-B mRNA expression in Mttpflox/flox (n = 8) and Mttpflox/floxMck-Cre+/o mice (n = 8), E) Cardiac MTP activity Mttpflox/flox (n = 6) and Mttpflox/floxMck-Cre+/o mice (n = 6), F) Cardiac triglycerides in Mttpflox/flox (n = 8) and Mttpflox/floxMck-Cre+/o mice (n = 8). Open bars: Mttpflox/flox mice and closed bars: Mttpflox/floxMck-Cre+/o mice. Values are mean±SEM. The p values for two-group comparisons are: ** P<0.01; *** P<0.005 compared to chow-fed controls.

https://doi.org/10.1371/journal.pone.0005300.g001

The ability of the MTP-B isoform to mediate triglyceride transfer was evaluated by incubating cardiac microsomal protein fractions from Mttpflox/floxMck-Cre+/o mice with 14C-trioleate-labeled lipid vesicles. Cardiac MTP activity was similar in Mttpflox/floxMck-Cre+/o mice and with Mttpflox/flox littermates (Fig. 1E). Moreover, cardiac triglyceride and cholesterol concentrations were similar in chow-fed, non-fasted Mttpflox/floxMck-Cre+/o mice and Mttpflox/flox littermates (Fig. 1F and data not shown). These results indicate that upregulation of MTP-B can compensate for the loss of MTP-A in the heart of lean chow-fed mice.

Effect of MTP-A deficiency on lipid accumulation and expression of lipid metabolizing genes in obese mouse heart

Previous studies suggest that mice with heart-specific MTP-A deficiency accumulate excess cardiac triglycerides after fasting [33]. Fasting increases the delivery of free fatty acids to the heart and causes cardiac triglyceride accumulation [16]. Thus, it is conceivable that the effect of MTP-A deficiency only becomes evident in the setting of surplus supplies of fatty acids and as such could be important in obese mice. We examined this possibility by comparing hearts from Mttpflox/floxMck-Cre+/o mice and Mttpflox/flox littermates that had been fed a chow diet and fasted for 18 hours or had been fed a fat-enriched diet for 3 months. After fasting or fat-feeding the cardiac triglyceride content was higher in Mttpflox/floxMck-Cre+/o mice compared with Mttpflox/flox littermates (P<0.05) (Fig. 2A). Notably, after fasting the cardiac MTP activity was increased in Mttpflox/flox mice (P = 0.009) but not in Mttpflox/floxMck-Cre+/o mice (Fig. 2B). Moreover, after fat feeding MTP-A and MTP-B expression increased in Mttpflox/flox control mice (Fig. 2C) whereas MTP-B expression was unaffected in Mttpflox/floxMck-Cre+/o mice (Fig. 2D). The present data thus imply that the excess cardiac triglyceride accumulation in Mttpflox/floxMck-Cre+/o mice occurs due to defect transcriptional activation of the ablated MTP gene locus after fasting- or fat-feeding. Moreover, the results thus support the notion that a compensatory increase in cardiac MTP expression protects against cardiac triglyceride accumulation in the obese mouse heart.

thumbnail
Figure 2. Effect of heart-specific MTP-A deficiency on cardiac lipid accumulation and MTP activity in fasted mice and fat-fed obese mice.

A) cardiac triglycerides in mice that were fasted for 18 hours and mice that were fat-fed for three months (n = 7–9 in each group). *P<0.05 compared to Mttpflox/flox mice. B) MTP activity in fasted versus chow-fed mice (n = 6 in each group). **P<0.01 compared to fed mice, ‡ P<0.01 fasted Mttpflox/flox compared to fasted Mttpflox/floxMck-Cre+/o mice A+B) Open bars: Mttpflox/flox mice and closed bars: Mttpflox/floxMck-Cre+/o mice. C) MTP-A and MTP-B mRNA expression in fat-fed (closed bars) versus chow-fed (open bars) Mttpflox/flox mice (n = 7 in each group). **P<0.01 and ***P<0.005 compared to chow-fed Mttpflox/flox mice. D) MTP-A and MTP-B mRNA expression in fat-fed (closed bars) versus chow-fed (open bars) Mttpflox/floxMck-Cre+/o mice (n = 7 in each group). Values are mean±SEM.

https://doi.org/10.1371/journal.pone.0005300.g002

Diet-induced obesity has been associated with impaired insulin-stimulated glucose uptake in skeletal muscle [35] and altered lipid metabolism in cardiac muscle [20]. There was no excess triglyceride accumulation in the hearts of Mttpflox/flox control mice after 3 months of fat-feeding (compare Fig. 1F and Fig 2A) which is in accord with previous findings by Somoza et al [13]. Nevertheless, insulin-stimulated glucose uptake was markedly reduced in the heart (as well as in skeletal muscle and adipose tissue) when insulin was injected intravenously together with 2-deoxy-[3H]glucose 25 minutes prior to removal of the mouse heart (Fig. 3A–C). The decrease in cardiac insulin-sensitivity occurred without changes in cardiac glucose transporter-4 (GLUT4) and (PFK) mRNA expression (involved in uptake and oxidation of glucose) (Fig 3D). Fat-feeding of the Mttpflox/flox control mice for 3 months also caused significant increases in the expression of several lipid metabolizing genes which mediate free fatty acid uptake, i.e. FAT/CD36, FATP1, FATP4, intracellular transport, i.e. heart-fatty acid binding protein (H-FABP), removal of the CoA group from acyl-CoA in the cytosol, i.e. cytosolic acyl-CoA thioesterase 1 (CTE1), mitochondrial uptake of fatty acids, i.e. carnitine palmitoyltransferase 1b (CPT1b), and shunting of fatty acids out the mitochondria, i.e. UCP3 (Fig. 3D). This suggests that diet-induced obesity causes alterations in cardiac glucose and lipid metabolism that precedes lipid accumulation in myocytes.

thumbnail
Figure 3. Effect of heart-specific MTP-A deficiency on insulin-stimulated glucose uptake and expression of lipid metabolizing genes in obese mouse heart.

The effect of heart-specific MTP-A deletion on A) insulin-stimulated glucose uptake in cardiac ventricles, B) skeletal muscle, and C) epididymal fat was determined after 3 months of fat-feeding. D) Cardiac mRNA expression was quantified with real-time PCR in fat-fed male Mttpflox/flox mice, Mttpflox/floxMck-Cre+/o mice and their lean controls. Values are after 3 months of diet. Open bars: chow-fed Mttpflox/flox mice (n = 7–8), closed bars: fat-fed Mttpflox/flox mice (n = 7–8), hatched bars: chow-fed Mttpflox/floxMck-Cre+/o mice (n = 7–8), squared bars: fat-fed Mttpflox/floxMck-Cre+/o mice (n = 7). Values are mean±SEM. The p values for two-group comparisons are: * P<0.05, ** P<0.01; *** P<0.005 compared to chow-fed controls; ‡ P<0.01 compared to fat-fed Mttpflox/floxMck-Cre+/o mice.

https://doi.org/10.1371/journal.pone.0005300.g003

There was no difference in cardiac insulin-stimulated glucose uptake between Mttpflox/floxMck-Cre+/o and Mttpflox/flox control mice (Fig 3A). However, the mRNA expression of diacylglycerol acyltransferase (DGAT) and PPARα was reduced in fat-fed Mttpflox/floxMck-Cre+/o mice compared with the fat-fed Mttpflox/flox mice (Fig. 3D), supporting the notion that rates of lipoprotein secretion may modulate cardiac triglyceride homeostasis in the setting of diet-induced obesity.

Cardiac triglyceride accumulation in fat-fed obese mice is prevented by overexpression of human apoB in the heart

Prolonged excess caloric intake increases cardiac triglyceride stores in mice [5]. We fed the fat-enriched diet to human apoB-transgenic male mice and litter-mate C57Bl/6 controls for ∼1 year to explore the impact of increased rates of cardiac lipoprotein formation in the setting of obesity and increased cardiac triglyceride stores. Fat-feeding induced equal increases in body weight, plasma insulin, leptin, and glucose in male C57Bl/6 mice and their apoB-transgenic littermates (Fig. S2A–F). Fasting plasma concentrations of free fatty acids after 9 months of fat-feeding were increased in to a similar extent in fat-fed C57Bl/6 mice and human apoB-transgenic littermates (0.80±0.03 mmol/L and 0.87±0.05 mmol/L) compared with chow-fed control mice (0.66±0.07 mmol/L and 0.34±0.01 mmol/L) (P<0.0001). After 12 months of fat-feeding, the heart triglyceride content was 110% increased in obese C57Bl/6 mice compared with lean C57Bl/6 mice (Fig. 4A). The obesity-induced increase in cardiac triglycerides was abolished in human apoB-transgenic mice (Fig. 4A). Neither cardiac cholesterol (data not shown) nor ceramide levels were affected by fat-feeding or overexpression of the human apoB transgene. The cardiac ceramide concentration was 20±1 nmol/mg wet weight (ww) in fat-fed versus 18±1 nmol/mg ww in chow-fed C57Bl/6 mice (P = 0.15), and 21±2 nmol/mg ww in fat-fed versus 23±2 nmol/mg ww in chow-fed apoB-transgenic mice (P = 0.61). These results suggest that increased lipoprotein formation in cardiac myocytes prevents triglyceride accumulation without affecting cholesterol or ceramide stores in hearts of long-term fat-fed obese mice.

thumbnail
Figure 4. Overexpression of human apoB in the heart affects cardiac triglyceride levels as well as expression of genes involved in cardiac stress and metabolism of free fatty acids in fat-fed C57Bl/6 mice.

The effect of cardiac overexpression of a human apoB-transgene in obese mice on A) cardiac triglycerides were determined using TLC, B) cardiac mRNA expression of lipid metabolising genes and C) cardiac mRNA expression of stress-related genes was quantified with real-time PCR in male fat-fed C57Bl/6 (n = 11) and C57Bl/6-apoB-Tg mice (n = 9) and their lean controls (n = 6). Values are after 12 months of diet. Open bars: chow-fed C57Bl/6 mice, closed bars: fat-fed C57Bl/6 mice, hatched bars: chow-fed C57Bl/6-apoB-Tg mice, squared bars: fat-fed C57Bl/6-apoB-Tg mice. Values are mean±SEM. The p values for two-group comparisons are: * P<0.05, ** P<0.01; *** P<0.005 compared to chow-fed controls; ‡ P<0.05 Fat-fed C57Bl/6 compared to fat-fed C57Bl/6-apoB-Tg.

https://doi.org/10.1371/journal.pone.0005300.g004

Overexpression of human apoB in the heart affects expression of genes involved in cardiac metabolism of free fatty acids

To judge whether increased lipoprotein formation affect metabolism of free fatty acids in hearts with excess lipid accumulation, we quantified the expression of selected genes controlling key steps in cardiac fatty acid metabolism. The mRNA levels of FAT/CD36, FATP1 and FATP4, CTE1, ACSL1 which couples the CoA group to fatty acids in the cytosol, CPT1b and LCAD which is involved in β-oxidation of fatty acids, were all increased in hearts from 1-yr fat-fed C57Bl/6 mice compared to chow-fed mice respectively (Fig. 4B). Notably, the magnitudes of the changes in gene expression were much more pronounced after fat-feeding for 1 year than after 3 months (compare Fig 3D and 4B). In contrast to the findings in C57Bl/6 mice, the cardiac expression of genes associated with fatty acid metabolizing showed less or no increase in fat-fed human apoB-transgenic mice (Fig. 4B). Thus, the expression levels of FAT/CD36, FATP1, FATP4, CTE1, and ACLS1 were significantly lower in hearts from fat-fed human apoB-transgenic than in the fat-fed C57Bl/6 littermates (Fig. 4B). These results suggest that increased lipoprotein formation attenuates changes in gene expression that increase uptake and metabolization of free fatty acids in the hearts of long-term fat-fed mice.

In accordance with previous results in obese ob/ob mice [5] and fa/fa Zucker rats [36] GLUT4 as well as PFK displayed increased mRNA expression in the fat-fed C57Bl/6 mouse hearts. In hearts from fat-fed versus chow-fed human apoB-transgenic mice we did not identify this difference in GLUT4 and PFK mRNA expression (Fig. 4B). This suggests that normalization of cardiac lipid stores not only affects lipid metabolism but also prevents changes in expression of essential genes governing glucose metabolism.

Overexpression of human apoB in the heart attenuates deterioration of cardiac function in fat-fed obese mice

Recent data demonstrated that fat-feeding of C57Bl/6 male mice results in deterioration of heart function [35]. This may result from cardiac lipid accumulation and increased uptake and metabolization of free fatty acids [37]. We therefore hypothesized that mitigated cardiac triglyceride accumulation and dampened uptake and metabolization of fatty acids in human apoB transgenic mice might alleviate adverse effects of fat-feeding on heart function. To test this idea, we examined in vivo heart function in human apoB-transgenic male mice and littermate C57Bl/6 controls that had been fat- or chow-fed for 11 months. Ejection fraction (EF) and the load-independent index, preload recruitable stroke work (PRSW) were decreased in fat-fed C57Bl/6 mice compared with chow-fed C57Bl/6 mice (Fig. 5A and 5B). In addition, the heart rate was reduced in the fat-fed versus chow-fed C57Bl/6 mice (Supplementary Table S1). This was in contrast to human apoB transgenic mice where neither of these variables was significantly altered in fat-fed compared to chow-fed mice (Fig. 5A and 5B and Supplementary Table S1). We did not observe any effects of fat-feeding or apoB overexpression on electrocardiographic recordings (data not shown). The results are compatible with the idea that increased lipoprotein formation in cardiac myocytes attenuates development of cardiac dysfunction in fat-fed obese mice.

thumbnail
Figure 5. Overexpression of human apoB in the heart attenuates deterioration of cardiac function in fat-fed obese mice.

Heart function was determined with a conductance catheter placed in the left cardiac ventricle A) EF, ejection fraction, B) PRSW, preload recruitable stroke work. Open bars: chow-fed C57Bl/6 mice (n = 7), closed bars: fat-fed C57Bl/6 mice (n = 7), hatched bars: chow-fed C57Bl/6-apoB-Tg mice (n = 11), squared bars: fat-fed C57Bl/6-apoB-Tg mice (n = 7). Values are mean±SEM after 11 months on the diets. The p values for two-group comparisons are: * P<0.05, ** P≤0.01.

https://doi.org/10.1371/journal.pone.0005300.g005

Obesity and type 2 diabetes increase intracellular stress promoting apoptosis and insulin resistance [38], [39]. Increased expression of uncoupling protein 2 (UCP2) and UCP3 (directing fatty acids fluxes away from oxidative pathways) and of the pro-apoptotic growth arrest and DNA-damage-inducible gene 34 (GADD34) mRNA are markers of myocardial stress. For all three genes, the expression was increased in the heart of long-term fat-fed versus chow-fed C57Bl/6 mice, but was less or unaffected in fat-fed versus chow-fed human apoB-transgenic mice (Fig. 4C). GADD34 expression can be induced by a number of pathways including stress in the endoplasmatic reticulum [40]. However, cardiac mRNA expression of two markers of ER stress (immunoglobulin heavy chain-binding protein (BiP) and C/EBP-homologous protein (CHOP)) was not affected by fat feeding (data not shown).

Discussion

The present data suggest that cardiac secretion of apoB-containing lipoproteins plays an integrated role in cardiac fatty acid metabolism and affects myocardial function in obese mice. Failure to upregulate MTP activity in fasted and short-term fat-fed mice with targeted MTP-A expression increased cardiac triglyceride stores. Overexpression of apoB attenuated triglyceride accumulation in the heart of long-term fat-fed obese mice. Thus, genes controlling lipoprotein synthesis, in addition to genes controlling rates of uptake and utilization of fatty acids in energy production, are significant determinants of cardiac triglyceride homeostasis in the obese heart.

Expression of MTP is mandatory for lipoprotein formation [41]. Both the canonical MTP-A isoform and the recently discovered MTP-B isoform are expressed in the murine heart. The expression of both MTP isoforms is increased in the heart of fat-fed obese mice suggesting that MTP is integrated in cardiac lipid metabolism. Complete MTP deficiency is embryonically lethal in mice [42]. We used mice with heart-specific deficiency of the MTP-A gene to study the role of MTP in the heart. Based on the comparison of the expression of total MTP and MTP-B mRNA in wild-type and MTP-A deficient mice, it can be estimated that ∼35% of the total amount of MTP mRNA represents the MTP-B transcript and ∼65% represents the MTP-A transcript in the mouse heart. The MTP-B isoform likely is functionally active in cardiac lipoprotein formation since the MTP-A deficient mouse hearts expressed the same MTP activity as control hearts and did not accumulate triglycerides when the mice were on a normal chow. Notably, direct evidence of MTP-B mediated lipoprotein formation in the heart was not provided by the present studies. Nevertheless, it is interesting that mice with heart-specific MTP-A deficiency, in contrast to wild type mice, could not upregulate MTP expression and accumulated excess triglycerides in the heart in response to fasting or 3 months of fat-feeding. This suggests that upregulation of cardiac MTP expression serves to protect against myocardial lipid accumulation when the supply of fatty acids exceeds the need for energy production. A promoter polymorphism in the MTP gene is associated with decreased MTP gene expression and excess cardiovascular mortality in patients with ischemic heart disease [43]. The present data thus warrants further studies to determine whether MTP promoter polymorphisms could also affect the risk of obesity-induced heart disease.

Triglyceride accumulation in skeletal muscle is associated with insulin resistance [44]. Recent studies, however, suggest that increased muscle uptake and metabolism of free fatty acids rather than fat accumulation in itself causes the insulin resistance in obesity [45], [46]. The present findings in the mouse heart are in accordance with this idea. Thus, the heart was insulin resistant in obese mice after 12 weeks of fat feeding. At this time point there was increased cardiac expression of lipid metabolizing genes but no excess cardiac triglyceride accumulation (cardiac triglycerides in Mttpflox/flox control mice were actually lower in the fat-fed group than in the chow-fed group, as seen in Fig. 1F and Fig. 2A). Moreover, cardiac insulin sensitivity was unaffected by the increased cardiac triglyceride stores in Mttpflox/floxMck-Cre+/o mice.

Several studies demonstrates that obesity-induced changes in cardiac energy metabolism involve increased uptake and utilization of free fatty acids that ultimatively lead to cardiac lipid accumulation [5], [47], [48]. Altered fatty acid metabolism leads to decreased metabolic flexibility which is detrimental to the function of the myocardium, e.g. due to increased formation of reactive oxygen species [49][51]. Interestingly, overexpression of apoB in the heart of long-term-fat-fed obese mice not only prevented cardiac triglyceride accumulation but also reduced the effect of fat-feeding and obesity on genes controlling fatty acid metabolism in the heart. The reduction of cardiac triglycerides in the fat-fed apoB transgenic mice was associated with partial normalization of the expression of genes that are key regulators of cardiac fatty acid uptake, cytosolic release of free fatty acids from acyl-CoA, and uncoupling of fatty acid β-oxidation in mitochondria. Increased expression of FAT/CD36, FATP1, UCP2 and UCP3 have all been implicated in development of lipotoxic heart disease [20], [52], [53]. Thus, the results are compatible with the idea that normalization of cardiac lipids via increased export in lipoproteins leads to decreased uptake and metabolism of fatty acids and confers at least partial resistance to the detrimental effect of obesity on myocardial function. Notably, normalization of triglyceride store also reduced the expression of genes involved with glucose metabolism and the GADD34 marker of cardiac stress. Thus, the data suggest that reduction of cardiac triglyceride stores have effects on cardiac energy utilization that exceeds lipid metabolism and, as such, is a key controlling factor in precipitation of lipotoxic heart disease in obesity. It should be noted, however, that this conclusion is based on mRNA expression data and further studies are needed to explore the impact of lipoprotein formation on cardiac energy metabolism in vivo.

In conclusion, the present results support that cardiac lipoprotein synthesis and secretion is important for controlling the triglyceride storage in the heart of mice when fatty acid supplies are increased, such as in obesity. In addition, the export of triglycerides in lipoproteins appears to reduce the effect of obesity on the otherwise perturbed expression profile of free fatty acid metabolizing genes and protect against adverse effects of obesity on myocardial function.

Materials and Methods

Mice

Mice with heart-specific MTP-A deficiency were obtained by breeding MTP loxp mice (stock no: 003902, Jackson Laboratories, USA) with Mck-Cre mice [54] to obtain Mttpflox/floxMck-Cre+/o mice and Mttpflox/flox littermate controls. For the study of cardiac MTP-activity and response to fasting the mice were kept on the standard chow diet. For the study of obesity-associated effects on the heart, 7-week-old mice were randomly assigned to either the high-fat diet or the standard chow diet for 12 weeks. At the end of the fat-feeding period the body weight was 43.4±1.7 g (n = 7) in fat-fed Mttpflox/flox, 45.5±1.8 g (n = 8) in Mttpflox/floxMck-Cre+/o, 31.2±0.6 g (n = 8) in chow-fed Mttpflox/flox, and 30.6±1.0 g (n = 8) in chow fed Mttpflox/floxMck-Cre+/o mice. Seven-week-old male C57Bl/6 and human apoB-transgenic mice (B6.SJL-Tg(APOB)1102Sgy-mice backcrossed to the C57Bl/6 background for >20 generations) littermates were randomized to a high-fat diet with 60% fat (D12492, Research Diets) or a standard chow diet with 12% fat (Altromin 1314, Brogaarden, Denmark). Heart function was studied after 45 weeks on the diet whereas cardiac gene expression and lipid accumulation was studied in separate mice after 52 weeks on the diet. The mice fed for 52 weeks were also used as a part of another study of atherosclerosis [55]. The animals were housed under temperature-controlled conditions with free access to food and water. The studies were approved by the Danish Animal Experiments Inspectorate (Dyreforsoegstilsynet).

Blood and tissue samples

Blood were drawn in pre-cooled tubes containing Na2-EDTA and centrifuged at 4000·g for 10 minutes at 4°C. The hearts were carefully cleaned from pericardial fat and atrial tissue before snap frozen in liquid nitrogen. Tissue and plasma samples were stored at −80°C until use.

Plasma biochemistry

Plasma glucose in tail blood was determined with a Medisense PCx glucose meter (Abbott Laboratories A/S, Gentofte, Denmark). Plasma free fatty acids concentrations were determined with an enzymatic kit (Wako NEFA C kit, TriChem Aps, Frederikssund, Denmark). Sandwich elisa assays were used to measure plasma insulin and leptin (catalogue no. EIA-3440, DRG, Germany, and catalogue no. RD291001200R, BioVendor, Heidelberg, Germany, respectively).

Cardiac triglycerides

Lipids were extracted, re-dissolved in toluene, and separated with TLC prior to quantification as previously described [56].

Cardiac ceramides

Cardiac ceramides were purified from lipid extracts after addition of C17-ceramide (Avanti Polar Lipids, Alabaster, AL, US) as internal standard. Ceramides were extracted by solid phase extraction (Strata NH2, Part Number: 8B-S009-HBJ), hydrolyzed to free sphingosine which was derivatized with O-phtalaldehyde (Sigma, Brøndby, Denmark) before separated on a C18 HPLC column (Gemini 5 µm C18 110 Å Column 250×3.0 mm) (Phenomenex, Allerød, Denmark) and detected as fluorescence at 450 nm (excitation at 340 nm).

Cardiac gene expression

Gene expression was measured with real-time PCR. mRNA was extracted using the Trizol reagent (Invitrogen, Taastrup, Denmark), quantified spectrophotometricly, and the quality was assessed with capillary electrophoresis (2100 bioanalyzer, Agilent Technologies). cDNA was made with M-MULV (Roche) and real-time PCR analyses were performed with SYBR-Green and the Light Cycler instrument (Roche). Primer sequences for mouse ACSL1, BiP, CHOP, CTE1, Cre, GADD34, MTP-A, MTP-B, MTP-A+MTP-B, UCP2, and UCP3 are shown in Supplementary Table S2. Sequences for human apoB, mouse apoB, CPT1b, DGAT, FAT/CD36, FATP1, FATP4, GLUT4, H-FABP, hypoxanthine-guanine phosphoribosyltransferase (HPRT), LCAD, LPL, PFK, PPARγ-coactivator-1α (PGC1α), and PPARα, have been published previously [5], [31], [57], [58]. The expression of the reference gene HPRT was used to normalize the expression of target genes.

In vivo evaluation of heart function

The left ventricular function was evaluated by analysis of the pressure-volume loops acquired by conductance catheter technique [59]. The animals were intubated and mechanically ventilated with 2.2% isoflurane in 50% O2 and 50% room air at 100 breaths/min and a tidal volume of 10 µl/g. Temperature was maintained at 37°C by a rectal temperature-controlled heating pad and a heating lamp. An intravenous line was established through the left jugular vein and used for continuous infusion of isotonic NaCl at an infusion rate of 0.05 ml·g−1·h−1 throughout the protocol. The infusion of NaCl was discontinued for 1–2 min, and the ventilator was stopped at end expiration for 2–3 s before every measurement. The chest was entered via an anterior thoracotomy, and a 1.4-F four-electrode conductance catheter for mice (SPR-839; Millar Instruments, Houston, TX) was inserted into the left ventricle through the apex and positioned along the cardiac longitudinal axis. Pressure-volume loops were acquired with a signal-conditioning box (MPCU-200; Millar Instruments) using a 20-kHz excitation frequency and sampling rate of 1,000 Hz. The parallel conductance was estimated by intra venous injection of a 5 µL bolus of 30% hypertonic saline over approximately 0.5 s. The volume signal was calibrated by measuring the specific conductance of blood from each mouse by the cylinder method as previously described [60]. Maximal and minimal LV volumes and pressures during the cardiac cycle were used as end diastolic volume (EDV) and end systolic volume (ESV) and as end systolic pressure (ESP) and end diastolic pressure (EDP) respectively.

MTP activity

MTP activity was assessed as previously described [5], [61]. Briefly, tissue biopsies of ∼50 mg were homogenised and the microsomal protein fraction was isolated by ultracentrifugation. Triglyceride transfer activity in the microsomal protein fraction was measured as transfer of 14C-trioleate-labeled lipid from donor vesicles to acceptor vesicle that contained only unlabeled triglycerides.

Insulin stimulated glucose uptake in vivo

Fed mice were anesthetized with a mixture of fentanyl:droperidol:midazolam (0.02∶1.38∶0.14 mg/ml, 0.2 ml/10 g body weight), and a mixture of 0.005 IU insulin, 1.6 µCi of 2-deoxy-[3H]glucose and 1.0 µCi of [14C]sucrose/10 g body weight in saline with 0.1% BSA was administered in the a vein. [14C]Sucrose was used to calculate extracellular space. After 25 minutes blood was drawn in pre-cooled Na2EDTA tubes and heart, soleus muscle and the epididymal fat pad were isolated and rinsed carefully in ice-cold isotonic saline and snap-frozen in liquid nitrogen. Uptake of 2-deoxy-[3H]glucose in heart, soleus muscle, and epididymal fat was detected in perchloric acid extracts after corrected for label in the extracellular space as determined by the [14C] counts for sucrose.

Statistics

Two-group comparison was performed with Student's t-test or non-parametric Mann-Whitney test when appropriate. P<0.05 was considered statistically significant. The effect of MTP-genotype on cardiac triglycerides was calculated with two-way ANOVA.

Supporting Information

Figure S1.

MTP-A and MTP-B expression in the obese mouse heart. Cardiac mRNA expression was quantified with real-time PCR in male fat-fed C57Bl/6 (n = 11) and and their lean controls (n = 6). Values are after 12 months of diet. Open bars: chow-fed C57Bl/6 mice, closed bars: fat-fed C57Bl/6 mice. Values are mean±SEM. The p values for two-group comparisons are: * P<0.05, *** P<0.005 compared to chow-fed controls.

https://doi.org/10.1371/journal.pone.0005300.s001

(0.13 MB TIF)

Figure S2.

Effect of prolonged fat-feeding on basic metabolic parameters in male C57Bl/6 and C57Bl/6-apoB-Tg mice. The effect of fat-feeding on A) bodyweight in C57Bl/6 mice, B) bodyweight in C57Bl/6-apoB-Tg mice, C) plasma glucose in C57Bl/6 and C57Bl/6-apoB-Tg mice, D) plasma insulin in C57Bl/6 and C57Bl/6-apoB-Tg mice, E) plasma leptin in C57Bl/6 and C57Bl/6-apoB-Tg mice. Values are after 11 months of diet and in overnight fasted mice. Open bars: chow-fed C57Bl/6 mice (n = 7), closed bars: fat-fed C57Bl/6 mice (n = 7), hatched bars: chow-fed C57Bl/6-apoB-Tg mice (n = 11), squared bars: fat-fed C57Bl/6-apoB-Tg mice (n = 7). Values are mean±SEM. The p values for two-group comparisons are indicated by: *** P<0.005 compared to chow-fed controls.

https://doi.org/10.1371/journal.pone.0005300.s002

(0.22 MB TIF)

Table S1.

Heart function in fat-fed C57Bl/6 and C57Bl/6-apoB-Tg mice. vData are from male mice chow- or fat-fed for 11 months. Values are mean±SEM, * P<0.05; ‡P<0.005 compared to lean controls. EDV indicates end-diastolic volume; ESV, end-systolic volume; ESP, end systolic pressure; EDP, end diastolic pressure; t, isovolumic relaxation time; EDVPR, end diastolic volume pressure relationship. P<0.05 is considered significant.

https://doi.org/10.1371/journal.pone.0005300.s003

(0.03 MB DOC)

Table S2.

Primers used for real-time PCR.

https://doi.org/10.1371/journal.pone.0005300.s004

(0.04 MB DOC)

Acknowledgments

The authors wish to thank Dr. Bo Porse, University of Copenhagen, for the Mck-Cre mice, and Maria Kristensen, Dr. Sara Elsoe Nielsen, Line Nolting, and Gerda Hau for technical assistance.

Author Contributions

Conceived and designed the experiments: EDB LBN. Performed the experiments: EDB JMN. Analyzed the data: EDB JMN LIH TP. Contributed reagents/materials/analysis tools: EDB JMN LIH TP LBN. Wrote the paper: EDB LBN.

References

  1. 1. Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, et al. (2002) Obesity and the risk of heart failure. N Engl J Med 347: 305–313.S. KenchaiahJC EvansD. LevyPW WilsonEJ Benjamin2002Obesity and the risk of heart failure.N Engl J Med347305313
  2. 2. Ford ES, Ajani UA, Croft JB, Critchley JA, Labarthe DR, et al. (2007) Explaining the decrease in U.S. deaths from coronary disease, 1980–2000. N Engl J Med 356: 2388–2398.ES FordUA AjaniJB CroftJA CritchleyDR Labarthe2007Explaining the decrease in U.S. deaths from coronary disease, 1980–2000.N Engl J Med35623882398
  3. 3. McGavock JM, Lingvay I, Zib I, Tillery T, Salas N, et al. (2007) Cardiac steatosis in diabetes mellitus: a 1H-magnetic resonance spectroscopy study. Circulation 116: 1170–1175.JM McGavockI. LingvayI. ZibT. TilleryN. Salas2007Cardiac steatosis in diabetes mellitus: a 1H-magnetic resonance spectroscopy study.Circulation11611701175
  4. 4. Szczepaniak LS, Dobbins RL, Metzger GJ, Sartoni-D'Ambrosia G, Arbique D, et al. (2003) Myocardial triglycerides and systolic function in humans: in vivo evaluation by localized proton spectroscopy and cardiac imaging. Magn Reson Med 49: 417–423.LS SzczepaniakRL DobbinsGJ MetzgerG. Sartoni-D'AmbrosiaD. Arbique2003Myocardial triglycerides and systolic function in humans: in vivo evaluation by localized proton spectroscopy and cardiac imaging.Magn Reson Med49417423
  5. 5. Christoffersen C, Bollano E, Lindegaard ML, Bartels ED, Goetze JP, et al. (2003) Cardiac lipid accumulation associated with diastolic dysfunction in obese mice. Endocrinology 144: 3483–3490.C. ChristoffersenE. BollanoML LindegaardED BartelsJP Goetze2003Cardiac lipid accumulation associated with diastolic dysfunction in obese mice.Endocrinology14434833490
  6. 6. Zhou YT, Grayburn P, Karim A, Shimabukuro M, Higa M, et al. (2000) Lipotoxic heart disease in obese rats: implications for human obesity. Proc Natl Acad Sci U S A 97: 1784–1789.YT ZhouP. GrayburnA. KarimM. ShimabukuroM. Higa2000Lipotoxic heart disease in obese rats: implications for human obesity.Proc Natl Acad Sci U S A9717841789
  7. 7. Lee Y, Naseem RH, Duplomb L, Park BH, Garry DJ, et al. (2004) Hyperleptinemia prevents lipotoxic cardiomyopathy in acyl CoA synthase transgenic mice. Proc Natl Acad Sci U S A 101: 13624–13629.Y. LeeRH NaseemL. DuplombBH ParkDJ Garry2004Hyperleptinemia prevents lipotoxic cardiomyopathy in acyl CoA synthase transgenic mice.Proc Natl Acad Sci U S A1011362413629
  8. 8. Lopaschuk GD, Folmes CD, Stanley WC (2007) Cardiac energy metabolism in obesity. Circ Res 101: 335–347.GD LopaschukCD FolmesWC Stanley2007Cardiac energy metabolism in obesity.Circ Res101335347
  9. 9. Szczepaniak LS, Victor RG, Orci L, Unger RH (2007) Forgotten but not gone: the rediscovery of fatty heart, the most common unrecognized disease in America. Circ Res 101: 759–767.LS SzczepaniakRG VictorL. OrciRH Unger2007Forgotten but not gone: the rediscovery of fatty heart, the most common unrecognized disease in America.Circ Res101759767
  10. 10. Stanley WC, Chandler MP (2002) Energy metabolism in the normal and failing heart: potential for therapeutic interventions. Heart Fail Rev 7: 115–130.WC StanleyMP Chandler2002Energy metabolism in the normal and failing heart: potential for therapeutic interventions.Heart Fail Rev7115130
  11. 11. Unger RH (2003) Minireview: weapons of lean body mass destruction: the role of ectopic lipids in the metabolic syndrome. Endocrinology 144: 5159–5165.RH Unger2003Minireview: weapons of lean body mass destruction: the role of ectopic lipids in the metabolic syndrome.Endocrinology14451595165
  12. 12. Stanley WC, Recchia FA, Lopaschuk GD (2005) Myocardial substrate metabolism in the normal and failing heart. Physiol Rev 85: 1093–1129.WC StanleyFA RecchiaGD Lopaschuk2005Myocardial substrate metabolism in the normal and failing heart.Physiol Rev8510931129
  13. 13. Somoza B, Guzman R, Cano V, Merino B, Ramos P, et al. (2007) Induction of cardiac uncoupling protein-2 expression and adenosine 5′-monophosphate-activated protein kinase phosphorylation during early states of diet-induced obesity in mice. Endocrinology 148: 924–931.B. SomozaR. GuzmanV. CanoB. MerinoP. Ramos2007Induction of cardiac uncoupling protein-2 expression and adenosine 5′-monophosphate-activated protein kinase phosphorylation during early states of diet-induced obesity in mice.Endocrinology148924931
  14. 14. Bamba V, Rader DJ (2007) Obesity and atherogenic dyslipidemia. Gastroenterology 132: 2181–2190.V. BambaDJ Rader2007Obesity and atherogenic dyslipidemia.Gastroenterology13221812190
  15. 15. Koutsari C, Jensen MD (2006) Thematic review series: patient-oriented research. Free fatty acid metabolism in human obesity. J Lipid Res 47: 1643–1650.C. KoutsariMD Jensen2006Thematic review series: patient-oriented research. Free fatty acid metabolism in human obesity.J Lipid Res4716431650
  16. 16. Hammer S, van der Meer RW, Lamb HJ, Schar M, de RA, et al. (2008) Progressive caloric restriction induces dose-dependent changes in myocardial triglyceride content and diastolic function in healthy men. J Clin Endocrinol Metab 93: 497–503.S. HammerRW van der MeerHJ LambM. ScharRA de2008Progressive caloric restriction induces dose-dependent changes in myocardial triglyceride content and diastolic function in healthy men.J Clin Endocrinol Metab93497503
  17. 17. Wisneski JA, Gertz EW, Neese RA, Mayr M (1987) Myocardial metabolism of free fatty acids. Studies with 14C-labeled substrates in humans. J Clin Invest 79: 359–366.JA WisneskiEW GertzRA NeeseM. Mayr1987Myocardial metabolism of free fatty acids. Studies with 14C-labeled substrates in humans.J Clin Invest79359366
  18. 18. Yagyu H, Chen G, Yokoyama M, Hirata K, Augustus A, et al. (2003) Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy. J Clin Invest 111: 419–426.H. YagyuG. ChenM. YokoyamaK. HirataA. Augustus2003Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.J Clin Invest111419426
  19. 19. Chiu HC, Kovacs A, Blanton RM, Han X, Courtois M, et al. (2005) Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy. Circ Res 96: 225–233.HC ChiuA. KovacsRM BlantonX. HanM. Courtois2005Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy.Circ Res96225233
  20. 20. Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, et al. (2002) The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus. J Clin Invest 109: 121–130.BN FinckJJ LehmanTC LeoneMJ WelchMJ Bennett2002The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus.J Clin Invest109121130
  21. 21. Chiu HC, Kovacs A, Ford DA, Hsu FF, Garcia R, et al. (2001) A novel mouse model of lipotoxic cardiomyopathy. J Clin Invest 107: 813–822.HC ChiuA. KovacsDA FordFF HsuR. Garcia2001A novel mouse model of lipotoxic cardiomyopathy.J Clin Invest107813822
  22. 22. Kurtz DM, Rinaldo P, Rhead WJ, Tian L, Millington DS, et al. (1998) Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation. Proc Natl Acad Sci U S A 95: 15592–15597.DM KurtzP. RinaldoWJ RheadL. TianDS Millington1998Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation.Proc Natl Acad Sci U S A951559215597
  23. 23. Wilson CR, Tran MK, Salazar KL, Young ME, Taegtmeyer H (2007) Western diet, but not high fat diet, causes derangements of fatty acid metabolism and contractile dysfunction in the heart of Wistar rats. Biochem J 406: 457–467.CR WilsonMK TranKL SalazarME YoungH. Taegtmeyer2007Western diet, but not high fat diet, causes derangements of fatty acid metabolism and contractile dysfunction in the heart of Wistar rats.Biochem J406457467
  24. 24. Saddik M, Lopaschuk GD (1991) Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts. J Biol Chem 266: 8162–8170.M. SaddikGD Lopaschuk1991Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts.J Biol Chem26681628170
  25. 25. Strawford A, Antelo F, Christiansen M, Hellerstein MK (2004) Adipose tissue triglyceride turnover, de novo lipogenesis, and cell proliferation in humans measured with 2H2O. Am J Physiol Endocrinol Metab 286: E577–E588.A. StrawfordF. AnteloM. ChristiansenMK Hellerstein2004Adipose tissue triglyceride turnover, de novo lipogenesis, and cell proliferation in humans measured with 2H2O.Am J Physiol Endocrinol Metab286E577E588
  26. 26. Nielsen LB, Veniant M, Boren J, Raabe M, Wong JS, et al. (1998) Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins. Circulation 98: 13–16.LB NielsenM. VeniantJ. BorenM. RaabeJS Wong1998Genes for apolipoprotein B and microsomal triglyceride transfer protein are expressed in the heart: evidence that the heart has the capacity to synthesize and secrete lipoproteins.Circulation981316
  27. 27. Adiels M, Boren J, Caslake MJ, Stewart P, Soro A, et al. (2005) Overproduction of VLDL1 driven by hyperglycemia is a dominant feature of diabetic dyslipidemia. Arterioscler Thromb Vasc Biol 25: 1697–1703.M. AdielsJ. BorenMJ CaslakeP. StewartA. Soro2005Overproduction of VLDL1 driven by hyperglycemia is a dominant feature of diabetic dyslipidemia.Arterioscler Thromb Vasc Biol2516971703
  28. 28. Mohler PJ, Zhu MY, Blade AM, Ham AJ, Shelness GS, et al. (2007) Identification of a novel isoform of microsomal triglyceride transfer protein. J Biol Chem 282: 26981–26988.PJ MohlerMY ZhuAM BladeAJ HamGS Shelness2007Identification of a novel isoform of microsomal triglyceride transfer protein.J Biol Chem2822698126988
  29. 29. Dougan SK, Rava P, Hussain MM, Blumberg RS (2007) MTP regulated by an alternate promoter is essential for NKT cell development. J Exp Med 204: 533–545.SK DouganP. RavaMM HussainRS Blumberg2007MTP regulated by an alternate promoter is essential for NKT cell development.J Exp Med204533545
  30. 30. Boren J, Veniant MM, Young SG (1998) Apo B100-containing lipoproteins are secreted by the heart. J Clin Invest 101: 1197–1202.J. BorenMM VeniantSG Young1998Apo B100-containing lipoproteins are secreted by the heart.J Clin Invest10111971202
  31. 31. Nielsen LB, Bartels ED, Bollano E (2002) Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice. J Biol Chem 277: 27014–27020.LB NielsenED BartelsE. Bollano2002Overexpression of apolipoprotein B in the heart impedes cardiac triglyceride accumulation and development of cardiac dysfunction in diabetic mice.J Biol Chem2772701427020
  32. 32. Yokoyama M, Yagyu H, Hu Y, Seo T, Hirata K, et al. (2004) Apolipoprotein B production reduces lipotoxic cardiomyopathy: studies in heart-specific lipoprotein lipase transgenic mouse. J Biol Chem 279: 4204–4211.M. YokoyamaH. YagyuY. HuT. SeoK. Hirata2004Apolipoprotein B production reduces lipotoxic cardiomyopathy: studies in heart-specific lipoprotein lipase transgenic mouse.J Biol Chem27942044211
  33. 33. Bjorkegren J, Veniant M, Kim SK, Withycombe SK, Wood PA, et al. (2001) Lipoprotein secretion and triglyceride stores in the heart. J Biol Chem 276: 38511–38517.J. BjorkegrenM. VeniantSK KimSK WithycombePA Wood2001Lipoprotein secretion and triglyceride stores in the heart.J Biol Chem2763851138517
  34. 34. Raabe M, Veniant MM, Sullivan MA, Zlot CH, Bjorkegren J, et al. (1999) Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice. J Clin Invest 103: 1287–1298.M. RaabeMM VeniantMA SullivanCH ZlotJ. Bjorkegren1999Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice.J Clin Invest10312871298
  35. 35. Park SY, Cho YR, Kim HJ, Higashimori T, Danton C, et al. (2005) Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice. Diabetes 54: 3530–3540.SY ParkYR ChoHJ KimT. HigashimoriC. Danton2005Unraveling the temporal pattern of diet-induced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice.Diabetes5435303540
  36. 36. Petersen S, Russ M, Reinauer H, Eckel J (1991) Inverse regulation of glucose transporter Glut4 and G-protein Gs mRNA expression in cardiac myocytes from insulin resistant rats. FEBS Lett 286: 1–5.S. PetersenM. RussH. ReinauerJ. Eckel1991Inverse regulation of glucose transporter Glut4 and G-protein Gs mRNA expression in cardiac myocytes from insulin resistant rats.FEBS Lett28615
  37. 37. Chess DJ, Stanley WC (2008) Role of diet and fuel overabundance in the development and progression of heart failure. Cardiovasc Res 79: 269–278.DJ ChessWC Stanley2008Role of diet and fuel overabundance in the development and progression of heart failure.Cardiovasc Res79269278
  38. 38. Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, et al. (2004) Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 306: 457–461.U. OzcanQ. CaoE. YilmazAH LeeNN Iwakoshi2004Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.Science306457461
  39. 39. Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov 7: 1013–1030.I. KimW. XuJC Reed2008Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities.Nat Rev Drug Discov710131030
  40. 40. Hollander MC, Sheikh MS, Yu K, Zhan Q, Iglesias M, et al. (2001) Activation of Gadd34 by diverse apoptotic signals and suppression of its growth inhibitory effects by apoptotic inhibitors. Int J Cancer 96: 22–31.MC HollanderMS SheikhK. YuQ. ZhanM. Iglesias2001Activation of Gadd34 by diverse apoptotic signals and suppression of its growth inhibitory effects by apoptotic inhibitors.Int J Cancer962231
  41. 41. Gordon DA, Jamil H, Sharp D, Mullaney D, Yao Z, et al. (1994) Secretion of apolipoprotein B-containing lipoproteins from HeLa cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability. Proc Natl Acad Sci U S A 91: 7628–7632.DA GordonH. JamilD. SharpD. MullaneyZ. Yao1994Secretion of apolipoprotein B-containing lipoproteins from HeLa cells is dependent on expression of the microsomal triglyceride transfer protein and is regulated by lipid availability.Proc Natl Acad Sci U S A9176287632
  42. 42. Raabe M, Flynn LM, Zlot CH, Wong JS, Veniant MM, et al. (1998) Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes. Proc Natl Acad Sci U S A 95: 8686–8691.M. RaabeLM FlynnCH ZlotJS WongMM Veniant1998Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes.Proc Natl Acad Sci U S A9586868691
  43. 43. Ledmyr H, McMahon AD, Ehrenborg E, Nielsen LB, Neville M, et al. (2004) The microsomal triglyceride transfer protein gene-493T variant lowers cholesterol but increases the risk of coronary heart disease. Circulation 109: 2279–2284.H. LedmyrAD McMahonE. EhrenborgLB NielsenM. Neville2004The microsomal triglyceride transfer protein gene-493T variant lowers cholesterol but increases the risk of coronary heart disease.Circulation10922792284
  44. 44. Jacob S, Machann J, Rett K, Brechtel K, Volk A, et al. (1999) Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects. Diabetes 48: 1113–1119.S. JacobJ. MachannK. RettK. BrechtelA. Volk1999Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects.Diabetes4811131119
  45. 45. Turner N, Bruce CR, Beale SM, Hoehn KL, So T, et al. (2007) Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents. Diabetes 56: 2085–2092.N. TurnerCR BruceSM BealeKL HoehnT. So2007Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents.Diabetes5620852092
  46. 46. Koves TR, Ussher JR, Noland RC, Slentz D, Mosedale M, et al. (2008) Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metab 7: 45–56.TR KovesJR UssherRC NolandD. SlentzM. Mosedale2008Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance.Cell Metab74556
  47. 47. Mazumder PK, O'Neill BT, Roberts MW, Buchanan J, Yun UJ, et al. (2004) Impaired cardiac efficiency and increased fatty acid oxidation in insulin-resistant ob/ob mouse hearts. Diabetes 53: 2366–2374.PK MazumderBT O'NeillMW RobertsJ. BuchananUJ Yun2004Impaired cardiac efficiency and increased fatty acid oxidation in insulin-resistant ob/ob mouse hearts.Diabetes5323662374
  48. 48. Peterson LR, Herrero P, Schechtman KB, Racette SB, Waggoner AD (2004) Effect of obesity and insulin resistance on myocardial substrate metabolism and efficiency in young women. Circulation 109: 2191–2196.LR PetersonP. HerreroKB SchechtmanSB RacetteAD Waggoner2004Effect of obesity and insulin resistance on myocardial substrate metabolism and efficiency in young women.Circulation10921912196
  49. 49. Park TS, Hu Y, Noh HL, Drosatos K, Okajima K, et al. (2008) Ceramide is a cardiotoxin in lipotoxic cardiomyopathy. J Lipid Res 49: 2101–2112.TS ParkY. HuHL NohK. DrosatosK. Okajima2008Ceramide is a cardiotoxin in lipotoxic cardiomyopathy.J Lipid Res4921012112
  50. 50. Ye G, Metreveli NS, Ren J, Epstein PN (2003) Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production. Diabetes 52: 777–783.G. YeNS MetreveliJ. RenPN Epstein2003Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production.Diabetes52777783
  51. 51. Finck BN, Han X, Courtois M, Aimond F, Nerbonne JM, et al. (2003) A critical role for PPARalpha-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: modulation by dietary fat content. Proc Natl Acad Sci U S A 100: 1226–1231.BN FinckX. HanM. CourtoisF. AimondJM Nerbonne2003A critical role for PPARalpha-mediated lipotoxicity in the pathogenesis of diabetic cardiomyopathy: modulation by dietary fat content.Proc Natl Acad Sci U S A10012261231
  52. 52. Buchanan J, Mazumder PK, Hu P, Chakrabarti G, Roberts MW, et al. (2005) 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 146: 5341–5349.J. BuchananPK MazumderP. HuG. ChakrabartiMW Roberts2005Reduced cardiac efficiency and altered substrate metabolism precedes the onset of hyperglycemia and contractile dysfunction in two mouse models of insulin resistance and obesity.Endocrinology14653415349
  53. 53. Yang J, Sambandam N, Han X, Gross RW, Courtois M, et al. (2007) CD36 deficiency rescues lipotoxic cardiomyopathy. Circ Res 100: 1208–1217.J. YangN. SambandamX. HanRW GrossM. Courtois2007CD36 deficiency rescues lipotoxic cardiomyopathy.Circ Res10012081217
  54. 54. Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, et al. (1998) A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 2: 559–569.JC BruningMD MichaelJN WinnayT. HayashiD. Horsch1998A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance.Mol Cell2559569
  55. 55. Bartels ED, Bang CA, Nielsen LB (2009) Early atherosclerosis and vascular inflammation in mice with diet-induced type 2 diabetes. Eur J Clin Invest 39: 190–199.ED BartelsCA BangLB Nielsen2009Early atherosclerosis and vascular inflammation in mice with diet-induced type 2 diabetes.Eur J Clin Invest39190199
  56. 56. Pedersen TX, Bro S, Andersen MH, Etzerodt M, Jauhiainen M, et al. (2009) Effect of treatment with human apolipoprotein A-I on atherosclerosis in uremic apolipoprotein-E deficient mice. Atherosclerosis 202: 372–381.TX PedersenS. BroMH AndersenM. EtzerodtM. Jauhiainen2009Effect of treatment with human apolipoprotein A-I on atherosclerosis in uremic apolipoprotein-E deficient mice.Atherosclerosis202372381
  57. 57. Bang CA, Bro S, Bartels ED, Pedersen TX, Nielsen LB (2007) Effect of uremia on HDL composition, vascular inflammation, and atherosclerosis in wild-type mice. Am J Physiol Renal Physiol 293: F1325–F1331.CA BangS. BroED BartelsTX PedersenLB Nielsen2007Effect of uremia on HDL composition, vascular inflammation, and atherosclerosis in wild-type mice.Am J Physiol Renal Physiol293F1325F1331
  58. 58. Lindegaard ML, Nielsen LB (2008) Maternal diabetes causes coordinated down-regulation of genes involved with lipid metabolism in the murine fetal heart. Metabolism 57: 766–773.ML LindegaardLB Nielsen2008Maternal diabetes causes coordinated down-regulation of genes involved with lipid metabolism in the murine fetal heart.Metabolism57766773
  59. 59. Georgakopoulos D, Mitzner WA, Chen CH, Byrne BJ, Millar HD, et al. (1998) In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol 274: H1416–H1422.D. GeorgakopoulosWA MitznerCH ChenBJ ByrneHD Millar1998In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry.Am J Physiol274H1416H1422
  60. 60. Nielsen JM, Kristiansen SB, Ringgaard S, Nielsen TT, Flyvbjerg A, et al. (2007) Left ventricular volume measurement in mice by conductance catheter: evaluation and optimization of calibration. Am J Physiol Heart Circ Physiol 293: H534–H540.JM NielsenSB KristiansenS. RinggaardTT NielsenA. Flyvbjerg2007Left ventricular volume measurement in mice by conductance catheter: evaluation and optimization of calibration.Am J Physiol Heart Circ Physiol293H534H540
  61. 61. Bartels ED, Lauritsen M, Nielsen LB (2002) Hepatic expression of microsomal triglyceride transfer protein and in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice. Diabetes 51: 1233–1239.ED BartelsM. LauritsenLB Nielsen2002Hepatic expression of microsomal triglyceride transfer protein and in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice.Diabetes5112331239