Original article
Post-translational modifications, a key process in CD36 function: Lessons from the spontaneously hypertensive rat heart

https://doi.org/10.1016/j.yjmcc.2011.04.001Get rights and content

Abstract

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of 13C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to β-oxidation (− 40%) and triglycerides (+ 2.8 folds), which was not explained by transcriptional changes or malonyl-CoA level, a recognized β-oxidation inhibitor, and (ii) membrane-associated CD36 protein level, but unchanged distribution. Other results demonstrate alterations in CD36 PTM in SHR hearts, specifically by N-glycosylation, and the importance of O-linked-β-N-acetylglucosamine for its membrane recruitment and role in LCFA use in the heart.

Research Highlights

►Alterations of CD36 in SHR are linked to reduced long chain fatty acid utilization. ►Post-translational modification of CD36 is crucial for its function. ►O-linked β-N-acetylglucosamine is involved in CD36 recruitment. ►N-glycosylation is a key process for CD36 function.

Introduction

Dysfunction of CD36, a trans-membrane multifunctional glycoprotein, has been linked to impaired fatty acid (FA) metabolism and increased cardiovascular risks as well as heart diseases in humans (for reviews see [1], [2], [3]). The Cd36 gene is highly conserved between species [4] and, hence, studies in transgenic (loss- and gain-of-function) mice have provided considerable insights into CD36 functions. However, the underlying molecular mechanisms for most reported functions, including those of long chain fatty acid (LCFA) transporter, remain to be clarified. In this regard, although useful, loss-of-function models do not reproduce the complexity of the situation in humans, whereby CD36 functional defects result from single nucleotide polymorphisms [5], [6], [7]. In fact, in most cases the functional impact of reported human Cd36 variants remains unclear. There exists, however, an animal model that offers a unique opportunity to unravel the regulation of CD36 function and its impact on LCFA uptake and utilization, namely, spontaneously hypertensive rats (SHR).

SHR are a widely-used genetic model of hypertension-induced left ventricular hypertrophy (LVH) and metabolic syndrome [8], [9], [10], [11]. In 1999, Aitman et al. [12] reported that the Cd36 cDNA of some SHR strains contains multiple sequence variants, a situation similar to that in humans (for review see [3]). In SHR, this is caused by unequal genomic recombination of a duplicated ancestral gene, which leads to a limited number of mutations within Cd36 mRNA [13]. Although the functional impact of this rearrangement within the Cd36 gene in SHR, particularly in the heart, remains to be elucidated, a recent study by Bonen et al. [14] clearly demonstrated that SHR expressed a different Cd36 mRNA and that the expression of CD36 protein at the plasma membrane was reduced in some tissues, including the heart. In parallel, we conducted a number of experiments in SHR with 13C-labeled LCFA and ex vivo perfusion of hearts in working mode, documenting a 3-fold lower contribution of exogenous LCFA to β-oxidation in 15-week-old SHR hearts compared to age-matched controls [9], [11].

Taken together, our afore-mentioned published data support the hypothesis of dysfunctional CD36 protein in SHR hearts, although several issues remain to be clarified, particularly with respect to the molecular mechanism(s) underlying this dysfunction. Admittedly, one issue that ought to be addressed is whether the previously-documented restricted LCFA utilization in SHR hearts could be due to changes associated with down-regulation of peroxisomal proliferator-activated receptor-alpha (PPARα), which are believed to be linked to recapitulation of the “fetal gene program” during LVH (for reviews see [15], [16]). Indeed, SHR develop LVH between 8 and 12 weeks of age, which is secondary to hypertension that appears as early as age 5 weeks [17], [18]. We believe, however, that the situation is likely to be much more complex given that SHR carry numerous mutations in the Cd36 gene. Hence, it may be viewed as a knock-in animal model, in which any existing mutations could impact on the structure of expressed CD36 protein and, hence, on its function. The issue is particularly important to consider since CD36 protein is known to be highly subject to post-translational modifications (PTM) by various processes, including phosphorylation and N-glycosylation (for reviews see [1], [2], [19]). However, much remains to be learned about the role of these PTM in CD36 folding, membrane targeting, and function as a LCFA transporter.

In this study, we used 7-week-old SHR and control Wistar rats to test whether SHR hearts display a reduced capacity for exogenous LCFA utilization for β-oxidation and triglyceride (TG) formation prior to developing LVH in our established model of ex vivo working hearts perfused with 13C-labeled substrates [9], [11], [20]. Further to our finding that such was the case, and was also not explained by changes in tissue levels of malonyl-CoA, a recognized regulator of β-oxidation [21], or in metabolic gene expression, we investigated the hypothesis that restricted LCFA utilization in SHR hearts may be explained by alterations in CD36 protein PTM. Collectively, data obtained via various approaches support this hypothesis and specifically the importance of changes in N-glycosylation.

Section snippets

Working rat heart perfusion in semi-recirculating mode

These animal experiments were approved by the local animal care committee in compliance with Canadian Council on Animal Care guidelines. Male SHR/Ncrl and Wistar rats (7-week-old; Charles River, Pointe Claire, QC, Canada) were provided with food and water ad libitum. Their mean body weight and heart wet weight were different (212 ± 7 vs. 171 ± 10 g; and 1.52 ± 0.05 vs. 1.25 ± 0.06 gww, respectively; p < 0.05), but their heart-to-body-weight ratio was similar (0.0074 ± 0.0004 vs. 0.0075 ± 0.0002; NS). The

Working hearts from 7-week-old SHR display a lower contribution of exogenous oleate to β-oxidation and triglycerides

Table 1 reports the various functional and physiological parameters measured in ex vivo working 7-week-old SHR and Wistar rat hearts perfused with a mixture of substrates and hormones mimicking the in vivo milieu in the fed state, while Fig. 1A enumerates the metabolic flux data assessed with 13C-labeled substrates. Working SHR hearts, perfused at an afterload of 70 and 80 mm Hg, presented functional and physiological parameters that closely matched those of Wistar rats perfused at 70 mm Hg.

Discussion

This study was undertaken in follow-up of previous experiments in which we documented restricted utilization of exogenous LCFA for β-oxidation in 15-week-old hypertrophied SHR hearts. Specifically, we postulated that the alteration in LCFA metabolism was also present in SHR hearts at a younger age, prior to the onset of LVH, and results from modifications in the Cd36 gene in SHR [12]. Collectively, the data from this study support these notions but, more importantly, provide evidence for the

Conclusion

In summary, beyond demonstrating, for the first time, that SHR (Ncrl strain) hearts display impaired oleate uptake linked to their Cd36 gene defect, the results from this study highlight the crucial role of PTM in CD36 protein recruitment and function. Specifically, we found that SHR hearts present reduced CD36 protein expression associated with normal localization, and have a reduced capacity to increase their exogenous free fatty acid utilization, as revealed by perfusion studies with

Disclosures

The authors have no conflicts of interest to disclose.

    Glossary

    3′UTR

    3′untranslation region

    AMP

    adenosine monophosphate

    AMPK

    AMP-dependent kinase

    CAC

    citric acid cycle

    CD36

    cluster of differentiation 36

    CHO

    carbohydrate

    CIP

    calf intestinal phosphatase

    FA

    fatty acid

    GCMS

    gas chromatography-mass spectrometry

    HBP

    hexosamine biosynthetic pathway

    HPLC

    high performance liquid chromatography

    HRP

    horseradish peroxidase

    LCFA

    long chain fatty acid

    LVH

    left ventricular hypertrophy

    MPE

    molar percent enrichment

    OAA

    oxaloacetate

    PNGase F

Acknowledgments

The present work was presented in part at meetings of the Society for Heart and Vascular Metabolism held in Semiahmoo in September 2006, in Boston in 2008, and in Kananaskis in 2010. This study was supported by the Canadian Institutes of Health Research (CIHR Grants #9575 to C.D.R. and #67053 to J.R.B.D.) and by the “Fondation Bettencourt Schueller” (B.L.). B.L. and C.M. are recipients of postdoctoral fellowships from “Fonds de la recherche en santé du Québec” and the Heart and Stroke

References (40)

  • B. Vistisen et al.

    Sarcolemmal FAT/CD36 in human skeletal muscle colocalizes with caveolin-3 and is more abundant in type 1 than in type 2 fibers

    J Lipid Res

    (2004)
  • N.S. Eyre et al.

    Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake

    J Lipid Res

    (2007)
  • J.F. Glatz et al.

    Membrane fatty acid transporters as regulators of lipid metabolism: implications for metabolic disease

    Physiol Rev

    (2010)
  • M.E. Rac et al.

    Molecular basis of human CD36 gene mutations

    Mol Med

    (2007)
  • T. Morii et al.

    CD36 single nucleotide polymorphism is associated with variation in low-density lipoprotein-cholesterol in young Japanese men

    Biomarkers

    (2009)
  • L. Love-Gregory et al.

    Variants in the CD36 gene associate with the metabolic syndrome and high-density lipoprotein cholesterol

    Hum Mol Genet

    (2008)
  • F. Labarthe et al.

    Fatty acid oxidation and its impact on response of spontaneously hypertensive rat hearts to an adrenergic stress: benefits of a medium-chain fatty acid

    Am J Physiol Heart Circ Physiol

    (2005)
  • T. Tanaka et al.

    Thiamine attenuates the hypertension and metabolic abnormalities in CD36-defective SHR: uncoupling of glucose oxidation from cellular entry accompanied with enhanced protein O-GlcNAcylation in CD36 deficiency

    Mol Cell Biochem

    (2007)
  • G. Vincent et al.

    Metabolic phenotyping of the diseased rat heart using 13 C-substrates and ex vivo perfusion in the working mode

    Mol Cell Biochem

    (2003)
  • T.J. Aitman et al.

    Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats

    Nat Genet

    (1999)
  • Cited by (45)

    • Post-translational modifications of CD36 (SR-B2): Implications for regulation of myocellular fatty acid uptake

      2016, Biochimica et Biophysica Acta - Molecular Basis of Disease
      Citation Excerpt :

      Hence, whether the fatty acid transport function of CD36 itself is altered by differences in glycosylation, is not known. In spontaneously hypertensive rats (SHR), CD36 is mutated at multiple sites, the mutations of which have been proposed to be responsible for the reduced cardiac fatty acid uptake seen in this model [21]. One of these mutations relates to Asp102, the only potential N-glycosylation site mutated in SHR.

    • Regulation of the subcellular trafficking of CD36, a major determinant of cardiac fatty acid utilization

      2016, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
    • The role of CD36 in the regulation of myocardial lipid metabolism

      2016, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
    View all citing articles on Scopus
    1

    Present address of S. Foisy: Diploide BioIT, 2347 Aubry, Suite 6, Montreal, QC H1L 4 G8, Canada.

    View full text