Original articlePost-translational modifications, a key process in CD36 function: Lessons from the spontaneously hypertensive rat 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.
- 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
Glossary
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
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