Effect of conjugated linoleic acid on inhibition of prolyl hydroxylase 1 in hearts of mice
© Zhang and Li; licensee BioMed Central Ltd. 2012
Received: 17 December 2011
Accepted: 7 February 2012
Published: 7 February 2012
Results from different trails have provided evidence of protective effects of cis- 9,trans-11-conjugated linoleic acid (CLA) on cardiovascular diseases. But the inhibition of prolyl hydroxylase 1 (PHD1) associated with induction of hypoxia inducible factors (HIFs) by CLA in these protective effects has never been reported before. The objective of this study was to evaluate if the two predominant cis- 9,trans-11 (c9, t11), trans-10,cis-12 (t10, c12) CLA isomers and mixture of these two isomers can inhibit PHD1 with induction of HIFs in myocardium in mice and subsequent effects on myocardium metabolism.
CLA mixture and c9, t11 CLA inhibited PHD1 protein expression and increased the levels of protein and mRNA in HIF-2α in myocardium in mice. Meanwhile, CLA mixture and c9, t11 CLA also elevated the expression of HIF related transcriptional factors like PDK4 and PPARα. The reprogramming of basal metabolism in myocardium in mice was shown on increasing of GLUT4 gene expression by c9, t11 CLA supplemented group. UCP2 was increased by CLA mixture and c9, t11 CLA for attenuating production of ROS.
CLA mixture and c9, t11 CLA could inhibit PHD1 and induce HIF-2α in myocardium in mice, which is associated with upregulation of PDK4 by activation of PPARα. This process also implies a reprogramming of basal metabolism and oxidative damage protection in myocardium in mice. All the effects shown in hearts of mice are due to c9, t11 CLA but not t10, c12 CLA.
Heart disease like myocardial infarction (MI) or acute myocardial infarction (AMI) and heart ischemia commonly are known as cardiovascular diseases (CVDs), which are the interruption of blood supply to part of the heart, causing heart cells to die. In 2008, an estimated 17.3 million people died from CVDs in the world, in which over 80% of CVD deaths take place in low-and middle-income countries .
Oxygen availability is insufficient when inadequate blood supply happens. Cells undergo adaptive changes in gene expression that promote survival in low oxygen (hypoxic) environment. Cellular adaptation to oxygen availability is mediated by the hypoxia inducible factors (HIFs), a member of the basic helix-loop-helix-PAS superfamily which transactivate a host of genes in the nucleus involved in the adaption of hypoxic stress . HIF consists of an unstable α subunit and a stable β subunit that binds DNA at specific locations termed hypoxia response elements (HERs) to regulate many genes expression related to hypoxia . HIF-α subunit is regulatory and unique to the hypoxic response. HIF-β subunit is constitutive and also involved in xenobiotic response. Three different genes encoding HIF-α subunit are found in mammals: HIF-1α, HIF-2α and HIF-3α . HIF-α proteins are maintained at low steady-state level under normoxic condition via hydroxylation by HIF prolyl hydroxylases (PHDs) . Among these three HIF-α isoforms, HIF-2α in particular shows a unique ability to induce metabolic reprogramming, which ultimately makes mitochondrion harmless but less active in certain conditions by regulating expression of numerous genes . PHDs are 2-oxoglutarate dioxygenases, which are present in three forms in mammals, designated PHD1, PHD2 and PHD3 . Hydroxylated HIF recruits the E3-ubiquitin ligase, von Hippel-Lindau protein (pVHL) [7, 8], which in turn tags HIF with ubiquitin groups and targets it for degradation by proteasome [9, 10].
Many cardiovascular diseases including anemia, myocardial infarction and stroke are linked to inadequate tissue oxygen. So, up-regulation of HIFs by inhibition of PHDs may have beneficial effect on therapy for hypoxia dependent process involved in cardiovascular disease . The availability of less cumbersome non-toxic inhibitors of PHDs has been proved very useful for therapeutic intervention [11–13].
Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of the essential fatty acid-linoleic acid (LA), which is produced by the bacterial biohydrogenation of linoleic acid in the gut of ruminant animals via an enzymatic isomerase reaction . CLA is found naturally in food products from these animals predominantly as the cis-9,trans-11 form, whereas synthetic CLA preparations consist of a few different isomers with approximately equal amount of cis-9,trans- 11 and trans-10,cis-12 CLA .
Since be found from 1980s, many research has been done with biological functions of these two predominant isomers of CLA. These isomers are both biologically active and known to have different physiological effects . The original discovery of CLA was as an anticancer component, which was proven to be an effective prevention tool in a number of animal cancer models, such as skin, colon, mammary, lung and liver [17, 18]. Both isomers have proven to be effective in prevention of cancer, while others reported differences in anti-cancer activities between these isomers [19, 20]. t10, c12 CLA was reported to prevent cardiovascular disease [21, 22] and reduce body fat in animals , which is one of the most important activities of CLA. While c9, t11 CLA was not responsible for these effects [24, 25]. There was also research about CLA that it can reduce inflammatory and improve immune response [26–28]. But the controversy was that t10, c12 CLA induced inflammatory responses in white adipose tissue . Besides positive effects induced by CLA, there were also negative effects reported for CLA, especially t10, c12 CLA. Mice fed with CLA mixture or t10, c12 CLA resulted in lipodystrophy, hyperinsulinemia and liver steatosis, whereas, c9, t11 CLA had no negative effect like t10,12 CLA [30–32].
There was research reported that c9, t11 CLA in adipose tissue was associated with a lower risk of MI in basic . But the mechanism of this protective effect is still not fully defined. We estimated that CLA may have influence on PHDs, which is related to the protective effect on myocardium. The mice model was used by supplementing with individual isomers of CLA and the mixture of these two isomers in the diet to determine if these effects are associated with role of prolyl hydroxylase inhibitor on myocardium.
Material and method
Semipurified diet (TD.04460) was from Harlan Teklad (Madison, WI). Antibodies for prolyl hydroxylase 1 (PHD1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Bethyl (Montgomery, TX). Antibodies for hypoxia inducible factor-2 alpha (HIF-2α), peroxisome proliferator-activated receptor alpha (PPARα) and secondary antibody coupled to horse radish peroxidase (HRP) were purchased from Abcam (Cambridge, MA). Other solvents used were purchased from either Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburg, PA).
Animal and diet
TD.04460 Basal Mix (94%)
Casein, "Vitamin-Free" Test
Mineral Mix, w/o Ca (04374)
Vitamin Mix, AIN-93-VX (94047)
Composition (%) of CLA Preparations Used in this studya
> 90% c9, t11
> 90% t10, c12
c9, t11 CLA
t10, c12 CLA
Oleic acid (18:1)
Sacrifice and tissue sample collection
After 8 weeks, animals were sacrificed by CO2 asphyxiation after 4 h fasting. Hearts were removed and frozen by immersion in liquid N2, stored at -80°C until needed for analysis.
mRNA expression analysis
Information of gene primers used for RT-PCR
Western blot analysis
Tissue lysates were prepared in homogenization buffer [50 mM Tris-HCl pH 7.5, 10%(v/v) glycerol, 5 mM magnesium acetate, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)] and centrifuged at 12,000 r.p.m. for 20 min at 4°C. Protein concentration was determined by Bio-Rad protein assay (Hercules, CA) using 1.4 mg/mL bovine serum albumin (BSA) as the standard. Proteins (25 μg) from each sample were separated on SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). After blocking with a specific primary antiserum in Tris buffered saline (TBS) containing 0.05% Tween-20 (TBS-T) and 5% non-fat dry milk at 4°C overnight, the membrane was incubated with each antibody (anti-PHD1, anti-HIF-2α, anti-PPARα, anti-GAPDH) at 4°C overnight. Finally, after three washes with TBS-T, the blots were incubated with secondary antibody coupled to HRP for 1 h at room temperature, visualized using ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and quantified by Kodak Image Station Software (Scion, Frederick, MD). Relative protein levels were determined by comparison to GAPDH band intensity, which was used as internal control for western blotting analysis.
Data are shown as means and standard errors. All analyses were carried out using SAS software (Version 9.2, SAS institute Inc., Cary, NC) by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.
Effect of individual isomers of CLA and CLA mixture on body weight in the mice
25.90 ± 0.65a
29.68 ± 0.44a
29.49 ± 0.51a
30.19 ± 0.60a
30.40 ± 0.64a
30.29 ± 0.41a
31.94 ± 0.44a
32.93 ± 0.52ab
33.17 ± 0.58ab
25.90 ± 0.65a
28.58 ± 0.46a
28.03 ± 0.58a
28.68 ± 0.68a
28.44 ± 0.77b
29.29 ± 0.77a
32.00 ± 0.73a
32.30 ± 0.71b
31.88 ± 0.79b
c9, t11 CLA
25.92 ± 0.65a
29.82 ± 0.61a
29.41 ± 0.68a
30.23 ± 0.62a
29.93 ± 0.67ab
30.04 ± 0.82a
32.36 ± 0.69a
34.36 ± 0.82a
34.29 ± 0.89a
25.63 ± 0.63a
29.31 ± 0.41a
28.92 ± 0.43a
29.79 ± 0.45a
29.20 ± 0.32ab
30.93 ± 0.32a
32.82 ± 0.41a
34.05 ± 0.44ab
33.26 ± 0.60ab
Gene expressions of HIFs and PHD1
Protein expressions of HIF-2α and PHD1
Gene and protein expressions of PPARα pathway
Gene expressions of glucose and lipid metabolism enzymes
In this study, CLA mixture and the two predominant isomers of CLA showed the ability to inhibit PHD1 protein expression. But interestingly, only CLA mixture and c9, t11 CLA played a role in stabilizing HIF-2α in the normoxia environment and affected expressions of HIF related transcriptional genes with inhibition of PHD1 in hearts of mice. This is a new discovery of well-studied CLA on myocardium protective effect.
One of the important effects of CLA is on decreasing body weight gain, which was proved in various animal models like mice and rats [25, 34]. However, these findings were shown when those animals were given high amount of CLA, in which t10, c12 CLA was contributed to this effect [35, 36]. When animals were supplemented with low amount of CLA mixture (≤ 0.5% in the diet) or the two predominant isomers of CLA, the body weights were not affected [37–39], which is consistent with our study.
Since PHD1 is a direct oxygen sensor, alternation of expression of PHD-1 is expected to change hypoxic adaptation . Most of the PHD inhibitors reported so far, e.g. DMOG , 3,4-DHB , FG-0041  are chemicals. None of them is from natural food like CLA which is contained in milk or beef. Inhibition of PHD1 associated with stabilized expression HIF-2α expression in normoxia condition was seen in CLA mixture and c9, t11 CLA fed mice, which is consistent with a former study that mice lacking PHD1 (PHD1-null mice) showed significant expression of HIF-2α in the absence of hypoxia .
Specifically, CLA mixture and c9, t11 CLA increased HIF-α expression like HIF-1α and HIF-2α in mRNA or protein level. The protective effect of HIFs within different tissues has already been noted before [5, 44, 45]. However, there is a debate about the expression of HIF-α in mRNA and in protein level. Research indicated that there was no significant increase in the mRNA expression of EPAS1 [46, 47]. This may be due to posttranscriptional regulation as found in specific cell types [48, 49]. From the results showing in the experiment, the regulation of expression of EGLN2 (PHD1) in different levels may share the same mechanism as HIF-α. Interesting, t10, c12 CLA also down-regulated PHD1 protein expression, but it was not as potent as 19, t11 CLA on EPAS1 expression, which may be the difference of bioactivity of these two isomers of CLA. In general, HIF-β is constitutively expressed and heterodimerizes with HIF-α subunit in the nucleus to form a complex, which binds to hypoxia-responsive elements in enhancers and promoters of oxygen-responsive genes under hypoxic conditions . Consistent with the former research, the expression of ARNT2 was not altered by all treatments in this study.
The possible mechanism inducing protective effect of HIFs, especially HIF-2α, is linked to an increase in the expression of PDK4 . In the normoxic environment, pyruvate enters the tricarboxylic acid (TCA) cycle inside the mitochondrion where it generates ATP in the presence of oxygen. But with the inhibition of PHD1 or in the hypoxia environment like heart ischemia, entry of pyruvate is restricted by expression of PDK4, which is associated with induction of HIF-2α [5, 51]. In this study, PDK4 gene expression was upregulated in mice fed with CLA mixture and c9, t11 CLA.
PPARα is known to activate the PDK4 gene . Research on the hibernating mammal model also showed the level of PDK4 mRNA increased greatly by activation of PPARα during hibernation . Hearts from PPAR agonist clofibrate-treated rats had an improved recovery of post-ischemic contractile function and reduced ischemia/reperfusion (I/R)-induced infarct size. The coincident upregulation of PPARα and PDK4 in PHD1-deficient was also demonstrated by knockdown of PPARα in muscles of PHD1-deficient mice in vivo and feeding wild-type mice with PPARα agonist . In this study, we found that CLA mixture and the two predominant isomers of CLA significantly increased PPARα protein expression. This result, combined with PDK4 gene expression and HIF-2α expression, suggests that PPARα activated by CLA mixture and c9, t11 CLA, with induction of HIF-2α, can initiate PDK4 gene expression.
Induction of HIF-2α by inhibition of PHD1 implies a reprogramming of basal metabolism in mice, especially in energy production and utilization like glucose metabolism and fatty acid metabolism. Glycolytic flux was increased in mouse muscle fiber in low oxygen conditions . As a marker for glucose metabolism, c9, t11 CLA fed mice increased GLUT4 gene expression significantly in this study. Combined, c9, t11 CLA may induce more glucose entering cell cytoplasm to generate ATP by glycolysis when PHD1 was inhibited. But for t10, c12 CLA, which promoted dysregulation of lipid and glucose metabolism in hepatic tissue while c9, t11 CLA had not effect . There was also another research reported that mice supplemented 0.5% CLA mixture showed different effect on GLUT4 gene expression in adipose and skeletal muscle tissue . All the evidences showed in experiments suggested that the two predominant isomers of CLA may have diverse effect on glucose metabolism in different tissues. CPT1b is known as a rate-limiting enzyme in fatty acid β-oxidation which transfers fatty acids through mitochondrial membrane to be oxidized within the mitochondrial matrix . A former research showed that fatty acid oxidation was not altered in PHD1-deficient mice , which is consistent with our research that CPT1 mRNA expression was not changed during PHD1 inhibition by CLA mixture and the two predominant isomers of CLA supplementation in the diet.
As an important energy expenditure parameter, the effect of uncoupling proteins (UCPs) has become prominent in the field of thermogenesis, especially UCP1 . But for UCP2 and UCP3, there is a consensus that the primary function of UCP2 and UCP3 is to attenuate mitochondrial production of free radical to protect against oxidative damage, degenerative disease and aging rather than to promote gross thermogenesis or energetic inefficiency [56, 57]. Oxidative damage caused by reactive oxygen species (ROS) is produced in mitochondrion, which can trigger the toxic effects living with oxygen. In contrast, oxygen depletion (hypoxia) also increases mitochondrial ROS that is detrimental to cells unless attenuated . In this study, the UCP2 gene expression in mice fed with CLA mixture and c9, t11 CLA was elevated greatly, which is consistent with former research done on effect of elucidating UCP2 on attenuating ROS production [59, 60].
To our knowledge, this study is the first to examine the effect of CLA on inhibition of PHD1 in vivo. One of the two predominant isomers of CLA- c9, t11 CLA showed more potent effect than the other CLA isomer- t10, c12 CLA. This inhibitory effect is associated with induction of HIF-2α. Several lines of evidence suggest the protective effect of c9, t11 CLA on inhibition of PHD1 plays a part by upregulation of PDK4 gene expression, which is activated by PPARα. This process can imply a reprogramming of basal metabolism in hearts of mice by increasing glycolysis. Meanwhile, c9, t11 CLA also increased UCP2 gene expression to attenuate the damage of ROS. This study provides a new interpretation of protective effect in myocardium in vivo by CLA, especially one of the predominant isomers-c9, t11 CLA.
The author is grateful for the suggestions during drafting manuscript by Dr. Linda Meccouri.
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