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Division of Foods and Nutrition, University of Utah, Salt Lake City, UT 84112
2To whom correspondence should be addressed. E-mail: thunder.jalili{at}m.cc.utah.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, ß1 and 
did not change. These results suggest that dietary fat type can alter PKC ß2 activation in the heart, and exert a mild hypertrophic effect on the heart.
KEY WORDS: • protein kinase C • heart • dietary fat
Protein kinase C (PKC),3 a family of 11 serine-threonine kinases, may play a central role in the modulation of cardiac hypertrophy in a variety of animal and cell culture models (1
–5
) through the modulation of transcription factors and gene expression (6
,7
). The cardiac-specific effects of two isoforms of PKC, ß2 and , have been investigated using transgenic mice with cardiac-specific overexpression. Both of these transgenic models possess a hypertrophic phenotype. PKC ß2 overexpression produces left ventricular dilation, fibrosis and cardiac failure, whereas PKC overexpression produces concentric hypertrophy and preserved contractile function (8
,9
). PKC is also thought to play a role in human cardiac hypertrophy and failure because humans with end-stage heart failure have increased translocation of cardiac PKC , ß1 and ß2 isoforms and elevated total PKC activity (10
). Taken together, cell culture, animal and human studies all indicate an important role for PKC in the development of cardiac hypertrophy.
PKC is activated through the guanine triphosphate regulatory binding protein q (Gq) coupled receptor. Hormones such as angiotensin, endothelin and phenylephrine bind to heterotrimeric guanine triphosphate regulatory binding protein (G protein) coupled receptors, causing a conformational shift and subsequent release of the Gq subunit. Gq can activate the membrane-associated enzyme phospholipase C ß1, which hydrolyzes membrane phospholipids to produce diacylglycerol and inositol triphosphate, both potent PKC activators. Many PKC isoforms can also be activated by free fatty acids (11
). Due to the link between lipids and PKC activation, dietary fat has been examined as a modulator of PKC activity in several tissue types and animal species. Feeding various types (saturated and unsaturated fat) and levels of fat in the diet of animals has produced alterations in PKC translocation and activation in a variety of tissue (12
–14
). Translocation of PKC, defined as redistribution of PKC from the cytosolic compartment to the membrane compartment, is concomitant with increased PKC activity and is generally used as an index of PKC activation. Given the role of PKC activation in mediating cardiac hypertrophy, coupled with previous literature reporting that dietary fat can modulate PKC activation, we investigated the association of dietary fat with PKC translocation and cardiac hypertrophy.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All protocols described were approved by the University of Utah Institutional Animal Care and Use Committee. To test the effect of fat source and amount on cardiac PKC activation, adult male Sprague-Dawley rats (n = 32; 250–275 g, 59–63 d old at delivery) were selected by random draw, placed into one of four groups (n = 8) and fed an AIN-93M purified diet that varied in fat source and amount (Harlan Teklad, Madison, WI) for a period of 40 d (15
). Diet treatment groups were initiated in a staggered fashion with a 2- to 3-d lag between each group. In this manner, not all rats had to be killed on the same day to maintain the 40-d treatment period. Dietary fat treatments consisted of the following: 1) 10% total energy intake (TEI) from corn oil defined as low fat unsaturated (LFU); 2) 40% TEI from corn oil defined as high fat unsaturated (HFU); 3) 10% TEI from coconut oil defined as low fat saturated (LFS); and 4) 40% TEI from coconut oil defined as high fat saturated (HFS). For coconut oil–based diets, a small amount of corn oil was included to attain a linoleic acid concentration 
1.3%, as specified for the AIN 93M diet. All diets maintained a 3.8:2.35:1 ratio of cornstarch/maltodextrin/sucrose. The low fat diets provided 15.15 kJ/g, whereas the high fat provided 18.5 kJ/g. An isoenergetic feeding protocol was used with metabolizable energy (ME) requirements calculated by the following formula: ME = 477 kJ (kg body wt)0.75. Rats were weighed every 3–4 d; adjustments to diet energy requirements were made as weight gain increased plus a small extra allowance to account for any food loss through the cage floor. Daily inspections usually found small (< 2 g) portions of diet remaining in feed dishes, indicating adequate provision of food. The micronutrient content was adjusted in high fat diets to ensure adequate micronutrient nutrition even though a smaller mass of energy-dense food was fed. Low fat diets contained 35 g/kg of mineral mix and 10 g/kg of vitamin mix. High fat diets contained 43.4 g/kg mineral mix and 12.4 g/kg vitamin mix.
After anesthesia with an intraperitoneal injection of 85:15 mg/kg body ketamine/xylazine, rats were killed by severing the aorta and removing the heart. Hearts were immediately rinsed in ice cold physiological saline, atria removed and weighed. Left ventricles were dissected out and also weighed. Hearts were then frozen in liquid nitrogen for subsequent PKC assessment.
Cardiac protein preparation and PKC immunoblotting.
Cardiac lysates containing cytosolic and membrane proteins were prepared as previously detailed (2
). All procedures and centrifugation were done at 4°C. Briefly, 0.3 g of the left ventricle was homogenized in 2 mL ice-cold buffer A (25 mmol/L Tris-HCl at pH 7.2 with 2 mmol/L EDTA, 5 mmol/L EGTA, 100 mmol/L NaF, and 10 µL/mL Sigma protease inhibitor cocktail) (Sigma, St. Louis, MO, cat. #P-8340) using a tissuemizer. Homogenates were centrifuged at 800 x g for 20 min; then the supernatant was removed and centrifuged at 100,000 x g to separate cytosolic and membrane-bound proteins. The supernatant was saved as the cytosolic cell fraction and used for PKC analysis. Pellets from previous centrifugation steps contained membrane-bound proteins and were rehomogenized in ice-bold buffer B (buffer A + 0.5% Triton X-100 and 5 mmol/L dithiothreitol), incubated on ice for 30 min and centrifuged at 100,000 x g for 1 h. The supernatant containing membrane-bound proteins was recovered and used for PKC analysis. The protein concentration of cell fractions was determined using a Bio-Rad Protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard.
PKC , ß1, ß2 and expression was assessed in cardiac membrane and cytosolic fractions using quantitative immunoblotting as previously detailed (2
). After electrophoresis and transfer to nitrocellulose membranes, membranes were stained with Ponceau S to confirm equal transfer efficiency and equal protein loading. Membranes were incubated with primary antibody directed against PKC , ß1, ß2 or (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C at a 1:1000 dilution, with secondary antibody conjugated to horseradish peroxidase for 1 h at 1:10,000 dilution, and then visualized using enhanced chemiluminescence (Amersham-Pharmacia, Piscataway, NJ). Quantification of relative density of immunoblots was carried out on a scanner using NIH 1.61 image software (NIH, Rockville, MD).
Statistical Analysis.
Two-way ANOVA was performed to detect differences between diet treatment groups using SPSS for Windows (Chicago, IL); significance was accepted at P 
0.05. The Least Significant Difference post-hoc test was used to detect individual group differences.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Initial body weights of LFS and HFS groups were greater than those of LFU and HFU groups because diet treatments were initiated in a staggered fashion as described (P < 0.01; Table 1
). After 14 d of diet consumption, rats fed the HFU diet had lower body weights compared with body weights from aged-matched Sprague-Dawley rats fed ad libitum (Table 1)
. After 28 d of diet consumption, rats fed HFU diets had body weights in line with reference values (Table 1)
. Rats consuming the LFS diet gained the least weight during the 40-d experimental period (P = 0.03) and had the lowest final body weight (P = 0.04) (Table 1)
.
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. However, the groups fed saturated fat did not differ (Table 1)
. PKC translocation.
PKC activation was assessed by quantitative immunoblot on cytosol and membrane fractions of ventricle homogenates. PKC isoform band density in membrane and cytosol fractions was measured and ratios of membrane to cytosol density calculated. No differences were detected in translocation of PKC , PKC ß1 or PKC among groups as evidenced by an unchanged membrane/cytosol ratio (Table 2
, Fig. 1
). Analysis of PKC ß2 translocation indicated significantly greater translocation in rats fed saturated fat than in those fed unsaturated fat (P < 0.01); however rats fed LFS and HFS did not differ (Table 2
, Fig. 1
).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The major finding of this study was increased translocation of PKC ß2 in rats fed dietary saturated fat from coconut oil. PKC ß2 was found to be activated in rats with cardiac hypertrophy induced by pressure overload and is thought to play an important role in mediating hypertrophy in rats and mice (3
,8
). In the present study, cardiac hypertrophy was observed in rats fed saturated fat, but was quite mild compared with more vigorous types of pathological insults such as pressure overload (hypertension) or myocardial infarction. To our knowledge, this is the first study demonstrating that dietary fat type, independent of pressure overload or myocardial infarction, can modulate PKC distribution in the heart. It was unexpected that within the two groups of rats fed different levels of saturated fat (10 and 40% of TEI), we did not observe any significant differences in PKC ß2 activation. These data suggest that the predominant fat type is more important for cardiac PKC activation than the overall amount consumed.
We attempted an isoenergetic feeding protocol among all diet groups to control for any possible effect of excess energy consumption upon heart size, particularly in the energy-dense high fat diets. After the first 4 d of dietary treatment, rats fed the LFS and HFU diets did not gain as much weight as expected. In the LFS group, this may have been due to the fragile nature of LFS feed pellets, which led to breakage and greater loss of food through the cage floor compared with the other diets. After a slight adjustment was made to compensate for the mass of the diet lost through the cage floor, rats of the LFS group gained weight normally and achieved final body weights in line with aged-matched rats consuming food ad libitum (Table 1)
. Rats fed HFU diets also had slower weight gain, but it did not appear to be due to food deprivation because daily inspections revealed small amounts of food left over in many feed dishes. It is possible that diet palatability may have been an issue when rats were switched from the standard AIN diet to the HFU diet. However, by d 28 of HFU diet consumption, these rats also achieved weights in line with reference values (Table 1)
. Rats from other treatment groups had normal body weights at all time points assessed.
With the exception of the present study, there are no studies to date that have examined the effect of dietary fat on PKC activation in the heart; however, several previous studies assessed PKC status in other tissue after feeding both saturated and unsaturated fat. Rats fed butter (
66% saturated fat) at 43% of energy intake had increased membrane PKC activity in the colonic mucosa (13
). Several other studies found that unsaturated fats can increase PKC activity. Epidermal PKC activity was increased in Sencar mice fed a diet containing 46% of total energy from corn oil compared with mice fed a control diet containing 11% of total energy from corn oil (12
). Hilakivi-Clarke and Clarke (16
) demonstrated that female Balb/c mice fed a diet containing 43% of total energy from corn oil had greater total PKC activity in mammary glands than mice fed a diet with 16% of total energy from corn oil. In rat skeletal muscle, a diet high in unsaturated fat (59% energy from safflower oil) increased PKC membrane translocation compared with controls; however, this was accompanied by a downregulation of PKC 
translocation (17
). Overall, the literature indicates that PKC activation can be modulated by feeding both saturated and unsaturated fats; however, the biological effect of PKC activation in these studies remains unknown. In the present study, we found that increased PKC ß2 translocation in the hearts of rats fed saturated fat was accompanied by mild cardiac hypertrophy, suggesting that this may be a possible biological effect of saturated fat consumption.
A limited number of studies have investigated the effect of dietary fat on the development of cardiac hypertrophy and have reported variable results. Additionally, these studies have not characterized cardiac PKC status. A recent study examining isolated cardiomyocytes from rats fed dietary fat based on lard and egg yolk reported increased myocyte volume and myocyte hypertrophy compared with controls (18
). It has also been reported that obesity prone Osborne-Mendel rats fed a high fat diet develop greater heart and ventricular weights than those fed standard diets (19
). This study, however, failed to normalize cardiac mass for the significant increase in body weight that occurred in rats given free access to the high fat, energy dense diet. An estimate of the heart/body weight ratio based on the reported data does not indicate that cardiac hypertrophy occurred in high fat–fed rats; however, this type of normalization may also be skewed due to the greater amount of body fat in Osborne-Mendel rats. In retrospect, normalization of heart weight for tibial length would have conclusively determined the presence of cardiac hypertrophy. Other animal models have also suggested a link between high fat diets and cardiac hypertrophy; rabbits fed a high fat diet for 12 wk had a significant increase in cardiac mass compared with controls fed low fat (20
). In contrast to both these findings and the current study, it was reported previously that rats fed 31% energy from a 2:1 saturated/unsaturated fat source had no increases in heart/body weight (21
). However, it should be noted that the fat type, amount and rat species were not the same as those used in the current study. These studies taken together with our current data, suggest that it is unclear whether dietary fat alone can induce cardiac hypertrophy. It seems likely that species-specific differences, genetic factors and the blend of saturated/unsaturated fat can affect dietary fat–induced cardiac hypertrophy.
In our study, a comparison of groups fed saturated and unsaturated fat indicated 6.6% greater heart/body weight in rats fed saturated fat for the 40-d experimental period. Although this is a significant increase in heart/body weight, it is mild compared with the hypertrophy produced by pathological insults such as pressure overload, produced by abdominal aortic constriction. Our laboratory has routinely found a 16% increase in heart/body after only 14 d of pressure overload in the same species of rat (data not shown). Further comparisons also indicate a dramatically greater degree of PKC ß2 translocation in pressure-overloaded rats compared with those fed saturated fat diets. Therefore, it appears that the mild degree of PKC ß2 translocation in the heart in rats fed saturated fat is in line with the resultant mild degree of cardiac hypertrophy.
The results of this study have implications for the development of cardiac hypertrophy in humans. In the current paradigm of hypertrophic signaling, PKC ß2 has been proposed to play a key role in mediating cardiac hypertrophy in response to pathologic stimuli such as pressure overload or myocardial infarction in rats and humans (3
,10
). Given the data in the current study, it remains unclear whether a diet rich in saturated fat in combination with a pathological stimulus, such as pressure overload, could exacerbate the degree of cardiac hypertrophy or increase the rate at which it develops. Future studies should investigate this possibility.
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FOOTNOTES |
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3 Abbreviations used: G protein, heterotrimeric guanine triphosphate regulatory binding protein; Gq, guanine triphosphate regulatory binding protein q; HFS, high saturated fat diet; HFU, high unsaturated fat diet; LFS, low saturated fat diet; LFU, low unsaturated fat diet; ME, metabolizable energy; PKC, protein kinase C; TEI, total energy intake.
Manuscript received 13 September 2002. Initial review completed 21 October 2002. Revision accepted 19 November 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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