Delay of Stroke Onset by Milk Proteins in Stroke-Prone Spontaneously Hypertensive Rats
Background and Purpose—There is an inverse association between dairy food consumption and the incidence of stroke in observational studies. However, it is unknown whether the relationship is causal or, if so, what components in milk are responsible for reducing the incidence of stroke.
Methods—Stroke-prone spontaneously hypertensive rats were fed diets comprising amino acids, proteins from different sources (casein, whey, soybean, or egg white), or fats from different sources (butter, beef tallow, or cocoa butter) and the onset of stroke and lifespan were examined.
Results—Increasing the amount of dietary casein (5% to 55% of caloric intake) markedly delayed the onset of stroke. However, when stroke-prone spontaneously hypertensive rats were fed diets containing 55% of caloric intake as protein, rats fed casein or whey protein, a major component of milk, displayed a delayed onset of stroke compared with rats fed soybean or egg white protein. Rats fed an amino acids diet containing the same amino acids composition as casein did not have a delay in the onset of stroke. Increasing dietary fats, including butter as well as beef tallow and cocoa butter, did not affect the onset of stroke. All diets did not affect blood pressure in the early stage.
Conclusions—These data suggest that the inverse association between dairy food consumption and incidence of stroke in epidemiological studies is causal and that peptides in milk protein, but not fat, might be responsible for this effect.
The consumption of milk and dairy food is associated with a significant reduction in stroke incidence. A recent meta-analysis of prospective cohort studies showed that the relative risk in subjects with the highest dairy food consumption relative to those with the lowest intake was 0.79 (0.68–0.91) for mostly ischemic stroke and 0.75 (0.60–0.94) for hemorrhagic stroke.1 Although ischemic and hemorrhagic stroke have different prevalences in different genetic and environmental backgrounds,2 milk consumption seems to reduce the incidence in both subtypes of stroke. However, because there is no large-scale intervention study, it is unknown whether the consumption of milk and dairy food is causative in the reduction of the incidence of both types of stroke. Other confounding factors such as differences in lifestyles of milk drinkers may reduce the incidence of stroke.
If we assumed that milk consumption could prevent stroke, the component(s) in milk, by which the incidence of stroke is reduced, has not been identified. Calcium in milk has been considered as a candidate mineral by reducing blood pressure; however, studies have shown no association between nondairy calcium intake and total stroke, intraparenchymal hemorrhage, and subarachnoid hemorrhage.3,4
In our previous study, increased dietary intake of casein delayed the onset of stroke in stroke-prone spontaneously hypertensive rats (SHRSP).5 SHRSP is a unique genetic model of stroke, especially of hemorrhage and lacunar infarction, due to its severe hypertension.6 Necrosis of smooth muscle cells and dysfunction of endothelial cells in brain arterioles might be initial events that lead to stroke.6,7 These changes lead to intramural infiltration of blood plasma in arterioles (hyaline and fibrinoid degeneration), which thickens the arterial wall (remodeling), narrows the lumen, and leads to occlusion and rupture.7 In our experimental conditions, the SHRSP rat showed both hemorrhage and infarction (28 of 38) and hemorrhage only (6 of 38); however, there were no rats that showed only infarction.5 These data suggested that brain hemorrhage was a primary cause of death of SHRSP; however, infarction to which arterial occlusion might lead was also accompanied by hemorrhage. Because similar pathological changes were observed in hypertensive intracerebral hemorrhage in humans,8 SHRSP is a useful model for studying environmental factors that render the wall of cerebral arteries vulnerable to prolonged hypertension.
To examine whether increasing milk intake may cause a reduction in the incidence of stroke, SHRSP were fed milk components (casein, whey protein, butter) and its metabolites (urea) and the onset of stroke was monitored.
Materials and Methods
Male SHRSP/Izm were purchased from Japan SLC Inc (Shizuoka, Japan) and maintained under specific pathogen-free conditions in a temperature-controlled room (22±2°C) with a 12-hour light/dark cycle. Animals had free access to laboratory chow (CE-2; CLEA Japan Inc, Tokyo, Japan) and water until 10 weeks of age. At 10 weeks of age, animals were fed diets of various protein or fat compositions (described in each experiment) ad libitum and given drinking water supplemented with 1% NaCl. Each dietary group comprised 7 to 8 animals, and each animal was kept in a separate steel cage to allow measurement of food intake. There was no dropout of animal numbers, and all animals were included in survival analysis. Food intake and body weight were measured daily at 10:00 am. Blood pressure was measured as described previously.5 All animal experiments were conducted with the approval of the National Institute of Health and Nutrition Ethics Committee on Animal Research.
All diets were purchased from Research Diet Inc (New Brunswick, NJ). The detailed composition of the control diet (caloric composition: 20% protein, 10% fat, and 70% carbohydrate from total calories) was described previously.5 Four independent experiments were conducted. In Experiment 1, effects of diets containing 5%, 20%, and 55% of total calories as casein (described as 5%, 20%, 55% casein diet) or soybean protein were examined. An independent experiment of 20% and 55% casein diets was conducted to measure changes in blood pressure during the experimental periods. In Experiment 2, the effects of various proteins (casein, whey, soybean, or egg white) at 55% of total calories were examined. An independent experiment of the same diet was conducted to examine inflammatory markers in brain. In Experiment 3, the effects of an amino acids diet that contains the same amino acids composition as a 55% casein diet were examined. In Experiment 4, effects of various fats (butter, beef tallow, or cocoa butter) were examined. Amino acids composition in proteins and fatty acids composition in fats are shown in online-only Supplemental Tables 1 and 2, respectively (http://stroke.ahajournals.org).
Please see Supplemental Methods at http://stroke.ahajournals.org.
Delayed Onset of Stroke in Rats by Increasing Dietary Casein Compared With Soybean Protein (Experiment 1)
Previously, we reported that protein (casein), rather than carbohydrate (starch and sucrose) and fat (soybean oil), was a primary determinant of the onset of stroke in SHRSP.5 To examine whether other proteins could delay the onset of stroke similarly to casein, SHRSP were fed varying amounts of casein or soybean protein (and a concomitant change in carbohydrate proportion) with fat intake fixed at 10% calories. As observed in the previous study, rats fed casein diets showed a marked delay in the onset of stroke (55% diet: P<0.001, 20% diet: P=0.016 versus 5% diet) and extended lifespan (55%: P<0.001, 20%: P=0.012 versus 5%) by increasing the amount of protein intake (Figure 1A). Rats fed soybean protein diets also had a delayed onset of stroke (55%: P=0.018, 20%: P=0.299 versus 5%); however, the effect of soybean protein was much weaker than casein (Figure 1B). At 55% calories, rats fed a casein diet showed a significant delay in the onset of stroke (P=0.019) and extended lifespan (P=0.036) compared with rats fed a soybean protein diet. When fed a 55% soybean protein diet, rats showed a decrease in food intake but did not change in body weight and blood pressure compared with other diets at 1 week after initiation of experiments (Table 1
To examine in detail the contribution of blood pressure to a delay in the onset of stroke in response to a 55% casein diet, an independent experiment of 2 groups of rats fed a 20% casein diet (control) or 55% casein diet was conducted to measure blood pressure before and every 1 week after the initiation of the experimental diet. Ten rats were used in each group; however, rats that died or showed any sign of stroke were excluded. Systolic blood pressure in rats fed a 20% casein diet (control) was 185±3 mm Hg (mean±SEM, n=10) before the initiation of the experimental diet, 194±3 mm Hg (n=10) at 1 week after the initiation of the experimental diet, 210±4 mm Hg (n=9) at 2 weeks, and 229 mm Hg (n=2) at 3 weeks, whereas those in rats fed a 55% casein diet were 184±3 mm Hg (n=10) before the initiation of the experimental diet, 192±2 mm Hg (n=10) at 1 week after the initiation of the experiment, 205±5 mm Hg (n=10) at 2 weeks, 215±5 mm Hg (n=9) at 3 weeks, 222±7 mm Hg (n=6) at 4 weeks, 221±6 mm Hg (n=3) at 5 weeks, and 226 mm Hg (n=2) at 6 weeks (Figure 1C). In the 2 weeks after the initiation of the experimental diet (comparable number of rats remained in control group), there was no significant difference in blood pressure between 20% casein diet and 55% casein diet (P=0.625 at 1 week, P=0.466 at 2 weeks). However, blood pressure was elevated with increasing feeding time in rats fed a 55% casein diet (P<0.001 before the initiation of the experimental diet versus 4 week after the initiation of the experimental diet).
These data suggest that dietary protein composition affects the onset of stroke in SHRSP independent of blood pressure.
Effects of Dietary Urea on the Onset of Stroke
Excess dietary protein is metabolized to urea in the liver. Therefore, we investigated whether increasing plasma urea concentrations might be a cause of delayed onset of stroke. As expected, fasting plasma urea concentrations at 1 week after initiation of experiments increased with both casein and soybean protein intake and to the same extent with both sources ([casein] 5%, 1.8±0.2; 20%, 5.4±0.4; 55%, 10.1±0.4; [soybean] 5%, 2.7±0.2; 20%, 5.1±0.2; 55%, 11.2±0.6 mmol/L, n=8). Considering there was a marked difference in the onset of stroke between casein and soybean protein, these data suggest that increasing urea concentration is unlikely to cause the delayed onset of stroke in response to casein.
To directly assess this issue, urea was administered orally and its effect on the onset of stroke was examined. SHRSP were fed the 5% casein diet (stroke-accelerated condition) augmented with 0%, 1%, or 5% (w/w) urea. After 1 week, rats fed urea showed increased plasma urea concentrations in a dose-dependent manner (0%, 1.8±0.2; 1%, 3.5±0.3; 5%, 5.9±0.6 mmol/L). Rats fed 5% (w/w) urea reached a similar plasma urea concentration to that of rats fed a 20% casein diet (5.4±0.4 mmol/L). However, dietary urea did not delay the onset of stroke (1%: P=0.958, 5%: P=0.804 versus 0% urea; data not shown). These data confirm that plasma urea is not a cause of the delayed onset of stroke in response to increased casein intake.
Delayed Onset of Stroke in Rats Fed Milk Proteins (Casein and Whey) Compared With Soybean and Egg White Proteins (Experiment 2)
At 55% calories, casein more effectively delayed the onset of stroke than soybean protein. To examine the effects of various dietary proteins on the onset of stroke, SHRSP were fed 55% calories as casein, whey, soybean, or egg white protein and the onset of stroke was monitored. Among dietary proteins, rats fed egg white protein showed slightly decreased food intake and body weight but did not differ in blood pressure compared with rats fed other proteins (Table 1). Rats fed casein or whey protein had delayed onset of stroke compared with rats fed soybean or egg white protein. The order of delaying effect is soybean <egg white ≪casein <whey (Figure 2A; online-only Supplemental Table 3). The order of survival rate is largely similar to the onset of stroke, whereby soybean <egg white=casein <whey (Figure 2B; online-only Supplemental Table 3).
An autopsy was conducted to determine the nature of cerebral lesions (Table 2). Hemorrhage was found to be greater in frequency and severity than infarction regardless of protein source. This data are consistent with the previous report that hemorrhage is the major type of stroke in SHRSP.5 However, mild and severe hemorrhage lesions (more than ++) were fewer in casein (6 instances in 8 rats) and whey protein (3 instances in 4 rats) than soybean (11 instances in 8 rats) and egg white (11 instances in 7 rats) proteins. Although the time when lesions occurred cannot be known because no preliminary euthanasia was made of animals, it was reported that pathological changes of autopsied brains were similar to those observed just after stroke.9 If we assumed that a similar trend of histological abnormality might occur in lived rats, these data suggested that milk proteins delay the onset of stroke by attenuating the severity of hemorrhage but not decreasing the incidence of hemorrhage or infarction.
In our previous study, dietary restriction was shown to suppress inflammation and delay the onset of stroke in SHRSP.10 However, there were no significant alterations in blood concentrations of inflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1) and monocyte chemoattractant protein-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 mRNA levels in cerebrovascular endothelial cells between dietary groups (online-only Supplemental Figure 1).
Effect of Amino Acids Diet on the Onset of Stroke (Experiment 3)
There are some differences in amino acids composition between dietary proteins (online-only Supplemental Table 1). To examine whether amino acid composition is responsible for the delayed onset of stroke in response to increasing casein, rats were fed an amino acid diet that contained the same amino acid composition of casein and were compared with rats fed casein diets. Rats fed the 55% amino acids diet showed suppression of body weight gain compared with rats fed a 20% casein diet but did not differ in blood pressure (Table 1). Rats fed the 55% casein diet showed a delay in the onset of stroke (P=0.002), but rats fed the 55% amino acids diet did not show a delay in the onset of stroke (P=0.069) compared with rats fed a 20% casein diet (Figure 3). These data suggest that the amino acid composition of casein is not causative of the delay in the onset of stroke in response to an increase in casein.
Effects of Fat (Butter) on the Onset of Stroke (Experiment 4)
Milk fat (butter) and other dietary fats that contain saturated fatty acids might delay the onset of stroke as suggested by epidemiological studies.11–14 To examine this possibility, the onset of stroke was monitored in SHRSP fed 50% of total calories as butter, beef tallow, or cocoa butter (all saturated fatty acid-rich) with 20% and 30% of total calories as casein and carbohydrate, respectively. For control, rats were fed 10%, 20%, and 70% total calories as butter, casein, and carbohydrate, respectively. Although the amount of dietary carbohydrate was different between test and control diets, it was shown in our previous study that the amount of dietary carbohydrate did not affect the onset of stroke.5 There was no significant difference in the onset of stroke and lifespan between rats fed 50% butter, beef tallow, and cocoa butter (Figure 4A–B). In addition, rats fed 50% fats appeared to have an accelerated onset of stroke compared with control rats, although not with statistical significance. These results indicate that increasing milk fat (butter) and other saturated-rich fats did not delay the onset of stroke in SHRSP.
Previously, we showed using casein that dietary protein was a primary determinant of the onset of stroke in SHRSP, a rat model of hemorrhagic stroke.5 The effects of casein were dose-dependent, ranging from 5% to 55% of calories, indicating that it may be applicable to prevent stroke in humans by increasing milk protein intake. In this report, we showed that milk protein (casein and whey) significantly delayed the onset of stroke compared with soybean and egg white proteins. Increasing milk fat (butter) as well as beef tallow and cocoa butter did not delay the onset of stroke. These data suggest that increasing milk protein intake, but not milk fat, is a cause of a delay in the onset of hemorrhagic stroke. However, it is unclear whether milk protein could delay the incidence of ischemic stroke.
Major epidemiological studies of Japanese populations suggested that a high consumption of animal protein and fat was associated with a reduced risk of stroke, largely confined to intraparenchymal hemorrhage.11,13,14 However, the results of large cohort studies in the United States are inconsistent. The Nurses' Health Study showed low intakes of saturated fat and animal protein were associated with increased risk of intraparenchymal hemorrhage and that the excess risk was confined to hypertensive women,12 whereas the Health Professionals Follow-Up Study did not show a significant association between total, animal, or vegetable protein and risk of stroke (total, ischemic, or hemorrhagic stroke) in this population of US men.15 These studies focused on animal protein and fats, but not on milk and dairy products. It will be interesting to see whether the Health Professional Follow-Up Study observes a reduction in intraparenchymal hemorrhage associated with milk consumption, as observed in other studies.1
In 2004, the aged-standardized cerebrovascular mortality rate in Japan was 42.2 per 100 000 population, whereas that in the United States was 30.4 (death rate standardized to the World Health Organization World Standard population).16 According to statistics in 2008 in Japan, mortality due to intracerebral hemorrhage was 26% of mortality by total stroke,17 whereas in the United States in 1995 to 1998, it was 14% and 19% in women and men, respectively.18 The average milk intake in the Japanese population is approximately one third that of the United States. The mean intake of total dairy products (mostly milk) in Japanese people <20 years of age in 2007 was 97 g/day.19 In the United States, the National Health and Nutrition Examination Survey (NHANES, 1999–2004) showed that the mean intake of total dairy products >18 years of age was 247 g/day.20 Lower consumption of milk in Japan may be a cause of the larger mortality rate of stroke, especially hemorrhagic stroke, relative to the United States.
Milk contains many nutrients, including carbohydrates, proteins, fats, minerals, and vitamins.21 It is unclear which nutrients are responsible for reducing the incidence of stroke. Our study of SHRSP suggests that milk proteins, casein, and whey protein could reduce the incidence of stroke. Rats fed urea, the metabolite of milk proteins, or amino acids themselves did not show a delay in the onset of stroke, suggesting that peptides in milk proteins might be causative of a delay in the onset of stroke. Bioactive peptides such as isoleucine-proline-proline and valine-proline-proline are known as antihypertensive peptides isolated from milk fermented with Lactobacillus helveticus and inhibit angiotensin-converting enzyme.22 However, addition of these peptides (0.01% w/w each) in a control casein diet did not delay the onset of stroke in SHRSP in our experimental conditions (Chiba T and Ezaki O, unpublished data, 2011).
Mechanistically, the delayed onset of stroke in response to milk protein may not be due to the suppression of systemic and local inflammation, which were observed in energy-restricted conditions.10 It is also unlikely that a reduction of blood pressure is a cause of delayed onset of stroke in response to milk protein, because there was no significant change in blood pressure until 2 weeks after the initiation of the experimental diet between the 20% casein diet and the 55% casein diet; rather, blood pressure gradually increased after initiation of 55% casein feeding. Currently, the mechanisms of delayed onset of stroke in response to milk protein are unknown.
Taken together, these rodent model data suggest that the inverse association between dairy food consumption and the incidence of stroke in epidemiological studies is causal. Further studies are warranted to identify the active component(s) in milk and to elucidate their mechanisms.
Milk protein, but not butter, delayed the onset of stroke in SHRSP. The data presented here suggest that an inverse association between dairy food consumption and the incidence of stroke in epidemiological studies is causal, and peptides in milk protein might be responsible for this effect. Further interventional studies to investigate increased milk consumption and active protein components are warranted.
Sources of Funding
This work was supported in part by research grants from the Skylark Food Science Institute; and by a grant-in-aid for scientific research (Kakenhi) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT, Tokyo, Japan).
We thank Yasumitsu Akahoshi for technical assistance with pathological analyses.
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.618496/-/DC1.
- Received February 21, 2011.
- Revision received October 25, 2011.
- Accepted October 26, 2011.
- © 2012 American Heart Association, Inc.
- Umesawa M,
- Iso H,
- Ishihara J,
- Saito I,
- Kokubo Y,
- Inoue M,
- et al.
- Abbott RD,
- Curb JD,
- Rodriguez BL,
- Sharp DS,
- Burchfiel CM,
- Yano K
- Chiba T,
- Itoh T,
- Tabuchi M,
- Satou T,
- Ezaki O
- Tagami M,
- Nara Y,
- Kubota A,
- Sunaga T,
- Maezawa H,
- Fujino H,
- et al.
- Takebayashi S,
- Kaneko M
- Iso H,
- Sato S,
- Kitamura A,
- Naito Y,
- Shimamoto T,
- Komachi Y
- Iso H,
- Stampfer MJ,
- Manson JE,
- Rexrode K,
- Hu F,
- Hennekens CH,
- et al.
- Sauvaget C,
- Nagano J,
- Allen N,
- Grant EJ,
- Beral V
- Sauvaget C,
- Nagano J,
- Hayashi M,
- Yamada M
- Preis SR,
- Stampfer MJ,
- Spiegelman D,
- Willett WC,
- Rimm EB
World Health Organization. WHO global infobase [online]. Available at: https://apps.who.int/infobase/comparisons.aspx. Accessed February 9, 2011.
Health and Welfare Statistics Association. Death rate by a cause of death, age, and sex [in Japanese]. Journal of Health and Welfare Statistics. 2010/2011;59:400.
- Ayala C,
- Croft JB,
- Greenlund KJ,
- Keenan NL,
- Donehoo RS,
- Malarcher AM,
- et al.
Ministry of Health Labour and Welfare. The National Health and Nutrition Survey in Japan, 2007 [in Japanese]. Tokyo: Daiich Shuppan Publishing; 2010.
- Beydoun MA,
- Gary TL,
- Caballero BH,
- Lawrence RS,
- Cheskin LJ,
- Wang Y
- Ohsawa K,
- Satsu H,
- Ohki K,
- Enjoh M,
- Takano T,
- Shimizu M