Dietary Protein, but Not Carbohydrate, Is a Primary Determinant of the Onset of Stroke in Stroke-Prone Spontaneously Hypertensive Rats
Background and Purpose—Previously, an inverse association has been found between the dietary proportion of protein or fat and incidence of intracerebral hemorrhage. A positive association has been found with respect to carbohydrate intake. To examine what changes in macronutrient intake are causative, animal studies were conducted.
Methods—Stroke-prone spontaneously hypertensive rats (SHRSP) were fed diets with varying ratios of macronutrients ad libitum, and the onset of stroke was examined.
Results—When 10% of calories were from fat, rats fed a high-protein/low-carbohydrate diet (55% calories from protein) had a delayed onset of stroke, whereas rats fed a low-protein/high-carbohydrate diet (5% calories from protein) had an accelerated onset of stroke. When 30% of calories were from carbohydrate, a marked delay in the onset of stroke was observed when the diet was high in protein. When 85% of calories were from carbohydrate, rats fed 7.5% of calories as protein displayed an accelerated onset of stroke. When 20% of calories were from protein, increased fat content did not affect the onset of stroke. However, with a fat-free diet, when 20% of calories were from protein, the onset of stroke was delayed, whereas when 10% of calories were from protein, the onset of stroke was accelerated.
Conclusions—The amount of protein, but not of carbohydrate and fat, is a primary determinant of the onset of stroke. However, when calories from protein are relatively low in the diet (10%), fat is necessary to delay the onset of stroke in SHRSP.
In epidemiological studies, an inverse association was found between animal protein (or saturated fatty acid) intake and incidence of intraparenchymal hemorrhage.1,2 In a male cohort of Japanese descent residing in Hawaii, the percentage of calories consumed as fat was inversely associated with total mortality from stroke.3 In contrast, carbohydrate intake has been positively associated with an increased risk of hemorrhagic stroke.4 These findings are consistent with results from ecological studies; stroke has been a principal cause of death in eastern Asian countries, which have traditionally had a high-carbohydrate and low-protein/fat intake. However, because a diet high in carbohydrate is usually associated with a diet low in protein and fat, and a diet high in saturated fatty acid is associated with a diet high in animal protein, it is difficult to determine from epidemiological studies which primary diet is more likely to lead to hemorrhagic stroke.
The stroke-prone spontaneously hypertensive rat (SHRSP) is a unique genetic model of stroke, especially of hemorrhage and lacunar infarction, because of its severe hypertension.5 Necrosis of smooth muscle cell and dysfunction of endothelial cell in brain arteriole might be initial events that lead to stroke.6,7 These changes lead to intramural infiltration of blood plasma in arteriole (hyaline and fibrinoid degeneration), which thickens the arterial wall (remodeling), narrows the lumen, and leads to occlusion and rupture. Around the lesions, brain edema develops.8 Because similar pathological changes were observed in hypertensive intracerebral hemorrhage in human,9,10 SHRSP is a useful model for studying environmental factors that render the wall of cerebral arteries vulnerable to prolonged hypertension.
Materials and Methods
SHRSP/Izm (male) 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 a laboratory chow (CE-2: CLEA Japan Inc) and water until 10 weeks of age. At 10 weeks of age, animals were fed differing diets of specific nutrient composition (described in each experiment) ad libitum and given drinking water supplemented with 1% NaCl. Each dietary group comprised 8 animals, and each animal was kept in a separate steel cage. 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 Incs. The detailed composition of the control diet (caloric composition: 20% protein, 10% fat, and 70% carbohydrate from total calories, #12450B Research Diet) is listed in Table 1. A group of rats fed the control diet was included with each experiment for comparison. Soybean oil contained 10.4% palmitic acid (16:0), 3.4% stearic acid (18:0), 24.3% oleic acid (18:1n-9), 0.1% palmitoleic acid (16:1n-7), 53.5% linoleic acid (18:2n-6), and 7.8% α-linolenic acid (18:3n-3). In total 5 independent experiments were conducted. In the first experiment, the caloric proportion from fat was fixed at 10% of total calories, and the effects of the proportion of protein and carbohydrate calories examined; in the second and third experiments, carbohydrate amount was fixed at 30% or 85% of calories, respectively, and the effects of protein and fat proportion were examined; in the fourth and fifth experiments, the protein proportion was fixed at 20% or 10% calories, respectively, and the effect of decreasing fat proportion was examined.
Food Intake, Body Weight, and Blood Pressure
Food intake and body weight were measured before and 1 week after initiation of the experimental diet, then measured every day during experimental periods. The mean food intake per week was estimated by subtracting the remaining weight of food each week from the initial weight of the previous week. Blood pressure was measured at before and 1 week after initiation of the experimental diet in the warmed, conscious, restrained state by using the photoelectric oscillometric tail-cuff method using a BP-98A machine (Softron Co Ltd). At each time point, at least 3 readings were obtained for each rat and the mean of these were used as the value.
Onset of Stroke
Onset of stroke was assessed by the appearance of neurological symptoms (hyperkinetic/hypokinetic behaviors, hyper/hyposensitivity [manipulation], or lift of proleg, piloerection) and physiological changes (decreasing body weight, decreased food intake, or increased drinking water intake). In most cases, these neurological symptoms and physiological changes in SHRSP are manifestations of cerebral lesions.11 Two people checked these symptoms at 10:00 am everyday.
At autopsy, the brain was fixed with 10% buffered formalin solution, sliced into 4 coronal sections, embedded in paraffin, cut into 4-μm-thick sections, and stained with hematoxylin-eosin. Lesions of infarction, hemorrhage, or edema were classified into 5 degrees: very slight (+), slight (++), mild (+++), severe (++++), or very severe (+++++).
Data are presented as the mean±SEM. Statistical analysis was conducted using 1-way ANOVA with Bonferroni post hoc testing for parametric data (Statview 5.0, Abacus Concepts). For the onset of stroke and survival curves, a Kaplan–Meier curve was obtained, and the comparison of groups was performed using the Log-Rank test. A probability value <0.05 was considered statistically significant.
Delayed Onset of Stroke in Rats Fed a High-Protein/Low-Carbohydrate Diet, and Accelerated Onset of Stroke in Rats Fed a Low-Protein/High-Carbohydrate Diet
To examine whether the amount of protein in the diet affects the onset of stroke, SHRSP were fed diets with varying amounts of protein (and a concomitant change in carbohydrate proportion) with fat intake fixed at 10% of calories (42.7 g/kg diet, Figure 1A). Fat amount was fixed at 10% of consumed calories because soybean oil is used at a concentration of 40 g/kg of feed diet (AIN 93M) for maintenance of rats.12 The onset of stroke was dependent on the amount of dietary protein (Figure 1B). Compared with rats fed a diet where 20% of calories were supplied as protein (control diet), rats fed a high-protein/low-carbohydrate diet (55% and 45% of calories as protein) showed a marked delay in the onset of stroke (P<0.001 and P=0.009, respectively), whereas rats fed a low-protein/high-carbohydrate diet (5% of calories as protein) showed an accelerated onset of stroke (P=0.004). Rats fed a diet of 20% of calories as protein (control) and 10% calories as protein showed a similar onset of stroke. The survival rate followed that for the onset of stroke (Figure 1C).
Food intake in the first 1 week (before onset of stroke) was significantly lower by 12% and 8% in rats fed 55% and 45% of calories as protein, respectively, than in control rats fed 20% of calories as protein (Table 2). In a preliminarily study, a reduction in energy intake by 25% did not affect the onset of stroke (T. Chiba and O. Ezaki, unpublished observation, 2008). At the day of the stroke, a marked decrease in food intake was observed, and this decrease continued until death for each group of rats. Therefore, mean food intake and body weight in each group throughout the study were affected by incidence of stroke (Figure 1D and 1E). There was no significant difference in blood pressure before the onset of stroke between groups (Table 2).
Rats that died were autopsied to determine the location and nature of the cerebral lesions (Table 3). Almost all rats showed cerebral lesions except 1 rat (Rat No. 1 in 20% protein). In rats fed 5% of calories as protein, 63% (5/8) of them showed substantial hemorrhage (more than +++) and 50% (4/8) of them edema, whereas in rats fed 55% and 45% of calories as protein, the incidence of substantial hemorrhage and edema was less (29% and 14% , respectively). In addition, subarachnoidal hemorrhage was observed only in rats fed 5% calories as protein (Rat Nos. 1, 3, and 7 in Table 3). There is no difference in the incidence of infarction between groups. Hyaline and fibrinoid degeneration and thickening in the arterial wall were observed in each group of rats (data not shown).
Effect of Altering the Carbohydrate Proportion of Calories in the Diet on the Onset of Stroke
When changing the protein proportion of calories in the diet with a fixed fat proportion, the total energy intake was adjusted by the proportion of carbohydrate. Therefore, it is conceivable that the proportion of carbohydrate may affect the onset of stroke. To examine which nutrient (protein or carbohydrate) was responsible for the accelerated onset of stroke, SHRSP were fed on different proportions of protein (with a concomitant change in fat proportion) with carbohydrate proportion fixed at 30% or 85% of calories (Figure 2A and 2B).
With calories from carbohydrate fixed at 30%, rats fed a high-protein/low-fat diet (60% of calories as protein) showed a marked delay in the onset of stroke, relative to rats fed the control diet (Figure 2A). However, when fed 20% of calories as protein, the onset of stroke did not differ between rats fed 70% of calories as carbohydrate (control diet) or 30% of calories as carbohydrate, suggesting that decreasing carbohydrate to 30% calories or increasing fat to 50% calories did not affect the onset of stroke. The survival rate followed that for the onset of stroke. Rats fed 20% of calories as protein (50% of calories as fat) showed an increase in the total energy intake and body weights, compared with control rats (Table 2). This might be attributable to increased fat intake. The total energy intake in rats fed a high-protein diet was slightly less than control rats, as observed in Figure 1.
When calories from carbohydrate were fixed at 85% of intake, rats fed 7.5% of calories as protein showed acceleration in the onset of stroke, relative to those fed 15% calories as protein, but this result was not significant (P=0.126; Figure 2B). The survival rate followed that for the onset of stroke. The total energy intake in rats fed 7.5% of calories as protein was slightly larger than control rats (Table 2).
Effect of a Fat-Free Diet on Onset of Stroke
The proportion of fat had no effect (within a range of 10% to 50% of calories) on the onset of stroke (Figure 2A). However, very low or absence of dietary fat may affect the onset of stroke. Because the amount of calories supplied as protein is a primary determinant of the onset of stroke, the effects of reduced fat content with 2 protein diets (20% [control] and 10% of calories as protein), in which the onset of stroke was not altered, were examined.
With calories from protein fixed at 20%, rats were fed at 10% (control), 5%, or 0% (fat-free diet) of calories as fat (Figure 3A). Rats fed a fat-free diet showed a marked delay in the onset of stroke compared with rats fed other diets (P=0.002, fat-free versus control; P=0.006, fat-free versus 5% fat). There was no difference in the onset of stroke between rats fed 10% and 5% of calories as fat (P=0.489). The survival rate followed that for the onset of stroke. Although rats fed a fat-free diet showed a slight increase in energy intake, they decreased their body weight relative to control rats (the reason for this is unclear).
However, when rats were fed 10% of calories as protein (Figure 3B), rats fed a fat-free diet showed an accelerated onset of stroke compared to rats fed 10% of calories as fat (P=0.015, fat-free versus 10% fat). Rats fed a diet of 20% of calories as protein (control) and 10% of calories as protein showed a similar onset of stroke, as shown in Figure 1. The survival rate followed that for the onset of stroke. Rats fed 5% of calories as fat showed a slight decrease in energy intake and body weight relative to control rats. Histological examination revealed that rats fed a fat-free diet showed an increased incidence of severe hemorrhage (4/8) compared to rats fed 10% of calories as fat (2/8) (data not shown).
In ad libitum feeding or iso-energy feeding conditions, when the amount of a specific macronutrient is changed to examine its effects, the amounts of the other macronutrients are obligatorily changed. Therefore, it is difficult to examine the specific effects of one macronutrient. To discern the macronutrients responsible for an increased incidence of stroke, the amount of one macronutrient was fixed, and the effect of different ratios of the other two macronutrients on stroke incidence compared. It was found that the proportion of protein in the diet, but not that of carbohydrate or fat, was a primary determinant of the onset of stroke. However, when protein proportion was low (10% of calories), dietary fat was required to delay the onset of stroke.
Previously, it was reported that SHRSP fed a Japanese rat diet revealed a much higher incidence of stroke than those fed an American rat diet.13 As the most important difference in the 2 diets was the protein content, it has been proposed that the increased protein proportion present in the American rat diet could prevent stroke in SHRSP, although fat and carbohydrate proportions that also differ were not considered.13 In humans, in the Nurses’ Health Study, animal protein intake or saturated fatty acid intake was inversely associated with intraparenchymal hemorrhage, and the excess risk associated with low saturated fat intake was observed primarily among women with a history of hypertension.1 In the same cohort, high carbohydrate intake was associated with an elevated risk of hemorrhagic stroke.4 However, the former study was not adjusted for the proportion of diet ingested as carbohydrate, and the latter study was not adjusted for the proportion of diet ingested as animal protein (or saturated fatty acids) by using Cox proportional hazards regression analysis. Although stroke in SHRSP under severe hypertension (induced by overloading of NaCl) may not be entirely relevant to stroke in humans, an increased incidence of hemorrhagic stroke observed in subjects with high carbohydrate intake might still be attributable to a lower intake of protein.
Substantial hemorrhage (including subarachnoidal hemorrhage) and edema in rats fed 5% of calories as protein were observed more frequently than for other groups of rats. Although the time when lesions occurred cannot be known as no preliminary sacrifice was made of animals, it was reported that pathological changes of autopsied brains were similar to those observed just after stroke.8 More severe forms of stroke might have occurred in rats fed a low protein diet. Although the association of protein intake and incidence of subarachnoidal hemorrhage have not been examined in humans, a lower intake of protein may increase incidence of subarachnoidal hemorrhage. High carbohydrate intake (maybe associated with low protein intake) was associated with an elevated risk of hemorrhagic stroke, in which both subarachnoidal and intracerebral hemorrhage are included.4
In this study casein and soybean oil were used as the sources of protein and fat, respectively, because casein and soybean oil are standard nutritional sources for rats.12 Casein was used because of its good balance of amino acids, and soybean oil was used for its abundance of essential fatty acids.12 However, other protein or fat sources might have different effects on the incidence in stroke. It was reported that among SHRSP fed various fatty acids, a diet containing 1% palmitoleic acid significantly prevented stroke, possibly by maintaining the metabolic integrity of the vascular smooth muscle.14 Because the soybean oil used in this study contain very low amounts of palmitoleic acid (0.1%), the effect of palmitoleic acid might not have been observed under the experimental conditions (even under high-fat diet). It was reported that some kinds of vegetable oils (canola oil, rapeseed oil, and olive oil) and a partially-hydrogenated oil shorten the survival of SHRSP, relative to perilla oil, soybean oil, lard, safflower oil, and butter, possibly due to minor components of the oils.15,16 It is possible that some specific oils may affect stroke incidence. However, in this study the replacement of soybean oil with butter (saturated fat rich oil) in control diets did not affect the onset of stroke (results not shown).
In summary, this current study clearly shows that a low protein diet is one of the causes of stroke in SHRSP. Conversely, a high-protein diet delayed the onset of stroke. These data in SHRSP concur well with previous epidemiological observations that a low-protein, low-fat, and high-carbohydrate diet is associated with an increased risk of hemorrhagic stroke. This study provides a rat model of stroke, induced by the interaction of hypertension and diet, and also suggests that in addition to control of blood pressure, a low-protein diet might be avoided for prevention of stroke in hypertensive patients.
The authors thank Lisa Shiraishi for assistance with care of animals and Yasumitsu Akahoshi for technical assistance with pathological analyses.
Sources of Funding
This work was supported in part by a grant-in-aid for scientific research Kakenhi from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT, Tokyo, Japan); by research grants from the Japanese Ministry of Health, Labor, and Welfare; and by a grant for the Promotion of Fundamental Studies in Health Sciences from the Organization for Pharmaceutical Safety and Research (OPSR, Osaka, Japan).
- Received April 16, 2009.
- Accepted April 27, 2009.
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