Cod and soy proteins compared with casein improve glucose tolerance and insulin sensitivity in rats

Charles Lavigne1,2, André Marette2,3, and Hélène Jacques1,2

1 Department of Food Science and Nutrition, Human Nutrition Research Group, 3 Department of Anatomy and Physiology, Laval University, Ste-Foy, Québec G1K 7P4; and 2 Lipid Research Centre, Laval University, Ste-Foy, Québec, Canada, G1V 4G2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to determine the effects of feeding various dietary proteins on insulin sensitivity and glucose tolerance in rats. Male Wistar rats were fed for 28 days with isoenergetic diets containing either casein, soy protein, or cod protein. Cod protein-fed and soy protein-fed rats had lower fasting plasma glucose and insulin concentrations compared with casein-fed animals. After intravenous glucose bolus, cod protein- and soy protein-fed rats induced lower incremental areas under glucose curves compared with casein-fed animals. Improved peripheral insulin sensitivity was confirmed by higher glucose disposal rates in cod protein- and soy protein-fed rats (15.2 ± 0.3 and 13.9 ± 0.6 mg · kg-1 · min-1, respectively) compared with casein-fed animals (6.5 ± 0.7 mg · kg-1 · min-1, P < 0.05). Moreover, test meal experiments revealed that, in the postprandial state, the lower plasma insulin concentrations in cod protein- and soy protein-fed animals could be also due to decreased pancreatic insulin release and increased hepatic insulin removal. In conclusion, the metabolic responses to three common dietary proteins indicate that cod and soy proteins, when compared with casein, improve fasting glucose tolerance and peripheral insulin sensitivity in rats.

triglycerides; intravenous glucose tolerance test; test meal; hyperinsulinemic-euglycemic clamp.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN RESISTANCE is characterized by an abnormally low response of the target cells to insulin, inducing high plasma insulin levels (6) and hypertriglyceridemia (27). Several studies have demonstrated that the macronutrient composition of the diet is an important determinant of insulin sensitivity. Although most studies have examined the role of high-fat (4, 13, 21, 23, 33, 35), low-soluble fiber (3) or high-sucrose diets (36) in the development of an impaired insulin action, relatively few studies have focused on the impact of dietary proteins. Up to now, a high-protein intake (60% of energy) has been reported to impair glucose metabolism in peripheral and hepatic tissues (29), but little information is available concerning the effect of different dietary protein sources. In this respect, when compared with casein, soy protein has been shown to decrease serum insulin concentrations in fasted normoglycemic rats (39). Moreover, postprandial studies in rats (12), and in humans (16), showed a reduced postprandial insulin response to a single test meal containing soy protein compared with casein. Iritani et al. (19) further reported that dietary soy protein, in a saturated high-fat diet, may help to improve insulin sensitivity by increasing insulin receptor mRNA levels in liver and adipose tissues. Interestingly, cod protein has also been shown to reduce fasting plasma glucose compared with casein in normoglycemic rats (17). Based on previous studies cited above, our working hypothesis was that cod and soy proteins exert beneficial effects on glucose tolerance, on peripheral insulin sensitivity, and on postprandial plasma glucose and insulin responses in rats maintained on controlled diets for a long-term period.

To test this hypothesis, rats were fed controlled diets containing either casein, cod, or soy protein for 28 days. Various parameters of glucose tolerance and insulin sensitivity were measured during 1) an intravenous glucose tolerance test (IVGTT), 2) a hyperinsulinemic-euglycemic clamp, and 3) a test meal. Physiological curve responses of plasma C-peptide, glucagon, and triglycerides were also determined after the test meal.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Wistar rats (Charles River, St. Constant, QC, Canada) weighing ~240 g on arrival were individually housed in wire-mesh cages in a temperature- and humidity-controlled room with a daily dephased 12:12-h light-dark cycle (lights on at 2200 to 1000). Upon arrival, all rats were fed a grounded nonpurified commercial diet (Purina rat chow; Ralston Purina, Lasalle, QC, Canada) for at least 6 days. At the end of this baseline period, rats were divided into three groups of the same average weights. Purified diets and tap water were provided ad libitum for 28 days. Food intake was estimated every day by subtracting the food spillage weight from the initial food weight, and body weight was measured weekly. The animal facilities met the guidelines of the Canadian Council on Animal Care, and the protocol was approved by the Animal Care Committee of Laval University.

Diets. After the baseline period, all animals were assigned to one of the three purified powdered diets varying in protein source, namely casein, cod protein, soy protein, and were fed for 28 days. The composition of each purified diet is detailed in Table 1. Those diets have been recognized to induce higher fasting plasma glucose and cholesterol in casein-fed rats compared with cod protein- and soy protein-fed rats (17). All diet ingredients, except vitamin mix (Teklad, Madison, WI) and cod protein, were purchased from ICN (Cleveland, OH). Cod protein was prepared in our laboratory by freeze-drying cod fillets, followed by a 24-h delipidation using diethylether as solvent in a Soxhlet-type apparatus (Canadawide Scientific, Montreal, QC, Canada). Ingredients for the purified diets were mixed and stored at -20°C until used. The energy content of the diets was measured with an automatic adiabatic calorimeter (model 1241; Parr Instruments, Moline, IL). Diets were found to be isoenergetic in the casein (19.91 kJ/g), soy protein (19.95 kJ/g), and cod protein (19.66 kJ/g) diets. The protein content (N × 6.25) was determined with a Kjeldahl Foss autoanalyzer (model 1612; Foss, Hillerod, Denmark). The level of protein in the purified diets was adjusted to an isonitrogenous basis at the expense of carbohydrates.

                              
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Table 1.   Composition of the purified diets

Experimental protocols. At day 25, rats were cannulated via the jugular vein (IVGTT and test meal experiments) and the carotid artery (hyperinsulinemic-euglycemic clamp experiment) under isoflurane anesthesia. Food intake was near normal postoperatively, and all rats were within 4% of surgery weight on the day of the study. Blood samplings were carried out in a 15 × 30-cm open plastic box to which rats were accustomed and in which they remained undisturbed during the experiment. Experiments 1, 2, and 3 were evaluated in separate groups of animals.

Experiment 1: IVGTT. At day 28, after a 12-h fast, 10 rats/dietary group were injected with 1.5 ml/kg body wt of a 35% glucose solution dissolved in saline as a bolus via the jugular catheter. The catheter was then flushed with saline. Blood samples (300 µl) were drawn through the catheter with EDTA-containing syringes (1.5 g/l blood) before (0 min) and 2, 5, 10, 20, and 50 min after the glucose load and were stored on ice. The plasma was separated by centrifugation and was stored at -80°C until analysis. All erythrocytes were pooled, resuspended in saline, and injected in the animals after the 20- and 50-min samples.

Experiment 2: Hyperinsulinemic-euglycemic clamp and in vivo 2-deoxy-D-glucose uptake. Whole body insulin action was determined by the hyperinsulinemic-euglycemic clamp procedure, as described previously (30). Briefly, rats were fasted overnight, transferred to a quiet isolated room, and weighed. Unrestrained conscious animals were allowed to rest for 40 min before the first blood sample (300 µl). A continuous intravenous infusion of purified human insulin (Humulin R; Eli Lilly, Indianapolis, IN) was then started at the rate of 4.0 mU · kg-1 · min-1 and was continued for 140 min with a syringe pump (Razel, Stamford, CT) to achieve plasma insulin concentrations in the physiological range. Dextrose solution (25%) was infused through the venous line at a variable rate to maintain blood glucose at the initial value. Blood samples (40 µl) were taken from the carotid artery catheter at 5-min intervals to monitor plasma glucose concentrations using an Elite glucometer (Bayer, Etobicoke, ON). Every 20 min, an additional 300 µl of blood was withdrawn for later determination of plasma insulin levels. Erythrocytes were suspended in saline and reinjected into the animals to prevent a fall in the hematocrit and minimize stress.

Insulin action within in vivo individual muscle was determined as described previously (30). Briefly, the nonmetabolizable glucose analog 2,6-[3H]2-deoxy-D-glucose (2-[3H]DG) and D-[14C]sucrose (NEN Du Pont, Boston, MA) were administered together in an intravenous bolus 20 min before the end of the clamp. Blood samples were drawn at 5, 7.5, 10, 12.5, 15, 17.5 and 20 min after bolus administration for determination of radiolabeled 2-[3H]DG and [14C]sucrose. The plasma concentrations of 2-[3H]DG after the single injection were plotted on a semilogarithmic scale, and the rate of 2-[3H]DG disappearance from plasma was calculated from the slope obtained by a linear regression analysis, as described previously (34, 40). At the completion of the clamp, rats were rapidly killed by decapitation, and the red gastrocnemius muscle was rapidly removed, frozen in liquid nitrogen, and stored at -80°C for subsequent analysis. Tissue samples (50-100 mg) were dissolved in 1 ml of Solvable (NEN) at 55°C for 16 h. Thereafter, hydrogen peroxide (30% solution) was added to decrease quenching, followed by the addition of 8 ml of scintillation fluid (BCS; Amersham, Mississauga, ON, Canada). The accumulation of 2-[3H]DG in muscle, corrected for extracellular space with [14C]sucrose, was used as an index of glucose uptake rates, as described by others (34, 40).

Experiment 3: Test meal. The experimental diet and jugular cannulation protocols were similar to those described in the IVGTT protocol. At day 28, after a 12-h fast, and at the beginning of the dark period, 10 rats/dietary group received 5 g of their assigned experimental diet for 30 min. After that time, any uningested food was removed. Blood samples were obtained before the beginning of the test meal (-30 min) and at 0 (end of the meal), 30, 60, 120, and 240 min. Blood samples for C-peptide and glucagon determinations (300 µl) were collected in tubes containing 250 kallikrein inhibitor units (Trasylol; Miles, Etobicoke, ON, Canada) at -30, 30, and 120 min only because of limited amounts of blood volumes. The plasma was separated by centrifugation and stored at -80°C until further analysis. All erythrocytes were pooled, resuspended in saline, and reinjected in the animals after the 30- and 120-min samples.

Analytical methods. Plasma glucose levels were analyzed using a glucose oxidase method (YSI 2700 Select; Yellow Springs Instruments, Yellow Springs, OH), and plasma insulin, C-peptide, and glucagon levels were measured with an RIA method (Linco Research, St. Charles, MO) using rat insulin, C-peptide, and glucagon standards. Triglycerides were assayed by an enzymatic method using a reagent kit from Boehringer Mannheim (Montreal, QC, Canada). Incremental areas under the curves obtained during IVGTT and test meals were calculated with a computer graphic program with 0- and -30-min time points as baseline values, respectively. IVGTT insulin response areas were distinguished as the first phase (0-10 min) and second phase (10-50 min) postinjection. 3H and 14C activities in aliquots of plasma and of dissolved tissue samples were determined by a liquid scintillation counter (Wallach 1409) using a dual-label counting program.

Statistical analyses. Data were analyzed with the general linear model program on the SAS statistical package for personal computers. Data obtained from serial sampling were analyzed using ANOVA with repeated measures, with time as the repeated variable. Physiological parameters, food intake, hepatic insulin extraction, and incremental areas under the curve were analyzed using one-way ANOVA. Individual between-group comparisons were performed using Duncan's new multiple range test after the ANOVA. Differences were considered significant at P < 0.05. All results are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Table 2 shows the amino acid composition of the tested dietary proteins. Casein contained the highest amounts of proline, tyrosine, and valine, whereas cod protein contained more alanine and lysine. Notably, the arginine level of cod and soy protein was two times that of casein. The levels of glycine and aspartic acid were also higher in soy protein and cod protein than in casein. The sum of branched-chain amino acids (leucine, isoleucine, and valine) was higher in casein than in cod and soy proteins, whereas the sum of essential amino acids was higher in the animal proteins, casein and cod protein, than in soy protein. Moreover, the lysine-to-arginine ratio was higher in casein (2.4) than in soy protein (0.9) and cod protein (1.5).

                              
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Table 2.   Amino acid composition of protein sources

After 28 days of dietary treatment, rats displayed comparable daily food intake and body weight gain regardless of the protein source (Table 3). The food intake for the last meal (experiment 3, test meal) was similar between the protein groups. After 4 wk of feeding, fasting plasma glucose and insulin were lower in cod protein- and soy protein-fed rats than in casein-fed rats (10 and 50%, respectively, P < 0.05; Table 3).

                              
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Table 3.   Physiological parameters and food intake of rats fed the purified diets

Plasma glucose and insulin responses and incremental areas under the glucose and insulin curves during IVGTT are shown in Fig. 1. Cod and soy protein diets resulted in significantly (P < 0.05) lower plasma glucose 10 and 20 min after the intravenous glucose load compared with the casein diet (Fig. 1A). Cod (24%, P < 0.05) and soy (22%, P < 0.05) proteins induced smaller incremental areas under the glucose curves than casein (Fig. 1B). Rats fed cod protein, when compared with casein, displayed a lower insulin response (P < 0.05) to the glucose load during the late-phase insulin secretion (10-50 min; Fig. 1C). Soy protein-fed rats displayed an intermediate response. However, the total incremental areas under the insulin curves (Fig. 1D) were similar between protein groups. We next divided the insulin response during IVGTT into an acute phase (first phase; 0-10 min) and a late phase (second phase; 10-50 min) to further dissect out beta -cell function (5). No significant differences were found between the experimental groups during the first phase (casein, 0.24 ± 0.04 arbitrary units; cod protein, 0.29 ± 0.03 arbitrary units; soy protein, 0.24 ± 0.04 arbitrary units). However, the second-phase incremental areas under the insulin curves were significantly higher in the casein group compared with the cod protein group (0.22 ± 0.04 vs. 0.07 ± 0.02 arbitrary units, respectively, P < 0.05), whereas soy protein-fed rats showed an intermediate response (0.15 ± 0.05 arbitrary units).


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Fig. 1.   A: changes in plasma glucose concentrations in the fasting state and after iv glucose bolus in rats fed either casein, cod protein, or soy protein diet for 28 days. B: glucose area responses to intravenous glucose tolerance tests (IVGTT) in arbitrary units. C: changes in plasma insulin concentrations in the fasting state and after iv glucose bolus in rats fed either casein, soy protein, or cod protein diet. D: total insulin area responses to IVGTT. Groups bearing different letters for a given time point are significantly different (P < 0.05). Areas are significantly different (P < 0.05) if they do not share a common letter. Values are means ± SE.

To see whether the improved glucose tolerance of cod protein- and soy protein-fed rats was associated with an increased peripheral insulin sensitivity, whole body and peripheral tissue glucose utilization was next measured during an hyperinsulinemic-euglycemic clamp. As depicted in Fig. 2A, both cod protein- and soy protein-fed rats displayed increased insulin sensitivity compared with rats fed casein. Plasma 2-[3H]DG disappearance rate was also higher in cod protein-fed (34%, P < 0.05) and soy protein-fed (32%, P < 0.05) rats than in casein-fed rats (Fig. 2B). In accordance with increased peripheral action of insulin in cod protein- and soy protein-fed rats, higher rates of insulin-stimulated glucose uptakes were observed in red gastrocnemius muscle of these rats (472 ± 26 and 412 ± 18 nmol · min-1 · g-1, respectively) compared with casein-fed animals (213 ± 24 nmol · min-1 · g-1, P < 0.05).


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Fig. 2.   A: glucose disposal rate (GDR) to maintain euglycemia during steady-state (60-120 min) insulin infusion in fasting state. B: plasma 2-deoxy-D-[3H]glucose disappearance rate (Kp). Rats were fed either casein, cod, or soy protein diet during 28 days. Bars represent means ± SE of data obtained from 3-4 rats/group. Groups without common letter differ at P < 0.05.

Figure 3 shows fasting and postprandial plasma glucose and insulin responses and incremental areas under the glucose and insulin curves of plasma collected before and after the test meal in animals fed the diets for 4 wk. Postprandial plasma glucose reached a peak response after 1 h (Fig. 3A), and the incremental areas under the glucose curves after the test meal (Fig. 3B) were similar regardless of the protein consumed. Postprandial plasma insulin concentrations were lower (P < 0.05) in soy protein-fed rats than in casein-fed rats at several time points (30, 60, and 120 min) after the test meal. Postprandial plasma insulin concentrations were lower (P < 0.05) in cod protein-fed rats compared with casein-fed rats immediately after the test meal (0 min) and 30 and 60 min after the test meal (Fig. 3C). The incremental areas under the insulin curves were significantly lower (P < 0.05) with cod protein (25%) and soy protein (35%) than with casein (Fig. 3D).


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Fig. 3.   A: changes in plasma glucose concentrations in the fasting state and after the test meal in rats fed either casein, cod protein, or soy protein diet for 28 days. Meals consisted of ~5 g of their current diet. B: glucose area responses to the test meal in arbitrary units. C: changes in plasma insulin concentrations in the fasting state and the test meal in rats fed either casein, soy protein, or cod protein diet. D: insulin area responses to the test meal in arbitrary units. Groups bearing different letters for a given time point are significantly different (P < 0.05). Areas are significantly different (P < 0.05) if they do not share a common letter. Values are means ± SE.

The plasma C-peptide curve responses are illustrated in Fig 4A. In the fasting state (-30 min), C-peptide concentrations were lower in rats fed soy (23%, P < 0.05) and cod proteins (30%, P < 0.05) compared with rats fed casein. In the postprandial state, plasma C-peptide concentrations were lower (28%, P < 0.05) at both 30 and 120 min after the test meal in soy protein- compared with casein-fed rats. Plasma C-peptide concentrations were intermediate at these two time points in cod protein-fed rats. The incremental areas under the C-peptide curves were similar whatever the dietary proteins consumed (Fig. 4B). Hepatic insulin extraction, as estimated by the molar C-peptide-to-insulin ratio, was significantly higher in the cod and soy protein groups in the fasting state and 30 min after the test meal than in the casein group (Table 4).


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Fig. 4.   A: changes in plasma C-peptide concentrations in the fasting state and after the test meal in rats fed either casein, soy protein, or cod protein diet for 28 days. B: plasma C-peptide area responses to the test meal in arbitrary units. C: changes in plasma glucagon concentrations in the fasting state and after the test meal in rats fed either casein, soy protein, or cod protein diet. D: plasma glucagon area responses to the test meal in arbitrary units. Groups bearing different letters for a given time point are significantly different (P < 0.05). Areas are significantly different (P < 0.05) if they do not share a common letter. Values are means ± SE.


                              
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Table 4.   Hepatic insulin extraction (C-peptide/insulin molar ratio) in rats fed experimental diets for 28 days

Plasma levels of the counterregulatory hormone glucagon are shown in Fig 4C. Fasting glucagon concentrations were comparable among protein groups. After the ingestion of cod and soy protein meals, there was no significant increase in plasma glucagon concentration. In rats fed casein, the postprandial glucagon concentrations increased with a peak after 30 min, which was 26% (P < 0.05) higher than that observed in soy protein-fed animals. Two hours after the test meal, the glucagon response was lower in cod protein- and soy protein-fed rats than in casein-fed rats by 20 and 25%, respectively (P < 0.05). The incremental area under the glucagon curve was greater with casein than with soy protein (P < 0.05; Fig. 4D). It is also noteworthy that the insulin-to-glucagon ratio was significantly (P < 0.05) higher in rats fed casein than in those fed cod or soy proteins before and 30 min after the test meal (Fig. 5A).


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Fig. 5.   Changes in plasma insulin-to-glucagon ratios (A) and plasma triglyceride concentrations (B) in the fasting state and after the test meal in rats fed either casein, soy protein, or cod protein diet for 28 days. Groups bearing different letters for a given time point are significantly different (P < 0.05). Values are means ± SE.

Plasma triglyceride responses are shown in Fig. 5B. In the fasting state, cod protein- and soy protein-fed rats had lower (30 and 25%, respectively, P < 0.05) plasma triglyceride concentrations compared with casein-fed rats. In the postprandial state, plasma triglyceride concentrations were lower 120 min after the test meal in cod protein- and soy protein-fed rats (25 and 40%, respectively, P < 0.05) than in casein-fed rats, whereas 240 min after the test meal, plasma triglycerides were lower only in soy protein-fed rats compared with casein-fed rats (37%, P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study shows that cod and soy proteins improve glucose tolerance and insulin sensitivity compared with casein in rats, as determined by indexes of glucose tolerance and insulin sensitivity during IVGTT, the test meal, and by direct measurement of peripheral insulin action using the hyperinsulinemic-euglycemic clamp technique.

Our observation that cod and soy protein diets produce lower fasting plasma glucose and insulin concentrations than the casein diet is consistent with previous results obtained in our laboratory (17) and with data published by Vahouny et al. (39), who showed lower serum insulin concentrations in fasted rats fed soy protein than in those fed casein. The reduction in both fasting glucose and insulin levels in cod- and soy protein-fed rats suggests improvement of insulin sensitivity. Similarly, the lower magnitude of the postprandial insulin response to cod and soy protein feeding in the present study is in good agreement with data from Hubbard and Sanchez (16) who reported lower blood insulin levels in humans fed a soy protein meal compared with those fed a casein meal. However, in the present study, the postprandial experiment did not allow us to distinguish between chronic (fasting) and acute (postprandial) effects of the different protein diets because the same diets were used for the acute test meal. However, the purpose of the test meal experiments was to examine the glucose, insulin, C-peptide, and glucagon responses to the dietary proteins under usual feeding conditions.

Our results strongly suggest that the reductions of plasma insulin concentrations in cod- and soy protein-fed rats during the IVGTT and the test meal were to a large extent associated with an enhanced peripheral insulin sensitivity, as assessed by the hyperinsulinemic-euglycemic clamp technique. The greater whole body insulin action, 2-[3H]DG disappearance rate, and skeletal muscle 2-[3H]DG uptake in cod- and soy protein-fed rats clearly indicate that those rats displayed improved peripheral insulin sensitivity compared with casein-fed rats. These results further suggest that skeletal muscle, the main site of insulin-stimulated peripheral glucose disposal, is a key target for dietary protein action.

On the other hand, the greater glucoregulation in rats fed cod or soy protein could also be attributed to either decreased pancreatic insulin release or increased hepatic insulin extraction. On the one hand, insulin and C-peptide are secreted in equimolar amounts by the pancreas (14), but, in contrast to insulin, very little C-peptide is catabolized by the liver (9), allowing determination of pancreatic insulin secretion. In the present study, a lower C-peptide peak response was observed after the soy protein meal than after the casein meal, suggesting a decrease in insulin secretion in the former. On the other hand, simultaneous assessment of C-peptide and insulin concentrations in peripheral blood enabled us to estimate hepatic insulin removal, as calculated from the C-peptide-to-insulin molar ratio (8). Hepatic insulin extraction was higher before (fasting state) and 30 min after the test meal in cod- and soy protein-fed rats than in casein-fed rats (Table 4). Therefore, it appears that, in the postprandial state, both a lower insulin secretion and a higher hepatic insulin extraction may have contributed to reduce plasma insulin concentrations in rats fed cod and soy proteins. Additional features, such as glucose absorption from the gut and non-insulin-mediated glucose uptake, could also be involved in the increased glucose tolerance and insulin sensitivity observed in rats fed cod and soy proteins.

Hypertriglyceridemia often accompanies the development of insulin resistance and impaired glucose tolerance associated with high-sucrose diets (2), but their causal relationship is still unclear (28). Insulin influences both the rate of hepatic triglyceride synthesis and subsequent very low density lipoprotein (VLDL) triglyceride secretion in the circulation and the rate of triglyceride disappearance from the blood stream through its action on lipoprotein lipase (LPL) activity (15). In the present study, feeding cod and soy proteins resulted in lowered fasting and postprandial circulating triglyceride concentrations. These results are in good agreement with those published in a study from our laboratory (17) and others (18, 39) showing the hypolipidemic effect of soy protein compared with casein in rats. According to Beynen and Sugano (1), increased insulin sensitivity, such as observed in our rats fed cod and soy proteins, may decrease tissue fatty acid mobilization and, in turn, decrease synthesis and secretion of VLDL triglycerides from the liver, reducing plasma triglyceridemia. Interestingly, Demonty et al. (7) recently demonstrated that LPL activity was lower in skeletal muscle of rats fed cod and soy proteins than of those fed casein. This is expected to decrease fatty acid availability at the muscle cells and thus reduce the ratio of fat to glucose oxidation in these cells (26). It is therefore possible that feeding cod or soy protein can reduce the supply of lipids and improve muscle insulin sensitivity compared with casein. However, whether the reduction of plasma triglycerides was the cause or the result of improved insulin action in cod- and soy protein-fed rats remains to be investigated.

Alterations in glucagon levels could contribute to the diet-induced changes in insulin sensitivity and triglyceridemia. Indeed, postprandial glucagon concentrations were higher in the casein group than in cod protein- and soy protein-fed animals, suggesting that the daily glucagon concentrations, which are more often in the postprandial state, could induce higher hepatic glucose output and higher fasting glucose levels (11). In addition, according to Hubbard and Sanchez (16), a high insulin-to-glucagon ratio can be considered as an early indicator of glucose intolerance. The present results corroborate this notion, since the insulin-to-glucagon ratio was higher in rats fed casein than in those fed cod or soy protein before and 30 min after the test meal.

The mechanisms by which cod and soy proteins improve glucose tolerance and insulin sensitivity are still unclear. Differences in the amino acid composition of dietary proteins have been proposed to mediate protein-dependent changes in glucose and insulin dynamics (31, 37). An early report (10) suggested that essential amino acids, either individually or in combination, can stimulate the pancreatic release of insulin. In that report (10), arginine given alone was the most potent stimulus for the release of insulin. However, at concentrations found in dietary proteins, arginine has been rather associated with a decrease of fasting insulin levels (37). This is in contrast with supraphysiological doses of arginine, which have been associated with increases in both insulin and glucagon concentrations (10, 24). Mulloy et al. (22) demonstrated that a diet containing 1.0% arginine, which is in close agreement with what is found in the cod protein (1.3%) and soy protein (1.5%) diets, induces lower plasma insulin concentrations 30 and 45 min after an IVGTT than a diet containing 0.5% arginine, which is closely equivalent to the arginine level found in our casein diet (0.7%; see Ref. 20). Furthermore, Vahouny et al. (39) showed that the addition of arginine to a casein diet, to mimic the lysine-to-arginine ratio of soy protein, resulted in serum fasting insulin levels similar to those measured in rats given a soy protein diet.

More recent reports (25, 38) further suggest that amino acids can diminish insulin's ability to stimulate peripheral glucose transport. Indeed, infusion of branched-chain amino acids (leucine, isoleucine, valine), which are predominantly metabolized in skeletal muscles, has been shown to inhibit insulin-mediated glucose uptake in the forearm muscle (32). Interestingly, the present study shows that the amount of branched-chain amino acids is slightly higher in casein than in cod and soy proteins. However, the mechanism by which dietary proteins induce insulin resistance at the cellular level is not yet well understood. Patti et al. (25) have proposed that a mixture of 20 amino acids can inhibit critical early steps in postreceptor insulin action for glucose transport, including decreased insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2, reduced binding of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2, and inhibition of insulin-stimulated phosphatidylinositol 3-kinase activity. It is therefore possible that specific amino acids of dietary proteins regulate skeletal muscle insulin sensitivity for glucose disposal by directly modulating the insulin signaling pathway.

In summary, the results of the present study show that, when compared with casein, soy protein and cod protein improve fasting and postprandial plasma insulin responses in rats. The beneficial effects of these proteins on glucose and insulin dynamics appear to be largely explained by an improved insulin sensitivity, as shown by an increased insulin action in skeletal muscle.


    ACKNOWLEDGEMENTS

We thank Roodly Archer and Christine Hurley for technical assistance.


    FOOTNOTES

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to H. Jacques) and the Canadian Diabetes Association (to H. Jacques and A. Marette). A. Marette was supported by scholarships from the Medical Research Council of Canada and the Fonds de la Recherche en Santé du Québec.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. Jacques, Dép. des sciences des aliments et de nutrition, FSAA, Pavillon Paul-Comtois, Université Laval, Ste-Foy, Québec, Canada G1K 7P4 (E-mail: helene.jacques{at}aln.ulaval.ca).

Received 8 March 1999; accepted in final form 11 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Beynen, A. C., and M. Sugano. Dietary protein as a regulator of lipid metabolism: state of the art and new perspectives. J. Nutr. Sci. Vitaminol. (Tokyo) 36, Suppl2: S185-S188, 1990[ISI][Medline].

2.   Boivin, A., and Y. Deshaies. Dietary rat models in which the development of hypertriglyceridemia and that of insulin resistance are dissociated. Metabolism 44: 1540-1547, 1995[ISI][Medline].

3.   Braaten, J. T., F. W. Scott, P. J. Wood, K. D. Riedel, M. S. Wolynetz, D. Brule, and M. W. Collins. High beta-glucan oat bran and oat gum reduce postprandial blood glucose and insulin in subjects with and without type 2 diabetes. Diabet. Med. 11: 312-318, 1994[ISI][Medline].

4.   Chisholm, K. W., and K. O'Dea. Effect of short-term consumption of a high fat diet on glucose tolerance and insulin sensitivity in the rat. J. Nutr. Sci. Vitaminol. (Tokyo) 33: 377-390, 1987[ISI][Medline].

5.   Collier, G. R., G. R. Greenberg, T. M. Wolever, and D. J. Jenkins. The acute effect of fat on insulin secretion. J. Clin. Endocrinol. Metab. 66: 323-326, 1988[Abstract].

6.   DeFronzo, R. A. Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidaemia and atherosclerosis. Neth. J. Med. 50: 191-197, 1997[ISI][Medline].

7.   Demonty, I., Y. Deshaies, and H. Jacques. Dietary proteins modulate the effects of fish oil on triglyceridemia in the rat. Lipids 33: 913-921, 1998[ISI][Medline].

8.   Faber, O. K., K. Christensen, H. Kehlet, S. Madsbad, and C. Binder. Decreased insulin removal contributes to hyperinsulinemia in obesity. J. Clin. Endocrinol. Metab. 53: 618-621, 1981[Abstract].

9.   Faber, O. K., H. Kehlet, S. Madsbad, and C. Binder. Kinetics of human C-peptide in man. Diabetes 27: 207-209, 1978[ISI][Medline].

10.   Fajans, S. S., J. C. Floyd, Jr., R. F. Knopf, and F. W. Conn. Effect of amino acids and proteins on insulin secretion in man. Recent Prog. Horm. Res. 23: 617-662, 1967[Medline].

11.   Felig, P., J. Wahren, R. Sherwin, and R. Hendler. Insulin, glucagon, and somatostatin in normal physiology and diabetes mellitus. Diabetes 25: 1091-1099, 1976[Abstract].

12.   Galibois, I., H. Jacques, C. Montminy, N. Bergeron, and C. Lavigne. Independent effects of protein and fibre sources on glucose and cholesterol metabolism in the rat. Nutr. Res. 12: 643-655, 1992[ISI].

13.   Harris, R. B., and H. Kor. Insulin insensitivity is rapidly reversed in rats by reducing dietary fat from 40 to 30% of energy. J. Nutr. 122: 1811-1822, 1992[ISI][Medline].

14.   Horwitz, D. L., J. I. Starr, M. E. Mako, W. G. Blackard, and A. H. Rubenstein. Proinsulin, insulin, and C-peptide concentrations in human portal and peripheral blood. J. Clin. Invest. 55: 1278-1283, 1975[ISI][Medline].

15.   Howard, B. V., and W. J. Howard. Dyslipidemia in non-insulin-dependent diabetes mellitus. Endocr. Rev. 15: 263-274, 1994[ISI][Medline] (published erratum appears in Endocr. Rev. 15: 438, 1994).

16.   Hubbard, R. W., and A. Sanchez. Dietary protein control of serum cholesterol by insulin and glucagon. Monogr. Atheroscler. 16: 139-147, 1990[Medline].

17.   Hurley, C., I. Galibois, and H. Jacques. Fasting and postprandial lipid and glucose metabolisms are modulated by dietary proteins and carbohydrates: role of plasma insulin concentrations. J. Nutr. Biochem. 6: 540-546, 1995[ISI].

18.   Iritani, N., H. Hosomi, H. Fukuda, K. Tada, and H. Ikeda. Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats. J. Nutr. 126: 380-388, 1996[ISI][Medline].

19.   Iritani, N., T. Sugimoto, H. Fukuda, M. Komiya, and H. Ikeda. Dietary soybean protein increases insulin receptor gene expression in Wistar fatty rats when dietary polyunsaturated fatty acid level is low. J. Nutr. 127: 1077-1083, 1997[Abstract/Free Full Text].

20.   Jacques, H., Y. Deshaies, and L. Savoie. Relationship between dietary proteins, their in vitro digestion products, and serum cholesterol in rats. Atherosclerosis 61: 89-98, 1986[ISI][Medline].

21.   Kraegen, E. W., P. W. Clark, A. B. Jenkins, E. A. Daley, D. J. Chisholm, and L. H. Storlien. Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats. Diabetes 40: 1397-1403, 1991[Abstract].

22.   Mulloy, A. L., F. W. Kari, and W. J. Visek. Dietary arginine, insulin secretion, glucose tolerance and liver lipids during repletion of protein-depleted rats. Horm. Metab. Res. 14: 471-475, 1982[ISI][Medline].

23.   Pagliassotti, M. J., P. A. Prach, T. A. Koppenhafer, and D. A. Pan. Changes in insulin action, triglycerides, and lipid composition during sucrose feeding in rats. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 271: R1319-R1326, 1996[Abstract/Free Full Text].

24.   Palmer, J. P., R. M. Walter, and J. W. Ensinck. Arginine-stimulated acute phase of insulin and glucagon secretion. I. in normal man. Diabetes 24: 735-740, 1975[Abstract].

25.   Patti, M. E., E. Brambilla, L. Luzi, E. J. Landaker, and C. R. Kahn. Bidirectional modulation of insulin action by amino acids. J. Clin. Invest. 101: 1519-1529, 1998[Abstract/Free Full Text].

26.   Randle, P. J., P. B. Garland, C. N. Hales, and E. A. Newsholme. The glucose fatty-acid cycle its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1: 785-789, 1963[ISI].

27.   Reaven, G. Hypertriglyceridemia in the central feature of syndrome X. Cardiovasc. Risk Factors 6: 29-35, 1996[ISI].

28.   Reaven, G. M., C. E. Mondon, Y. D. Chen, and J. L. Breslow. Hypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene are neither insulin resistant nor hyperinsulinemic. J. Lipid Res. 35: 820-824, 1994[Abstract].

29.   Rossetti, L., D. L. Rothman, R. A. DeFronzo, and G. I. Shulman. Effect of dietary protein on in vivo insulin action and liver glycogen repletion. Am. J. Physiol. Endocrinol. Metab. 257: E212-E219, 1989[Abstract/Free Full Text].

30.   Roy, D., M. Perreault, and A. Marette. Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Am. J. Physiol. Endocrinol. Metab. 274: E692-E699, 1998[Abstract/Free Full Text].

31.   Sanchez, A., and R. W. Hubbard. Plasma amino acids and the insulin/glucagon ratio as an explanation for the dietary protein modulation of atherosclerosis. Med. Hypotheses 35: 324-329, 1991[ISI][Medline].

32.   Schwenk, W. F., and M. W. Haymond. Effects of leucine, isoleucine, or threonine infusion on leucine metabolism in humans. Am. J. Physiol. Endocrinol. Metab. 253: E428-E434, 1987[Abstract/Free Full Text].

33.   Sevilla, L., A. Guma, G. Enrique-Tarancon, S. Mora, P. Munoz, M. Palacin, X. Testar, and A. Zorzano. Chronic high-fat feeding and middle-aging reduce in an additive fashion Glut4 expression in skeletal muscle and adipose tissue. Biochem. Biophys. Res. Commun. 235: 89-93, 1997[ISI][Medline].

34.   Shibata, H., F. Perusse, A. Vallerand, and L. J. Bukowiecki. Cold exposure reverses inhibitory effects of fasting on peripheral glucose uptake in rats. Am. J. Physiol. Regulatory Integrative Comp. Physiol. 257: R96-R101, 1989[Abstract/Free Full Text].

35.   Storlien, L. H., L. A. Baur, A. D. Kriketos, D. A. Pan, G. J. Cooney, A. B. Jenkins, G. D. Calvert, and L. V. Campbell. Dietary fats and insulin action. Diabetologia 39: 621-631, 1996[ISI][Medline].

36.   Storlien, L. H., E. W. Kraegen, A. B. Jenkins, and D. J. Chisholm. Effects of sucrose vs starch diets on in vivo insulin action, thermogenesis, and obesity in rats. Am. J. Clin. Nutr. 47: 420-427, 1988[Abstract].

37.   Sugano, M., N. Ishiwaki, and K. Nakashima. Dietary protein-dependent modification of serum cholesterol level in rats. Significance of the arginine/lysine ratio. Ann. Nutr. Metab. 28: 192-199, 1984[ISI][Medline].

38.   Traxinger, R. R., and S. Marshall. Role of amino acids in modulating glucose-induced desensitization of the glucose transport system. J. Biol. Chem. 264: 20910-20916, 1989[Abstract/Free Full Text].

39.   Vahouny, G. V., I. Adamson, W. Chalcarz, S. Satchithanandam, R. Muesing, D. M. Klurfeld, S. A. Tepper, A. Sanghvi, and D. Kritchevsky. Effects of casein and soy protein on hepatic and serum lipids and lipoprotein lipid distributions in the rat. Atherosclerosis 56: 127-137, 1985[ISI][Medline].

40.   Vallerand, A. L., F. Perusse, and L. J. Bukowiecki. Cold exposure potentiates the effect of insulin on in vivo glucose uptake. Am. J. Physiol. Endocrinol. Metab. 253: E179-E186, 1987[Abstract/Free Full Text].


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