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Glucose and insulin responses in healthy women after intake of composite meals containing cod-, milk-, and soy protein



To evaluate the metabolic effect of three different kinds of dietary proteins as part of composite meals with similar macronutrient composition in healthy subjects.


A randomised meal study.


Metabolic ward.

Subjects and methods:

In total, 17 healthy women, 30–65 years old, consumed three meals in randomised order. The meals consisted of foodstuffs with similar nutrient composition but different types of protein (cod, cottage cheese, or soy protein isolate). The distribution of energy from protein, fat and carbohydrates was 33, 26, and 41 energy percent, respectively. Total amount of energy was 2300 kJ. Blood samples were drawn for assay of B-glucose, S-insulin, S-free fatty acids, S-triglycerides, and C-peptide in the fasting state and at seven times (20, 40, 60, 90, 120, 180, and 240 min) after starting to eat the test meal.


The blood glucose response after the cod protein meal differed from that of the soy protein meal, with a larger area under the curve (AUC) calculated up to 120 min. The serum insulin response after the milk protein meal differed from that of the cod protein meal with a larger AUC calculated up to 240 min. The insulin/C-peptide and the insulin/glucose ratios differed between the meals; the insulin/C-peptide ratio was higher after the milk protein meal compared to the cod, and soy protein meal at 120 min. The insulin/glucose ratio was lower after the cod protein meal compared to the milk, and soy protein meals at 120 min. The results showed that the metabolic responses differed after meals with similar macronutrient composition containing cod-, milk-, or soy protein.


The effects of dietary fat and carbohydrate on the metabolic control in humans have been widely studied. However, knowledge about the effects of protein subtypes is scant and has recently become a new research area (Storlien et al., 2000).

The metabolic effects of dietary protein have mostly been studied in animals; for example, fish protein, soy protein, and casein (the main part of milk protein) have been compared to each other to detect whether differences in the amino-acid composition might affect metabolic responses (Zhang and Beynen, 1993). In high-fat fed rats given different kinds of protein for 28 days, intake of cod protein did not, as compared to diets containing soy protein or casein, lead to insulin resistance (Lavigne et al., 2001). In an earlier study, rats fed fish protein or soy protein showed improved glucose tolerance and insulin sensitivity compared to casein fed rats (Lavigne et al., 2000). Other studies suggest that changing the protein source in synthetic diets, with identical distribution of protein, fat and carbohydrates, may directly influence insulin sensitivity in rats fed a high-fat diet (Iritani et al., 1997). A recent study indicated that cod protein improves translocation of GLUT4 (glucose transporters) in skeletal muscle, possibly through a direct effect of specific amino acids on insulin stimulated glucose uptake in the skeletal muscle cells (Tremblay et al., 2003). Yahia et al. (2003) reported that fish protein, but not casein, attenuated the development of hypertension and decreased plasma total cholesterol concentration in spontaneously hypertensive rats.

To date, few controlled studies in humans show the effects of dietary protein type on glucose response, insulin secretion, insulin sensitivity and other metabolic responses. In six healthy males, the postprandial plasma insulin level increased, with a higher insulin/glucagon ratio, when fed beef rather than a fish meal (Soucy and Le Blanc, 1999). In another group of healthy men, modification of the protein source in mixed meals containing either casein, gelatin or soy protein was associated with diverging kinetics of postprandial blood glucose (Lang et al., 1999).

The aim of this study was therefore to investigate whether the type of dietary protein affected metabolic control (blood glucose, insulin, free fatty acids, triglycerides, and C-peptide) in humans, when administered as a part of an ordinary meal. We compared the effects of fish protein, supplied as lean fish (cod), with milk protein (cottage cheese 1% fat) and soy protein (soy protein isolate) when given as equivalent amounts of protein in meals with otherwise the same nutrient composition.

Materials and methods


Subjects were recruited through an advertisement in a local newspaper. Inclusion criteria were age between 30 and 65 and a body mass index (BMI) between 22 and 32 kg/m2. A questionnaire was sent to the responding subjects, where they had to answer questions about, for example, illnesses, medication, smoking, and alcohol habits. Subjects with diabetes mellitus and subjects using lipid lowering drugs, thiazide diuretics, beta blockers, and corticosteroids were excluded. Six men and 26 women were invited to a screening test that included anthropometry, serum lipids, plasma glucose, kidney, liver, thyroid function, and a medical questionnaire. Other causes for exclusion were allergy or not being able to eat certain kinds of foodstuffs, difficulty of being off from work and not wanting to participate. In total, 17 healthy subjects fulfilled the inclusion criteria, all women, with a mean age of 46 years (range 32–64 years). The mean body weight was 68.8±7.5 kg and body mass index was 24.7±2.5 kg/m2. The subjects acted as their own control. All participants were informed about the study protocol. They had given their informed consent before entering the study and the study design was approved by the Ethical Committee of the Medical Faculty of Uppsala University, Uppsala, Sweden (02-099).

Meals and nutrient calculation

Three test meals with different types of protein (either cod protein, milk protein or soy protein) were given to all participants in randomised order, with 1-week in between. Besides type of protein, the nutrient composition of all meals was as similar as possible, with an energy content of 2300 kJ (550 kcal) (Table 1). Food stuffs included in the meals are shown in Table 2. The amounts of cod, cottage cheese, and soy protein isolate are adjusted to get equal an amount of protein (45 g) in all the meals. In cottage cheese, the proportion of casein and whey protein is 80% and 20%, respectively. A very small amount of bouillon was added to the cod protein meal to make it more palatable. Lactose was added to the cod and soy protein meals to equalise the carbohydrate content in all meals. The amount of fluid (water), both in food stuffs and added for preparation of meals, was in all meals 440 g. For energy and nutrient calculations, the database from the Swedish National Food Administration was used (PC-kost version 2002:1). The meal was eaten at 0800 and was completed within 15 min. At 120 min, the participants had a glass of water (200 ml). Subjects were asked not to change their dietary and exercise habits during the study period.

Table 1 Calculated amount of energy, protein, fat, carbohydrates, and fluid in the three different test meals
Table 2 Food stuffs included in the meals

Biochemical measurements

Blood samples were drawn for assay of blood glucose, serum insulin, serum triglycerides, serum nonesterified fatty acids (NEFAs), and plasma C-peptide in the fasting state and at 20, 40, 60, 90, 120, 180, and 240 min after starting to eat the test meal.

Blood glucose was determined directly by the glucose dehydrogenase-based reaction in a HemoCue® blood-glucose photometer (HemoCue AB, Sweden). Serum insulin was measured by an enzyme immunoessay, ELISA-kit (Mercordia AB, Uppsala, Sweden in a Coda Automated EIA Analyser (Bio-Rad Laboratories AB, Scandinavia). Nonesterified fatty acids were determined with a Wako NEFA C-kit (994-75409) (Wako, Neuss, Germany), modified for use in a Monarch apparatus (Instrumentation Laboratories, Lexington, MA, USA). Triglyceride was measured enzymatically in serum, using the IL Test triglyceride enzymatic-colorimetric method 181610-60 in a Monarch apparatus (Instrumentation Laboratories). Plasma C-peptide was measured by the two-site enzyme immunoassay (Mercodia C-peptide ELISA Enzyme immunoassay, Mercodia AB, Uppsala, Sweden).

Statistical analysis

Data are presented as means±standard deviations (s.d.). Results are expressed as incremental Area under the curve (AUC) FAO/WHO, 1997. The AUC was calculated according to the trapezoidal model (Bryant, 1983; Allison et al., 1995). Incremental areas of the curve from baseline (fasting) values over time were calculated up to 90, 120, and 240 min after the meal. For comparisons between the three meals, an analysis of variance model with factors for meal and subject was used. All meal comparisons were made within subjects. If the test for the meal factor in this model was significant at the 5% level, all pair-wise comparisons were performed.


The mean AUC for blood glucose calculated from 0 to 240 min after the cod-, milk-, and soy protein meal were 135, 131, and 117 area units, respectively. No significant differences could be seen up to that time. The AUC from 0 to 120 min was significantly larger after the cod protein meal compared to the soy protein meal (P<0.01). The AUC from 0 to 90 min was larger after consuming cod protein compared to intake of both soy, and milk protein (+82%, P<0.001 and +39%, P<0.05, respectively). The results are shown in Figure 1.

Figure 1

The mean blood glucose after consumption of cod-, milk-, and soy protein meals. AUC (area units) 0–90 min: cod 78±33, milk 56±29, and soy 43±23. AUC (area units) 0–120 min: cod 90±34, milk 72±34, and soy 60±28. P<0.05 cod vs milk AUC 0–90 min. P<0.01 cod vs soy AUC 0–120 min, and P<0.001 cod vs soy AUC 0–90 min.

The mean AUC calculated up to 240 min was for serum insulin significantly larger after the milk protein meal compared to the cod protein meal (P<0.001). There were no significant differences in S-insulin between any of the meals if tested from 0 up to 90 and 0 up to 120 min (Figure 2).

Figure 2

Mean serum insulin after consumption of cod-, milk-, and soy protein meals. AUC (area units) 0–240 min: cod 2328±1391, milk 2942±1483, and soy 2841±1780. P<0.001, milk vs cod AUC 0–240 min.

The insulin/glucose ratio was significantly lower after the cod protein meal compared to the milk-, and soy protein meals 40 min after the beginning of test meal intake.

After 120 min, the milk protein meal resulted in a higher insulin/glucose ratio compared to the two other meals (Table 3).

Table 3 Insulin and glucose concentrations (mean; s.d.), and insulin/glucose ratio at 0, 40, and 120 min after intake of cod-, milk-, and soy protein meals

No differences were seen in the AUC neither for serum triglycerides nor serum FFAs at 90, 120, or up to 240 min between the three meals. The increase of serum triglycerides started at approximately 90 min after test meal intake. The decrease of FFA began directly after intake of test meal and reached the lowest point at 90 min.

The mean AUC were for C-peptide calculated from 0 to 40 and 0 to 120 min and no significant differences could be seen at any times.

The insulin/C-peptide ratio was after 40 min significantly lower after ingesting the cod protein meal compared to both the milk, and soy protein meal. After 120 min, the milk protein meal resulted in significantly higher insulin/C-peptide ratio compared to the both other meals (Table 4).

Table 4 Insulin and C-peptide concentrations (mean; s.d.), and insulin/C-peptide ratio at 0, 40, and 120 min after intake of cod-, milk-, and soy protein meals


There were three main findings in the present study. Firstly, there was a significantly larger area under the first part of the glucose curve in subjects after consuming cod protein compared to intake of soy, and milk protein (Figure 1). Secondly, there was a significantly larger area under the insulin curve for subjects consuming milk protein rather than cod protein, especially after the first hour of the test period (Figure 2). Finally, there were higher insulin/C-peptide and insulin/glucose ratios in the subjects after the milk protein meal compared to especially the cod protein meal (Tables 3 and 4).

It has been demonstrated that ingestion of proteins only, results in little or no increase in circulating glucose concentration in non-diabetic people or in people with type 2 diabetes mellitus (Gannon et al., 2001). In a study of type 2 diabetic patients, 50 g glucose without or with the addition of 25 g of seven different kinds of protein (lean beef, turkey, gelatin, egg white, cottage cheese, fish, and soy) were given in single breakfast meals. The highest blood glucose levels were seen after glucose alone or glucose + egg white. Protein from turkey, gelatin, and cottage cheese resulted in lower blood glucose levels (Gannon et al., 1988).

A few studies on dietary fatty acids and metabolic responses in subjects with type 2 diabetes show slightly elevated blood glucose levels after intake of n-3 fatty acids. When two diets rich in either saturated or polyunsaturated fats were given over two consecutive 3-week periods, the polyunsaturated fat diet resulted in a higher blood glucose concentration, while HbA1c did not differ significantly between the two dietary periods (Vessby et al., 1992). In another study, in which non-insulin dependent diabetes subjects were given supplementation with either olive oil or n-3 fatty acid capsules during two consecutive 8-week-periods, the blood glucose concentration tended to increase during the n-3 fatty acid period (Vessby and Boberg, 1990). However, this has not been a consisting finding. A recent meta-analysis of 18 trials of fish oil supplementation in diabetes with a mean follow-up of 12 weeks concluded that there was no statistically significant effect on glycaemic control (Farmer et al., 2001). If the slightly higher blood glucose response in certain studies of diets containing fish or fish products is due to different protein composition or due to other components of fish ought to be further investigated. Whether the residual amount of 0.7% fat (of which 1.28 g are n-3 fatty acids) in this study could contribute to the increased blood glucose levels is an open question.

In the present study, the intention was to use as ordinary food stuffs as possible. This explains why we used cottage cheese (milk protein). In other studies, pure casein has been used when comparing different kinds of protein. A mixed meal study of Lang et al. of healthy men ingesting casein, gelatin or soy protein resulted in no difference in the satiety effect. However, there were significant effects on the kinetics of metabolic response (Lang et al., 1999). Glucose and insulin responses following casein-enriched meal lagged 1–1.5 h behind responses following soy protein-enriched meal. In an investigation of rats, it was concluded that the metabolic responses of cod and soy proteins, when compared to casein, improved fasting glucose tolerance, and peripheral insulin sensitivity (Lavigne et al., 2000).

Earlier studies report an inconsistent behavior of milk products giving a normal glycemic index but a higher insulinemic index. Liljeberg et al. (2001) showed that inclusion of milk in energy-equivalent cereal-based breakfasts did not affect the glycemic index but resulted in significantly higher insulinemic indices. In a study of glycemic and insulinemic responses to regular and fermented milk products, despite low glycemic indices, all of the milk products produced high insulinemic indices (Östman et al., 2001). Milk products appear to be an exception in that the insulinemic index cannot be predicted from the glycemic index. Björck et al. suggested that the insulinotrophic effect could be related to the protein fraction in milk and/or that milk products may have the capacity to stimulate the incretin hormones (Björck et al., 2000; Nilsson et al., 2004).

In diabetic patients, a meal with glucose and cottage cheese resulted in higher insulin response than a corresponding meal with various other proteins, for example fish, or soy protein in spite of a low blood glucose area. Gannon et al. (1988) suggested that the major stimulus for insulin secretion is an increase in incretion hormones by the intestine in response to the presence of protein or digestion products from protein in the intestine. This implies that the insulin response may also be related to the rate of digestion of various proteins. In six healthy males, who were given either a beef or a fish meal, plasma insulin levels increased significantly more with the beef steak meal than with the cod fillet (Soucy and Le Blanc, 1999). The beef meal increased the plasma amino levels of histidine, while the fish meal gave higher levels of arginine and lysine. The authors concluded that a possible reason for increased insulin levels could depend on the histidine and/or differences in absorption and digestibility.

Different amino acids seem to affect insulin secretion in different ways. Reportedly, the branched-chain amino acids leucine, isoleucine, and valine increase insulin secretion more than other amino acids. Lavigne et al. (2000) demonstrated that these kinds of amino acids were represented in higher amounts in casein than in cod, and soy protein.

In the present study, the insulin response after the milk protein meal was similar to that in these earlier studies, giving a later insulin response and significantly larger AUC compared to the insulin response after intake of the cod protein meal.

The predominant amino acids in cod protein are lysine, methionine, alanine, and glycine; in soy protein, aspartic acid, arginine, cysteine, and serine; and in casein glutamine acid, isoleucine, leucine, valine, proline, tyrosine, and histidine, respectively. As earlier stated, the branched-chained amino acids isoleucine, leucine, and valine are suggested to be most closely related to the insulin response (Calbet and MacLean, 2002), which could explain that milk protein with a high amount of these amino acids gives a higher insulin response. Gannon et al. (2002) reported that arginine in an amount likely to be ingested in a high protein meal did not stimulate insulin secretion. This is compatible with our study that both the cod and soy protein meal with a higher amount of arginine gave a lower insulin response compared to the milk protein meal.

There were no significant differences between the meals for C-peptide concentrations at the time points 0, 40, or 120 min. However, the cod protein meal resulted in the lowest, and the milk protein meal in the highest insulin/C-peptide and insulin/glucose ratios. The high insulin/C-peptide ratio may be due to delayed elimination of insulin through extraction in the liver. Lavigne et al. (2000) suggested that in the postprandial state, both a lower insulin secretion and a higher hepatic extraction may have contributed to reduce plasma insulin concentrations in rats fed cod and soy proteins.

The long-term health consequenses of the slightly increased blood glucose levels, lower insulin levels and reduced insulin/C-peptide and insulin/glucose ratios after cod protein remain to be determined. This also goes for the increased insulin levels with milk protein with simultaneously increased insulin/C-peptide ratio compatible with delayed extraction of insulin. If anything, this may be a cause of concern with regard to possible health effects. Increased insulin levels due to increased insulin secretion or delayed clearance with an increased insulin/glucose ratio may hypothetically contribute to atherogenesis (Stout, 1996).

Different glucose and insulin responses in healthy subjects after intake of meals with different kinds of protein could be caused by either the effects on the insulin secretion and/or to the insulin extraction rates in the liver. This may be due to the specific amino-acid composition or other properties in the food stuffs. More studies, preferably during a longer period of time, are needed to further elucidate the metabolic effects of different proteins, as part of a composite meal, in the human body.


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This study was supported by grants from the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning (Formas). We thank the staff at the Center for Clinical Testing of Foodstuffs (KPL), Uppsala University, where blood samples were taken.

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Correspondence to M von Post-Skagegård.

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Guarantor: B Karlström.

Contributors: BV originally designed the study. MvP-S and BK performed the study and processed the material. MvP-S, BV, and BK all contributed to writing the paper.

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von Post-Skagegård, M., Vessby, B. & Karlström, B. Glucose and insulin responses in healthy women after intake of composite meals containing cod-, milk-, and soy protein. Eur J Clin Nutr 60, 949–954 (2006).

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  • amino acids
  • dietary protein
  • blood glucose
  • serum insulin

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