Acute differential effects of milk-derived dietary proteins on ... - Nature

European Journal of Clinical Nutrition (2012) 66, 32–38

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ORIGINAL ARTICLE

Acute differential effects of milk-derived dietary proteins on postprandial lipaemia in obese non-diabetic subjects J Holmer-Jensen1, ML Hartvigsen1, LS Mortensen1, A Astrup2, M de Vrese3, JJ Holst4, C Thomsen1 and K Hermansen1 1

Department of Endocrinology and Metabolism MEA, Aarhus University Hospital, Aarhus, Denmark; 2Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark; 3Federal Research Institute for Nutrition and Food, Institute for Physiology and Biochemistry of Nutrition, Kiel, Germany and 4Department of Biomedical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

Background/Objectives: Postprandial lipaemia is an established risk factor for atherosclerosis. To investigate the acute effect of four milk-derived dietary proteins (alpha-lactalbumin, whey isolate, caseinoglycomacropeptide and whey hydrolysate) on postprandial lipaemia, we have conducted a randomized, acute, single-blinded clinical intervention study with crossover design. Subjects/Methods: A total of 11 obese non-diabetic subjects (age: 44–74, BMI: 30–41.4 kg m-2) were included. On 4 different days the subjects ingested a high-fat meal with the following energy distribution: 66% energy from fat (100 g of butter), 15% of energy from carbohydrate (90 g of white wheat bread) and 19% of energy from protein (45 g of pure protein). Our primary variable was plasma triglyceride measured in the 8-h postprandial period. Secondarily, retinyl palmitate, non-esterified free fatty acids, glucose, insulin, glucagon, GLP-1 and GIP, active and total grehlin and cholecystokinin were measured. Results: We observed no statistically significant (P ¼ 0.8) differences between meals on our primary variable that is, triglycerides. Whey hydrolysate was associated with a significantly (P ¼ 0.02) smaller postprandial suppression of non-esterified free fatty acids compared with the other dietary proteins. Conclusion: We did not observe significant differences in postprandial lipaemia to the four milk-derived dietary proteins. Whey hydrolysate caused less postprandial suppression of free fatty acids.

European Journal of Clinical Nutrition (2012) 66, 32 – 38; doi:10.1038/ejcn.2011.142; published online 27 July 2011 Keywords: postprandial lipaemia; dietary protein; obesity; atherosclerosis; triglycerides; whey

Introduction Obesity is at epidemic proportions worldwide. Obesity is a significant risk factor for and contributor to increased morbidity and mortality, most importantly from cardiovascular disease and diabetes (Yusuf et al., 2005). Thus, obesity is strongly associated with an increased risk of all-cause mortality as well as cardiovascular mortality (Teucher et al., 2010). The increased risk of cardiovascular

Correspondence: Dr J Holmer-Jensen, Department of Endocrinology and Metabolism MEA, Aarhus University Hospital, Tage-Hansens Gade 2, Aarhus C DK-8000, Denmark. E-mail: [email protected] Received 31 January 2011; revised 23 June 2011; accepted 23 June 2011; published online 27 July 2011

disease linked to obesity is caused by a combination of silent risk factors such as hypertension, insulin resistance, inflammation and dyslipidaemia (Sehested et al., 2010). In relation to dyslipidaemia, postprandial lipaemia (PPL) is a key contributor to cardiovascular disease risk and progression (Bansal et al., 2007; Nordestgaard et al., 2007). Specifically, delayed clearance of chylomicrons (CM) and their remnants increase the delivery of triglyceride and cholesteryl ester to the vessel wall and can accelerate the progression of atherosclerosis, which may be particularly pertinent to individuals with obesity and insulin resistance (Guerci et al., 2000). An exaggerated PPL response in both increment and duration is observed in both type 2 diabetes (T2DM) (Madhu et al., 2008) and obesity (Couillard et al., 1998; Guerci et al., 2000; van Wijk et al., 2003).

Postprandial lipaemia, milk protein and obesity J Holmer-Jensen et al

33 The extent of PPL is primarily correlated to the diet in general and fat content of meals in particular (Lairon, 2008). Interestingly, it has been demonstrated that the addition of refined carbohydrates to a fat-rich meal increases PPL in T2DM (Brader et al., 2010). However, this observation could not be demonstrated in healthy subjects (Westphal et al., 2004; Lairon et al., 2007). In recent years, increased scientific interest has been paid to the acute effects of dietary protein on PPL. Thus, the unfavorable effect of carbohydrate on PPL seen in T2DM can be reduced by adding casein to the meal (Westphal et al., 2004; Brader et al., 2010). However, casein added to a fat-rich meal without carbohydrate did not affect PPL compared with a fat-rich meal per se in T2DM (Brader et al., 2010). In addition, Nilsson et al. (2004) showed that milk proteins in general and whey protein in particular have the most insulinotropic abilities among a wide range of dietary proteins. Regarding the importance of insulin on postprandial glucose and fat metabolism, it occurs that dietary proteins have differential impact. Thus, Mortensen et al. (2009) recently demonstrated that whey protein reduce PPL in T2DM compared with casein, gluten and cod protein, and Akhavan et al. (2010) and Frid et al. (2005) found lower postprandial glycaemia in young, healthy adults and T2DM following premeal consumption of whey protein. The impact of dietary protein on PPL in obese non-diabetic subjects is unknown. We hypothesize, that whey protein has a beneficial effect on PPL in obese non-diabetic subjects. The proposed beneficial effect is thought to be caused by a subfraction of whey. In this study, we have compared the effects on PPL of a structurally modified derivate of whey isolate (WI) and two subfractions of whole whey. The structurally modified derivate is whey hydrolysate (WH), that primarily consists of di- and tripeptides from extensively hydrolysed WI. The two subfractions are alpha-lactalbumin (ALPH) and caseinoglycomacropeptide (CGMP), both major fractions of whey but here given as ALPH-enhanced whey protein and CGMP-enhanced whey protein. As comparator we have used WI.

Subjects and methods Subjects A total of 11 obese Caucasian subjects (six postmenopausal women and five men) were recruited by advertising in local newspapers. All subjects had a BMI above 30 and were non-diabetics as determined by fasting glucose levels o7.0 mmol/l. Subjects with impaired fasting glucose were subjected to an oral glucose tolerance test before enrolment, and were excluded if diabetic (one subject). No participant took lipid-lowering drugs and all participants were nonsmokers. Subject characteristics are shown in Table 1. No change in concomitant medication was allowed during the trial. All subjects gave written informed consent, and the study was approved by the Committees on Biomedical Research Ethics for the Central Region of Denmark. This study is registered on Clinicaltrials.gov ID: NCT00809874.

Study design In accordance with a randomized, single-blinded, acute study with crossover design, all subjects ingested four different meals at 2 weeks interval between meals. Each subject was randomized to one of four test meal sequences according to a Latin square. Each subject was given a standard diet that is to be consumed on the day preceding each study day. The diet had the following energy distribution: 56% energy from carbohydrate, 24% energy from fat and 20% energy from protein. The energy content was 7000 and 9000 kJ for female and male participants, respectively. In the morning after a 12-h fasting period the subjects were instructed to minimize their physical activity. Upon arrival at the research facility baseline samples were drawn after 15 min of rest. The test meal was ingested within 20 min and during the 8-h postprandial period blood samples were drawn regularly. Insulin, glucagon like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) and glucose were measured at 0, 15, 30, 45, 60, 120, 240, 360 and 480 min. Glucagon, active grehlin, total grehlin and cholecystokinin (CCK) were measured at 0, 30, 60, 120, 240, 360

Table 1 Clinical characteristics of the eleven (six females and five males) obese non-diabetic subjectsa Group Age (year) Weight (kg) BMI (kg/m2) Waist (cm) Waist-to-hip ratio HbA1c (%) Fasting P-glucose (mmol/l) HOMA2 (IR) Fasting P-triglyceride (mmol/l)

60.4±10.2 101.0±10.4 35.3±3.6 109.5±11.9 0.93±0.10 5.6±0.2 5.9±0.6 1.0±0.4 1.4±0.5

(44–74) (83.5–120.2) (30.0–41.4) (87–128) (0.76–1.17) (5.4–6.0) (4.8–6.9) (0.5–1.7) (0.5–2.3)

Women 55.5±3.7 94.5±3.3 36.2±1.6 100.7±7.7 0.82±0.04 5.6±0.1 5.9±0.3 0.8±0.1 1.3±0.2

(44–66) (83.5–107) (30.0–41.4) (87–109) (0.76–0.87) (5.4–5.8) (4.8–6.9) (0.5–1.2) (0.5–1.9)

Men 66.2±3.9 108.8±3.2 34.2±1.4 120.0±5.0 1.06±0.08 5.6±0.1 6.0±0.2 1.3±0.2 1.4±0.3

(53–74) (100.5–120.2) (31–39.4) (116–128) (0.98–1.17) (5.4–6.0) (5.6–6.8) (0.9–1.7) (0.7–2.3)

a

All values are means±s.d.; range in parentheses.

European Journal of Clinical Nutrition


34 and 480 min. Non-esterified free fatty acids (NEFA) were measured at 0, 60, 120, 240, 360 and 480 min. Triglycerides and retinyl palmitate (RP) were measured at 0, 120, 240, 360, 420 and 480 min. Plasma was separated immediately by centrifugation at 2000 g for 20 min at 4 1C. Plasma samples were stored at 80 1C until analyzed. The subjects were allowed to drink tap water ad libitum.

Test meals All test meals consisted of an energy-free soup added 100 g of butter (Lurpak; Arla Foods amba, Viby J, Denmark) corresponding to 80 g of fat (68% of energy as saturated fat). A measure of 45 g of carbohydrate was added as white wheat bread (La¨ntmann Schulstad A/S, Hvidovre, Denmark) and finally, 45 g of protein was dissolved in 200 ml cold water as a milkshake containing one of the four milk-derived proteins of interest: alpha-lactalbumin (LACPRODAN- ALPHA-10) (ALPH-meal), whey isolate (LACPRODAN- DI-9224) (WImeal), caseinoglycomacropeptide (LACPRODAN CGMP-10) (CGMP-meal) and whey hydrolysate (LACPRODANDI-3065) (WH-meal). All four milk-derived proteins (kindly produced, tested and provided by Arla Foods Ingredients) were applied in a spray-dried formulation. Before serving the meals, 25 g of raw leek was added to the soup making it more palatable. Butter was added and the soup heated to a serving temperature of 65 1C. With each test meal 100 000 IU of vitamin A was taken with the first spoonful of soup. Vitamin A causes retinyl ester labeling of chylomicrons (Blomhoff et al., 1991) and is used as a marker of lipoproteins of intestinal origin, that is, chylomicrons and chylomicron remnants (Chen and Reaven, 1991) in the postprandial period. Macronutrient distribution and energy content of the four meals is shown in Table 2. Lactose content of

Table 2 Energy content, macronutrient distribution and protein distribution in the four test meals consumed by eleven (six women and five men) obese non-diabetic subjectsa

Total energy (kJ) Fat (% of energy) Carbohydrate (% of energy) Protein (% of energy) a-la, % of proteinc b-la, % of proteind CGMP, % of protein

ALPH

WI

CGMP

WHb

4986 66 15 19 B42 B26 B32

4971 66 15 19 B25 B52 B24

4981 66 15 19 B13 B10 B77

4976 66 15 19 o0.0001 o0.0001 o0.0001

Abbreviations: ALPH, alpha-lactalbumin; CGMP, caseinoglycomacropeptide; WH, whey hydrolysate; WI, whey isolate. a Meals: An energy-free soup plus 100 g butter and 45 g carbohydrate consumed with either 45 g ALPH, 45 g WI, 45 g caseinoglycomacropeptide CGMP or 45 g WH. b WH is composed of di- and tripeptides from hydrolysed whey protein concentrate (LACPRODAN DI-8090) with a protein distribution of: a-la ¼ 20% of protein; b-la ¼ 47% of protein; CGMP ¼ 20% of protein. c a-la: alpha-lactalbumin. d b-la: beta-lactoglobulin.


each meal did not differ. The total volume of each meal did not differ.

Ultracentrifugation To isolate chylomicrons from lower density lipoproteins, that is, VLDL, IDL, LDL and chylomicron remnants, we performed a single step of ultracentrifugation on our plasma samples. Plasma samples were defrosted at 4C. Plasma (4 ml) was overlayered with 2 ml of a saline solution with a density of 1006 g/ml in a quick seal tube (number 344 619; Beckman Instruments, Palo Alto, CA, USA) and then centrifuged for 30 min at 26 000 g at 25 1C. The chylomicron-rich supernatant (Svedberg flotation (Sf) 41000) was aspirated and brought to a final volume of 4 ml with saline. The chylomicron-poor infranatant contained the more dense lipoproteins (Sf o1000). Blood analyses Plasma glucose was measured with a glucose oxidase method (coefficient of variation (CV): 1.8%). Serum insulin was measured with an enzyme-linked immunosorbent assay method (CV: 1.7%) (K6219; Dako, Cambridgeshire, UK). Concentrations of triglyceride (CV: 1.8%), high-density lipoprotein (HDL)-cholesterol and NEFA were measured with standard enzymatic colorimetric assays using commercial kits (Roche Diagnostics GmbH and Wako Chemicals GmbH, Neuss, Germany). Total GIP was measured as described previously (Krarup et al., 1983). The plasma concentrations of total GLP-1 were measured using antiserum code number 89 390 (Krarup et al., 1983). Thus, it mainly reacts with GLP-1 of intestinal origin. For both assays, sensitivity was o1 pmol/l and the interassay CV was o6% at 20 pmol/l. Glucagon of mainly pancreatic origin was measured by radioimmunoassay using antibody number 4305 in ethanol-extracted plasma as described previously (Holst, 1982; Orskov et al., 1991). RP was extracted and measured by isocratic absorption high-performance liquid chromatography as described previously (Biesalski, 1990). CCK was measured using the EURIA-CCK kit (EURO¨ , Sweden) (CV 13.7%). Active DIAGNOSTICA AB, Malmo grehlin was estimated with the Grehlin RIA kit (GHRA-88HK, Linco Research, Billerica, MA, USA) (CV 9.6%). Total grehlin was measured with the Grehlin RIA kit (GHRT-89HK, Millipore, Billerica, MA, USA) (CV 14.7%).

Statistical analysis and calculations To achieve a statistical power of 80%, the number of subjects needed was 10 based on power calculations to detect a difference of 30% between meals on our primary variable, triglyceride. Comparisons of the iAUC’s were based on a mixed-effects model (Rabe-Hesketh, 2008) (STATA/IC 10.1), using treatment group as fixed variable and participant ID as random variable. All statistical comparisons were adjusted


35 for treatment order, waist-to-hip ratio, gender and baseline values. F-test or Wald-test was used as appropriate. A P-value o0.05 was considered statistical significant. Any statistically significant main effect of treatment group was followed up by Tukey’s post hoc adjustment for multiple comparisons. Response data were given as net incremental area under the curve after 480 min (net iAUC-480 min) using trapezoidal rule. However, as previously shown, plasma insulin reaches baseline at 240 min after a fat-rich mixed meal in nondiabetic subjects (Thomsen et al., 1999). Therefore, response data on insulin were given at 240 min. To describe initial responses of insulin, glucagon, GLP-1 and GIP data were also presented as net iAUC after 30 min (net iAUC-30 min) in the results section. Whenever data were not normally distributed, a log transformation was performed and the statistical analyses were carried out on the normal distributed log data and results given as medians with interquartile ranges. Otherwise data were given as means±s.d. unless otherwise stated.

was significantly (P ¼ 0.0219) smaller compared with the three other meals (Table 4). No statistically significant differences were observed in postprandial glucose net iAUC-480 min (Table 4) or in glucose net iAUC-30 min (data not shown).

Hormone responses There were no overall significant differences in postprandial insulin net iAUC-480 min between meals (Table 4). However, WH-meal induced a significantly (P ¼ 0.0112) larger net iAUC-30 min of insulin compared with the three other meals (Table 4). The insulin net iAUC-30 min of WH-meal was 6928 pmol/l per 30 min;±5476. The insulin net iAUC-30 min of ALPH-, WI-, and CGMP-meal accounted for 46%, 59% and 62% of that to WH-meal, respectively. No significant differences were observed in postprandial responses of glucagon, incretin hormones, CCK or grehlin (Table 4).

Discussion Results All 11 obese non-diabetic participants completed the four test meals according to the protocol. We found no significant differences in body weight or fasting concentrations of the measured variables between test days (Table 3). Lipaemic responses No significant differences in plasma triglycerides, supernatant triglycerides or infranatant triglycerides were observed between meals (Table 4). Concordantly, we observed no significant differences in RP supernatant or RP infranatant (Table 4). NEFA and glucose All meals suppressed postprandial NEFA concentrations. However, the overall suppression of NEFA after WH-meal Table 3

The present study evaluated the acute effects of milk-derived dietary proteins on postprandial lipaemia in obese nondiabetic subjects. We compared four different isocaloric meals with the same nutrient distribution but with four different protein sources, that is, alpha-lactalbumin, whey isolate, caseinoglycomacropeptide and whey hydrolysate— all milk derived. The most important findings were a smaller postprandial suppression of NEFA after WH-meal compared with the other meals and a larger insulin increment during the initial 30 min after WH-meal compared with the other meals. We did not observe significant differences between meals in our primary outcome that is, postprandial lipaemia. As expected, we found a suppression of NEFA for all four meals. The suppression observed after WH-meal was only about one third of what we saw after the other meals. In the postprandial state the adipose tissue shifts from triglyceride

Plasma and serum concentrations in the fasting state in the eleven (six women and five men) obese non-diabetic subjects on the 4 test daysa

Triglycerides, plasma (mmol/l) NEFA, plasma (mmol/l) HDL, plasma (mmol/l) Glucose, plasma (mmol/l) Insulin, plasma (pmol/l) Glucagon, plasma (pmol/l) GLP-1, plasma (pmol/l) GIP, plasma (pmol/l) CCK, plasma (pmol/l) Total ghrelin, plasma (pg/ml) Active ghrelin, plasma (pg/ml)

ALPH

WI

CGMP

WH

1.32±0.66 0.55±0.15 1.08±0.21 5.86±0.62 53.2±18.7 9.73±3.61 16.3±5.7 9.82±4.29 0.411±0.546 747±218 45.1±32.0

1.57±0.66 0.52±0.15 1.08±0.29 5.77±0.61 56.5±20.9 9.36±4.41 20.9±10.1 10.91±4.80 0.265±0.157 789±297 44.4±26.3

1.41±0.44 0.54±0.15 1.06±0.26 5.83±0.57 51.6±32.8 8.27±2.87 21.4±10.5 9.18±2.60 0.413±0.288 774±253 52.6±33.4

1.41±0.50 0.46±0.14 1.07±0.24 5.84±0.41 47.4±18.9 8.00±2.28 16.1±4.8 10.81±5.13 0.306±0.217 783±358 53.5±29.7

Abbreviations: ALPH, alpha-lactalbumin; CCK, cholecystokinin; CGMP, caseinoglycomacropeptide; GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide 1; HDL, high density lipoprotein; NEFA, non-esterified free fatty acids; RP, retinyl palmitate; WH, whey hydrolysate; WI, whey isolate. a All values are means±s.d. (normal distributions; mixed-effects model; F-test or Wald-test; Tukey’s post hoc adjustment). Meals: energy-free soup plus 100 g butter and 45 g carbohydrate consumed with either 45 g ALPH, 45 g WI, 45 g CGMP or 45 g WH.



36 Table 4 Net incremental areas under the curve after 480 min (net iAUC-480 min) and P-value for main effect of treatment groups in 11 (6 women and 5 men) obese non-diabetic subjects in response to the four test mealsa

TG plasma (mmol/l 480 min) TG supernatant (mmol/l 480 min) TG infranatant (mmol/l 480 min) RP supernatant (mg/l 480 min) RP infranatant (mg/l 480 min) NEFA (mmol/l 480 min) Glucose (mmol/l 480 min) Insulin (pmol/l 240 min) Glucagon (pmol/l 480 min) GLP-1 (pmol/l 480 min) GIP (pmol/l 480 min) CCK (pmol/l 480 min) Total ghrelin (pg/ml 480 min) Active ghrelin (pg/ml 480 min)

ALPH

WI

CGMP

WH

P main effect

206±105 76.2±50.2 59.4±44.6 3 308 (171–27 953) 6 248 (2 262–19 019) 62.3±68.1y 119±188 57 722±26 454 3 662±1 047 13 260 (7 847–23 376) 12 406±4 235 195±244 64 890±32 050 5 867±12 623

222±158 93.3±72.8 53.8±53.2 13 831 (6 451–17 415) 17 466 (14 411–30 537) 55.0±65.2y 74±219 67 247±30 566 3 706±1 192 11 901 (7 987–17 961) 11 638±3 102 257±100 91 135±78 779 5 295±8 467

204±139 58.7±63.6 31.9±39.6 2 816 (761–12 770) 3 042 (1 146–15 641) 73.5±56.8y 141±156 58 013±33 746 4 096±1 765 8 742 (5 692–14 858) 11 843±3 074 261±211 98 110±77 883 9 002±11 194

208±153 65.1±72.7 38.8±30.4 4 458 (3 987–11 011) 6 779 (5 843–30 776) 20.0±50.2x 125±104 63 216±32 852 4 058±1 396 11 342 (8 219–15 997) 13 075±4 026 269±168 80 307±98 763 9 834±10 984

0.85 0.11 0.15 0.40 0.30 0.02 0.68 0.37 0.98 0.61 0.53 0.71 0.38 0.93

Abbreviations: ALPH, alpha-lactalbumin; CCK, cholecystokinin; CGMP, caseinoglycomacropeptide; GIP, glucose-dependent insulinotropic peptide; GLP-1, glucagon-like peptide 1; HDL, high density lipoprotein; NEFA, non-esterified free fatty acids; RP, retinyl palmitate; TG, triglycerides; WH, whey hydrolysate; WI, whey isolate. a All values are means±S.d. (normal distributions; mixed-effects model; F-test or Wald-test; Tukey’s post hoc adjustment) or medians with interquartile ranges in parentheses (skewed distribution; mixed-effects model on normal distributed log-transformed data, F-test or Wald-test; Tukey’s post hoc adjustment). Meals: energy-free soup plus 100 g butter and 45 g carbohydrate consumed with either 45 g ALPH, 45 g WI, 45 g CGMP or 45 g WH. Values in a row with different superscript letters are significantly different, P o0.05.

lipolysis to NEFA re-esterification. This process is mainly driven by substrate availability and insulin inhibition of adipose tissue lipolysis. Increased substrate availability is associated to a process designated ‘spillover’. ‘Spillover’ occurs when endothelial lipoprotein lipase releases NEFA from chylomicrons by hydrolysis of chylomicron triglyceride. Approximately half of the NEFA is taken up by adipocytes and the other half is released to circulation (Ruge et al., 2009). In the present study, however, we found no differences in substrate availability, that is, triglycerides, to explain the differences in postprandial NEFA concentrations. We did demonstrate, although, that the insulin response was initially higher after WH-meal compared with the other meals. However, higher insulin concentrations would physiologically lead to larger postprandial suppression of NEFA unless the meal had caused a state of acute insulin resistance. NEFA derived from serum triglycerides are known to increase hepatic glucose production and induce hepatic insulin resistance (Lam et al., 2003). However, it occurs unlikely that WH-meal would induce acute postprandial insulin resistance within the initial 30 min. The insulinotropic properties of whey protein are described earlier. Thus, whey protein is rich in essential amino acids and branched chained amino acids that possess strong insulinotropic properties (Floyd et al., 1970). Furthermore, whey hydrolysate has been found to elicit higher insulin responses compared with whole milk (Calbet and Maclean, 2002). Interestingly, although, comparison of oral administration of native whey protein and hydrolysed whey protein or hydrolysed casein revealed no differences in gastric emptying or appearance of amino acids in peripheral blood (Calbet and Holst, 2004). The different European Journal of Clinical Nutrition

insulinotropic properties may on the other hand be attributed to differential stimulation of intestinal GIP secretion as demonstrated by Calbet and Holst (2004). In the present study, we observed a tendency for WH-meal to elicit larger GIP responses compared with the other meals. Thus, our findings regarding incretin hormones, glucagon and insulin corroborate well with the findings of Calbet and Holst (2004) and Claessens et al. (2008). Furthermore, Power et al. (2009) demonstrated augmented insulinotropic effects of WH compared with WI following ingestion of a protein solution, but did not see any differences in gastric emptying. We did not observe acute differential effects of the four test meals on postprandial lipaemia. However, in both supernatant and infranatant triglyceride net iAUC-480 min tended to be lower following CGMP-meal compared with the other meals. This corroborates with the tendency of lower concentrations of incretin hormones after CGMP-meal as this may reflect a slower rate of gastric emptying. However, there are limitations that need to be acknowledged and addressed regarding the present study. Because of the relatively small sample size, we may not have been able to detect a potential treatment effect in the triglyceride supernatant and infranatant. Furthermore, it is debatable whether an ultracentrifugation time of 30 min is sufficient to secure all chylomicron triglyceride as it has been argued that a few hours of centrifugation is needed. Moreover, we did not carry out measurements of gastric emptying, fecal lipid content or lipoprotein lipase concentrations, which could have helped elucidating postprandial kinetics of the four protein meals. Also, it appears puzzling that the recovery percentage of triglyceride in the two ultracentrifuged plasma fractions was smaller after CGMP-meal compared with the other meals.


37 Plasma samples were stored and handled in order of the randomized test sequence, which would otherwise prevent meal categorical differences in blood analyses and ultracentrifugation techniques. It is to be expected that the sum of the two ultracentrifuged fractions do not add up to total triglyceride. However, we cannot exclude that the inter-meal differences in recovery percentage can explain some of the variation observed. The variation in HOMA between men and women in the present study could potentially cause gender differences to outcomes. However, post hoc analyses did not demonstrate any gender differences to outcomes (data not shown). We labeled the chylomicrons with vitamin-A to get an approximation of the chylomicron concentration at a given time. It has, although, been demonstrated that RP is not exclusively associated to chylomicrons but is interchangeable to other lipoproteins in the late postprandial phase. Furthermore, RP concentrations in each lipoprotein are correlated to the size of the particle with more RP in larger particles (Karpe et al., 1995). In both supernatant and infranatant RP tended to be lower after CGMP meal. During the postprandial period chylomicrons undergo lipolysis leaving smaller and theoretically atherogenic (Karpe et al., 2001) chylomicron remnant particles. The long-term clinical impact of fewer chylomicrons and less triglyceride in the blood during the postprandial period may consequently be a reduction in cardiovascular risk. We found no differences between meals in grehlin or CCK. Several studies have shown diminished or absent postprandial suppression of grehlin in obese subjects (Neary and Batterham, 2009) compared with lean subjects following meals with energy content ranging from 250 to 3000 kcal. Even so, the physiological and potentially antiobesity effect of grehlin and CCK in obese subjects is still debated (Field et al., 2010). In the present study, we have demonstrated differential acute effects of whey-derived dietary proteins on PPL. The detrimental effect of excess NEFA, the so called lipotoxicity, contributes to insulin resistance (Bergman and Ader, 2000) and therefore, the weaker suppression of NEFA by WH-meal may relatively enhance long-term insulin resistance in obese non-diabetic individuals compared with the other meals. The high-fat test meals were based on the recommended method (Kolovou et al., 2011) to study postprandial lipaemia but do obviously not reflect a normal diet. Consequently, there is a need for further insight into mechanisms and pathways to understand the observed disparity, as well as long-term studies on ordinary diets to assess the clinical implications of protein supplementation in obese nondiabetic subjects.

Conflict of interest J Holmer-Jensen, LS Mortensen and K Hermansen received a shared research grant from Arla. A Astrup is a scientific

member of Global Dairy Platform (Chicago) and has received speaker’s honoraria and research funding from the Danish Dairy Foundation, Arla and Danish Meat Association. None of the other authors had a conflict of interest to disclose.

Acknowledgements We wish to thank Tove Skrumsager and Lene Trudsø for excellent technical assistance. This work is carried out as a part of the research program of the Danish Obesity Research Centre (DanORC, see http://www.danorc.dk) and is supported by Nordic Centre of Excellence (NCoE) programme (Systems biology in controlled dietary interventions and cohort studies—SYSDIET, P number, 070014). Supported by ‘Marie Krogh Center for Metabolic Research’, Life Sciences, Copenhagen University. Supported by a grant from Arla Foods Ingredients amba, Viby, Denmark.

References Akhavan T, Luhovyy BL, Brown PH, Cho CE, Anderson GH. (2010). Effect of premeal consumption of whey protein and its hydrolysate on food intake and postmeal glycemia and insulin responses in young adults. Am J Clin Nutr 9, 966–975. Bansal S, Buring JE, Rifai N, Mora S, Sacks FM, Ridker PM. (2007). Fasting compared with nonfasting triglycerides and risk of cardiovascular events in women. JAMA 298, 309–316. Bergman RN, Ader M. (2000). Free fatty acids and pathogenesis of type 2 diabetes mellitus. Trends Endocrinol Metab 11, 351–356. Biesalski HK. (1990). Separation of retinyl esters and their geometric isomers by isocratic adsorption high-performance liquid chromatography. Methods Enzymol 189, 181–189. Blomhoff R, Green MH, Green JB, Berg T, Norum KR. (1991). Vitamin A metabolism: new perspectives on absorption, transport, and storage. Physiol Rev 71, 951–990. Brader L, Holm L, Mortensen L, Thomsen C, Astrup A, Holst JJ et al. (2010). Acute effects of casein on postprandial lipemia and incretin responses in type 2 diabetic subjects. Nutr Metab Cardiovasc Dis 20, 101–109. Calbet JA, Holst JJ. (2004). Gastric emptying, gastric secretion and enterogastrone response after administration of milk proteins or their peptide hydrolysates in humans. Eur J Nutr 43, 127–139. Calbet JA, MacLean DA. (2002). Plasma glucagon and insulin responses depend on the rate of appearance of amino acids after ingestion of different protein solutions in humans. J Nutr 132, 2174–2182. Chen YD, Reaven GM. (1991). Intestinally-derived lipoproteins: metabolism and clinical significance. Diabetes Metab Rev 7, 191–208. Claessens M, Saris WH, van Baak MA. (2008). Glucagon and insulin responses after ingestion of different amounts of intact and hydrolysed proteins. Br J Nutr 100, 61–69. Couillard C, Bergeron N, Prud’homme D, Bergeron J, Tremblay A, Bouchard C et al. (1998). Postprandial triglyceride response in visceral obesity in men. Diabetes 47, 953–960. Field BC, Chaudhri OB, Bloom SR. (2010). Bowels control brain: gut hormones and obesity. Nat Rev Endocrinol 6, 444–453. Floyd Jr JC, Fajans SS, Pek S, Thiffault CA, Knopf RF, Conn JW. (1970). Synergistic effect of essential amino acids and glucose upon insulin secretion in man. Diabetes 19, 109–115. ¨ rck IM. (2005). Effect of whey on Frid AH, Nilsson M, Holst JJ, Bjo blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects. Am J Clin Nutr 82, 69–75.



38 Guerci B, Verge`s B, Durlach V, Hadjadj S, Drouin P, Paul JL. (2000). Relationship between altered postprandial lipemia and insulin resistance in normolipidemic and normoglucose tolerant obese patients. Int J Obes Relat Metab Disord; 24, 468–478. Holst JJ. (1982). Evidence that enteroglucagon II is identical with the C-terminal sequence residues 33-69 of glicentin. Biochem J 207, 381–388. ¨ rkegren J, Hamsten A. (1995). Quantification of Karpe F, Bell M, Bjo postprandial triglyceride-rich lipoproteins in healthy men by retinyl ester labeling and simultaneous measurement of apolipoproteins B-48 and B-100. Arterioscler Thromb Vasc Biol 15, 199–207. Karpe F, Boquist S, Tang R, Bond GM, de Faire U, Hamsten A. (2001). Remnant lipoproteins are related to intima-media thickness of the carotid artery independently of LDL cholesterol and plasma triglycerides. J Lipid Res 42, 17–21. Kolovou GD, Mikhailidis DP, Kovar J, Lairon D, Nordestgaard BG, Ooi TC et al. (2011). Assessment and clinical relevance of nonfasting and postprandial triglycerides: an expert panel statement. Curr Vasc Pharmacol 9, 258–270. Krarup T, Madsbad S, Moody AJ, Regeur L, Faber OK, Holst JJ et al. (1983). Diminished immunoreactive gastric inhibitory polypeptide response to a meal in newly diagnosed type I (insulindependent) diabetics. J Clin Endocrinol Metab 56, 1306–1312. Lairon D, Play B, Jourdheuil-Rahmani D. (2007). Digestible and indigestible carbohydrates: interactions with postprandial lipid metabolism. J Nutr Biochem 18, 217–227. Lairon D. (2008). Macronutrient intake and modulation on chylomicron production and clearance. Atheroscler Suppl 9, 45–48. Lam TK, van de Werve G, Giacca A. (2003). Free fatty acids increase basal hepatic glucose production and induce hepatic insulin resistance at different sites. Am J Physiol Endocrinol Metab 284, E281–E290. Madhu SV, Kant S, Srivastava S, Kant R, Sharma SB, Bhadoria DP. (2008). Postprandial lipaemia in patients with impaired fasting glucose, impaired glucose tolerance and diabetes mellitus. Diabetes Res Clin Pract 80, 380–385. Mortensen LS, Hartvigsen ML, Brader LJ, Astrup A, Schrezenmeir J, Holst JJ et al. (2009). Differential effects of protein quality on postprandial lipemia in response to a fat-rich meal in type 2 diabetes: comparison of whey, casein, gluten, and cod protein. Am J Clin Nutr 90, 41–48. Neary MT, Batterham RL (2009). Gut hormones: implications for the treatment of obesity. Pharmacol Ther 124, 44–56.


¨ rck IM. (2004). Glycemia Nilsson M, Stenberg M, Frid AH, Holst JJ, Bjo and insulinemia in healthy subjects after lactose-equivalent meals of milk and other food proteins: the role of plasma amino acids and incretins. Am J Clin Nutr 80, 1246–1253. Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. (2007). Nonfasting triglycerides and risk of myocardial infarction, ischemic heart disease, and death in men and women. JAMA 298, 299–308. Orskov C, Jeppesen J, Madsbad S, Holst JJ. (1991). Proglucagon products in plasma of noninsulin-dependent diabetics and nondiabetic controls in the fasting state and after oral glucose and intravenous arginine. J Clin Invest 87, 415–423. Power O, Hallihan A, Jakeman P. (2009). Human insulinotropic response to oral ingestion of native and hydrolysed whey protein. Amino Acids 37, 333–339. Rabe-Hesketh S. (2008). Multilevel and Longitudinal Modeling Using Stata. Stata Press: TX. Ruge T, Hodson L, Cheeseman J, Dennis AL, Fielding BA, Humphreys SM et al. (2009). Fasted to fed trafficking of Fatty acids in human adipose tissue reveals a novel regulatory step for enhanced fat storage. J Clin Endocrinol Metab 94, 1781–1788. Sehested TS, Hansen TW, Olsen MH, Abildstrøm SZ, Rasmussen S, Ibsen H et al. (2010). Measures of overweight and obesity and risk of cardiovascular disease: a population-based study. Eur J Cardiovasc Prev Rehabil 17, 486–490. Teucher B, Rohrmann S, Kaaks R. (2010). Obesity: focus on all-cause mortality and cancer. Maturitas 65, 112–116. Thomsen C, Rasmussen O, Lousen T, Holst JJ, Fenselau S, Schrezenmeir J. (1999). Differential effects of saturated and monounsaturated fatty acids on postprandial lipemia and incretin responses in healthy subjects. Am J Clin Nutr; 69, 1135–1143. van Wijk JP, Halkes CJ, Erkelens DW, Castro Cabezas M. (2003). Fasting and daylong triglycerides in obesity with and without type 2 diabetes. Metabolism 52, 1043–1049. Westphal S, Ka¨stner S, Taneva E, Leodolter A, Dierkes J, Luley C. (2004). Postprandial lipid and carbohydrate responses after the ingestion of a casein-enriched mixed meal. Am J Clin Nutr 80, 284–290. Yusuf S, Hawken S, Ounpuu S, Bautista L, Franzosi MG, Commerford P et al. (2005). Obesity and the risk of myocardial infarction in 27 000 participants from 52 countries: a case-control study. Lancet 366, 1640–1649.