2016 Volume 22 Issue 5 Pages 631-638
Whey protein consumption is reported to reduce serum lipids, however the responses to derived peptides have not yet been fully characterized. In this study, we evaluated the effects of whey protein-hydrolyzed peptides (WPP) on serum and hepatic lipid levels, as well as hepatic lipogenic gene expression in SD rats consuming a high-sucrose AIN-76 based diet. After a 14-day ad libitum consumption of diet containing WPP, serum and hepatic triglycerides, and total cholesterol levels were significantly decreased when compared with the control group. A similar trend was observed in the time-restricted feeding groups, in which food was provided for 2 h at the beginning and the end of the dark period cycle during the 14-day treatment period. Additionally, hepatic gene expression linked to triglyceride and cholesterol biosynthesis, and their enzymatic activities were downregulated in the WPP group. In conclusion, daily consumption of WPP appears to decrease lipid levels in the blood and liver, and potentially protect against dyslipidemia.
Whey is a byproduct of casein removal from milk, which is an important step in the manufacture of cheese (Balagtas, et al. 2003). Whey is rich in proteins, which have biologically active components that provide additional benefits to enhance biological functions (Hoffman and Falvo 2004) such as antioxidative and antigenotoxic effects, and improve insulin-resistance and stimulation of muscle protein synthesis (Deminice, et al. 2015, Kim, et al. 2013, Tong, et al. 2014). Furthermore, it has also been suggested that whey protein consumption may regulate physiological lipid metabolism (Morifuji, et al. 2005). In contrast to rats fed a casein-rich diet, a whey protein diet significantly decreased serum triglycerides due to the downregulation of liver lipogenic enzyme activities, including malic enzyme (ME) and fatty acid synthase (FASN). Furthermore, unlike other protein sources, such as soy protein, whey protein also decreased liver triglycerides (Aparicio, et al. 2013). These studies suggest that whey protein might be a beneficial ingredient in dietary therapy aiming at preventing dyslipidemia.
In addition to the protein, whey hydrolysates have also been found to exert similar biological functions. When administered orally in rats, whey protein hydrolysates reach a maximum plasma amino acid concentration 7 min after administration, while the whole protein reaches similar levels 30 min after administration (Nakano, et al. 1994), suggesting that whey hydrolysates are a more digestible and absorbable nitrogen source than the whole protein. Several peptides present in whey hydrolysates have shown a potent radical scavenging activity (Contreras, et al. 2011). Interestingly, whey hydrolysates were shown to be more effective in preventing colon tumor development than the whole protein (Attaallah, et al. 2012). Furthermore, hydrolysates have also shown superior muscle protein synthesis (Kanda, et al. 2013). However, there is limited information about the effects of daily consumption of whey protein hydrolysates on the regulation of dyslipidemia.
In this study, we evaluated the effects of ad libitum consumption of whey protein-hydrolyzed peptides (WPP) on serum and hepatic lipid levels, as well as liver lipogenic enzyme activity on rats. We also evaluated the effects of time-restricted feeding, in which food was supplied for 2 h at the beginning and the end of the dark cycle period. Several recent animal studies have used an AIN-93 based purified diet, the formula of which has been published by the American Institute of Nutrition (AIN) for experimental rodents (Reeves, et al. 1993). This diet was formulated to improve animal performance in experimental models, and the major difference with the previous AIN-76 based diets lays in the substitution of cornstarch for sucrose. High dietary concentrations of sucrose have been associated with several metabolic complications, including dyslipidemia and fatty liver (Gerber and Berneis 2012, Reeves 1997). In this study, we utilized the AIN-76 diet (sucrose contents, 50%) as our control diet in which rats presented a mild dyslipidemia.
Reagents The whey protein-hydrolyzed peptides (WPP) used in this study were whey peptide HW-3 obtained from MEGMILK SNOW BRAND Co. Ltd. (Tokyo, Japan). WPP consisted of 3.4 g water, 80.2 g protein, 0.1 g lipid, 5.5 g mineral, and 10.8 g carbohydrates per 100 g material, and the average molecular weight was approximately 400 Da, in which the main components were di- and tri-peptides. Its primary amino acids were branched-chain amino acid (valine, leucine, isoleucine) and lysine, as shown in Appendix Table 1. Soy protein-hydrolyzed peptides (SPP) was purchased from Fuji Oil Co. Ltd. (Osaka, Japan). Cellulose, β-cornstarch, sucrose, vitamin mixture (AIN-76), mineral mixture (AIN-76) and nicotinamide-adenine dinucleotide phosphate (NADPH) were purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). Casein, corn oil, DL-methionine, and choline bitartrate were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Malonyl-CoA was from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest grade available.
Animal housing, diets and experiments All animal maintenance and experiments were conducted in accordance with the care and use of laboratory animals policy of the University of Miyazaki (Miyazaki, Japan). The experimental protocol was registered under number 2008-007-6. In this study, we conducted two animal studies according to the following protocols:
Protocol I: seventeen male Sprague-Dawley (SD) rats (4 weeks old; Kyudo Co. Ltd., Saga, Japan) were housed in individual stainless-steel cages in air/temperature-controlled rooms (temperature, 23 ± 1°C; relative humidity, 55 ± 5%) respecting a 12-h dark/light cycle; light on 07:00 i.e. Zeitgeber time (ZT0) and light off 19:00 (ZT12) with free access to commercial diet (Type CE-2, Clea Japan, Tokyo, Japan) and deionized water ad libitum. After 1 week acclimatization, animals were randomly divided into the following three groups: the first group consumed an AIN-76 based purified powder diet (American Institute of Nutrition, 1977); and the other two groups consumed 10% or 20% of WPP containing diets, as shown in Table 1 (groups: Control, WPP10, WPP20). Two weeks later, blood was taken from the abdominal vein under anesthesia with sodium pentobarbital (30 mg/kg body weight) at ZT2 without starvation. Serum was separated by centrifugation at 1,000 g for 20 min, 30 min after resting at room temperature. Liver samples were removed, and weighed. All samples were stored at −80°C for subsequent analysis.
Protocol II: the food consumption patterns were analyzed at 2-h intervals throughout the 12-h dark/light cycle (Appendix Figure 1). Under the ad libitum feeding condition, food consumption during the light period (inactive phase) was low, and dramatically increased just before the onset of the dark period (ZT12). The active food consumption exhibited two peaks at the beginning and the end of the dark period. These results are in agreement with reports published previously (Liu, et al. 2004). Twenty-two male SD rats (4 weeks old; Japan SLC, Shizuoka, Japan) were housed in individual stainless-steel cages in air and temperature controlled rooms with free access to CE-2 diet and deionized water ad libitum. One week after acclimatization to the housing environment, animals were further acclimated to time-restricted feeding using CE-2 diets for one week. Subsequently, the animals were randomly divided into three groups: AIN-76 based purified powder diet (Control), 20% WPP containing diet (WPP20), and 20% SPP containing diet (SPP20). All groups were given access to food for 2 h twice a day, corresponding to the active food consumption period (ZT23-1 and ZT11-13). The WPP20 and SPP20 groups were allowed to consume individual diets freely during this period. However, the control group was given a set amount of AIN-76 based food, which was based on the previous day's average intake in the WPP20 and SPP20 groups, according to the modified method reported by Stevanovic et al (Stevanovic, et al. 2012). After the 14-day trial, blood was collected from the tail vein under anesthesia with sodium pentobarbital (30 mg/kg body weight) at ZT23 following a 10-h fast. Serum was separated by centrifugation at 1,000 g for 20 min as described above, and stored at −80°C until analysis.
Diurnal food consumption patterns on Sprague-Dawley rats.
Twenty rats were housed in individual stainless-steel cages in air and temperature controlled rooms (temperature, 23 ± 1°C; relative humidity, 55 ± 5%) under a 12-h light cycle: on at 07:00 corresponding to Zeitgeber time (ZT0) and off at 19:00 (ZT12) with free access to AIN-76 based purified powder diets and deionized water ad libitum. Food consumption was assessed at 2 h intervals for 36 h. Values are expressed as means ± standard error (n=20).
Serum lipid analyses Three serum lipid parameters, including triglyceride (TG), total cholesterol (T-chol) and phospholipid (PL), were analyzed using individual E-test Wako kits, obtained from Wako Pure Chemical Industries, Ltd. All standards were used in accordance with the manufacturer's instructions.
Hepatic lipid analyses Lipids contained in the liver samples were extracted as described previously by Folch and colleagues (Folch, et al. 1957). The extracts were saponified for the measurement of T-chol amounts as described by Sperry and Webb (Sperry and Webb 1950). TG and PL amounts were analyzed using the individual classical methods (Fletcher 1968, Rouser, et al. 1966).
Enzymatic activities Cytosolic fractions were prepared from the liver samples using the modified method described by Graham (Graham 1997). Briefly, liver samples were placed in 10 volumes of buffer containing 0.25 mol/L sucrose, 0.01 mol/L Tris-HCl (pH 7.4) and 1 μmol/L EDTA-2Na, and then homogenized at 4°C. Homogenates were centrifuged at 700 g for 10 min at 4°C, and supernatants were further centrifuged (10,000 g, 4°C, 10 min). Finally, the cytosolic fractions were obtained by centrifugation at 126,000 g for 60 min at 4°C. Enzymatic activities of FASN and ME were evaluated according to the methods by Kelley and colleagues (Kelley, et al. 1986) and Ochoa (Ochoa 1955), respectively. The protein contents in the cytosolic fractions were quantified using a Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA).
Gene expression analysis Total RNA was extracted from each liver (100 mg each) according to the modified method of Chomczynski and colleagues (Chomczynski and Sacchi 1987). RNA optical density ratios were measured at 260 nm and 280 nm to evaluate nucleic acid purity, and total RNA concentrations were determined based on absorbance at 260 nm. Total RNA quality was further determined by the integrity of the 28S and 18S rRNAs. Two hundred micro gram of total RNA was reverse transcribed in 10 µL final volume using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. A total of 1 µL of cDNA solution was added to 19 µL of the PCR mixture containing TaqMan Universal PCR Master Mix (10 µL, Applied Biosystems), DNase/RNase-free water (8 µL), house-keeping or individual target primers (1 µL). All primers used in this study were obtained from Applied Biosystems as follows: 18s, Assay ID, Hs_99999901_s1; Cyp7a1, Rn00564065_m1; Fasn, Rn00569117_m1; Srebf1, Rn01495769_m1; Srebf2, Rn01502638_m1; Hmgcr, Rn00565598_m1. Quantitative RT-PCR was performed on MiniOpticon real-time PCR detection system (Bio-Rad Laboratories). The relative expression level of the target gene was calculated by the comparative automatic threshold cycles method using 18s as a calibrator. The relative differences in expression levels between groups were expressed using cycle time (Ct) values and the relative differences between groups expressed as relative increases, setting the control group, which are consuming AIN-76 based purified diets, to 100. Each experiment was carried out in duplicate.
Statistical analyses Statistical analyses were undertaken using the software program Stat View for Windows (Version 5.0, SAS Institute, Cary, NC, USA). Statistical analyses of differences between the control and treated groups were carried out using one-way ANOVA, followed by the Williams or Tukey's test. Results were considered significant if the probability of error was <5%.
Ad libitum feeding of WPP containing diets In this study, we evaluated the effects of WPP on serum and hepatic lipid levels, and on liver lipogenic enzyme activity in rats. Male SD rats were allowed to consume ad libitum a 20% casein diet (normal control AIN-76 diet; control group), a 10% WPP + 10% casein diet (WPP10 group) or a 20% WPP diet (WPP20 group) for 14 days. It has been previously reported that the consumption of whey protein rich diets decreases food intake without changing the rats body weight when compared with other protein sources, such as casein and soy protein (Morifuji, et al. 2005, Zhou, et al. 2011). However, the WPP containing diets did not appear to affect overall food intake in this study (Table 2). The difference could originate from the better digestibility and absorption of WPP compared to that of whey protein (Nakano, et al. 1994). Furthermore, body and liver weights were not changed upon WPP consumption (Table 2).
Rats were fed respective diets ad libitum for 14 days.
Serum levels of TG, total T-chol and PL in non-fasting rats consuming WPP10 or WPP20 showed a decrease when compared with control (Figure 1A–C). In particular, serum T-chol and PL levels in rats consuming WPP20 were significantly decreased compared with the control. Hepatic TG in the WPP10 and WPP20 groups was significantly lower than that of the control (Figure 1D). Hepatic T-chol was dose-dependently decreased, and was significantly reduced in the WPP20 group (Figure 1E). Conversely, hepatic PL levels were not affected by consumption of WPP (Figure 1F). Our findings are in agreement with Morifuji and associates who reported that serum TG levels decreased in SD rats after a 3-h fasting period (from 182 mg/dL in casein-based diet to 150 mg/dL in whey protein-based diet), although they did not analyze the hepatic lipid levels (Morifuji, et al. 2005). They also observed a significant decrease in the activity and gene expression of the hepatic lipogenic enzymes ME and FASN in rats fed whey protein when compared with casein as the protein source. In the present study, ad libitum feeding of WPP significantly inhibited hepatic FASN and ME activity, even in the WPP10 group, which received a lower amount of WPP (Figure 2). Furthermore, hepatic gene expression of Fasn in the WPP20 group was significantly decreased in a dose-dependent manner when compared with the control group (Figure 3A). Additionally, hepatic gene expression of Srebf1, Srebf2 and Hmgcr, responsible for the catalysis of the rate limiting step of cholesterol biosynthesis (Brown and Goldstein 1997), showed significant or slight decrease (Figure 3B–D). Activation of AMP-activated protein kinase (AMPK) has been reported to be observed in muscle collected from mice consuming whey peptides containing diets (Ichinoseki-Sekine, et al. 2015). Activation of AMPK can reduce SREBF1c activity (Deng, et al. 2015). Furthermore, Fasn expression is recognized to be affected through the modulation of the expression and/or nuclear maturation of the transcription factor SREBF1c, which binds to and activates sterol regulatory elements in the promoter region of Fasn (Menendez and Lupu 2007). Therefore, our findings imply that a WPP-based diet downregulates the expression of genes involved in the biosynthesis of triglyceride and cholesterol, such as Fasn through the AMPK pathway, and consequently lowers total lipid levels in both the liver and blood.
Effects of ad libitum whey protein hydrolyzed peptides feeding on serum and hepatic lipid levels.
Rats were given a control diet (AIN-76), 10% or 20% of whey protein peptides based diets ad libitum for 14 days. Serum and liver samples were collected at ZT2 in the absence of fasting, and lipid levels were analyzed. (A) serum triglyceride, (B) serum total cholesterol, (C) serum phospholipid, (D) hepatic triglyceride, (E) hepatic total cholesterol, (F) hepatic phospholipid. Values indicate mean ± standard error (n = 6). #P < 0.05 vs. control group (Williams's test).
Effects of ad libitum whey protein-hydrolyzed peptides feeding on hepatic enzyme activity according to fatty acid synthesis.
Rats were given the control diet (AIN-76), 10% or 20% of whey protein peptides containing diets ad libitum for 14 days. Liver samples were collected at ZT2 in the absence of fasting, and hepatic enzyme activity was analyzed: (A) fatty acid synthase (FASN) and (B) malic enzyme (ME). Values indicate mean ± standard error (n = 6). #P < 0.05 vs. control group (Williams's test).
Effects of ad libitum whey protein-hydrolyzed peptides feeding on lipid metabolism related gene expression in rat liver.
Rats were given a control diet (AIN-76), 10% or 20% of whey protein peptides containing diets ad libitum for 14 days. Liver samples were collected at ZT2 in the absence of fasting, and hepatic enzyme activity was analyzed: (A) Fasn, (B) Srebf1, (C) Srebf2, (D) Hmgcr and (E) Cyp7a1. Values indicate mean ± standard error (n = 6). #P < 0.05 vs. control group (Williams's test).
Time-restricted feeding of WPP containing diets Using a rat model of nonalcoholic fatty liver disease (NAFLD), in which rats were fed a high carbohydrate, fat free diet for 28 days, Hamad and associates reported that oral administration of 0.15 g/day/rat of whey protein hydrolysates once daily significantly decreased serum T-chol and hepatic TG levels (Hamad, et al. 2011). In Protocol I, the rats consumed diets ad libitum. Furthermore, we evaluated the effect of time-restricted feeding, which regulates the consumption timing, of WPP-based diets on lipid levels in Protocol II.
Male SD rats were given a 20% casein diet (AIN-76 diet, control group), a 20% WPP diet (WPP20 group), or a 20% SPP diet (SPP20 group) for 14 days. As described above, the rats exhibited two peaks of active feeding periods, at the beginning and end of the dark period, corresponding to ZT11-13 and ZT23-1 respectively, times at which rats were given free access to food (Appendix Figure 1). After 14 days following this regimen, TG and T-chol levels in the serum collected from the control group at ZT23 after a 10-h fast were 102 g/dL and 99 mg/dL, respectively (Figure 4). Interestingly, blood TG and T-chol levels from fasted male SD rats (less than 10 weeks old) administered a normal diet, such as AIN-93G, have been quantified at between 30 – 50 mg/dL and 60 – 90 mg/dL, respectively (Shrestha, et al. 2009). In the present study, the AIN-76 diet, in which cornstarch is substituted with sucrose (Table 1), was used to induce a mild dyslipidemia via a nutritional consequence (Gerber and Berneis 2012, Reeves 1997). Consequently, the serum TG and T-chol levels observed here in fasted control rats confirmed that we successfully induced mild dyslipidemia following a time-restricted sucrose-rich regimen. However, rats fed WPP-based diets showed significantly decreased serum TG and T-chol levels when compared with the control, with an average value of 64 mg/dL and 77 mg/dL, respectively (Figure 4), suggesting that WPP possesses serum TG and T-chol lowering effects similar to SPP (Tamaru, et al. 2007). Body and liver weights, and food consumption were not affected by consumption of WPP and SPP containing diets during the experimental period (Table 3). Additionally, our results obtained after 10-h fasting also imply that these effects are not likely to be due to inhibition of lipid absorption.
Effects of time-restricted feeding of whey and soy protein-hydrolyzed peptides on serum lipid levels.
Rats were given a control diet (AIN-76), 20% of whey protein peptides or soy protein peptides containing diets daily during ZT23-1 and ZT11-13 for 14 days. Quantities among all groups were homogenized. Serum samples were collected at ZT23 after a 10-h fast, and lipid levels were analyzed. (A) serum triglyceride, (B) serum total cholesterol. Values indicate mean ± standard error (control group, n=7; WPP group, n = 7; SPP group, n = 8), and a different alphabetical superscript indicates a significant difference at P < 0.05 (Tukey's test).
Rats were given access to equal quantities of food daily on ZT23-1 and ZT11-13 for 14 days.
Lactostatin is a penta-peptides (isoleucine- isoleucine- alanine-glutamic acid-lysine) derived from β-lactoglobulin in bovine milk. Nagaoka et al. reported that the tryptic hydrolysate of β-lactoglobulin exerts hypocholesterolemic activity (Nagaoka, et al. 2001). Although we have not been able to identify the active ingredients in the WPP used in this study, lactostatin is unlikely to be the main component for diminishing blood and hepatic lipid levels, because the main components in WPP were di- and tri-peptides (approximate molecular weight, 400 Da). Interestingly, SPP with bound phospholipids were reported to exhibit a higher level of efficacy at lowering cholesterol than intact SPP (Nagaoka, et al. 1999). A low-molecular weight fraction of SPP displayed more effective serum TG and T-chol lowering effects than a low-and high-molecular weight fraction mixture (Tamaru, et al. 2007). Therefore, further investigation is required to clarify the role of individual peptides and/or amino acids present in whey protein-hydrolyzed peptides, including the exploration of active peptides for alteration of the hepatic lipid metabolism.
We have shown for the first time that ad libitum consumption of whey protein-hydrolyzed peptides significantly decreased hepatic triglyceride and cholesterol levels, suggesting that WPP can attenuate circulatory lipid levels, and consequently dyslipidemia. We have also shown that these effects might be mediated via downregulation of hepatic gene expression linked to triglyceride and cholesterol biosynthesis in a similar fashion to whey protein.
Appendix Table 1. Amino acid composition of whey and soy protein peptides.
Appendix Fig. 1. Diurnal food consumption patterns on Sprague-Dawley rats.