Whey protein protects liver mitochondrial function against oxidative stress in rats exposed to acrolein

AC was purchased from Sigma-Aldrich (Steinheim, Germany). Unflavoured WP was purchased as food supplement from the General Nutrition Centre (GNC, Istanbul, Turkey). A 1-ounce (28 g) serving of WP contains 21 g of proteins (493 mg of L-arginine, 3558 mg of L-glutamine, 1212 mg of valine, 631 mg of tyrosine, 355 mg of tryptophan, 1350 mg of threonine, 995 mg of serine, 1163 mg of proline, 719 mg of phenylalanine, 443 mg of methionine, 1025 mg of alanine, 2237 mg of aspartic acid, 522 mg of cysteine, 384 mg of glysine, 414 mg of histidine, 1301 mg of isoleucine, 2296 mg of leucine, and 1902 mg of lysine), 1 g of saturated and 2 g of unsaturated fat, 2 g of carbohydrate, and 2 g of sugar. All other (analytical grade) chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Twenty-four healthy Sprague Dawley male rats, each weighing 250–300 g, were obtained from the Experimental Animal Centre of Ondokuz Mayıs University (Samsun, Turkey). The animals were housed in polycarbonate cages under standard laboratory conditions (21±1 °C temperature, 40–55 % humidity, 12:12 h light-dark cycle) and had free access to tap water and standard rodent pellet diet throughout the experiment.

For the experiment the rats were randomly divided into four groups of six. The control group was receiving 1 mL of 0.9 % NaCl solution through oral gavage a day because AC and WP were dissolved in 0.9 % NaCl solution for the experimental treatment. The other three groups were orally gavaged with AC alone (5 mg/ kg bw a day), WP alone (200 mg/ kg bw a day) and AC+WP (5 mg/ kg + 200 mg/kg bw a day) in 1 mL of 0.9 % NaCl solution. All groups were receiving treatment six days a week for 30 days. The choice of AC and WP doses was based on maximal daily human consumption as described in our earlier articles (1, 2).

The study was approved by Medical Research Ethics Committee of Ondokuz Mayıs University (HADYEK 2014/24) and all procedures followed the guidelines provided by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (15).

The livers were placed in ice cold 0.15 mol/ L NaCI solution, perfused with the same solution to remove red blood cells, blotted on filter paper, weighed, and stored at -80 °C until use. Mitochondria were isolated from the liver by differential centrifugation as described elsewhere (16, 17). Briefly, liver tissues were homogenised in buffer A (250 mmol/L sucrose, 10 mmol/L Tris-HCI, 1 mmol/L EGTA, pH 7.6), centrifuged at 1000 × g for 5 min, the obtained supernatant strained through gauze, and then centrifuged again at 7000 × g for 10 min. After washing with buffer B (250 mmol/L sucrose, 10 mmol/L Tris-HCI, and 0.1 mmol/L EGTA, pH 7.6), the obtained mitochondrial pellets were then centrifuged twice at 4 °C, first at 1000 × g and then 7000 × g, both times for 15 min Mitochondrial fractions were stored at -80 °C for further analysis and mitochondrial protein content determined using the method described by Lowry et al. (18).

Mitochondrial super oxide dismutase (MnSOD) activity was determined according to the method described by Beauchamp and Fridovich (19). One unit of SOD activity was defined as the amount of enzyme required to inhibit nitroblue tetrazolium photoreduction rate by 50 %. Glutathione peroxidase (GPx) activity was determined according to Flohe and Gunzler (20). Glutathione (GSH) levels were measured as described by Moron et al. (21) and lipid peroxidation (LP) and protein carbonyls (PCs) levels as described by Esterbauer and Chessman (22) and Levine et al(23), respectively.

Complex I (NADH ubiquinone oxidoreductase) and complex II (succinate ubiquinone oxidoreductase) activities were determined as described by Janssen et al. (24). Complex IV (cytochrome c oxidase) activity was determined as described by Trounce et al. (25). Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (ICDH) activity was determined according to Fatania et al. (26). Alpha-ketoglutarate dehydrogenase (α-KGDH) and malate dehydrogenase (MDH) activities were defined as described by Lucas et al. (27) and Gelpi et al. (28), respectively.

ATP concentrations were determined using the ATP colourimetric/fluorometric assay kit (K354-100, Biovision, Milpitas, CA, USA) and calculated against the ATP standard curve following the manufacturer’s instructions. The results are expressed as nmol/mg of protein.

Data are expressed as means ± standard deviations (SD). For all group comparisons we used one-way analysis of variance (ANOVA) followed by the post hoc Tukey’s test for comparison of means, with p<0.05 taken as significant difference. All analyses were run on IBM SPSS Statistics for Windows, version 22.0 (IBM Corp., Armonk, NY, USA)

As expected, AC treatment significantly decreased MnSOD (17.71 %), GPx (32.84 %), and GSH (28.14 %) levels compared to the control group. Compared to the AC group, WP and WP+AC groups had significantly higher respective MnSOD (28.9 % and 8.53 %), GPx (32.27 % and 22.54 %), and GSH (42.38 % and 20.65 %) levels (Figure 1), which clearly shows that treatment with WP improved all these enzyme activities.

Compared to control, AC treatment also significantly increased LP (72.81 %) and PC (29.52 %) levels, but WP, alone or in combination with AC, significantly restored their levels by decreasing LP 35.97 % and PCs 12.50 % compared to the AC group (Figure 2).

Figure 1

Figure 2

Decreases in OXPHOS complex II (38.28 %) and IV (23.82 %) levels in AC-treated rats were significant compared to control, and both were significantly improved by treatment with WP alone or in combination with AC, which increased complex II by 55.48 % and 42.87 %, respectively and complex IV by 43.95 % and 39.84 %, respectively compared to the AC group (Figure 3).

Figure 3

A similar pattern is observed with ICDH (Figure 4), whose activity in the AC-treated group was significantly lower than in control but was restored by WP and AC+WP treatments (38.59 % and 12.86 % higher than in the AC group, respectively). AC did not significantly affect α-KGDH and MDH activity, but WP alone increased both significantly (26.55 % and 48.78 %, respectively) compared to control. In combination with WP it also resulted in significantly higher MDH activity (35.59 %) than in control (Figure 4).

Figure 4

ATP levels dropped significantly (25.95 %) with AC treatment compared to control, whereas WP in combination with AC was not as successful in restoring ATP as it was with other parameters, considering that ATP levels remained 16.27 % lower than control (Figure 5).

Figure 5