Whey protein in cow’s milk is a nutritionally beneficial source of protein, which has well-balanced amino acid ratios. It is also known that whey protein efficiently enhances protein synthesis in skeletal muscles    by activating a pathway regulated by mechanistic target of rapamycin (mTOR)   , and its ability to enhance protein synthesis is more potent than casein or soy protein  . Additionally, whey protein exhibits various physiological functions including suppression of inflammation.
The anti-inflammatory function of whey protein was shown in animal inflammatory models such as the galactosamine-induced hepatitis model  , which reproduces chemical-induced hepatitis, and the ConA-induced hepatitis model  , caused by immunological reactions. The anti-inflammatory effect of whey protein was shown in human-beings as well. In chronical obstructive pulmonary disease (COPD) patients, a liquid diet comprising whey protein peptides as a protein source suppressed inflammatory reactions  . It also reduced the risk of bacteremia post hepatic transplantation surgery  .
These data suggest that whey protein might have therapeutic applications in clinical problems where protein deficiency and inflammatory reactions cause symptoms, for instance, sarcopenia, which is characterized by age-related loss of muscle mass and quality. Because loss of muscle protein is enhanced by declined protein intake and chronic low-grade inflammation associated with the aged  , the prerequisites for clinical therapy of sarcopenia should be protein supplementation and suppression of inflammation. Whey protein has both these properties making it a potential therapeutic candidate for sarcopenia.
On the other hand, sarcopenia is often associated with type 2 diabetes   , as loss of muscle mass and chronic inflammation enhance the progression of insulin resistance leading to the onset of diabetes. A stronger association of type 2 diabetes with sarcopenia is observed in patients with sarcopenia obesity, a comorbidity of sarcopenia and obesity  . Accumulated mass of adipose tissues in sarcopenia obesity is thought to be a cause of aggravation of insulin resistance, as it causes adipose tissue-related inflammation and systemic dyslipidemia exacerbating insulin resistance  .
For the therapeutic application of whey protein for sarcopenia and sarcopenia obesity, it is important to know how whey protein affects lipid metabolism. This study describes how whey protein intake affects expression patterns of genes related to lipid metabolism in mice. A pathway for the regulation of triglyceride degradation by the transcription factors, peroxisome proliferator-activated receptors (PPARs), and their target enzymes, hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL), and a pathway for the regulation of fatty acid synthesis, the transcription factor, sterol regulatory element-binding transcription factor 1 (SREBP1c), and its target enzymes, acetyl-CoA carboxylase alpha (ACCα) and fatty acid synthase (FAS), were analyzed.
2. Materials and Methods
2.1. Animal Experiments
Animal experiments were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments (The Science Council of Japan) after approval by the Animal Ethical Care Committee of Kanagawa Institute of Technology.
Male, 6-week-old, C57BL mice were purchased from Charles River Laboratories (Japan) and were separated into two groups (n = 5). They were fed AIN-93G (Oriental Yeast, Japan), comprising casein as the protein source for 5 days to acclimate to environment and diet. For the next 4 weeks, one group was fed AIN-93G composed of casein and the other AIN-93G composed of whey protein (Arla Foods Ingredients, Japan) as the protein source. Mice were maintained at constant room temperature of 20˚C - 22˚C with free access to water and diet under a 12:12 h light-dark cycle with lights on at 7:00 AM.
The gastrocnemius muscle, liver, and epididymal adipose tissue of these mice were excised, immersed, and stored in RNALater (Thermo Fischer Scientific, USA) at −20˚C.
2.2. Real-Time PCR
The protocol provided with ReliaPrep RNA Miniprep Systems (Promega, USA) was used to separate total RNA from tissues. Reverse transcription of RNA to cDNA was performed using ReverTra Ace qPCR RT Master Mix (TOYOBO, Japan) on ABI Geneamp 9700 PCR-Thermal Cycler (Applied Biosystems, USA).
Real-time PCR was performed using KOD-Plis-Ver.2 (TOYOBO, Japan) on ABI Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, USA). Primers used to detect and quantify gene expression were synthesized using TKARA’s (Japan) database. Sequences are shown in Table 1.
Table 1. List of DNA primer sets for quantitative real-time PCR.
Gene expression levels were normalized to the expression level of the internal control gene, 18S rRNA, and expressed as the mean with the standard error of mean in figures.
2.3. Statistical Analysis
The results were statistically analyzed using a Student’s t-test.
3.1. Body Weight
Body weights of mice fed casein or whey protein were 25.57 ± 0.6 g and 25.1 ± 0.8 g (mean ± standard error), respectively. No significant difference was detected between them.
3.2. Gastrocnemius Muscle
Because triglyceride degradation is regulated by PPAR pathways, the mRNA expression levels of genes associated with these pathways were measured. And since gene expression levels of the enzymes for lipid degradation, HSL and LPL, are upregulated by the activation of PPARs, the expression levels of these enzymes were also measured. In whey protein fed mice, the mRNA expression of PPARα increased, while that of PPARγ decreased. Significant decrease in mRNA expression of HSL and significant increase in LPL were observed (Figure 1).
Fatty acid synthesis is regulated by SREBP1c pathways. Activated SREBP1c upregulates gene expression levels of ACCα and FAS, two major enzymes for the fatty acid synthesis. In whey protein fed mice, the mRNA expression of SREBP1c
Figure 1. Effect of whey protein on mRNA levels of the enzymes related to triglyceride breakdown in the gastrocnemius muscle. The mRNA expression levels were normalized to the casein control. Ɨ: P < 0.1, *: P < 0.05, ***: P < 0.001.
was significantly decreased. Consequently, the mRNA expression levels of ACCα and FAS were also significantly decreased (Figure 2).
In whey protein fed mice, the mRNA expression of PPARα was significantly decreased, while PPARγ was significantly increased. Whey protein significantly increased the mRNA expression of HSL, while it did not significantly change that of LPL (Figure 3).
Compared to casein fed mice, the mRNA expression of SREBP1c was significantly decreased in whey fed mice. The mRNA expression levels of ACCα and FAS were also significantly decreased in whey protein fed mice (Figure 4).
3.4. Epididymal Adipose Tissue
In the epididymal adipose tissue of whey protein fed mice, the mRNA expression
Figure 2. Effect of whey protein on mRNA levels of the enzymes related to fatty acid synthesis in the gastrocnemius muscle. The mRNA expression levels were normalized to the casein control. **: P < 0.01, ***: P < 0.001, Ɨ: P < 0.01.
Figure 3. Effect of whey protein on mRNA levels of the enzymes related to triglyceride breakdown in the liver. The mRNA expression levels were normalized to the casein control. Ɨ: P < 0.1, *: P < 0.05.
Figure 4. Effect of whey protein on mRNA levels of the enzymes related to fatty acid synthesis in the liver. The mRNA expression levels were normalized to the casein control. **: P < 0.01, ***: P < 0.001.
of PPARγ was significantly increased while that of PPARα was not changed. The mRNA expression of HSL was significantly increased by whey protein, and that of LPL was not significantly changed (Figure 5).
The mRNA expression of SREBP1c decreased. A significant decrease in the mRNA expression levels of FAS was observed, while that of ACCα was not changed (Figure 6).
In this study, the gene expression patterns of the transcription factors, PPARs and SREBP1c, and their downstream enzymes related to lipid metabolism in the gastrocnemius muscle, liver and epididymal adipose tissue of mice fed whey protein, were analyzed.
PPARα is a transcription factor that regulates lipid metabolism by modulating the gene expression of enzymes related to energy production from triglyceride    . For example, PPARα upregulates the gene expression of the two major enzymes for triglyceride breakdown, HSL and LPL. In this study, it was shown that PPARα was transcriptionally upregulated by whey protein in the gastrocnemius muscle. Consequently, the gene expression of LPL was also upregulated. These results suggest that the supply of fatty acids by triglyceride breakdown is enhanced in the gastrocnemius muscle probably for energy production.
In the liver and epididymal adipose tissue, PPARγ, not PPARα, was observed to be transcriptionally upregulated, suggesting that the regulatory mechanisms for lipid metabolism might be different between the gastrocnemius muscle and the liver and adipose tissue of mice fed whey protein. In this study, it was observed that the expression levels of PPARγ and HSL in the liver were concomitantly increased. In the epididymal adipose tissue also, PPARγ was observed to be transcriptionally upregulated by whey protein together with HSL, coinciding with the report that the expression of HSL was enhanced by PPARγ in adipose tissue of rosiglitazone-treated mice  . The same expression pattern of PPARγ and HSL might indicate that similar, if not identical, metabolic configuration for
Figure 5. Effect of whey protein on mRNA levels of the enzymes related to triglyceride breakdown in the epididymal adipose tissue. The mRNA expression levels were normalized to the casein control. *: P < 0.05.
Figure 6. Effect of whey protein on mRNA levels of the enzymes related to fatty acid synthesis in the epididymal adipose tissue. The mRNA expression levels were normalized to the casein control. Ɨ: P < 0.1, *: P < 0.05.
triglyceride breakdown takes place in the liver and epididymal adipose tissue of mice fed whey protein.
Major enzymes in the pathway of fatty acid synthesis are ACCα, which catalyzes the conversion of acetyl CoA to malonyl CoA, and FAS, which catalyzes the synthesis of palmitic acid from acetyl CoA and malonyl CoA. These enzymes are transcriptionally regulated by the transcriptional factor, SREBP1c   . The results in this study indicate that the gene expression of SREBP1c was suppressed in the gastrocnemius muscle and liver by whey protein, subsequently lowering the expression levels of ACCα and FAS. In the epididymal adipose tissue, the expression of SREBP1c tended to be decreased less potently than those in the other two tissues. These results implicate that regulation modes of fatty acid synthesis vary between tissues. That is, the fatty acid synthesis in the gastrocnemius muscle and liver of mice fed whey protein might be more efficiently suppressed, whereas the adipose tissue might be more potently directed toward fatty acid synthesis than other tissues.
The food component, resveratrol, modulates lipid metabolism by activating PPARs    . Resveratrol directly interacts with PPARs and enhance their transcriptional activity  . It has also been reported that soy protein decreases blood glucose and triglyceride levels via the modulation of PPARα pathways  and suppresses the expression of genes related to lipid synthesis by downregulating the gene expression of SREBP1c  . Although these food components and whey protein share similar properties with respect to modulating PPARs and SREBP1c, only whey protein shows potency to stimulate protein synthesis, differentiating it from resveratrol and soy protein.
Beneficial properties of whey protein, a protein source with balanced amino acid ratios, abilities to stimulate muscle protein synthesis and suppress inflammatory reactions, suggests its potential use in the prevention of sarcopenia or delay of its progress. The results of this study might add a new aspect to the properties of whey protein. Namely, whey protein modulates lipid metabolism directing toward enhancement of triglyceride breakdown and suppression of fatty acid synthesis.
This property might help patients with sarcopenia especially with a morbidity of obesity improve lipid metabolism by directing it toward lipid catabolism. On the other hand, this property might not disturb usage of whey protein for patients with sarcopenia without a morbidity of obesity, because it is thought that whey protein, as a dairy food component, moderately modulates lipid metabolism in these patients without adverse outcomes. All of these properties of whey protein might become an incentive to promote its therapeutic usage for sarcopenia with and without a morbidity of obesity.
The current study demonstrated that whey protein intake affected the mRNA expression of the transcription factors, PPARα, PPARγ and SREBP1c, in the gastrocnemius muscle, liver and epididymal adipose tissue of mice, concomitantly affecting the expression of their downstream enzymes, HSL, LPL, ACCα and FAS in these tissues. The expression patterns of these factors and enzymes indicated that whey protein transcriptionally modulated lipid metabolism directing it toward enhancement of triglyceride breakdown and suppression of fatty acid synthesis. Together with beneficial properties of whey protein to enhance protein synthesis and to suppress inflammation, these results implicate that whey protein may be a potent candidate for dietary therapies for sarcopenia and sarcopenia obesity.
This work was supported by JSPS KAKENHI Grant Number JP15K00890.
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