The combination of green tea extract and exercise reduced the severity of obesity-related fatty liver disease by 75% in mice fed a high-fat diet, according to Penn State researchers, whose recent study may point to a potential health strategy for people.
The outcome is important, explained Joshua Lambert, associate professor of food science, because nonalcoholic fatty liver disease is a significant global health problem that is expected to worsen.
Because of the high prevalence of risk factors such as obesity and type 2 diabetes, fatty liver disease is forecast to afflict more than 100 million people by 2030.
And there are currently no validated therapies for the disease.
In the study, mice fed a high-fat diet for 16 weeks that consumed green tea extract and exercised regularly by running on a wheel were found to have just a quarter of the lipid deposits in their livers compared to those seen in the livers of a control group of mice.
Mice that were treated with green tea extract alone or exercise alone had roughly half as much fat in their livers as the control group.
In addition to analyzing the liver tissues of mice in the study, which was published recently in the Journal of Nutritional Biochemistry, researchers also measured the protein and fat content in their feces.
They found that the mice that consumed green tea extract and exercised had higher fecal lipid and protein levels.
“By examining the livers of these mice after the study concluded and by screening their feces during the research, we saw that the mice that consumed green tea extract and exercised actually were processing nutrients differently – their bodies were handling food differently,” Lambert said.
“We think the polyphenols in green tea interact with digestive enzymes secreted in the small intestine and partially inhibit the breakdown of carbohydrates, fat and protein in food,” he added.
“So, if a mouse doesn’t digest the fat in its diet, that fat and the calories associated with it pass through the mouse’s digestive system, and a certain amount of it ends up coming out in its feces.”
It may be significant, Lambert explained, that mice treated with both green tea extract and exercise had higher expression of genes related to the formation of new mitochondria.
That gene expression is important, he said, because it provides markers that will help researchers understand the mechanism by which green tea polyphenols and exercise might work together to mitigate fatty liver deposits.
“We measured the expression of genes that we know are related to energy metabolism and play an important role in energy utilization,” Lambert said.
“In the mice that had the combination treatment, we saw an increase in the expression of genes that wasn’t there before they consumed green tea extract and exercised.”
More research is needed to see if there is a synergy created by green tea extract and exercise working together to reduce fat deposited in the liver, or if the effects are simply additive, Lambert pointed out.
His research group in the College of Agricultural Sciences for 12 years has studied the health benefits of polyphenols – often called antioxidants – from green tea, cocoa, avocados and other sources.
In previous related research, Lambert and colleagues demonstrated that green tea extract and exercise together sharply reduced body mass and improved cardiovascular health of high-fat-fed mice.
But because no human trials assessing the health benefits and risks of green tea combined with exercise have been conducted, he urges caution for people who decide to experiment with the health strategy on their own.
“I believe people should engage in more physical activity, and replacing high-calorie beverages with decaffeinated, diet green tea – which has no calories – is a smart move,” he said. “Combining the two might have health benefits for people, but we don’t have the clinical data yet.”
Metabolic Syndrome (MetS) is a group of chronic metabolic disorders including obesity, hyperglycemia, dyslipidemia and hypertension. The risk for developing MetS is closely related to dietary and lifestyle, such as consumption of a high-fat diet, less exercise. MetS has been a strong risk factor for cardiovascular disease, diabetes and cancer [1].
Over the past decades, rapid development of economy and technology has resulted in increasing food supply and declining physical activity [2, 3]. Sedentary lifestyle and over eating habits are considered to be primarily responsible for the growing rate of obesity, diabetes and other MetS associated diseases across the world [4, 5].
The data from International Diabetes Federation (IDF) showed that the global diabetes population reaches 425 million, and undiagnosed diabetes has 212 million in 2017. According to the World Health Organization (WHO) in 2016, nearly 2 billion adults worldwide were overweight and, of these, more than half a billion were obese.
Although the great progress has been made in the treatment of MetS by surgery and pharmacology, some potential side effects are inevitable all the time [6, 7]. At present, lifestyle improvement, diet intervention and exercise therapy are the basic strategies for prevention of MetS. Recent findings indicated that extended sedentary time in individuals with type 2 diabetes is associated with glucose intolerance and higher risk for development of cardiovascular diseases (CVD) [8, 9].
Aerobic exercise reduces blood pressure in both hypertensive and normotensive persons, increase insulin sensitivity and enhance immune function [10, 11].
Regular exercise training increases oxidative capacity, lipid metabolism, reduces serum triglycerides, blood pressure, insulin resistance [12, 13].
Tea has been very popular in the world. And its consumption is only second to water [14]. It has been widely recognized that tea has healthy effects on lipid lowing and anti-obesity [15, 16].
In the processing of green tea, polyphenol oxidase is inactivated by either steaming or pan-frying fresh tea leaves to save the polyphenols. Green tea contains high amount of a monomeric polyphenolic compound known as catechin and (−)-epigallocatechin gallate (EGCG), which is a major component of catechins and was the most studied in recent years.
Many reports have previously demonstrated that catechins, especially EGCG, improve insulin resistance [17, 18], promote fat oxidation, lower blood total cholesterol and triglycerides, and reduce body weight [14, 19, 20].
Yunkang 10 (Camellia sinesis var. assamica cv. Yun Kang 10), is a widely cultivated tea cultivar in Southwestern China. It bears the advantages of wide range of adaptability, low-cost cultivation, and quick growth rate over other’s cultivars.
The genome, chemical profiling, volatile components, entophytic bacteria of Yunkang 10 have been reported [21–24]. Until now, there is no report to evaluate its healthy values against MetS.
Because teas are consumed by drinking water infusion or grinding powders of whole-leaf teas, it is very curious to know the beneficial effects and underlying mechanism by dietary supplement of whole tea powder using HFD induced obese mice.
Previous studies reported that a combination of green tea and exercise facilitates sports performance and endurance capacity, and effectively prevents obesity [25–27].
However, there is limited information on therapeutic effect of green tea supplement combined with aerobic exercise for ameliorating excisting MetS.
The goal of this study is to investigate if the combination of Yunkang 10 green tea supplement and physical exercise has synergistically therapeutic effects on MetS induced by HFD in C57BL/6 J mice, and if so, what are underlying mechanisms?
Yunkang 10 has been widely cultivated in Southwestern China. However, the potential health effects and relevant molecular mechanisms of YKGT are still unknown. Preventive effects of MetS by green tea and catechins, especially EGCG have been extensively investigated [14–19, 34].
Until today, there is only limited information on therapeutic effects of green tea or EGCG intervention on ameliorating existed MetS [35–37]. During recent years, several studies have reported that a combination of green tea and exercise facilitates sports performance and endurance capacity, and effectively prevents obesity [25–27, 38, 39].
However, the underlying mechanisms are still unclear. The present study investigated therapeutic effects and molecular mechanisms of the combination of YKGT supplement and physical exercise using 10 week HFD induced MetS mice model, which mostly represented MetS of human population.
Obesity, hyperlipidemia and insulin resistance are the major features of the MetS. The mice fed with HFD for10 weeks showed typical MetS phenotype, which include obese, hyperglycemia, hyperlipidemia and hyperinsulinemia, as well as fatty liver.
The HFD mice were then treated with YKGT, Ex or YKGT plus Ex for additional 8 weeks. The results showed that treatment with YKGT alone for 8 weeks did not prevent the body weight and liver weight gain, and triglyceride (TG) increase (Fig. (Fig.11 a, b, e), but did ameliorate the increase of serum glucose, insulin and total cholesterol level (Fig. (Fig.1c,1c, d, f), compared to continuous HFD feeding group mice.
Ex alone did restrain the body weight and liver weight gain (Fig. (Fig.1a,1a, b), prevented the increase of insulin and total cholesterol level (Fig. (Fig.1d,1d, f), and did not inhibit the increase of plasma TG level compared to continuous HFD feeding group mice. However, YKGT plus Ex prevented all these index increases.
A randomized trial reported the effect of green tea supplement and interval sprinting exercise (ISE) on the body composition of overweight males, and found that ingestion of green tea by itself did not result in a significant decrease in body or abdominal fat, but increased fat utilization during submaximal exercise.
And the combination of 12 weeks of GT ingestion and ISE did not result in greater total and abdominal fat reduction compared to 12 weeks of ISE alone [26]. This result indicated that GT ingestion might not contribute to fat reduction in overweight males.
This finding is close correlated to our data that YKGT supplement did not prevent the body weight and liver weight gain, and TG increase. Another randomized control trial suggested that GT catechin consumption enhances exercise-induced deduction in abdominal fat and serum TG [27].
Martin et al. reported that short-term GT supplementation did not affect glucose kinetics following ingestion of an oral glucose load post exercise. However, GT was associated with attenuated insulinemia [40].
Recently, Amozadeh et al. reported a randomized trial for overweight and obese females who participating aerobic training (AT) and green tea supplementation on cardio metabolic risk factors, and found that GT plus AT had better effects on decreasing body weight, body fat percentage, body mass index (BMI), TG, LDL, blood pressure, and heart rate (HR) than that of GT or AT alone [41]. These results are consistent with our finding.
Next, we investigated the molecular mechanism underlying YKGT and Ex prevented MetS induced by HFD. Sterol regulatory element-binding protein 1 (SREBP1), Fatty acid synthase (FAS) and Acetyl-CoA carboxylase (ACC) are critical enzymes for fatty acid synthesis.
Our data showed that the either mRNA expression of SREBP1, FAS, or protein expression of SREBP1, FAS were altered by intervention of YKGT or Ex alone in the liver of mice. However, treatment of mice with YKGT plus Ex significantly prevented the increase of gene expression of Srebp1, Fas and ACC(Fig. (Fig.3a-c),3a-c), as well as protein expression of SREBP1 and FAS (Fig. (Fig.4a-d).4a-d).
Previous studies reported that GT ameliorated hyperglycemia and improved blood lipid parameters by regulating the expression of FAS, ACC, SREBP-1 genes [42–44]. PI3K-Akt-mTOR signaling pathway was activated in the liver of HFD mice, and YKGT and Ex intervention block this activation by phosphorylation (Fig. (Fig.4e-j).4e-j).
PI3K-Akt signaling pathway modulated lipid synthesis genes by regulating transcriptional factor l SREBP-1 [45]. In addition, PI3K-Akt-mTOR1 participated lipid and glucose metabolism by regulating the secretion of very-low-density lipoprotein (VLDL) cholesterol, oxidation of fatty acids and hepatic gluconeogenesis [46].
Tea intake prevented lipid accumulation in the liver and adipose tissues [47, 48]. Exercise can effectively increase fat oxidation and energy consumption [49, 50]. Previous studies reported that GT supplement combined with exercise enhanced fat oxidation in HFD mice and obese adults [39, 51].
In addition to promote energy expenditure, this study demonstrated that YKGT plus Ex also effectively suppressed fatty acid synthesis in the liver of HFD mice.
Inflammatory response plays a momentous role in the development of MetS [52, 53]. Numerous inflammatory cytokines are involved in the process of MetS developing into diabetes and cardiovascular disease [54].
Our results showed that YKGT, Ex and YKGT plus Ex significantly prevented the over-expression of pro-inflammatory cytokines IL-6, TNF-α, IL-1β and MCP1 induced by HFD in the mice liver (Fig. (Fig.5a-d).5a-d). And RNAseq data from skeleton muscle tissue also showed that inflammatory related genes, Cd163, Cfh, Il33, C3, Hp, Lbp were all down-regulated by YKGT, Ex or YKGT plus Ex treatment (Fig. (Fig.8a-f).8a-f). Ma S et al. found that Ketogenic Diet and exercise significantly decreased IL-6 concentration in gastrocnemius and plasma [55]. Recently, Cialdella-Kam et al. reported that four-week supplementation of quercetin or green tea extract (GTE) or quercetin plus GTE did not altered inflammatory cytokine level of IL-1β, IL-6, TNF-αand IFN-γin the plasma of 12-week HFD mice [56]. The difference between our data and this report might due to the different intervention period (8 weeks vs. 4 weeks).
Proinflammatory cytokines are directly regulated by the activation of NFκB. The activation of NF-κB pathway is a central part of the complex network of inflammatory response. The NF-κB proteins contain multiple subunits, p50 and p65 dimer bounds to IκBα localized in the cytosol under unstimulated condition.
The inhibitor of κB kinase (IKK) complex is phosphorylated once stimulated, and then activated IKK phosphorylated IκBα proteins, which releases p50 and p65 dimer from complex, p65 transfers to cell nucleus, where binds to specific sites of DNA in promoter region, and induce the synthesis of pro-inflammatory cytokines in the cells [57]. Therefore, the phosphorylation of IKK and IκBα is a key event for NFκB activation.
In this study, YKGT, Ex and YKGT plus Ex all blocked the phosphorylation of IKKα/β and IκBα, and thus inhibited the transcript of pro-inflammatory cytokines. GT polyphenol, major EGCG has found to have an anti-inflammation function. Ueno T et al. found that EGCG decreased the lipid droplets in hepatic cells and prevented liver injury by the inhibition of NF-kB pathway [42].
In addition, Tipoe et al. illustrated that EGCG decreased the DNA-binding activity of NF-kB and the expression of TNF-α, which ameliorated liver inflammation and fibrosis in carbon tetrachloride (CCl4)-induced liver injury in mice [58].
Besides generating force for movement, skeletal muscle also contributes to health through the use and storage of macronutrients. RNAseq data from soleus muscle tissues showed that treatment with YKGT, Ex and YKGT plus Ex significantly down-regulated most of genes induced by HFD (Additional file 3: Figure S1).
KEGG pathway analysis of DEGs revealed that nutrients metabolic pathways (regulation of lipolysis, PPAR signaling pathway, and protein digestion and absorption) and inflammatory pathways (chemokine signaling pathway, NF-κB signaling pathway and phagosome) are most modulated pathways response to YKGT, Ex or YKGT plus Ex intervention. Hodgson et al. suggested that long term GTE intake accelerates fat metabolism at rest, while facilitates the expression of fat metabolism enzyme genes in the skeletal muscles during exercise, thus decreases adipogenic genes in the liver [19].
Previous studies also demonstrated that a combination of tea catechin intake and frequent exercise prevents obesity efficiently by accelerating fat oxidation in the liver and skeletal muscles and facilitates energy expenditure [27, 59].
Recently, a report found that theabrownin and swinging exercise prevented obesity and insulin resistance by accelerating metabolism of glucose and lipid [60]. Emerging data have revealed that obesity may also negatively alter muscle protein turnover, or the breaking down and rebuilding of functional proteins [61].
Feeding and resistance exercise reduced stimulation of myofibrillar protein synthesis in obese people [62]. One paper reported that GT catechins suppress muscle inflammation and hasten performance recovery after exercise [63].
Type 2 diabetes results from defects in glucose transport in skeletal muscle. And the glucose transport gene 4 (GLU4) is abundant present in the skeleton muscles [64]. Tsai et al. reported that 8-week oral GTE supplementation increased post exercise systemic fat oxidation and exercise-induced muscle GLUT4 protein content [65].
From RNAseq data, we found that glucose metabolic related genes Prkcd and Slc2a3 were promoted by YKGT, Ex or YKGT plus Ex. PRKCD is activated by diacylglycerol (DAG) and involves in glucose metabolic pathway. SLC2A3 is a glucose transmembrane transporter, and facilitates glucose transport in the muscle cell [66].
Obesity decreased sensitivity to leptin, developed leptin resistance [67]. Our RNAseq data showed that YKGT, Ex or YKGT plus Ex significantly decreased leptin gene expression in the skeletal muscle. Murase et al. reported that combination of tea-catechin intake and regular swimming significantly decreased serum leptin level in diet induced obesity C57BL/6 mice [68].
Conclusion
YKGT is characterized by high concentration of total catechins and caffeine. YKGT supplement only significantly prevented the increase of plasma glucose, insulin and TC level, and Ex alone ameliorated body weight and liver weight increase, and decreased insulin and TC level.
However, YKGT plus Ex prevented all these parameters increase, and had better therapeutic effects than that of YKGT or Ex alone for HFD induced MetS mice. HE staining showed that YKGT plus Ex reversed fatty liver, and decreased the serum ALT activity.
Mechanistic studies revealed that combination of YKGT and Ex significantly suppressed the key lipid synthesis genes and protein expression in the liver, and significantly upregulated glucose transport genes expression in the skeletal muscles, when compared to the HFD group mice.
In addition, the hepatic pro-inflammatory gene expression was mitigated significantly by inhibition of NF-kB activation through decreasing the phosphorylation of IKKα/β and IkBα by YKGT, Ex and YKGT plus Ex intervention.
RNAseq data from soleus muscle tissues showed that treatment with YKGT, Ex and YKGT plus Ex significantly down-regulated most of genes induced by HFD. KEGG pathway analysis indicated that nutrients metabolic pathways and inflammatory pathways are the most modulated pathways response to YKGT, Ex or YKGT plus Ex intervention compared to HFD mice. The schematic diagram of YKGT plus Ex reversed HFD induced MetS of C57BL/6 J mice is showing in Fig. 10.
This study demonstrated that combination of YKGT supplement and aerobic exercise appeared to reverse preexisting MetS, and effectively relieved the fatty liver and hepatic inflammatory response induced by HFD. YKGT supplement and aerobic exercise together might be a beneficial strategy for ameliorating MetS of human population. However, adequate intensity and appropriate period of exercise intervention, and YKGT dosage for treatment of MetS require further investigation.
More information: Weslie Y. Khoo et al, Mitigation of nonalcoholic fatty liver disease in high-fat-fed mice by the combination of decaffeinated green tea extract and voluntary exercise, The Journal of Nutritional Biochemistry (2019). DOI: 10.1016/j.jnutbio.2019.108262
