**6. Clinical Studies**

The number of clinical studies evaluating skeletal muscle mass and function for patients with chronic/non-communicable diseases and following grape polyphenols administration is strongly limited compared to in vitro and animal studies. This relative absence of clinical trials is particularly difficult to understand considering the encouraging results obtained in vitro, in animal models, and during the scarce clinical studies yet realized some years ago [126,193–195]. In 2011, Brasnyo et al. studied the effect of supplementation of resveratrol (10 mg/d) in a group of 10 T2D subjects for four weeks [193]. This low dose of resveratrol improved insulin sensitivity, decreased blood glucose levels, decreased blood oxidative stress, and increased Akt phosphorylation in platelets without any data on muscles. The same year, Timmers et al. demonstrated the beneficial effect of nutritional supplementation of 150 mg/d of resveratrol for 30 d in 11 healthy obese men [194]. This supplementation clearly induced metabolic changes in obese humans, mimicking the effects of calorie restriction or endurance exercise. In fact, Timmers observed activation of AMPK in muscle biopsies of the resveratrol-treated subjects, increased PGC-1α and citrate synthase activity, attesting to an increased muscle mitochondrial activity. Interestingly, resveratrol decreased hepatic lipid content but increased intra-myocellular lipid content. Hokayem et al. showed that eight weeks of supplementation with a natural mixture of grape polyphenols at nutritional doses (2 g/d), efficiently prevents fructose-induced oxidative stress and IR in healthy overweight/obese first-degree relatives of T2D patients [126]. At the skeletal muscle level, grape polyphenols supplementation protected mitochondrial function, prevented oxidative stress, and tended to increase insulin sensitivity after fructose challenge Goh et al. showed that supplementation with 3 g resveratrol daily for 12 weeks regulates energy expenditure through increased skeletal muscle SIRT1 and AMPK protein expression in patients with T2D [195]. These interesting results indicate that resveratrol may have beneficial exercise-mimetic effects in patients with T2D [195]. However, no significant modification in systemic insulin sensitivity was observed during this study whereas a decrease in glycated hemoglobin was observed, suggesting an improvement in glucose tolerance. The number of volunteers included in this trial, 10 subjects, could be not enough to have a clear demonstration of grape polyphenols' effects on systemic insulin sensitivity. In seventeen well-controlled T2D subjects, supplementation of 150 mg/d of resveratrol for 30 d was also not able to change insulin sensitivity whereas it increased lipidderivate mitochondrial respiration in muscle [196]. An 8-week supplementation of obese subjects with red wine polyphenols (600 mg/day) does not also improve obesity-associated IR [197]. The main limitation of this study was that total polyphenols intake was similar between the treated and placebo groups limiting the conclusions on the supplementation. Overall, the improvement of insulin sensitivity with grape polyphenols (resveratrol or polyphenols mixture) was mainly found in metabolically stressed patients at a systemic level but not in a normal state of health [198], whereas the improvement in mitochondrial

function and oxidative stress were evident whatever the tissues and the metabolic status of patients. These data underline that in obese/T2D humans, grape polyphenols seem to act mainly as antioxidants.

Clinical trials for investigating polyphenols supplementation on aged muscle are even more limited. In an attempt to decrease functional limitations, most studies on elderly subjects have combined exercise training and polyphenols supplementation in order to amplify the benefits of exercise alone. One study found the beneficial effect of 500 mg/d of resveratrol supplementation associated with exercise on muscle force with higher mitochondrial density and muscle fatigue resistance in elderly subjects allowing to potentially reverse sarcopenia [199]. However, most of the studies report no effect of the combination [200] or even detrimental effect on muscle [201,202]. Recently, in a pilot study on elderly community-dwelling adults, Harper et al. found that 1000 mg/d of resveratrol permits a 33.1 m improvement in the 6-min walk distance associated with higher mitochondrial function and decreased inflammation [203]. This data could be related to the epidemiologic studies where an association between the Mediterranean diet with gait speed is found [204]. However, the beneficial effect of grape polyphenols supplementation on sarcopenia needs to be investigated. To date, no prospective study with data on sarcopenic or frailty subjects is available but some clinical trials are ongoing [124].

#### **7. Conclusions**

Restoration of skeletal muscle mass and function is vital to cure the comorbidities associated with obesity and aging. Grape polyphenols have therapeutic potential for such diseases mediated by oxidative stress and inflammation. Nevertheless, large-scale clinical trials are still necessary to better investigate the activity of these natural compounds at the skeletal muscle level. Moreover, the majority of clinical trials have studied the effect of one purified polyphenol, resveratrol, and not the activity of a crude extract or of a mixture of several molecules of polyphenols. Yet in vitro studies or animal models, even not concerning skeletal muscle, showed an interest in using a mixture of polyphenols. In fact, such a mixture of polyphenols is more active than each individual molecule alone as a synergy effect occurs between the molecules [205,206].

Another important point in using a mixture of polyphenols is that an extract in which many polyphenolic molecules are blended together in low concentrations has the advantage of reducing the toxicity that could be derived from the use of a single molecule in higher concentrations [207].

Additionally, the advantage of grapes over other sources of polyphenols is that grapes, after strawberries and lychees, are among the fruits with the highest polyphenols content (anthocyanins/anthocyanidins, flavonols) [208]. As shown in Table 1, moreover grape boasts a wide variety of phenolic molecules. Although stilbenes, such as resveratrol, have a lower relative abundance than the previously mentioned molecules in grapes, it is at least 10–100 times higher than in other berries [209], making grapes one of its primary sources. Grapes are consumed worldwide. They are the fourth most abundant fruit crop cultivated all over the world [210], so their supply as a source of extraction is easy and affordable for many countries. Moreover, there is an environmental and eco-friendly aspect to using grape polyphenols. Indeed, grape skins and grape seeds are waste by-products of wine or grape juice production. They are rich sources of polyphenols and could be used in the form of food supplements.

Translational data confirming the benefit of grape polyphenols in human health at the muscle level would allow the development of grape polyphenols supplementation in therapy and in the management of obese and aging people on a routine basis. This would improve the quality of life and decrease the economic cost of medical care for obese and elderly patients.

**Author Contributions:** A.C., C.B., C.S. and K.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Region Occitanie: Green2Grape project and PhD grant (for Adriana Capozzi) from University of Montpellier (ED GAIA).

**Conflicts of Interest:** The authors declare no conflict of interest.
