## 4 Discussion
We can speculate a disrupted p62 activation leading to a decreased expression of HO-1, as has already been observed in obesity [40]. Treatment with GPP extracts increased HO-1 expression, overcoming the blockage and triggering this crucial cellular defense mechanism, essential for maintaining muscle health. Conversely, changes in cytosolic sod1 and catalase proteins levels were not observed. These data highlight the complexity and specificity of the extracts' effects on the antioxidant system during metabolic stress due to 24 h palmitate treatment on SM cells. The main effects exerted by GPP extracts were therefore on muscle lipid peroxidation and mitochondrial enzymes' expression and function. HO-1 has recently been identified as a regulator of mitochondrial function and biogenesis [14; 15]. After treatment with all GPP extracts, a notable enhancement in both mitochondrial activity and quantity was observed, as evidenced by the increased activity of citrate synthase (CS) and the expression levels of both CS and CoxIV proteins. Furthermore, treatment with GPP extracts facilitated the normalization of the ratio between CS activity and CS protein levels. In human palmitate-treated SM cells, a decrease in CS protein expression was observed, while CS activity remained unchanged, suggesting an increase in activity per mitochondrion, as indicated by the elevated ratio of CS activity to CS protein. This elevation might signify an overactivity of mitochondria that increased the production of ROS, which was subsequently mitigated by GPP treatment. Remarkably, Sd-BvPP demonstrated a reduction in lipid peroxidation, an enhancement in mitochondrial function and a reduction in oxidative stress, although without a reversal of IR caused by lipid overload in human SM cells. These data question the classical relationship between oxidative stress and IR, at least in SM. While this outcome may seem perplexing, it aligns with previous findings that have indicated grape Sd extracts could potentially impair insulin signaling pathways [41]. We can hypothesize various mechanisms to explain this phenomenon, such as another biological activity not yet identified regulating insulin response or the potential presence in the extract of a specific inhibitor of insulin signaling, yet not inducing SM cell toxicity as analyzed and shown in Section 3.2 (Figure 2C). Our observations suggest that GPP extracts not only at the M stage, as extensively demonstrated in previous decades, but also those from the Bv stage enhance mitochondrial activity and biogenesis, as evidenced in palmitate-treated SM cells [20; 21]. The qualitative and quantitative analysis conducted by means of UPLC revealed that the primary subclass of polyphenols in Sd extracts are flavan-3-ols (in monomer or polymerized form). Given the absence of anthocyanins and stilbenes in grape Sd extracts, molecules often attributed to the health-promoting properties of grapes [42; 43; 44], we can conclude that other polyphenolsor classes of compounds are certainly present as bioactive molecules and require further exploration to profile them. It is now well known that PPs could be metabolized before their absorption and/or excreted from the body, although this is highly dependent on their molecular structures and GPPs show considerable variability. Furthermore, Sk-BvPP showed some cell toxicity at a high concentration, which could limit their use in vivo. Therefore, future evaluations of GPP toxicity, metabolism and bioavailability within a living organism will be necessary to validate our findings and ascertain the therapeutic potential and toxicity of the extract in a more physiologically relevant context. However, it is noteworthy that we observed metabolic and antioxidant effects at a low dosage in vitro, suggesting plausible translatability to in vivo settings, given our prior findings demonstrating the beneficial effects on humans induced by treatment with a mixture of GPPs from the M stage at the dose of 2 g/kg/d [20].