Obesity is a potent risk factor for kidney disease as it increases the possibility of developing diabetes and hypertension, and it has a direct impact on the development of chronic kidney disease and end-stage renal disease. In this study, we tested the effect of bergamot polyphenolic fraction in a cafeteria with diet-fed rats, an excellent experimental model for studying human metabolic syndrome, as it is able to induce severe obesity with insulin resistance and high plasma triglyceride levels more efficiently than a traditional lard-based high-fat diet used in rodent models. We analyzed the plasmatic oxidative balance by photometric tests, and the expression of cytoplasmic antioxidant enzymes (superoxide dismutase 1 and glutatione S-tranferasi P1) and apoptotic markers (Caspase 8 and 9) in kidney tissues by Western blot analysis. Our results clearly showed that the cafeteria diet induces a marked pro-oxidant effect: significant reduction of plasmatic antioxidant capacity; downregulation of cytoplasmic antioxidant enzymes expression; and activation of apoptotic pathways. All these hallmarks of redox disequilibrium were mitigated by treatment with polyphenolic fraction of bergamot, highlighting its antioxidant effect in the metabolic syndrome. Our data show that the link between obesity and renal damage could be represented by oxidative stress.

Bergamot flavonoids counteract dyslipidemia and hyperglycemia but fail to induce a significant weight loss. Here, we evaluated the efficacy of bergamot polyphenol extract complex (BPE-C), a novel bergamot juice-derived formulation enriched with flavonoids and pectins, on several metabolic syndrome parameters. Obese patients with atherogenic index of plasma (AIP) over 0.34 and mild hyperglycemia were recruited to a double-blind randomized trial comparing two doses of BPE-C (650 and 1300 mg daily) with placebo. Fifty-two subjects met the inclusion criteria and were assigned to three experimental groups. Fifteen subjects per group completed 90 days-trial. BPE-C reduced significantly fasting glucose by 18.1%, triglycerides by 32% and cholesterol parameters by up to 41.4%, leading to a powerful reduction of AIP (below 0.2) in the high dose group. The homeostasis model assessment of insulin resistance (HOMA-IR) and insulin levels were also reduced. Moreover, BPE-C decreased body weight by 14.8% and body mass index by 15.9% in BPE-C high group. This correlated with a significant reduction of circulating hormones balancing caloric intake, including leptin, ghrelin and upregulation of adiponectin. All effects showed a dose-dependent tendency. This study suggests that food supplements, containing full spectrum of bergamot juice components, such as BPE-C efficiently induce a combination of weight loss and insulin sensitivity effects together with a robust reduction of atherosclerosis risk.

Wrong alimentary behaviors and so-called “junk food” are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The “junk food” toxicity can be studied in “cafeteria” (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot (Citrus bergamia Risso et Poiteau) flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC±BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 (Il6) mRNA expression and increasing anti-inflammatory Il10, which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.

Microphysiological systems (MPS), also referred to as tissue chips, incorporating 3D skeletal myobundles are a novel approach for physiological and pharmacological studies to uncover new medical treatments for sarcopenia. We characterize a MPS in which engineered skeletal muscle myobundles derived from donor‐specific satellite cells that model aged phenotypes are encapsulated in a perfused tissue chip platform containing platinum electrodes. Our myobundles were derived from CD56⁺ myogenic cells obtained via percutaneous biopsy of the vastus lateralis from adults phenotyped by age and physical activity. Following 17 days differentiation including 5 days of a 3 V, 2 Hz electrical stimulation regime, the myobundles exhibited fused myotube alignment and upregulation of myogenic, myofiber assembly, signaling and contractile genes as demonstrated by gene array profiling and localization of key components of the sarcomere. Our results demonstrate that myobundles derived from the young, active (YA) group showed high intensity immunofluorescent staining of α‐actinin proteins and responded to electrical stimuli with a ~1 μm displacement magnitude compared with non‐stimulated myobundles. Myobundles derived from older sedentary group (OS) did not display a synchronous contraction response. Hypertrophic potential is increased in YA‐derived myobundles in response to stimulation as shown by upregulation of insulin growth factor (IGF‐1), α‐actinin (ACTN3, ACTA1) and fast twitch troponin protein (TNNI2) compared with OS‐derived myobundles. Our MPS mimics disease states of muscle decline and thus provides an aged system and experimental platform to investigate electrical stimulation mimicking exercise regimes and may be adapted to long duration studies of compound efficacy and toxicity for therapeutic evaluation against sarcopenia.

Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.

Bergamot flavonoids have been shown to prevent metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and stimulate autophagy in animal models and patients. To investigate further the mechanism of polyphenol-dependent effects, we performed a RT2-PCR array analysis on 168 metabolism, transport and autophagy-related genes expressed in rat livers exposed for 14 weeks to different diets: standard, cafeteria (CAF) and CAF diet supplemented with 50 mg/kg of bergamot polyphenol fraction (BPF). CAF diet caused a strong upregulation of gluconeogenesis pathway (Gck, Pck2) and a moderate (>1.7 fold) induction of genes regulating lipogenesis (Srebf1, Pparg, Xbp1), lipid and cholesterol transport or lipolysis (Fabp3, Apoa1, Lpl) and inflammation (Il6, Il10, Tnf). However, only one β-oxidation gene (Cpt1a) and a few autophagy genes were differentially expressed in CAF rats compared to controls. While most of these transcripts were significantly modulated by BPF, we observed a particularly potent effect on lipogenesis genes, like Acly, Acaca and Fasn, which were suppressed far below the mRNA levels of control livers as confirmed by alternative primers-based RT2-PCR analysis and western blotting. These effects were accompanied by downregulation of pro-inflammatory cytokines (Il6, Tnfa, and Il10) and diabetes-related genes. Few autophagy (Map1Lc3a, Dapk) and no β-oxidation gene expression changes were observed compared to CAF group. In conclusion, chronic BPF supplementation efficiently prevents NAFLD by modulating hepatic energy metabolism and inflammation gene expression programs, with no effect on β-oxidation, but profound suppression of de novo lipogenesis.