Natural Products and Diabetes: (-)-Epicatechin and Mechanisms Involved in the Regulation of Insulin Sensitivity.

Type 2 diabetes (T2D) is a disease that occurs when cells do not respond normally to insulin, a condition called insulin resistance, which leads to high blood glucose levels. Although it can be treated pharmacologically, dietary habits beyond carbohydrate restriction can be highly relevant in the management of T2D. Emerging evidence supports the possibility that natural products (NPs) could contribute to managing blood glucose or counteract the undesirable effects of hyperglycemia and insulin resistance. This chapter summarizes the relevant preclinical evidence involving the flavonoid (-)-epicatechin (EC) in the optimization of glucose homeostasis, reducing insulin resistance and/or diabetes-associated disorders. Major effects of EC are observed on (i) intestinal functions, including digestive enzymes, glucose transporters, microbiota, and intestinal permeability, and (ii) redox homeostasis, including oxidative stress and inflammation. There is still a need for further clinical studies to confirm the in vitro and rodent data, allowing recommendations for EC, particularly in prediabetic and T2D patients. The collection of similar data and the lack of clinical evidence for EC is also applicable to other NPs.

Marginal Zinc Deficiency during Gestation and Lactation in Rats Affects Oligodendrogenesis, Motor Performance, and Behavior in the Offspring.

BACKGROUND: Oligodendrocytes are responsible for myelin production in the central nervous system (CNS). Hypomyelination may slow saltatory nerve signal conduction and affect motor performance and behavior in adults. Gestational marginal zinc deficiency in rats significantly decreases proliferation of neural stem cells (NSCs) in the offspring brain. OBJECTIVES: Given that NSCs are precursors of oligodendrocytes, this study investigated if marginal zinc deficiency during early development in rats affects oligodendrogenesis in the offspring's CNS. METHODS: Rat dams were fed an adequate (25 µg zinc/g diet) (C) or a marginal zinc diet (MZD) (10 µg zinc/g diet), from gestation day zero until postnatal day (P) 20, and subsequently all offspring was fed the control diet until P60. Oligodendrogenesis was evaluated in the offspring at P2, P5, P10, P20, and P60, by measuring parameters of oligodendrocyte progenitor cells (OPCs) proliferation, differentiation, maturation, and of myelination. RESULTS: The expression of 1) proteins that regulate OPC proliferation (Shh, Sox10, Olig2); 2) OPC markers (NG2, PDGFRα); 3) myelin proteins (MBP, MAG, MOG, PLP) were lower in the brain cortex from MZD than C offspring at various stages in development. The amount of myelin after zinc replenishment continued to be low in the MZD young adult at P60. Accordingly, parameters of motor performance and behavior [grip strength, rotarod, elevated T-maze (ETM), and open-field tests] were impaired in the MZD offspring at P60. CONCLUSIONS: Results support the concept that maternal and early postnatal exposure to MZD affects oligodendrogenesis causing long-lasting effects on myelination and on motor performance in the young adult offspring.

Di-2-ethylhexyl phthalate affects zinc metabolism and neurogenesis in the developing rat brain.

We previously observed that developmental marginal zinc deficiency affects neurogenesis. Maternal phthalate exposure could disrupt fetal zinc homeostasis by triggering an acute phase response, causing maternal liver zinc retention that limits zinc availability to the fetus. Thus, we currently investigated whether exposure to di-2-ethylhexyl phthalate (DEHP) during gestation in rats alters fetal brain neurogenesis by impairing zinc homeostasis. Dams consumed an adequate (25 µg zinc/g diet) (C) or a marginal zinc deficient (MZD) (10 µg zinc/g diet) diet, without or with DEHP (300 mg/kg BW) (C + DEHP, MZD + DEHP) from embryonic day (E) 0 to E19. To evaluate neurogenesis we measured parameters of neural progenitor cells (NPC) proliferation and differentiation. Maternal exposure to DEHP and/or zinc deficiency lowered fetal brain cortical tissue (CT) zinc concentrations. Transcription factors involved in NPC proliferation (PAX6, SOX2, EMX1), differentiation (TBR2, TBR1) and mature neurons (NeuN) were lower in MZD, MZD + DEHP and C + DEHP than in C E19 brain CT, being the lowest in the MZD + DEHP group. VGLUT1 levels, a marker of glutamatergic neurons, showed a similar pattern. Levels of a marker of GABAergic neurons, GAD65, did not vary among groups. Phosphorylated ERK1/2 levels were reduced by both MZD and DEHP, and particularly in the MZD + DEHP group. MEHP-treated human neuroblastoma IMR-32 cells and E19 brains from DEHP-treated dams showed that the zinc-regulated phosphatase PP2A can be in part responsible for DEHP-mediated ERK1/2 downregulation and impaired neurogenesis. Overall, gestational exposure to DEHP caused secondary zinc deficiency and impaired neurogenesis. These harmful effects could have long-term consequences on the adult offspring brain structure and function.

Proanthocyanidins at the gastrointestinal tract: mechanisms involved in their capacity to mitigate obesity-associated metabolic disorders.

The prevalence of overweight and obesity is continually increasing worldwide. Obesity is a major public health concern given the multiple associated comorbidities. Finding dietary approaches to prevent/mitigate these conditions is of critical relevance. Proanthocyanidins (PACs), oligomers or polymers of flavan-3-ols that are extensively distributed in nature, represent a major part of total dietary polyphenols. Although current evidence supports the capacity of PACs to mitigate obesity-associated comorbidities, the underlying mechanisms remain speculative due to the complexity of PACs' structure. Given their limited bioavailability, the major site of the biological actions of intact PACs is the gastrointestinal (GI) tract. This review discusses the actions of PACs at the GI tract which could underlie their anti-obesity effects. These mechanisms include: i) inhibition of digestive enzymes at the GI lumen, including pancreatic lipase, α-amylase, α-glucosidase; ii) modification of gut microbiota composition; iii) modulation of inflammation- and oxidative stress-triggered signaling pathways, e.g. NF-κB and MAPKs; iv) protection of the GI barrier integrity. Further understanding of the mechanisms and biological activities of PACs at the GI tract can contribute to develop nutritional and pharmacological strategies oriented to mitigate the serious comorbidities of obesity.

(-)-Epicatechin and Anthocyanins Modulate GLP-1 Metabolism: Evidence from C57BL/6J Mice and GLUTag Cells.

BACKGROUND: Generated in intestinal L cells through cleavage of proglucagon (Gcg), glucagon-like peptide 1 (GLP-1) is secreted and rapidly inactivated by dipeptidyl peptidase IV (DPP-IV). GLP-1 regulates insulin secretion and overall glucose homeostasis. The capacity of dietary bioactives to increase GLP-1 circulating levels, and therefore increase insulin secretion and glucose metabolism, has gained significant interest of late. OBJECTIVES: We evaluated the effects of (-)-epicatechin (EC) and different anthocyanins (ACs) and AC metabolites on GLP-1 metabolism in mice and on GLUTag cells. METHODS: We fed 6-week-old C57BL/6J male mice a control diet or a control diet supplemented with either 40 mg AC or 20 mg EC/kg body weight for 14 weeks (AC) or 15 weeks (EC). Intestinal mRNA levels of Gcg and Dpp-iv were measured. In vitro, GLUTag cells were incubated in the presence or absence of different ACs, the AC metabolite protocatechuic acid (PCA), and EC. GLP-1 secretion and the main pathways involved in its release were assessed. RESULTS: Long-term supplementation with EC or AC increased mouse GLP-1 plasma concentrations (55% and 98%, respectively; P < 0.05). In mice, 1) EC and AC increased Gcg mRNA levels in the ileum (91%) and colon (41%), respectively (P < 0.05); and 2) AC lowered ileum Dpp-iv mRNA levels (35%), while EC decreased plasma DPP-IV activity (15%; P < 0.05). In GLUTag cells, 1) cyanidin, delphinidin, PCA, and EC increased GLP-1 secretion (53%, 33%, 53%, and 68%, respectively; P < 0.05); and 2) cyanidin, delphinidin, EC, and PCA increased cyclin adenosine monophosphate levels (25-50%; P < 0.05) and activated protein kinase A (PKA; 100%, 50%, 80%, and 86%, respectively; P < 0.05). CONCLUSIONS: In mice, EC and ACs regulated different steps in GLP-1 regulation, leading to increased plasma GLP-1. Cyanidin, delphinidin, PCA, and EC promoted GLP-1 secretion from GLUTag cells by activating the PKA-dependent pathway. These findings support the beneficial actions of these flavonoids in sustaining intestinal and glucose homeostasis through the modulation of the GLP-1 metabolism.

(-)-Epicatechin and the comorbidities of obesity.

Obesity has major adverse consequences on human health contributing to the development of, among others, insulin resistance and type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease, altered behavior and cognition, and cancer. Changes in dietary habits and lifestyle could contribute to mitigate the development and/or progression of these pathologies. This review will discuss current evidence on the beneficial actions of the flavan-3-ol (-)-epicatechin (EC) on obesity-associated comorbidities. These benefits can be in part explained through EC's capacity to mitigate several common events underlying the development of these pathologies, including: i) high circulating levels of glucose, lipids and endotoxins; ii) chronic systemic inflammation; iii) tissue endoplasmic reticulum and oxidative stress; iv) insulin resistance; v) mitochondria dysfunction and vi) dysbiosis. The currently known underlying mechanisms and cellular targets of EC's beneficial effects are discussed. While, there is limited evidence from human studies supplementing with pure EC, other studies involving cocoa supplementation in humans, pure EC in rodents and in vitro studies, support a potential beneficial action of EC on obesity-associated comorbidities. This evidence also stresses the need of further research in the field, which would contribute to the development of human dietary strategies to mitigate the adverse consequences of obesity.

(-)-Epicatechin and its metabolites prevent palmitate-induced NADPH oxidase upregulation, oxidative stress and insulin resistance in HepG2 cells.

While diets rich in fruit and vegetables can decrease the risk for type 2 diabetes (T2D), diets rich in fat and carbohydrates can increase it. The flavanol-3-ol (-)-epicatechin (EC) can improve insulin sensitivity both in humans and animal models of T2D. NADPH oxidases and oxidative stress can contribute to the development of insulin resistance. This study investigated the capacity of EC and EC metabolites (ECM) to downregulate NADPH oxidases and oxidative stress, and its association to an improvement of insulin sensitivity. This was studied in in vivo (high fat-fed mice) and in vitro (HepG2 cells) conditions of hepatic lipid overload. EC decreased NOX3/NOX4 liver expression and mitigated oxidative stress in high fat-fed mice. In HepG2 cells, incubation with palmitate increased: i) lipid deposition, ii) NOX3/NOX4 expression, iii) NADPH oxidase activity, and iv) oxidative stress; promoting v) the activation of redox-sensitive kinases (JNK and IKK), and vi) impaired insulin responses. Physiological concentrations of EC and ECM, and NADPH oxidase inhibitors (apocynin, VAS2870) prevented all those deleterious effects of palmitate. The obtained results points to NADPH oxidases as an important target in the capacity of EC to improve insulin sensitivity in conditions of liver lipid overload, as those associated with Western-style diets.

Aldosterone activates the oncogenic signals ERK1/2 and STAT3 via redox-regulated mechanisms.

Epidemiological studies found an increased risk for kidney cancer in hypertensive patients, of which a subgroup has high aldosterone (Ald) levels. We recently showed that Ald is genotoxic both in kidney tubular cells and in rats with mineralocorticoid-mediated hypertension. The present work investigated in vitro and in vivo, if the oxidative stress-mediated activation of the ERK1/2 pathway, and its downstream target STAT3, could be one mechanism involved in the potential oncogenic capability of excess Ald exposure. The effects of excess Ald were investigated in LLC-PK1 cells and in Ald-induced hypertensive rats. Ald caused cRaf, MEK1/2, and ERK1/2 phosphorylation both in LLC-PK1 cells and in rat kidneys. ERK1/2 activation led to an increased phosphorylation of MSK1, p90RSK, and STAT3. The involvement of ERK1/2 in the activation of STAT3 was evidenced by the capacity of the MEK inhibitor U0126 to prevent Ald-mediated ERK1/2 and STAT3 phosphorylation. Both in vitro and in vivo, the activation of ERK1/2 and STAT3 by Ald was dependent on the mineralocorticoid receptor and was triggered by an increase in cellular oxidants. Ald-mediated oxidant increase was in part due to the activation of the enzymes NADPH oxidase and NO synthase. Proliferation was significantly enhanced and apoptosis decreased in Ald-treated rat kidneys and/or LLC-PK1 cells. Results support the concept that the oxidant-mediated long-term activation of ERK1/2/STAT3 by persistently high Ald levels could trigger proliferative and prosurvival events. Ald-mediated promotion of cell survival and DNA damage could result in kidney cell transformation and initiation of cancer in hypertensive patients with hyperaldosteronism.

The PI3K/Akt pathway is involved in procyanidin-mediated suppression of human colorectal cancer cell growth.

Colorectal cancer (CRC) has the third highest incidence worldwide. Epidemiological studies showed that the consumption of fruit and vegetables containing procyanidins (PCA), polymers of flavan-3-ols, is associated with lower CRC risk. However, the molecular mechanisms supporting this positive association are unclear. This study investigated the capacity of PCA with different degrees of polymerization to reduce CRC cell growth, characterizing the underlying mechanisms. Compared to the monomer ((-)-epicatechin) and the trimer, the hexamer (Hex) was the most active at reducing CRC cell viability. Hex caused a concentration- (2.5-50 µM) and time- (24-72 h) dependent decrease in the viability of six human CRC cell lines in culture. Hex caused CRC apoptotic Caco-2 cell death within 24 h, as evidenced by caspase 3 and caspase 9 activation, DNA fragmentation, and changes in nuclear morphology/staining. Hex-induced apoptosis occurs through the mitochondrial pathway, as evidenced by an increased Bad mitochondrial translocation, and cytochrome c release from the mitochondria to the cytosol. Hex also arrested the Caco-2 cell cycle at G2 /M phase and upregulated genes involved in autophagy. Mechanistically, in Caco-2 cells Hex inhibited the PI3K/Akt signaling pathway, causing the downstream downregulation of proteins involved in the regulation of cell survival (Bad, GSK-3ß). Accordingly, the Akt inhibitor MKK-2206 decreased Bad and GSK-3ß phosphorylation. MKK-2206 decreased cell growth, having an additive effect with Hex. In conclusion, our results show that large PCA can inhibit CRC cell growth via the Akt kinase pathway, demonstrating a mechanism to explain the epidemiological evidence linking PCA-rich diets with lower CRC risk. © 2016 Wiley Periodicals, Inc.

(-)-Epicatechin improves insulin sensitivity in high fat diet-fed mice.

Obesity constitutes a major public health concern, being frequently associated with type 2 diabetes (T2D). Evidence from studies in humans and experimental animals suggest that consumption of the flavan-3-ol (-)-epicatechin (EC) and of EC-rich foods may improve insulin sensitivity. To further understand the potential benefits of dietary EC consumption on insulin resistance, this study investigated the capacity of EC supplementation to prevent high fat diet (HFD)-induced insulin resistance in mice. To assess the underlying mechanisms, the effects of HFD and EC consumption on the activation of the insulin cascade and of its negative modulators were evaluated. HFD consumption for 15 w caused obesity and insulin resistance in C57BL/6J mice as evidenced by high fasted and fed plasma glucose and insulin levels, and impaired ITT and GTT tests. This was associated with alterations in the activation of components of the insulin-triggered signaling cascade (insulin receptor, IRS1, ERK1/2, Akt) in adipose and liver tissues. EC supplementation prevented/ameliorated all these parameters. EC acted improving insulin sensitivity in the HFD-fed mice in part through a downregulation of the inhibitory molecules JNK, IKK, PKC and protein tyrosine phosphatase 1B (PTP1B). Thus, the above results suggest that consumption of EC-rich foods could constitute a dietary strategy to mitigate obesity-associated insulin resistance.

In vitro measurements and interpretation of total antioxidant capacity.

BACKGROUND: One of the strategies most commonly used to assess a free radical-antioxidant balance in chemical and biological systems is the determination of the total antioxidant capacity (TAC). A large amount of research has been published using TAC. However, it remains unclear which is the significance of these investigations for understanding the biological importance of free radical reactions. SCOPE OF REVIEW: This review discusses the relevance and limitations of TAC for the assessment of the antioxidant activities present in food and food derivatives, and in body tissues and fluids. MAJOR CONCLUSIONS: TAC determinations are simple, inexpensive, and able to evaluate the capacity of known and unknown antioxidants and their additive, synergistic and/or antagonistic actions, in chemical and biological systems. However, different TAC assays correlate poorly with each other, since each TAC assay is sensitive to a particular combination of compounds, but exclude many others. The TAC values for foods cannot be translated to the in vivo (human) antioxidant defenses, and furthermore, to health effects provided by that food. GENERAL SIGNIFICANCE: Up to date, conclusions that can be drawn from the extensive amount of research done using TAC of foods or populations should not be considered when used for making decisions affecting population health. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

(-)-Epicatechin in the prevention of tumor necrosis alpha-induced loss of Caco-2 cell barrier integrity.

An increased permeability of the intestinal barrier is proposed as a major event in the pathophysiology of inflammatory bowel diseases (IBD). Tumor necrosis alpha (TNFα) plays a central role in IBD pathogenesis, in part promoting tight function (TJ) barrier dysfunction. Food extracts enriched in (-)-epicatechin (EC) prevent the development or improve the progression of IBD in animal models. This study investigated the capacity of EC to inhibit TNFα-induced permeabilization of Caco-2 cell monolayers, characterizing the underlying mechanisms. Caco-2 cells differentiated into intestinal epithelial cells were incubated in the absence/presence of TNFα, with or without the addition of 0.5-5 µM EC. TNFα triggered cell monolayer permeabilization, decreasing transepithelial electrical resistance (TEER) and increasing the paracellular transport of fluorescein sulfonic acid. The permeabilizing effects of TNFα were not due to Caco-2 cell apoptosis as evaluated by DNA fragmentation, caspase 3 and 9 activation, and cell morphology. EC prevented TNFα-triggered Caco-2 monolayer permeabilization and acted inhibiting the associated: (i) NADPH oxidase (NOX)-mediated increased oxidant production, (ii) NF-κB (IκBα phosphorylation, p50 and RelA nuclear transport, and nuclear NF-κB-DNA binding) and ERK1/2 activation, (iii) increased myosin light kinase expression, and decreased TJ protein ZO-1 levels. In summary, EC prevented TNFα-mediated Caco-2 cell barrier permeabilization in part through the inhibition of NOX/NF-κB activation and downstream TJ disruption. Diets rich in EC could contribute to ameliorate IBD-associated increased intestinal permeability.

Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-ß-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.

The anthocyanin metabolites gallic acid, 3-O-methylgallic acid, and 2,4,6-trihydroxybenzaldehyde decrease human colon cancer cell viability by regulating pro-oncogenic signals.

Anthocyanins are a class of polyphenols abundant in the skins of red grapes, and have been shown to have anti-cancer effects in models of colon cancer [Cooke et al. Int J Cancer 2006;119:2213-2220; Jing et al. J Agric Food Chem 2008;56:9391-9398]. Gut microflora metabolize anthocyanins to phenolic acids and aldehydes. These metabolites may explain the relationship between anthocyanin consumption and reduced incidence of colorectal cancer (CRC). Previously, gallic acid (Gal), 3-O-methylgallic acid (Megal), and 2,4,6-trihydroxybenzaldehyde (THBA) were found to decrease Caco-2 cell viability to a larger extent than other anthocyanin metabolites. To better understand the potential anti-CRC action of these compounds, this paper investigated their capacity to modulate the cell cycle, and induce apoptotic cell death. Dividing Caco-2 cells were incubated for 24-72 h in the presence of 10-100 µM Gal, Megal, THBA, and malvidin-3-glucoside (M3g). THBA reduced cell viability only at 100 µM, while Gal and Megal (10-100 µM) caused a time- and dose-dependent decrease in cell viability. After 72 h incubation, the metabolites caused cell cycle arrest at G0 /G1 . The activation of the apoptotic pathway by Megal, Gal, and THBA was evidenced by the activation of caspase-3. However, only Megal and Gal caused DNA fragmentation and nuclear condensation. Megal, Gal, and THBA inhibited transcription factors NF-κB, AP-1, STAT-1, and OCT-1 which are known to be activated in CRC. In conclusion, the anti-cancer effects of Megal and Gal occurs as a consequence of both the inhibition of cell proliferation and induction of apoptosis. The inhibition of transcription factors that promote cell proliferation and survival can in part underlie the observed effects.

Effects of (-)-epicatechin on hepatic triglyceride metabolism.

(-)-Epicatechin (EC) consumption is associated with an improvement of hyperlipemia and other metabolic changes linked to obesity and western-style diets. This work investigated the effects of EC on triglyceride (TG) metabolism both in vivo, where mice were supplemented with EC (2 and 20 mg EC per kg body weight), and in vitro, when human HepG2 hepatocytes were incubated in the presence of EC and the main EC metabolites found in human plasma. Increased hepatic TG levels were only observed after 24 weeks supplementation with EC (20 mg per kg body weight), with a preserved liver structure and absence of inflammation or oxidative stress. EC caused increased expression of diacylglycerol acyltransferases (DGAT2), key enzymes in TG synthesis, and the upregulation of PPARα, which promotes free fatty acid (FFA) oxidation. On the other hand, incubation of HepG2 cells in the presence of high concentrations of EC (1-10 µM) did not affect TG deposition nor DGAT2 expression. In summary, in mouse liver, EC upregulated mechanisms that can neutralize the potential toxicity of FFA, i.e. TG synthesis and FFA ß-oxidation. Results in mouse liver and HepG2 cells stress the safety of EC in terms of TG metabolism and development of hepatopathies in doses within the limits given by a rational time and dose for human consumption.

Anthocyanins and their metabolites promote white adipose tissue beiging by regulating mitochondria thermogenesis and dynamics.

High-fat diet (HFD) consumption and excess nutrient availability can cause alterations in mitochondrial function and dynamics. We previously showed that anthocyanins (AC) decreased HFD-induced body weight gain and fat deposition. This study investigated: i) the capacity of AC to mitigate HFD-induced alterations in mitochondrial dynamics, biogenesis, and thermogenesis in mouse subcutaneous white adipose tissue (sWAT), and ii) the underlying mechanisms of action of cyanidin-3-O-glucoside (C3G), delphinidin-3-O-glucoside (D3G), and their gut metabolites on mitochondria function/dynamics in 3T3-L1 adipocytes treated with palmitate. Mice were fed control or HFD diets, added or not with 40 mg AC/kg body weight (BW). Compared to control and AC-supplemented mice, HFD-fed mice had fewer sWAT mitochondria that presented alterations of their architecture. AC supplementation prevented HFD-induced decrease of proteins involved in mitochondria biogenesis (PPARγ, PRDM16 and PGC-1α), and thermogenesis (UCP-1), and decreased AMPK phosphorylation. AC supplementation also restored the alterations in sWAT mitochondrial dynamics (Drp-1, OPA1, MNF-2, and Fis-1) and mitophagy (BNIP3L/NIX) caused by HFD consumption. In mature 3T3-L1, C3G, D3G, and their metabolites protocatechuic acid (PCA), 4-hydroxybenzaldehyde (HB), and gallic acid (GA) differentially affected palmitate-mediated decreased cAMP, PKA, AMPK, and SIRT-1 signaling pathways. C3G, D3G, and metabolites also prevented palmitate-mediated decreased expression of PPARγ, PRDM16, PGC-1α, and UCP1. Results suggest that consumption of select AC, i.e. cyanidin and delphinidin, could promote sWAT mitochondriogenesis and improve mitochondria dynamics in the context of HFD/obesity-induced dysmetabolism in part by regulating PKA, AMPK, and SIRT-1 signaling pathways.

NADPH oxidase 1: A target in the capacity of dimeric ECG and EGCG procyanidins to inhibit colorectal cancer cell invasion.

Colorectal cancer (CRC) is prevalent worldwide. Dietary consumption of procyanidins has been linked to a reduced risk of developing CRC. The epidermal growth factor (EGF) receptor (EGFR) signaling pathway is frequently dysregulated in CRC. Our earlier research showed that the procyanidin dimers of epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), through their interaction with lipid rafts, inhibit the EGFR signaling pathway and decrease CRC cell growth. The process of cancer cell invasion and metastasis involves matrix metalloproteinases (MMPs), which are partially EGFR-regulated. This study investigated whether ECG and EGCG dimers can inhibit EGF-induced CRC cell invasion by suppressing the redox-regulated activation of the EGFR/MMPs pathway. Both dimers mitigated EGF-induced cell invasion and the associated increase of MMP-2/9 expression and activity in different CRC cell lines. In Caco-2 cells, both dimers inhibited the activation of the EGFR and downstream of NF-κB, ERK1/2 and Akt, which was associated with decreased MMP-2/9 transcription. EGF induced a rapid NOX1-dependent oxidant increase, which was diminished by both ECG and EGCG dimers and NOX inhibitors (apocynin, Vas-2870, DPI). Both dimers inhibited NOX1 gene expression, as well as NOX1 activity with evidence of direct binding to NOX1. Both dimers, all NOX chemical inhibitors and NOX1 silencing inhibited EGF-mediated activation of the EGFR signaling pathway and the increased MMP-2/9 mRNA levels and activity. Pointing to the relevance of NOX1 on ECG and EGCG dimer effects on CRC invasiveness, silencing of NOX1 also inhibited EGF-stimulated Caco-2 cell invasion. In summary, ECG and EGCG dimers can act inhibiting CRC cell invasion/metastasis both, by downregulating MMP-2 and MMP-9 expression via a NOX1/EGFR-dependent mechanism, and through a direct inhibitory effect on MMPs enzyme activity.