A Ketogenic Diet in Combination with Gemcitabine Mitigates Pancreatic Cancer-Associated Cachexia in Male and Female KPC Mice.

Cancer-associated cachexia (CAC) is a critical contributor to pancreatic ductal adenocarcinoma (PDAC) mortality. Thus, there is an urgent need for new strategies to mitigate PDAC-associated cachexia; and the exploration of dietary interventions is a critical component. We previously observed that a ketogenic diet (KD) combined with gemcitabine enhances overall survival in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. In this study, we investigated the effect and cellular mechanisms of a KD in combination with gemcitabine on the maintenance of skeletal muscle mass in KPC mice. For this purpose, male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD), a KD, a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. We observed that a KD or a KG-mitigated muscle strength declined over time and presented higher gastrocnemius weights compared CD-fed mice. Mechanistically, we observed sex-dependent effects of KG treatment, including the inhibition of autophagy, and increased phosphorylation levels of eIF2α in KG-treated KPC mice when compared to CG-treated mice. Our data suggest that a KG results in preservation of skeletal muscle mass. Additional research is warranted to explore whether this diet-treatment combination can be clinically effective in combating CAC in PDAC patients.

A ketogenic diet in combination with gemcitabine increases survival in pancreatic cancer KPC mice.

Pancreatic ductal adenocarcinoma (PDAC) continues to be a major health problem. A ketogenic diet (KD), characterized by a very low carbohydrate and high fat composition, has gained attention for its anti-tumor potential. We evaluated the effect and mechanisms of feeding a strict KD alone or in combination with gemcitabine in the autochthonous LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx1-Cre (KPC) mouse model. For this purpose, both male and female pancreatic tumor-bearing KPC mice were allocated to a control diet (CD; %kcal: 70% carb, 14% protein, 16% fat), a KD (%kcal: 14% protein, 1% carb, 85% fat), a CD + gemcitabine (CG), or a KD + gemcitabine (KG) group. Mice fed a KD alone or in combination with gemcitabine showed significantly increased blood ß-hydroxybutyrate levels compared to mice fed a CD or CG. KPC mice fed a KG had a significant increase in overall median survival compared to KPC mice fed a CD (increased overall median survival by 42%). Interestingly, when the data was disaggregated by sex, the effect of a KG was significant in female KPC mice (60% increase in median overall survival), but not in male KPC mice (28% increase in median overall survival). Mechanistically, the enhanced survival response to a KD combined with gemcitabine was multifactorial, including inhibition of ERK and AKT pathways, regulation of fatty acid metabolism and the modulation of the gut microbiota. In summary, a KD in combination with gemcitabine appears beneficial as a treatment strategy in PDAC in KPC mice, deserving further clinical evaluation.

Phospho-Aspirin (MDC-22) inhibits pancreatic cancer growth in patient-derived tumor xenografts and KPC mice by targeting EGFR: Enhanced efficacy in combination with irinotecan.

Novel therapeutic strategies are needed in the fight against pancreatic cancer. We have previously documented the chemopreventive effect of MDC-22 in preclinical models of pancreatic cancer. In the present work, we examined the therapeutic effects of MDC-22 in patient-derived tumor xenografts (PDTXs) and in LSL-KrasG12D/+, LSL-Trp53R172H/+, Pdx1-Cre (KPC) genetically engineered mice, two complementary and clinically relevant animal models of pancreatic cancer. In addition, we evaluated whether MDC-22 could synergize with current chemotherapeutic drugs used in the clinic. MDC-22 reduced the growth of various human pancreatic cancer cell lines in a concentration-dependent manner. In vivo, MDC-22 strongly reduced patient-derived pancreatic tumor xenograft growth by 50%, and extended survival of LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) mice by over a month (5.3 months versus 7.0 months). In both models, MDC-22 inhibited EGFR activation and its downstream signals, including ERK and FAK phosphorylation. In human pancreatic cancer cell lines, MDC-22 enhanced the growth inhibitory effect of irinotecan, and to a lesser degree those of gemcitabine and nab-paclitaxel. Normal human pancreatic epithelial cells were more resistant to the cytotoxic effects of, both, MDC-22 alone or in combination with irinotecan, indicating selectivity. Furthermore, MDC-22 enhanced irinotecan's effect on cell migration, in part, by inhibiting EGFR/FAK signaling. Collectively, our results indicate that MDC-22 is an effective anticancer drug in preclinical models of pancreatic cancer, and suggest that MDC-22 plus irinotecan as drug combination strategy for pancreatic cancer treatment, which warrants further evaluation.

Quercetin attenuates adipose hypertrophy, in part through activation of adipogenesis in rats fed a high-fat diet.

An impaired capacity of adipose tissue expansion leads to adipocyte hypertrophy, inflammation and insulin resistance (IR) under positive energy balance. We previously showed that a grape pomace extract, rich in flavonoids including quercetin (Q), attenuates adipose hypertrophy. This study investigated whether dietary Q supplementation promotes adipogenesis in the epididymal white adipose tissue (eWAT) of rats consuming a high-fat diet, characterizing key adipogenic regulators in 3T3-L1 pre-adipocytes. Consumption of a high-fat diet for 6 weeks caused IR, increased plasma TNFα concentrations, eWAT weight, adipocyte size and the eWAT/brown adipose tissue (BAT) ratio. These changes were accompanied by decreased levels of proteins involved in angiogenesis, VEGF-A and its receptor 2 (VEGF-R2), and of two central adipogenic regulators, i.e. PPARγ and C/EBPα, and proteins involved in mature adipocyte formation, i.e. fatty acid synthase (FAS) and adiponectin. Q significantly reduced adipocyte size and enhanced angiogenesis and adipogenesis without changes in eWAT weight and attenuated systemic IR and inflammation. In addition, high-fat diet consumption increased eWAT hypoxia inducible factor-1 alpha (HIF-1α) levels and those of proteins involved in adipose inflammation (TLR-4, CD68, MCP-1, JNK) and activation of endoplasmic reticulum (ER) stress, i.e. ATF-6 and XBP-1. Q mitigated all these events. Q and quercetin 3-glucoronide prevented TNFα-mediated downregulation of adipogenesis during 3T3-L1 pre-adipocytes early differentiation. Together, Q capacity to promote a healthy adipose expansion enhancing angiogenesis and adipogenesis may contribute to reduced adipose hypertrophy, inflammation and IR. Consumption of diets rich in Q could be useful to counteract the adverse effects of high-fat diet-induced adipose dysfunction.

Grape pomace extract supplementation activates FNDC5/irisin in muscle and promotes white adipose browning in rats fed a high-fat diet.

Irisin is a myokine regulated by peroxisome proliferator-activated receptor gamma co-activator-1α (PGC-1α) in the exercising skeletal muscle and released into the bloodstream after cleavage of FNDC5. Circulating irisin can up-regulate UCP-1 expression in white adipose tissue (WAT) promoting the formation of brown-like adipocytes. The aim of this study was to evaluate if supplementation with a grape pomace extract (GPE) could activate the FNDC5/irisin pathway via PGC-1α in rats fed a high fat diet (HFD). For this purpose we characterized the activation of: i. the FNDC5/irisin pathway and AMPK in skeletal muscle and ii. proteins involved in the formation of brown-like cells in epididymal WAT (eWAT). Consumption of the GPE activated the FNDC5/irisin pathway, increased AMPK phosphorylation in skeletal muscle and enhanced irisin plasma levels. In eWAT, the GPE increased the level of proteins involved in WAT browning, i.e. PGC-1α, PPARγ, PRDM16 and UCP-1. The GPE also prevented HFD-induced adipocyte hypertrophy and systemic insulin resistance. Consistently, in L6 myotubes, (-)-epicatechin (EC), a flavonoid abundant in the GPE, prevented palmitate-mediated downregulation of FNDC5/irisin protein expression and secretion, in part via PGC-1α activation. Consumption of the GPE, a winemaking residue rich in bioactive compounds, could be a beneficial strategy to counteract the adverse effects of Western style diets through the promotion of WAT browning.

Dietary (-)-epicatechin affects NF-κB activation and NADPH oxidases in the kidney cortex of high-fructose-fed rats.

Inflammation involves the activation of redox-sensitive transcription factors, e.g., nuclear factor κB (NF-κB). Administration of (-)-epicatechin to high-fructose-fed rats prevented NF-κB activation and up-regulation of the NADPH oxidase 4 (NOX4) in the kidney cortex. These results add mechanistic insights into the action of (-)-epicatechin diminishing inflammatory responses.

Phenolic metabolites in plasma and tissues of rats fed with a grape pomace extract as assessed by liquid chromatography-tandem mass spectrometry.

Grape pomace extract (GPE) is a rich and relatively low-cost source of phenolic compounds. However, little is known about the main GPE metabolites in mammals, which could help explain the observed health-promoting effects. This study investigated the presence of parent compounds from flavanol, flavonol and stilbene families and their metabolites in rat plasma and tissues after an acute intake of GPE in doses of 300 and 600 mg kg/body weight. The measurement of free compounds and their metabolites was performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Results showed the presence of epicatechin, epicatechin methyl-glucuronide, epicatechin methyl-sulphate, catechin, catechin-glucuronide, quercetin methyl-glucuronide, resveratrol-3-glucuronide, resveratrol-4-glucuronide and resveratrol-3-sulphate in plasma, which was dose dependent. The most abundant measured compound in plasma was epicatechin-glucuronide. The presence of glucuronidated and methyl-glucuronidated forms of catechin were observed in the liver at both doses, while epicatechin-glucuronide and methyl-glucuronide were detected only upon intake of 600 mg GPE/kg body weight. At this dose epicatechin-glucuronide and methyl-glucuronide were also detected in muscle, and catechin methyl-glucuronide in adipose tissue. Results show the main GPE metabolites present in rat tissues after oral consumption, contributing to better understand the health benefits of GPE and its potential utilization as a functional ingredient.

Grape pomace extract induced beige cells in white adipose tissue from rats and in 3T3-L1 adipocytes.

This study investigated the effects of a grape pomace extract (GPE) rich in phenolic compounds on brown-like adipocyte induction and adiposity in spontaneously hypertensive (SHR) and control normotensive Wistar-Kyoto (WKY) rats fed a high-fat diet (HFD). HFD consumption for 10 weeks significantly increased epididymal white adipose tissue (eWAT) in WKY but not in SHR rats. Supplementation with GPE (300 mg/kg body weight/day) reduced adipocyte diameter and increased levels of proteins that participate in adipogenesis and angiogenesis, i.e., peroxisome-proliferator activated receptor gamma (PPARγ), vascular endothelial grow factor-A (VEGF-A) and its receptor 2 (VEGF-R2), and partially increased the uncoupling protein 1 (UCP-1) in WKY. In both strains, GPE attenuated adipose inflammation. In eWAT from SHR, GPE increased the expression of proteins involved in adipose tissue "browning," i.e., PPARγ-coactivator-1α (PGC-1α), PPARγ, PR domain containing 16 (PRDM16) and UCP-1. In primary cultures of SHR adipocytes, GPE-induced UCP-1 up-regulation was dependent on p38 and ERK activation. Accordingly, in 3T3-L1 adipocytes treated with palmitate, the addition of GPE (30 µM) activated the ß-adrenergic signaling cascade (PKA, AMPK, p38, ERK). This led to the associated up-regulation of proteins involved in mitochondrial biogenesis (PGC-1α, PPARγ, PRDM16 and UCP-1) and fatty acid oxidation (ATGL). These effects were similar to those exerted by (-)-epicatechin and quercetin, major phenolic compounds in GPE. Overall, in HFD-fed rats, supplementation with GPE promoted brown-like cell formation in eWAT and diminished adipose dysfunction. Thus, winemaking residues, rich in bioactive compounds, could be useful to mitigate the adverse effects of HFD-induced adipose dysfunction.

Grape pomace reduced reperfusion arrhythmias in rats with a high-fat-fructose diet.

Metabolic syndrome (MetS) is a risk factor for sudden cardiac death in humans, but animal models are needed for the study of this association. Grape pomace (GP), obtained from the winemaking process, contains phenolic compounds with potential cardioprotective effects. The aim of this study was to evaluate if a high-fat-fructose (HFF) diet facilitates the occurrence of arrhythmias during the reperfusion, and if a GP supplementation could counteract these effects. Wistar rats were fed with control (Ctrl), HFF diet and HFF plus GP (1 g kg-1 day-1) for six weeks. The HFF diet induces characteristic features of MetS (higher systolic blood pressure, dyslipidemia and insulin resistance) which was attenuated by GP supplementation. In addition, HFF induced increased reperfusion arrhythmias that were reduced upon GP supplementation. GP also reduced the non-phosphorylated form of connexin-43 (Cx43) while enhancing heart p-AKT and p-eNOS protein levels and reducing Nox4 levels enhanced by the HFF diet, indicating that GP may increase NO bioavailability in the heart. We found a murine model of MetS with increased arrhythmogenesis and translational value. Furthermore, GP prevents diet-induced heart dysfunction and metabolic alterations. These results highlight the potential utilization of winemaking by-products containing significant amounts of bioactive compounds to prevent/attenuate MetS-associated cardiovascular pathologies.

Effects of salicylic acid-induced wine rich in anthocyanins on metabolic parameters and adipose insulin signaling in high-fructose fed rats.

We evaluated the effects of Syrah red wine treated with salicylic acid (RW SA) and its control red wine (RW) on metabolic parameters, systolic blood pressure and adipose tissue insulin signaling in high-fructose (F) fed rats. Grape treated with SA increased the anthocyanin (ANTs) levels in RW. F induced increased systolic blood pressure, dislipidemia and insulin resistance (HOMA:IR). F rats treated with RW significantly prevented these alterations while RW SA partially attenuated triglycerides levels and HOMA:IR without modifications in HDL cholesterol levels. F impaired the adipose tissue response to insulin. Supplementation with RW and RW SA partially attenuated these alterations. Rats supplemented with RW SA had lesser beneficial effects on metabolic alterations than control RW, while both RW and RW SA attenuated altered adipose response to insulin. More studies are necessary to deeply evaluate the effect on SA-induced RW rich in ANTs levels on metabolic alterations associated to MetS.

Grape pomace and grape pomace extract improve insulin signaling in high-fat-fructose fed rat-induced metabolic syndrome.

In this study the effect of diet supplementation with grape pomace (GP) and grape pomace extract (GPE) on insulin sensitive tissues (adipose, liver and muscle) was evaluated in an experimental model of metabolic syndrome (MetS). MetS was developed by giving a high-fat-fructose (HFF) diet to Wistar rats. Six weeks of HFF diet induced weight gain, which was partially attenuated by GP (1 g per kg per day) and GPE (300 mg per kg per day) supplementation. HFF diet increased systolic blood pressure, triglycerides, insulin resistance (HOMA:IR) and inflammation (c-reactive protein (CRP)). Supplementation with GP prevented SBP, triglycerides and CRP increased and partially attenuated insulin resistance. On the other hand, GPE partially reduced SBP and triglycerides and significantly prevented insulin resistance and inflammation. Also, HFF diet induced higher triglycerides content and enhanced NADPH oxidase activity in the liver. Also, HFF diet increased the epididymal adipose tissue weight, enlarged adipocyte size, and c-jun N-terminal kinase (JNK) activation, probably contributing to a pro-inflammatory cytokine pattern (higher resistin) and lower adiponectin protein expression. These alterations may result in an impairment of insulin signaling cascade observed in adipose, liver and muscle tissue (IRS1, Akt, and extracellular signal-regulated kinases (ERK1/2)) from HFF rats. Supplementation with GP and to a greater extent GPE attenuated liver triglyceride content and adiposity and restored adipose, liver and muscle response to insulin. These findings show that supplementation with GP and GPE to a greater extent can counteract adiposity, inflammation, liver damage and impaired insulin signaling associated to MetS, supporting the utilization of winemaking residues in food industry/human health due to their high amount of bioactive compounds.

Catechin and quercetin attenuate adipose inflammation in fructose-fed rats and 3T3-L1 adipocytes.

SCOPE: This study evaluated the capacity of dietary catechin (C), quercetin (Q), and the combination of both (CQ), to attenuate adipose inflammation triggered by high fructose (HFr) consumption in rats and by tumor necrosis factor alpha (TNF-α) in 3T3-L1 adipocytes. METHODS AND RESULTS: In rats, HFr consumption for 6 wk caused dyslipidemia, insulin resistance, reduced plasma adiponectin, adiposity, and adipose tissue inflammation. Dietary supplementation with 20 mg/kg/day of C, Q, and CQ improved all these parameters. In 3T3-L1 adipocytes, C and Q attenuated TNF-α-induced elevated protein carbonyls, increased proinflammatory cytokine expression (MCP-1, resistin), and decreased adiponectin. The protective effects of C and Q on adipose inflammation are in part associated with their capacity to (i) decrease the activation of the mitogen-activated kinases (MAPKs) JNK and p38; and (ii) prevent the downregulation of PPAR-γ. In summary, C and Q, and to a larger extent the combination of both, attenuated adipose proinflammatory signaling cascades and regulated the balance of molecules that improve (adiponectin) or impair (TNF-α, MCP-1, resistin) insulin sensitivity. CONCLUSION: Together, these findings suggest that dietary Q and C may have potential benefits in mitigating MetS-associated adipose inflammation, oxidative stress, and insulin resistance.

(-)-Epicatechin mitigates high-fructose-associated insulin resistance by modulating redox signaling and endoplasmic reticulum stress.

We investigated the capacity of dietary (-)-epicatechin (EC) to mitigate insulin resistance through the modulation of redox-regulated mechanisms in a rat model of metabolic syndrome. Adolescent rats were fed a regular chow diet without or with high fructose (HFr; 10% w/v) in drinking water for 8 weeks, and a group of HFr-fed rats was supplemented with EC in the diet. HFr-fed rats developed insulin resistance, which was mitigated by EC supplementation. Accordingly, the activation of components of the insulin signaling cascade (insulin receptor, IRS1, Akt, and ERK1/2) was impaired, whereas negative regulators (PKC, IKK, JNK, and PTP1B) were upregulated in the liver and adipose tissue of HFr rats. These alterations were partially or totally prevented by EC supplementation. In addition, EC inhibited events that contribute to insulin resistance: HFr-associated increased expression and activity of NADPH oxidase, activation of redox-sensitive signals, expression of NF-κB-regulated proinflammatory cytokines and chemokines, and some sub-arms of endoplasmic reticulum stress signaling. Collectively, these findings indicate that EC supplementation can mitigate HFr-induced insulin resistance and are relevant for defining interventions that can prevent/mitigate MetS-associated insulin resistance.

Garlic and onion attenuates vascular inflammation and oxidative stress in fructose-fed rats.

This study evaluates the antioxidant and the anti-inflammatory properties of garlic (G) and onion (O) in fructose-fed rats (FFR). Thirty-day-old male Wistar rats were assigned to control (C), F (10% fructose in drinking water), F+T (tempol 1 mM as control antioxidant), F+G, and F+O. Aqueous G and O extracts were administered orally in doses of 150 and 400 mg/kg/d respectively, and along with tempol, were given during the last 8 weeks of a 14-week period. At the end of the study, FFR had developed insulin resistance, aortic NADPH oxidase activity, increased SBP, plasma TBARS and vascular cell adhesion molecule-1 (VCAM-1) expression in mesenteric arteries, and a decrease in heart endothelial nitric oxide synthase (eNOS). Garlic and onion administration to F rats reduced oxidative stress, increased eNOS activity, and also attenuated VCAM-1 expression. These results provide new evidence showing the anti-inflammatory and antioxidant effect of these vegetables.