Fasting in combination with the cocktail Sorafenib:Metformin blunts cellular plasticity and promotes liver cancer cell death via poly-metabolic exhaustion.

PURPOSE: Dual-Interventions targeting glucose and oxidative metabolism are receiving increasing attention in cancer therapy. Sorafenib (S) and Metformin (M), two gold-standards in liver cancer, are known for their mitochondrial inhibitory capacity. Fasting, a glucose-limiting strategy, is also emerging as chemotherapy adjuvant. Herein, we explore the anti-carcinogenic response of nutrient restriction in combination with sorafenib:metformin (NR-S:M). RESULTS: Our data demonstrates that, independently of liver cancer aggressiveness, fasting synergistically boosts the anti-proliferative effects of S:M co-treatment. Metabolic and Cellular plasticity was determined by the examination of mitochondrial and glycolytic activity, cell cycle modulation, activation of cellular apoptosis, and regulation of key signaling and metabolic enzymes. Under NR-S:M conditions, early apoptotic events and the pro-apoptotic Bcl-xS/Bcl-xL ratio were found increased. NR-S:M induced the highest retention in cellular SubG1 phase, consistent with the presence of DNA fragments from cellular apoptosis. Mitochondrial functionality, Mitochondrial ATP-linked respiration, Maximal respiration and Spare respiratory capacity, were all found blunted under NR-S:M conditions. Basal Glycolysis, Glycolytic reserve, and glycolytic capacity, together with the expression of glycogenic (PKM), gluconeogenic (PCK1 and G6PC3), and glycogenolytic enzymes (PYGL, PGM1, and G6PC3), were also negatively impacted by NR-S:M. Lastly, a TMT-proteomic approach corroborated the synchronization of liver cancer metabolic reprogramming with the activation of molecular pathways to drive a quiescent-like status of energetic-collapse and cellular death. CONCLUSION: Altogether, we show that the energy-based polytherapy NR-S:M blunts cellular, metabolic and molecular plasticity of liver cancer. Notwithstanding the in vitro design of this study, it holds a promising therapeutic tool worthy of exploration for this tumor pathology.

Short-term periodic restricted feeding elicits metabolome-microbiome signatures with sex dimorphic persistence in primate intervention.

Dietary restriction has shown benefits in physiological, metabolic, and molecular signatures associated with aging but is a difficult lifestyle to maintain for most individuals. In mice, a less restrictive diet that allows for cyclical periods of reduced calories mitigates aging phenotypes, yet the effects of such an intervention in a genetically heterogenous, higher-order mammal has not been examined. Here, using middle-aged rhesus macaques matched for age and sex, we show that a regimen of 4 days of low-calorie intake followed by 10 days of ad libitum feeding (4:10 diet) performed in repeating cycles over 12 weeks led to significant loss of weight and fat percentage, despite the free access to food for most of the study duration. We show the 4-day restriction period is sufficient to drive alterations to the serum metabolome characterized by substantial differences in lipid classes. These phenotypes were paralleled by changes in the gut microbiome of restricted monkeys that highlight the involvement of a microbiome-metabolome axis. This regimen shows promising phenotypes, with some sex-dimorphic responses, including residual memory of the diet. As many calorie restriction interventions are difficult to sustain, we propose that this short-term diet may be easier to adhere to and have benefits directly relevant to human aging.

Obesity and lifespan, a complex tango.

Obesity and aging share comorbidities, phenotypes, and deleterious effects on health that are associated with chronic diseases. However, distinct features set them apart, with underlying biology that should be explored and exploited, especially given the demographic shifts and the obesity epidemic that the world is facing.

Hepatocyte PPARγ contributes to the progression of non-alcoholic steatohepatitis in male and female obese mice.

Non-alcoholic steatohepatitis (NASH) is associated with obesity and increased expression of hepatic peroxisome proliferator-activated receptor γ (PPARγ). However, the relevance of hepatocyte PPARγ in NASH associated with obesity is still poorly understood. In this study, hepatocyte PPARγ was knocked out (PpargΔHep) in male and female mice after the development of high-fat diet-induced obesity. The diet-induced obese mice were then maintained on their original diet or switched to a high fat, cholesterol, and fructose (HFCF) diet to induce NASH. Hepatic PPARγ expression was mostly derived from hepatocytes and increased by high fat diets. PpargΔHep reduced HFCF-induced NASH progression without altering steatosis, reduced the expression of key genes involved in hepatic fibrosis in HFCF-fed male and female mice, and decreased the area of collagen-stained fibrosis in the liver of HFCF-fed male mice. Moreover, transcriptomic and metabolomic data suggested that HFCF-diet regulated hepatic amino acid metabolism in a hepatocyte PPARγ-dependent manner. PpargΔHep increased betaine-homocysteine s-methyltransferase expression and reduced homocysteine levels in HFCF-fed male mice. In addition, in a cohort of 102 obese patients undergoing bariatric surgery with liver biopsies, 16 cases were scored with NASH and were associated with increased insulin resistance and hepatic PPARγ expression. Our study shows that hepatocyte PPARγ expression is associated with NASH in mice and humans. In male mice, hepatocyte PPARγ negatively regulates methionine metabolism and contributes to the progression of fibrosis.

Influence of Protein Carbonylation on Human Adipose Tissue Dysfunction in Obesity and Insulin Resistance.

BACKGROUND: Obesity is characterized by adipose tissue dysregulation and predisposes individuals to insulin resistance and type 2 diabetes. At the molecular level, adipocyte dysfunction has been linked to obesity-triggered oxidative stress and protein carbonylation, considering protein carbonylation as a link between oxidative stress and metabolic dysfunction. The identification of specific carbonylated proteins in adipose tissue could provide novel biomarkers of oxidative damage related to metabolic status (i.e prediabetes). Thus, we aimed at characterizing the subcutaneous and omental human adipose tissue carbonylome in obesity-associated insulin resistance. METHODS: 2D-PAGE was used to identify carbonylated proteins, and clinical correlations studies and molecular biology approaches including intracellular trafficking, reactive oxygen species assay, and iron content were performed using in vitro models of insulin resistance. RESULTS: The carbonylome of human adipose tissue included common (serotransferrin, vimentin, actin, and annexin A2) and depot-specific (carbonic anhydrase and α-crystallin B in the subcutaneous depot; and α-1-antitrypsin and tubulin in the omental depot) differences that point out the complexity of oxidative stress at the metabolic level, highlighting changes in carbonylated transferrin expression. Posterior studies using in vitro prediabetic model evidence alteration in transferrin receptor translocation, linked to the prediabetic environment. Finally, ligand-receptor molecular docking studies showed a reduced affinity for carbonylated transferrin binding to its receptor compared to wild-type transferrin, emphasizing the role of transferrin carbonylation in the link between oxidative stress and metabolic dysfunction. CONCLUSIONS: The adipose tissue carbonylome contributes to understanding the molecular mechanism driving adipocyte dysfunction and identifies possible adipose tissue carbonylated targets in obesity-associated insulin resistance.

Fatty acids homeostasis during fasting predicts protection from chemotherapy toxicity.

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity.

Understanding the adipose tissue acetylome in obesity and insulin resistance.

Obesity is a widely prevalent pathology with a high exponential growth worldwide. Altered lipid accumulation by adipose tissue is one of the main causes of obesity and exploring lipid homeostasis in this tissue may represent a source for the identification of possible therapeutic targets. The study of the proteome and the post-translational modifications of proteins, specifically acetylation due to its involvement in energy metabolism, may be of great interest to understand the molecular mechanisms involved in adipose tissue dysfunction in obesity. The objective of this study was to characterize the subcutaneous and omental adipose tissue acetylome in conditions of obesity and insulin resistance and to describe the importance of acetylation of key molecules in adipose tissue to use them as therapeutic targets. The results describe for the first time the acetylome of subcutaneous and omental adipose tissue under physiological and physiopathological conditions such as obesity and insulin resistance. New evidence showed different acetylation patterns between two main depots and highlight the molecular complexity of adipose tissue. Results showed changes in FABP4 acetylation in subcutaneous fat in relation to insulin resistance, thus unveiling a potential marker of depot-specific dysfunctional expansion in obesity-associated metabolic disease. Furthermore, it is shown that the acetylation of FABP4 affects its function, modulating the capacity of differentiation in adipocytes. In conclusion, this study demonstrates a profound, depot-specific alteration of adipose tissue acetylome, wherein the acetylation of FABP4 may play a key role in adipocyte differentiation and lipid accumulation.

Deprogramming metabolism in pancreatic cancer with a bi-functional GPR55 inhibitor and biased ß2 adrenergic agonist.

Metabolic reprogramming contributes to oncogenesis, tumor growth, and treatment resistance in pancreatic ductal adenocarcinoma (PDAC). Here we report the effects of (R,S')-4'-methoxy-1-naphthylfenoterol (MNF), a GPR55 antagonist and biased ß2-adrenergic receptor (ß2-AR) agonist on cellular signaling implicated in proliferation and metabolism in PDAC cells. The relative contribution of GPR55 and ß2-AR in (R,S')-MNF signaling was explored further in PANC-1 cells. Moreover, the effect of (R,S')-MNF on tumor growth was determined in a PANC-1 mouse xenograft model. PANC-1 cells treated with (R,S')-MNF showed marked attenuation in GPR55 signal transduction and function combined with increased ß2-AR/Gαs/adenylyl cyclase/PKA signaling, both of which contributing to lower MEK/ERK, PI3K/AKT and YAP/TAZ signaling. (R,S')-MNF administration significantly reduced PANC-1 tumor growth and circulating L-lactate concentrations. Global metabolic profiling of (R,S')-MNF-treated tumor tissues revealed decreased glycolytic metabolism, with a shift towards normoxic processes, attenuated glutamate metabolism, and increased levels of ophthalmic acid and its precursor, 2-aminobutyric acid, indicative of elevated oxidative stress. Transcriptomics and immunoblot analyses indicated the downregulation of gene and protein expression of HIF-1α and c-Myc, key initiators of metabolic reprogramming in PDAC. (R,S')-MNF treatment decreased HIF-1α and c-Myc expression, attenuated glycolysis, shifted fatty acid metabolism towards ß-oxidation, and suppressed de novo pyrimidine biosynthesis in PANC-1 tumors. The results indicate a potential benefit of combined GPR55 antagonism and biased ß2-AR agonism in PDAC therapy associated with the deprogramming of altered cellular metabolism.

Diet composition influences the metabolic benefits of short cycles of very low caloric intake.

Diet composition, calories, and fasting times contribute to the maintenance of health. However, the impact of very low-calorie intake (VLCI) achieved with either standard laboratory chow (SD) or a plant-based fasting mimicking diet (FMD) is not fully understood. Here, using middle-aged male mice we show that 5 months of short 4:10 VLCI cycles lead to decreases in both fat and lean mass, accompanied by improved physical performance and glucoregulation, and greater metabolic flexibility independent of diet composition. A long-lasting metabolomic reprograming in serum and liver is observed in mice on VLCI cycles with SD, but not FMD. Further, when challenged with an obesogenic diet, cycles of VLCI do not prevent diet-induced obesity nor do they elicit a long-lasting metabolic memory, despite achieving modest metabolic flexibility. Our results highlight the importance of diet composition in mediating the metabolic benefits of short cycles of VLCI.

Daily caloric restriction limits tumor growth more effectively than caloric cycling regardless of dietary composition.

Cancer incidence increases with age and is a leading cause of death. Caloric restriction (CR) confers benefits on health and survival and delays cancer. However, due to CR's stringency, dietary alternatives offering the same cancer protection have become increasingly attractive. Short cycles of a plant-based diet designed to mimic fasting (FMD) are protective against tumorigenesis without the chronic restriction of calories. Yet, it is unclear whether the fasting time, level of dietary restriction, or nutrient composition is the primary driver behind cancer protection. Using a breast cancer model in mice, we compare the potency of daily CR to that of periodic caloric cycling on FMD or an isocaloric standard laboratory chow against primary tumor growth and metastatic burden. Here, we report that daily CR provides greater protection against tumor growth and metastasis to the lung, which may be in part due to the unique immune signature observed with daily CR.

Rosiglitazone Requires Hepatocyte PPARγ Expression to Promote Steatosis in Male Mice With Diet-Induced Obesity.

Thiazolidinediones (TZD) are peroxisome proliferator-activated receptor γ (PPARγ) agonists that may reduce hepatic steatosis through their effects in adipose tissue and therefore have been assessed as potential therapies to treat nonalcoholic fatty liver disease (NAFLD) in humans. However, some studies suggest that expression and activation of hepatocyte PPARγ promotes steatosis and that would limit the benefits of TZD as a NAFLD therapy. To further explore this possibility, we examined the impact of short-term rosiglitazone maleate treatment after the development of moderate or severe diet-induced obesity, in both control and adult-onset hepatocyte-specific PPARγ knockout (PpargΔHep) mice. Independent of the level of obesity and hepatic PPARγ expression, the TZD treatment enhanced insulin sensitivity, associated with an increase in white adipose tissue (WAT) fat accumulation, consistent with clinical observations. However, TZD treatment increased hepatic triglyceride content only in control mice with severe obesity. Under these conditions, PpargΔHep reduced diet-induced steatosis and prevented the steatogenic effects of short-term TZD treatment. In these mice, subcutaneous WAT was enlarged and associated with increased levels of adiponectin, while hepatic levels of phosphorylated adenosine 5'-monophosphate-activated protein kinase were also increased. In addition, in mice with severe obesity, the expression of hepatic Cd36, Cidea, Cidec, Fabp4, Fasn, and Scd-1 was increased by TZD in a PPARγ-dependent manner. Taken together, these results demonstrate that hepatocyte PPARγ expression offsets the antisteatogenic actions of TZD in mice with severe obesity. Therefore, in obese and insulin resistant humans, TZD-mediated activation of hepatocyte PPARγ may limit the therapeutic potential of TZD to treat NAFLD.

Hepatocyte-Specific Loss of PPARγ Protects Mice From NASH and Increases the Therapeutic Effects of Rosiglitazone in the Liver.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is commonly observed in patients with type 2 diabetes, and thiazolidinediones (TZD) are considered a potential therapy for NASH. Although TZD increase insulin sensitivity and partially reduce steatosis and alanine aminotransferase, the efficacy of TZD on resolving liver pathology is limited. In fact, TZD may activate peroxisome proliferator-activated receptor gamma (PPARγ) in hepatocytes and promote steatosis. Therefore, we assessed the role that hepatocyte-specific PPARγ plays in the development of NASH, and how it alters the therapeutic effects of TZD on the liver of mice with diet-induced NASH. METHODS: Hepatocyte-specific PPARγ expression was knocked out in adult mice before and after the development of NASH induced with a high fat, cholesterol, and fructose (HFCF) diet. RESULTS: HFCF diet increased PPARγ expression in hepatocytes, and rosiglitazone further activated PPARγ in hepatocytes of HFCF-fed mice in vivo and in vitro. Hepatocyte-specific loss of PPARγ reduced the progression of HFCF-induced NASH in male mice and increased the benefits derived from the effects of TZD on extrahepatic tissues and non-parenchymal cells. RNAseq and metabolomics indicated that HFCF diet promoted inflammation and fibrogenesis in a hepatocyte PPARγ-dependent manner and was associated with dysregulation of hepatic metabolism. Specifically, hepatocyte-specific loss of PPARγ plays a positive role in the regulation of methionine metabolism, and that could reduce the progression of NASH. CONCLUSIONS: Because of the negative effect of hepatocyte PPARγ in NASH, inhibition of mechanisms promoted by endogenous PPARγ in hepatocytes may represent a novel strategy that increases the efficiency of therapies for NAFLD.

NQO1 protects obese mice through improvements in glucose and lipid metabolism.

Chronic nutrient excess leads to metabolic disorders and insulin resistance. Activation of stress-responsive pathways via Nrf2 activation contributes to energy metabolism regulation. Here, inducible activation of Nrf2 in mice and transgenesis of the Nrf2 target, NQO1, conferred protection from diet-induced metabolic defects through preservation of glucose homeostasis, insulin sensitivity, and lipid handling with improved physiological outcomes. NQO1-RNA interaction mediated the association with and inhibition of the translational machinery in skeletal muscle of NQO1 transgenic mice. NQO1-Tg mice on high-fat diet had lower adipose tissue macrophages and enhanced expression of lipogenic enzymes coincident with reduction in circulating and hepatic lipids. Metabolomics data revealed a systemic metabolic signature of improved glucose handling, cellular redox, and NAD+ metabolism while label-free quantitative mass spectrometry in skeletal muscle uncovered a distinct diet- and genotype-dependent acetylation pattern of SIRT3 targets across the core of intermediary metabolism. Thus, under nutritional excess, NQO1 transgenesis preserves healthful benefits.

Adipose tissue depot-specific intracellular and extracellular cues contributing to insulin resistance in obese individuals.

Adipose tissue dysregulation in obesity strongly influences systemic metabolic homeostasis and is often linked to insulin resistance (IR). However, the molecular mechanisms underlying adipose tissue dysfunction in obesity are not fully understood. Herein, a proteomic analysis of subcutaneous (SC) and omental (OM) fat from lean subjects and obese individuals with different degrees of insulin sensitivity was performed to identify adipose tissue biomarkers related to obesity-associated metabolic disease. Our results suggest that dysregulation of both adipose tissue extracellular matrix (ECM) organization and intracellular trafficking processes may be associated with IR in obesity. Thus, abnormal accumulation of the small leucine-rich proteoglycan, lumican, as observed in SC fat of IR obese individuals, modifies collagen I organization, impairs adipogenesis and activates stress processes [endoplasmic reticulum and oxidative stress] in adipocytes. In OM fat, IR is associated with increased levels of the negative regulator of the Rab family of small GTPases, GDI2, which alters lipid storage in adipocytes by inhibiting insulin-stimulated binding of the Rab protein, Rab18, to lipid droplets. Together, these results indicate that lumican and GDI2 might play depot-dependent, pathogenic roles in obesity-associated IR. Our findings provide novel insights into the differential maladaptive responses of SC and OM adipose tissue linking obesity to IR.

The road ahead for health and lifespan interventions.

Aging is a modifiable risk factor for most chronic diseases and an inevitable process in humans. The development of pharmacological interventions aimed at delaying or preventing the onset of chronic conditions and other age-related diseases has been at the forefront of the aging field. Preclinical findings have demonstrated that species, sex and strain confer significant heterogeneity on reaching the desired health- and lifespan-promoting pharmacological responses in model organisms. Translating the safety and efficacy of these interventions to humans and the lack of reliable biomarkers that serve as predictors of health outcomes remain a challenge. Here, we will survey current pharmacological interventions that promote lifespan extension and/or increased healthspan in animals and humans, and review the various anti-aging interventions selected for inclusion in the NIA's Interventions Testing Program as well as the ClinicalTrials.gov database that target aging or age-related diseases in humans.

A Central Role for the Gasotransmitter H2S in Aging.

Conserved mechanisms of aging often illuminate the development of novel therapeutic interventions to delay aging and the onset of age-related diseases and functional decline. Recently in Cell Metabolism, Zivanovic et al. (2019) demonstrated a decline during aging of the protective H2S-dependent protein persulfidation across species and its reversal by targeted strategies.

Energy Restriction and Colorectal Cancer: A Call for Additional Research.

: Colorectal cancer has the second highest cancer-related mortality rate, with an estimated 881,000 deaths worldwide in 2018. The urgent need to reduce the incidence and mortality rate requires innovative strategies to improve prevention, early diagnosis, prognostic biomarkers, and treatment effectiveness. Caloric restriction (CR) is known as the most robust nutritional intervention that extends lifespan and delays the progression of age-related diseases, with remarkable results for cancer protection. Other forms of energy restriction, such as periodic fasting, intermittent fasting, or fasting-mimicking diets, with or without reduction of total calorie intake, recapitulate the effects of chronic CR and confer a wide range of beneficial effects towards health and survival, including anti-cancer properties. In this review, the known molecular, cellular, and organismal effects of energy restriction in oncology will be discussed. Energy-restriction-based strategies implemented in colorectal models and clinical trials will be also revised. While energy restriction constitutes a promising intervention for the prevention and treatment of several malignant neoplasms, further investigations are essential to dissect the interplay between fundamental aspects of energy intake, such as feeding patterns, fasting length, or diet composition, with all of them influencing health and disease or cancer effects. Currently, effectiveness, safety, and practicability of different forms of fasting to fight cancer, particularly colorectal cancer, should still be contemplated with caution.

Benefits of Caloric Restriction in Longevity and Chemical-Induced Tumorigenesis Are Transmitted Independent of NQO1.

Caloric restriction (CR) is the most potent nonpharmacological intervention known to both protect against carcinogenesis and delay aging in laboratory animals. There is a growing number of anticarcinogens and CR mimetics that activate NAD(P)H:quinone oxidoreductase 1 (NQO1). We have previously shown that NQO1, an antioxidant enzyme that acts as an energy sensor through modulation of intracellular redox and metabolic state, is upregulated by CR. Here, we used NQO1-knockout (KO) mice to investigate the role of NQO1 in both the aging process and tumor susceptibility, specifically in the context of CR. We found that NQO1 is not essential for the beneficial effects of CR on glucose homeostasis, physical performance, metabolic flexibility, life-span extension, and (unlike our previously observation with Nrf2) chemical-induced tumorigenesis.

Adult-Onset Hepatocyte GH Resistance Promotes NASH in Male Mice, Without Severe Systemic Metabolic Dysfunction.

Nonalcoholic fatty liver disease (NAFLD), which includes nonalcoholic steatohepatitis (NASH), is associated with reduced GH input/signaling, and GH therapy is effective in the reduction/resolution of NAFLD/NASH in selected patient populations. Our laboratory has focused on isolating the direct vs indirect effects of GH in preventing NAFLD/NASH. We reported that chow-fed, adult-onset, hepatocyte-specific, GH receptor knockdown (aHepGHRkd) mice rapidly (within 7 days) develop steatosis associated with increased hepatic de novo lipogenesis (DNL), independent of changes in systemic metabolic function. In this study, we report that 6 months after induction of aHepGHRkd early signs of NASH develop, which include hepatocyte ballooning, inflammation, signs of mild fibrosis, and elevated plasma alanine aminotransferase. These changes occur in the presence of enhanced systemic lipid utilization, without evidence of white adipose tissue lipolysis, indicating that the liver injury that develops after aHepGHRkd is due to hepatocyte-specific loss of GH signaling and not due to secondary defects in systemic metabolic function. Specifically, enhanced hepatic DNL is sustained with age in aHepGHRkd mice, associated with increased hepatic markers of lipid uptake/re-esterification. Because hepatic DNL is a hallmark of NAFLD/NASH, these studies suggest that enhancing hepatocyte GH signaling could represent an effective therapeutic target to reduce DNL and treat NASH.