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Fructose intake has increased substantially throughout the developed world and is associated with obesity, type 2 diabetes and non-alcoholic fatty liver disease. Currently, our understanding of the metabolic and mechanistic implications for immune cells, such as monocytes and macrophages, exposed to elevated levels of dietary fructose is limited. Here, we show that fructose reprograms cellular metabolic pathways to favour glutaminolysis and oxidative metabolism, which are required to support increased inflammatory cytokine production in both LPS-treated human monocytes and mouse macrophages. A fructose-dependent increase in mTORC1 activity drives translation of pro-inflammatory cytokines in response to LPS. LPS-stimulated monocytes treated with fructose rely heavily on oxidative metabolism and have reduced flexibility in response to both glycolytic and mitochondrial inhibition, suggesting glycolysis and oxidative metabolism are inextricably coupled in these cells. The physiological implications of fructose exposure are demonstrated in a model of LPS-induced systemic inflammation, with mice exposed to fructose having increased levels of circulating IL-1β after LPS challenge. Taken together, our work underpins a pro-inflammatory role for dietary fructose in LPS-stimulated mononuclear phagocytes which occurs at the expense of metabolic flexibility., Myeloid cells are able to utilize a variety of monosaccharides from our diet, including fructose. Here the authors show that when monocytes are reliant on fructose as a carbon energy source they are reprogrammed towards oxidative metabolism, glutamine anaplerosis and a pro-inflammatory phenotype owing to excess pro-inflammatory cytokine production.

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet., Dietary serine and glycine starvation has emerged as a potential therapy for cancer. Here, the authors show that inhibition of PHGDH, which mediates the first step in the serine synthesis pathway, improves the therapeutic efficacy of serine depletion diet in mouse xenograft models.

Summary Loss of p53 function contributes to the development of many cancers. While cell-autonomous consequences of p53 mutation have been studied extensively, the role of p53 in regulating the anti-tumor immune response is still poorly understood. Here, we show that loss of p53 in cancer cells modulates the tumor-immune landscape to circumvent immune destruction. Deletion of p53 promotes the recruitment and instruction of suppressive myeloid CD11b+ cells, in part through increased expression of CXCR3/CCR2-associated chemokines and macrophage colony-stimulating factor (M-CSF), and attenuates the CD4+ T helper 1 (Th1) and CD8+ T cell responses in vivo. p53-null tumors also show an accumulation of suppressive regulatory T (Treg) cells. Finally, we show that two key drivers of tumorigenesis, activation of KRAS and deletion of p53, cooperate to promote immune tolerance., Graphical Abstract, Highlights • Tumor-specific loss of p53 delays tumor rejection in immune-competent hosts • p53 loss increases myeloid infiltration through enhanced cytokine secretion • The increase in Treg cells in response to loss of p53 is independent of Kras mutation • Kras mutations coordinate with p53 loss to regulate myeloid recruitment, TP53 is one of the most frequently mutated genes in cancer; however, its significance in controlling tumor-immune crosstalk is not fully understood. Blagih et al. highlight a key role for tumor-associated loss of p53, a common oncogenic event, in regulating myeloid and T cell responses.

Background & Aims Phosphatidylinositides-3 kinases (PI3Ks) are promising drug targets for cancer therapy, but blockage of PI3K-AKT signalling causes hyperglycaemia, hyperinsulinaemia, and liver damage in patients, and hepatocellular carcinoma (HCC) in mice. There are 4 PI3Ks: PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ. The role of PI3Kγ in HCC is unknown. Methods We performed histopathological, metabolic, and molecular phenotyping of mice with genetic ablation of PI3Kγ using models where HCC was initiated by the carcinogen diethylnitrosamine (DEN) and promoted by dietary or genetic obesity (ob/ob). The role of PI3Kγ in leucocytes was investigated in mice lacking PI3Kγ in haematopoietic and endothelial cells. Results Loss of PI3Kγ had no effects on the development of DEN-induced HCC in lean mice. However, in mice injected with DEN and placed on an obesogenic diet, PI3Kγ ablation reduced tumour growth, which was associated with reduced insulinaemia, steatosis, and expression of inflammatory cytokines. ob/ob mice lacking PI3Kγ, and mice with diet-induced obesity lacking PI3Kγ in leucocytes and endothelial cells did not display improved insulin sensitivity, steatosis, metabolic inflammation, or reduced tumour growth. However, these mice showed a reduced number of tumours, reduced liver infiltration by neutrophils, and reduced hepatocyte proliferation acutely induced by DEN. Conclusions Loss of PI3Kγ reduces tumour development in obesity-promoted HCC through multiple cell types and mechanisms that include improved insulinaemia, steatosis, and metabolic inflammation as well as the regulation of acute neutrophil infiltration and compensatory hepatocyte proliferation. PI3Kγ-selective inhibition may represent a novel therapeutic approach to reduce HCC initiation and slow HCC progression. Lay summary Class-1 phosphatidylinositides-3 kinases (PI3Ks) are critical targets in cancer therapy, but complete inhibition of all isoforms causes liver damage, hyperglycaemia, and insulinaemia. Here we show that selective ablation of the PI3Kγ isoform dampens tumour initiation and growth in a mouse model of carcinogen-initiated and obesity-promoted hepatocellular carcinoma (HCC). The effect of PI3Kγ ablation on reduced tumour growth was explained by reduced tumour cell proliferation, which was associated with reduced insulin levels, liver lipids, and reduced expression of tumour-promoting cytokines. PI3Kγ ablation in leucocytes of obese mice had no effects on tumour size. However, it reduced tumour number in association with reduced carcinogen-induced neutrophil infiltration and hepatocyte proliferation in livers of obese mice. Inhibition of PI3Kγ may thus reduce HCC initiation and growth in obese subjects by a mechanism involving reduced metabolic stress and insulinaemia and reduced carcinogen-induced neutrophil infiltration to the fatty liver., Graphical abstract, Highlights • PI3Kγ ablation does not affect carcinogen-induced liver cancer in lean mice. • PI3Kγ ablation reduces carcinogen-induced liver cancer in obese mice. • Systemic PI3Kγ ablation reduces hyperinsulinaemia, steatosis, metabolic inflammation, and growth of liver tumours. • PI3Kγ ablation in leucocytes and endothelial cells reduces neutrophil infiltration and hepatocyte proliferation acutely induced by carcinogen in the fatty liver.

Regulatory T cells (Tregs) are essential for mitigating inflammation. Tregs are found in nearly every tissue and play either beneficial or harmful roles in the host. The availability of various nutrients can either enhance or impair Treg function. Mitochondrial oxidative metabolism plays a major role in supporting Treg differentiation and fitness. While Tregs rely heavily on oxidation of fatty acids to support mitochondrial activity, they have found ways to adapt to different tissue types, such as tumors, to survive in competitive environments. In addition, metabolic by-products from commensal organisms in the gut also have a profound impact on Treg differentiation. In this review, we will focus on the core metabolic pathways engaged in Tregs, especially in the context of tissue nutrient environments, and how they can affect Treg function, stability and differentiation.

PI3Kγ has emerged as a promising target for the treatment of obesity and insulin resistance; however, previous studies have indicated that PI3Kγ activity in pancreatic β cells is required for normal insulin secretion in response to glucose. Hence, a possible deterioration of insulin secretion capacity in patients who are predisposed to the failure of pancreatic β-cell function is a major concern for the pharmacologic inhibition of PI3Kγ. To address this issue, we investigated the effects of PI3Kγ ablation in db/db diabetic mice, a genetic model of obesity-driven β-cell failure and diabetes. Mice that lacked PI3Kγ were backcrossed into db/+ mice C57BL/KS (>10 generations) to obtain db/db-PI3Kγ-/- mice. db/db-PI3Kγ-/- mice and control db/db mice were phenotyped for glucose homeostasis, insulin sensitivity, insulin secretion, steatosis, metabolic inflammation, pancreatic islet morphometry, islet cellular composition, and inflammation. Pancreatic β-cell apoptosis and proliferation were also evaluated. db/db-PI3Kγ -/- mice and control db/db mice developed similar body weight, steatosis, glycemia, and insulin levels after a glucose load; however, db/db-PI3Kγ-/- mice displayed improved insulin tolerance, higher levels of fasting serum insulin, and lower pancreatic insulin content. In db/db-PI3Kγ-/- mice, the number of adipose tissue macrophages was similar to control, but displayed reduced adipose tissue neutrophils and M2-polarized adipose tissue gene expression. Finally, db/db-PI3Kγ-/- mice have more pancreatic β cells and larger islets than db/db mice, despite displaying similar islet inflammation. This phenotype could be explained by reduced β-cell apoptosis in db/db-PI3Kγ-/- mice compared with control db/db mice. Our results are consistent with the concept that the beneficial action of PI3Kγ ablation in obesity-driven glucose intolerance is largely a result of its leptin-dependent effects on adiposity and, to a lesser extent, the promotion of adipose tissue neutrophil recruitment and M1 polarization of gene expression. Of importance, our data challenge the concept that PI3Kγ is required for insulin secretion in response to glucose in vivo, and indicate that PI3Kγ ablation protects db/db mice from β-cell apoptosis and improves fasting insulin levels. We conclude that PI3Kγ inhibition in obese patients who are predisposed to β-cell failure is not expected to produce adverse effects on insulin secretion.-Breasson, L., Sardi, C., Becattini, B., Zani, F., Solinas, G. PI3Kγ ablation does not promote diabetes in db/db mice, but improves insulin sensitivity and reduces pancreatic β-cell apoptosis.

p53 is an important tumour suppressor gene, with loss of p53 contributing to the development of most human cancers. However, the activation of p53 in response to stress signals underpins a role for p53 in diverse aspects of health and disease. Activities of p53 that regulate metabolism can play a role in maintaining homeostasis and protecting cells from damage - so preventing disease development. By contrast, either loss or over-activation of p53 can contribute to numerous metabolic pathologies, including aging, obesity and diabetes.

Ketogenic diets (KDs) have increasingly gained attention as effective means for weight loss and potential adjunctive treatment of cancer. The metabolic benefits of KDs are regularly ascribed to enhanced hepatic secretion of fibroblast growth factor 21 (FGF21) and its systemic effects on fatty-acid oxidation, energy expenditure (EE) and body weight. Ambiguous data from Fgf21-knockout animal strains and low FGF21 concentrations reported in humans with ketosis have nevertheless cast doubt regarding the endogenous function of FGF21. We here aimed to elucidate the causal role of FGF21 in mediating the therapeutic benefits of KDs on metabolism and cancer. We established a dietary model of increased vs decreased FGF21 by feeding C57BL/6J mice with KDs, either depleted of protein or enriched with protein. We furthermore used wild-type and Fgf21-knockout mice that were subjected to the respective diets, and monitored energy and glucose homeostasis as well as tumour growth after transplantation of Lewis lung carcinoma cells. Hepatic and circulating, but not adipose tissue, FGF21 levels were profoundly increased by protein starvation, independent of the state of ketosis. We demonstrate that endogenous FGF21 is not essential for the maintenance of normoglycaemia upon protein and carbohydrate starvation and is therefore not needed for the effects of KDs on EE. Furthermore, the tumour-suppressing effects of KDs were independent of FGF21 and, rather, driven by concomitant protein and carbohydrate starvation. Our data indicate that the multiple systemic effects of KD exposure in mice, previously ascribed to increased FGF21 secretion, are rather a consequence of protein malnutrition.

The phosphoinositide 3-kinase γ (PI3Kγ) plays a major role in leukocyte recruitment during acute inflammation and has been proposed to inhibit classical macrophage activation by driving immunosuppressive gene expression. PI3Kγ plays an important role in diet-induced obesity and insulin resistance. In seeking to determine the underlying molecular mechanisms, we showed that PI3Kγ action in high-fat diet– induced inflammation and insulin resistance depended largely on its role in the control of adiposity, which was due to PI3Kγ activity in a nonhematopoietic cell type. However, PI3Kγ activity in leukocytes was required for efficient neutrophil recruitment to adipose tissue. Neutrophil recruitment was correlated with proinflammatory gene expression in macrophages in adipose tissue, which triggered insulin resistance early during the development of obesity. Our data challenge the concept that PI3Kγ is a general suppressor of classical macrophage activation and indicate that PI3Kγ controls macrophage gene expression by non–cell-autonomous mechanisms, the outcome of which is context-dependent.

ObjectivesObesity-related cancers represent public health burdens of the first order. Nevertheless, suitable mouse models to unravel molecular mechanisms linking obesity to human cancer are still not available. One translational model is the immunocompromised Foxn1 (winged-helix/forkead transcription factor) nude mouse transplanted with human tumor xenografts. However, most xenograft studies are conducted in nude mice on an in-bred BALB/c background that entails protection from diet-induced obesity. To overcome such resistance to obesity and its sequelae, we here propose the dual strategy of utilizing Foxn1 nude mice on a C57BL/6 background and housing them at their thermoneutral zone.MethodsC57BL/6 nude and corresponding wild type mice, housed at 23 °C or 33 °C, were subjected to either low fat diet or high fat diet. Energy expenditure, locomotor activity, body core temperature, respiratory quotient as well as food and water intake were analyzed using indirect calorimetry. Immune function at different housing temperatures was assessed by using an in vivo cytokine capture assay.ResultsOur data clearly demonstrate that conventional housing protects C57BL/6 nude mice from high fat diet (HFD)-induced obesity, potentially via increased energy expenditure. In contrast, HFD-fed C57BL/6 nude mice housed at thermoneutral conditions develop adiposity, increased hepatic triglyceride accumulation, adipose tissue inflammation, and glucose intolerance. Moreover, increased circulating levels of lipopolysaccharide (LPS)-driven cytokines suggest a greatly enhanced immune response in C57BL/6 nude mice housed at thermoneutrality.ConclusionOur data reveals mild cold stress as a major modulator for energy and body weight homeostasis as well as immune function in C57BL/6 nude mice. Adjusting housing temperatures to the thermoneutral zone may ultimately be key to successfully study growth and progression of human tumors in a diet-induced obese environment.

Catch-up growth, a risk factor for type 2 diabetes, is characterized by hyperinsulinemia and accelerated body fat recovery. Using a rat model of semistarvation-refeeding that exhibits catch-up fat, we previously reported that during refeeding on a low-fat diet, glucose tolerance is normal but insulin-dependent glucose utilization is decreased in skeletal muscle and increased in adipose tissue, where de novo lipogenic capacity is concomitantly enhanced. Here we report that isocaloric refeeding on a high-fat (HF) diet blunts the enhanced in vivo insulin-dependent glucose utilization for de novo lipogenesis (DNL) in adipose tissue. These are shown to be early events of catch-up growth that are independent of hyperphagia and precede the development of overt adipocyte hypertrophy, adipose tissue inflammation, or defective insulin signaling. These results suggest a role for enhanced DNL as a glucose sink in regulating glycemia during catch-up growth, which is blunted by exposure to an HF diet, thereby contributing, together with skeletal muscle insulin resistance, to the development of glucose intolerance. Our findings are presented as an extension of the Randle cycle hypothesis, whereby the suppression of DNL constitutes a mechanism by which dietary lipids antagonize glucose utilization for storage as triglycerides in adipose tissue, thereby impairing glucose homeostasis during catch-up growth.

The interplay between hepatic glycogen metabolism and blood glucose levels is a paradigm of the rhythmic nature of metabolic homeostasis. Here we show that mice lacking a functional PER2 protein (Per2 (Brdm1) ) display reduced fasting glycemia, altered rhythms of hepatic glycogen accumulation, and altered rhythms of food intake. Per2 (Brdm1) mice show reduced hepatic glycogen content and altered circadian expression during controlled fasting and refeeding. Livers from Per2 (Brdm1) mice display reduced glycogen synthase protein levels during refeeding, and increased glycogen phosphorylase activity during fasting. The latter is explained by PER2 action on the expression of the adapter proteins PTG and GL, which target the protein phosphatase-1 to glycogen to decrease glycogen phosphorylase activity. Finally, PER2 interacts with genomic regions of Gys2, PTG, and G L . These results indicate an important role for PER2 in the hepatic transcriptional response to feeding and acute fasting that promotes glucose storage to liver glycogen.

Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 μL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.

Introduction Pancreatic cancer is one of the poorest understood cancers with a in vivo. A model of pancreatic ductal adenocarcinoma (PDAC) was generated using a Cre-recombinase system with the following combinations KRas G12D ;p53 +/+ or KRas G12D ;p53 f/f . Using a technique of tumour rejection, we demonstrate a role for p53 in PDAC cells in modulating the immune response. Material and methods In vivo studies were done using PDAC cell lines derived from GEMMs with ectopic expression of iRFP. FVB or CD1 nu/nu mice were injected subcutaneously with PDAC lines and subcutaneous growth was measured using the Licor Pearl Imager. Immunophenotyping was performed using flow cytometry (i.e. intracellular cytokine staining and surface staining) and acquired on the BD Symphony Fortessa. In vitro studies were conducted on the PDAC cell lines, bone marrow derived macrophages (BMDM) and primary T cells. Results and discussions Genetic ablation of p53 in PDAC cells provided immunological advantage and delayed tumour rejection. Immunophenotyping results demonstrated a suppression of Th1-like responses in vivo as well as changes in myeloid populations. In vitro studies provided evidence of alterations in macrophage priming and consequential changes in T cell polarisation. Recent studies have demonstrated roles for oncogenes, such as c-Myc and KRas in influencing anti-tumourgenic responses, which supports our data of p53-dependent changes. Conclusion Over the last 50 years immunoediting of the tumour has been shown to be a powerful mechanism of a tumour selection. Loss and mutation of p53 occurs quite frequently in human cancers and understanding the underlying immunological mechanisms of escape in the context of p53 is crucial in developing immunotherapies.

Obesity and insulin resistance are associated with oxidative stress, which may be implicated in the progression of obesity-related diseases. The kinase JNK1 has emerged as a promising drug target for the treatment of obesity and type 2 diabetes. JNK1 is also a key mediator of the oxidative stress response, which can promote cell death or survival, depending on the magnitude and context of its activation. In this article, we describe a study in which the long-term effects of JNK1 inactivation on glucose homeostasis and oxidative stress in obese mice were investigated for the first time. Mice lacking JNK1 (JNK1−/−) were fed an obesogenic high-fat diet (HFD) for a long period. JNK1−/− mice fed an HFD for the long term had reduced expression of antioxidant genes in their skin, more skin oxidative damage, and increased epidermal thickness and inflammation compared with the effects in control wild-type mice. However, we also observed that the protection from obesity, adipose tissue inflammation, steatosis, and insulin resistance, conferred by JNK1 ablation, was sustained over a long period and was paralleled by decreased oxidative damage in fat and liver. We conclude that compounds targeting JNK1 activity in brain and adipose tissue, which do not accumulate in the skin, may be safer and most effective.— Becattini, B., Zani, F., Breasson, L., Sardi, C., D’Agostino, V. G., Choo, M.-K., Provenzani, A., Park, J. M., Solinas, G. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.

Background & Aims Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. Methods We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2−negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. Results Peptide GPC3 367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3 367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3 367 multimer and secreted interferon-γ when cultured with GPC3 367 , but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8 + T cells that expressed the transgenic T-cell receptor specifically bound GPC3 367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. Conclusions We identified a GPC3 367 -specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.

Obesity is associated with a chronic low-grade inflammation, and specific antiinflammatory interventions may be beneficial for the treatment of type 2 diabetes and other obesity-related diseases. The lipid kinase PI3Kγ is a central proinflammatory signal transducer that plays a major role in leukocyte chemotaxis, mast cell degranulation, and endothelial cell activation. It was also reported that PI3Kγ activity within hematopoietic cells plays an important role in obesity-induced inflammation and insulin resistance. Here, we show that protection from insulin resistance, metabolic inflammation, and fatty liver in mice lacking functional PI3Kγ is largely consequent to their leaner phenotype. We also show that this phenotype is largely based on decreased fat gain, despite normal caloric intake, consequent to increased energy expenditure. Furthermore, our data show that PI3Kγ action on diet-induced obesity depends on PI3Kγ activity within a nonhematopoietic compartment, where it promotes energetic efficiency for fat mass gain. We also show that metabolic modulation by PI3Kγ depends on its lipid kinase activity and might involve kinase-independent signaling. Thus, PI3Kγ is an unexpected but promising drug target for the treatment of obesity and its complications.