Objective Regulation of food intake and energy balance depends on a group of hypothalamic neurons that release anorexigenic melanocortins encoded by the Pomc gene. Although the physiological importance of central melanocortins is well appreciated, the genetic program that defines the functional identity of melanocortin neurons and assures high levels of hypothalamic Pomc expression is only beginning to be understood. Here, we interrogated whether the transcriptional regulator PRDM12, identified as a highly expressed gene in adult mouse POMC neurons, plays an important role in the identity and function of melanocortin neurons. Methods We first determined the cellular distribution of PRDM12 in the developing hypothalamus. Then, we studied mutant mice with constitutively inactivated Prdm12 to evaluate possible alterations in hypothalamic Pomc expression. In addition, we characterized conditional mutant mice specifically lacking Prdm12 in ISL1-positive or POMC neurons during development. Finally, we measured food intake, body weight progression up to 16 weeks of age, adiposity and glucose tolerance in adult mice lacking Prdm12 selectively from POMC neurons. Results We found that PRDM12 coexpressed with POMC in mouse hypothalamic neurons from early development to adulthood. Mice lacking Prdm12 displayed greatly reduced Pomc expression in the developing hypothalamus. Selective ablation of Prdm12 from ISL1 neurons prevented hypothalamic Pomc expression. The conditional ablation of Prdm12 limited to POMC neurons greatly reduced Pomc expression in the developing hypothalamus and in adult mice led to increased food intake, adiposity and obesity. Conclusions Altogether, our results demonstrate that PRDM12 plays an essential role in the early establishment of hypothalamic melanocortin neuron identity and the maintenance of high expression levels of Pomc. Its absence in adult mice greatly impairs Pomc expression and leads to increased food intake, adiposity and obesity.

Objectives Nutrient sensing by hypothalamic neurons is critical for the regulation of food intake and energy expenditure. We aimed to identify long- and medium-chain fatty acid species transported into the brain, their effects on energy balance and mechanisms by which they regulate activity of hypothalamic neurons. Methods Simultaneous blood and cerebrospinal fluid (CSF) sampling was undertaken in rats and metabolic analyses using radiolabelled fatty acid tracers were performed on mice. Electrophysiological recording techniques were used to investigate signalling mechanisms underlying fatty acid-induced changes in activity of pro-opiomelanocortin (POMC) neurons. Results The medium chain fatty acid (MCFA) octanoic acid (C8:0), unlike long chain fatty acids, was rapidly transported into the hypothalamus of mice and almost exclusively oxidized, giving rise to rapid, transient reductions in food intake and increased energy expenditure. Octanoic acid differentially regulates excitability of POMC neurons, activating these neurons directly via GPR40 and inducing inhibition via an indirect non-synaptic, purine and adenosine receptor-dependent mechanism. Conclusions The MCFA octanoic acid is a centrally signalling nutrient that targets POMC neurons via distinct direct and indirect signal transduction pathways to instigate changes in energy status. These results could explain the beneficial health effects that accompany MCFA consumption.

Objective It is well established that the liver specific miR-122, a bona fide tumor suppressor, plays critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine is a critical energy and anaplerotic source for mammalian cells, we assessed glutamine metabolism in the control (WT) and miR-122 knockout mice (KO) by SIRM (Stable Isotope Resolved Metabolomics) studies. Methods To this end, 6-8-week-old WT and KO mice and 12-15-month-old liver tumor-bearing mice were injected with [U-13C5,15N2]-L-glutamine (Gln) and polar metabolites from the liver tissues were analyzed by NMR (Nuclear Magnetic Resonance) and IC-MS (Ion Chromatography-Mass Spectrometry). Gln-metabolism was also assessed in a glutamine-dependent HCC cell line (EC4). Expressions of glutaminases (Gls and Gls2) and Slc1a5, a glutamine transporter were analyzed in mouse livers and human primary HCC samples. Results The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of 13C- and/or 15N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3’-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased 13C-Gln, 13C-α-ketoglutarate, 13C-isocitrate and 13C-citrate levels. In contrast, 13C-phosphoenolpyruvate and 13C-G6P levels were elevated in cells expressing ectopic miR-122 suggesting enhanced gluconeogenesis. Finally, TCGA-LIHC (The Cancer Genome Atlas Liver Hepatocellular Carcinoma) database analysis revealed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared to those expressing lower levels of these proteins. Conclusions Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.

Background The diminished glucose lowering effect of insulin in obesity, denoted “insulin resistance”, is associated with glucose intolerance, type 2 diabetes and other serious maladies. Many thousands of publications on the topic have suggested dozens of hypotheses on the molecular and cellular disruptions that contribute to the syndrome. Yet there is still deep uncertainty on the mechanisms of its initiation and long term maintenance. Scope of review To simplify analysis of insulin resistance, this review focusses on the unifying concept that adipose tissue is a central regulator of systemic glucose homeostasis by controlling liver and skeletal muscle metabolism. Key aspects of adipose function related to insulin resistance reviewed here are: 1.) the modes by which specific adipose tissues control hepatic glucose output and systemic glucose disposal, 2.) recently acquired understanding of the underlying mechanisms of these modes of regulation, and 3.) the steps in these pathways adversely affected by obesity that give rise to insulin resistance. Major Conclusions Adipocyte heterogeneity is required to mediate the multiple pathways that control systemic glucose tolerance. White adipocytes specialize in sequestering triglyceride away from liver, muscle and other tissues to limit toxicity. In contrast, brown/beige adipocytes are very active in directly taking up glucose in response to β adrenergic signaling and insulin and enhancing energy expenditure. Nonetheless, white, beige and brown adipocytes all share the common feature of secreting factors and perhaps exosomes that act on distant tissues to control glucose homeostasis. Obesity exerts deleterious effects on each of the above adipocyte functions to cause insulin resistance.

Background Leptin acts via its receptor, LepRb, on specialized neurons in the brain to modulate energy balance and glucose homeostasis. LepRb→STAT3 signaling plays a crucial role in leptin action, but LepRb also mediates an additional as-yet unidentified signal (“Signal 2”) that is important for leptin action. Signal 2 requires LepRb regions additional to those required for JAK2 activation, but operates independently of STAT3 and LepRb phosphorylation sites. Methods To identify LepRb sequences that mediate Signal 2, we used CRISPR/Cas9 to generate five novel mouse lines containing COOH-terminal truncation mutants of LepRb. We analyzed metabolic phenotype and measures of hypothalamic function for these mouse lines. Results We found that deletion of LepRb sequences between residues 921 and 960 dramatically worsens metabolic control and alters hypothalamic function relative to smaller truncations. We also found that deletion of the regions including residues 1013-1053 and 960-1013 each decreased obesity compared to deletions that include additional COOH-terminal residues. Conclusions LepRb sequences between residues 921 and 960 mediate the STAT3 and LepRb phosphorylation-independent second signal that contributes to the control of energy balance and metabolism by leptin/LepRb. In addition to confirming the inhibitory role of the region (residues 961-1013) containing Tyr985, we also identified the region containing residues 1013-1053 (which contains no Tyr residues) as a second potential mediator of LepRb inhibition. Thus, the intracellular domain of LepRb mediates multiple Tyr-independent signals.

Objective A widely recognized metabolic side-effect of glucocorticoid (GC) therapy is steroid-induced diabetes mellitus (DM). However, studies on the molecular basis of GC-induced pancreatic beta cell dysfunction in human beta cells are lacking. Meanwhile, the significance of non-coding RNAs in various cellular processes is emerging. Here we aimed to show the direct negative impact of GC on beta cell function and elucidate the role of the riborepressor GAS5 lincRNA in GC signaling pathway in human pancreatic beta cells. Methods Patients undergoing two weeks high-dose prednisolone therapy were monitored for c-peptide levels. Human pancreatic islets and the human beta cell line, EndoC-βH1, were incubated in pharmacological concentrations of dexamethasone. GAS5 level was modulated using anti-sense LNA gapmer or using short oligonucleotides with GAS5 HREM (hormone response element motif). Immunoblotting and/or real-time PCR were used to assess changes in protein and RNA expression, respectively. Functional characterization included glucose-stimulated insulin secretion and apoptosis assays. Correlation analysis was performed on RNAseq data of human pancreatic islets. Results We found reduced c-peptide levels in patients undergoing high dose GC therapy. Human islets and the human beta cell line, EndoC-βH1, exposed to GC exhibited reduced insulin secretion and increased apoptosis. Concomitantly, reduced expression of important beta cell transcription factors, PDX1 and NKX6.1, as well as the exocytotic protein, SYT13 was observed. Furthermore, the expression of the glucocorticoid receptor was decreased, while SGK1 (serum and glucocorticoid-regulated kinase 1) was elevated. The expression of these genes was found to significantly correlate with GAS5 in human islet transcriptomics data. Increasing GAS5 levels using GAS5 HREM alleviated the inhibitory effects of dexamethasone on insulin secretion. Conclusions The direct adverse effect of glucocorticoid in human beta cell function is mediated via important beta cell proteins and components of the GC-signaling pathway in an intricate interplay with the GAS5 lincRNA, a potentially novel therapeutic target to counter GC-mediated beta cell dysfunction.

Background The microbiota in our gut is an important component of normal physiology that has co-evolved with us from the earliest multicellular organisms. It is, therefore, not surprising that there is an intimate crosstalk between the microbial world in the gut and the host. Genome regulation through microbiota-host interactions not only affect the host’s immunity, but also metabolic health and resilience against cancer. Chromatin dynamics of the host epithelium involving histone modifications and other facets of the epigenetic machinery play an important role in this process. Scope of Review In this review we will discuss recent findings relevant to how chromatin dynamics shape the crosstalk between the microbiota and its host, with special focus on the role of histone modifications. Major Conclusions Host-microbiome interactions are important evolutionary drivers and are, thus, expected to be hardwired into and mould the epigenetic machinery in multicellular organisms. Microbial derived short chain fatty acids (SCFA) emerge as a dominant determinant in microbiome-host interaction and the inhibition of histone deacetylases (HDACs) by SCFA is a key mechanism in this process. The discovery of alternative histone acylations, such as crotonylation, in addition to the canonical histone acetylation reveals a new layer of complexity in this crosstalk.

Background An organism’s metabolic phenotype is primarily affected by its genotype, its lifestyle, and the nutritional composition of its food supply. In addition, it is now clear from studies in many different species that ancestral environments can also modulate metabolism in at least one to two generations of offspring. Scope of review We limit ourselves here to paternal effects in mammals, primarily focusing on studies performed in inbred rodent models. Although hundreds of studies link paternal diets and offspring metabolism, the mechanistic basis by which epigenetic information in sperm programs nutrient handling in the next generation remains mysterious. Our goal in this review is to provide a brief overview of paternal effect paradigms and the germline epigenome. We then pivot to exploring one key mystery in this literature: how do epigenetic changes in sperm, most of which are likely to act transiently in the early embryo, ultimately direct a long-lasting physiological response in offspring? Major conclusions Several potential mechanisms exist by which transient epigenetic modifications, such as small RNAs or methylation states erased shortly after fertilization, could be transferred to more durable heritable information. A detailed mechanistic understanding of this process will provide deep insights into early development, and could be of great relevance for human health and disease.

Objective Among obesity-associated metabolic diseases, non-alcoholic fatty liver disease (NAFLD) represents an increasing public health issue due to its emerging association with atherogenic dyslipidemia and cardiovascular diseases (CVDs). The lower prevalence of NAFLD in pre-menopausal women compared to men or post-menopausal women led us to hypothesize that the female-inherent ability to counteract this pathology might strongly rely on estrogen signaling. In female mammals, Estrogen Receptor alpha (ERα) is highly expressed in the liver, where it acts as a sensor of the nutritional status and adapts the metabolism to the reproductive needs. As in the male liver this receptor is little expressed, we here hypothesize that hepatic ERα might account for sex differences in the ability of males and females to cope with an excess of dietary lipids and counteract the accumulation of lipids in the liver. Methods Through liver metabolomics and transcriptomics we analyzed the relevance of hepatic ERα in the metabolic response of males and females to a diet highly enriched in fats (HFD) as a model of diet-induced obesity. Results The study shows that the hepatic ERα strongly contributes to the sex-specific response to a fat-enriched diet and its action accounts for opposite consequences for hepatic health in males and females. Conclusion This study identified hepatic ERα as a novel target for the design of sex-specific therapies against fatty liver and its cardio-metabolic consequences.

Objective Post-bariatric surgery hypoglycemia (PBH) is defined as the presence of neuroglycopenic symptoms accompanied by postprandial hypoglycemia in bariatric surgery patients. Recent clinical studies using continuous glucose monitoring (CGM) technology revealed that PBH is more frequently observed in vertical sleeve gastrectomy (VSG) patients than previously recognized. PBH cannot be alleviated by current medication. Therefore, a model system to investigate the mechanism and treatment is required. Methods We utilized CGM in a rat model of VSG and monitored the occurrence of glycemic variability and hypoglycemia in various meal conditions for 4-weeks after surgery. Another cohort of rat VSG with CGM was utilized to investigate whether the blockade of glucagon-like peptide-1 receptor (GLP-1R) signaling alleviates these symptoms. A mouse VSG model was utilized to investigate whether the impaired glucose counterregulatory system causes postprandial hypoglycemia. Results Like in humans, rats have increased glycemic variability and hypoglycemia after VSG. Postprandial hypoglycemia specifically detected after liquid vs. solid meals. Further, the blockade of GLP-1R signaling raises the glucose nadir but does not affect glycemic variability. Conclusions Rat bariatric surgery duplicates many features of human post bariatric surgery hypoglycemia including postprandial hypoglycemia and glycemic variability, while blockade of GLP-1 receptor signaling prevents the hypoglycemia but the variability.

Objective Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle are confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of the study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development. Methods A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKOism). Moreover, ERαshRNA was used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese-insulin resistant women. Results Twelve weeks of HFD exposure had no differential effects on body composition, VO2, VCO2, RER, energy expenditure, and activity counts across genotypes. Although ERαKOism exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in ERαKOism. Expression of pro-inflammatory genes were altered in skeletal muscle of ERαKOism, but concentrations of these inflammatory markers in systemic circulation were either lower or remained similar to WT. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2O2 emission potential was not affected in ERαKOism. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity. Conclusions Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.

Objective Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome wide association studies (GWAS) have identified >400 independent signals that encode genetic predisposition. More than 90% of the associated single nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, and so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity. Methods We present genome-wide single cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell-combinatorial-indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on the U-Net architecture to accurately predict open chromatin peak calls in rare cell populations. Results We show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, we find that T2D GWAS SNPs are significantly enriched in beta cell-specific and cross cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep-learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals. Conclusions Collectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways.

Background Cancer cell metabolism can be characterised by adaptive metabolic alterations which support abnormal proliferative cell growth with high energetic demand. De novo nucleotide biosynthesis is essential for providing nucleotides for RNA and DNA synthesis, drugs targeting this biosynthetic pathway have proven to be e↵ective anticancer therapeutics. Nevertheless, cancers are often able to circumvent chemotherapeutic interventions and become therapy resistant. Our understanding of the changing metabolic profile of the cancer cell, and the mode of action of therapeutics, is dependent on technological advances in biochemical analysis. Scope of Review This review starts with information about carbon and nitrogen donating pathways to build purine and pyrimidine moieties in the course of nucleotide biosynthesis. We discuss the application of stable isotope resolved metabolomics to investigate the dynamics of cancer cell metabolism, and outline the benefits of high resolution accurate mass spectrometry that enables multiple tracer studies. Conclusion With the technological advances in mass spectrometry that allows for the analysis of the metabolome in high resolution, the application of stable isotope resolved metabolomics became an important technique to investigate biological processes. The literature in the area of isotope labeling is dominated by 13C tracer studies. Metabolic pathways have to be considered as complex interconnected networks and should be investigated as such. Moving forward to simultaneous tracing of di↵erent stable isotopes will help elucidate the interplay between carbon and nitrogen flow and the dynamics of de novo nucleotide biosynthesis within the cell.

Objective Bone morphogenetic protein 4 (BMP4) adeno-associated viral vectors of serotype 8 (AAV8) gene therapy targeting the liver prevents the development of obesity in initially lean mice by browning the large subcutaneous white adipose tissue (WAT) and enhancing energy expenditure. Here, we examine whether this approach could also reduce established obesity. Methods Dietary-induced obese C57BL6/N mice received AAV8 BMP4 gene therapy at 17–18 weeks of age. They were kept on a high-fat diet and phenotypically characterized for an additional 10–12 weeks. Following termination, the mice underwent additional characterization in vitro. Results Surprisingly, we observed no effect on body weight, browning of WAT, or energy expenditure in these obese mice, but whole-body insulin sensitivity and glucose tolerance were robustly improved. Insulin signaling and insulin-stimulated glucose uptake were increased in both adipose cells and skeletal muscle. BMP4 also decreased hepatic glucose production and reduced gluconeogenic enzymes in the liver, but not in the kidney, in addition to enhancing insulin action in the liver. Conclusions Our findings show that BMP4 prevents, but does not reverse, established obesity in adult mice, while it improves insulin sensitivity independent of weight reduction. The BMP antagonist Noggin was increased in WAT in obesity, which may account for the lack of browning.

Objective Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents, and can extend lifespan in model organisms. An undesirable side effect of these drugs is hyperlipidemia. Despite multiple roles that have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo. Methods We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in adipocytes. Results Mice lacking mTORC1 activity in adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in adipose tissue restored normal levels of triglycerides and cholesterol in the fed state, as well as the ability to clear triglycerides in an oral fat tolerance test. Conclusions Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis.

Objectives Cerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key-mediators of reperfusion injury remain unclear and there is a great interest in therapeutically targeting metabolism and the cellular response to oxidative stress. Methods The objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatments to mitigate reperfusion injuries in in vitro and in vivo model systems. We performed metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of treated/untreated mice. Results We demonstrate that the “immunometabolite” itaconate slows tricarboxylic acid (TCA) cycle metabolism and buffers redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. Addition of itaconate to reperfusion fluids after mouse cerebral IR injury elevates glutathione levels and reduces reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids are increased post-reperfusion injury, while administration of itaconate normalizes these metabolites. In mouse cranial window models, itaconate significantly improves hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increases survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock. Conclusions We hypothesize that itaconate transiently inhibits SDH to gradually “awaken” mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.

Objective The glycoprotein hormone erythropoietin (EPO) is required for erythropoiesis, and the kidney is the primary site of adult EPO synthesis. Limited evidence has suggested that EPO could be detectable in the brain under certain conditions, but it remains unknown if the brain might have its own EPO system for biological functions that are independent of peripheral EPO production and action. Here, we performed this study to address this question using mice under normal conditions versus pathophysiological conditions including aging or dietary obesity. Methods EPO expression was measured in different brain regions as well as the cerebrospinal fluid. Hypothalamic ventricular EPO administration was performed for physiologically examining possible therapeutic effects in the conditions of aging or dietary obesity. Body weight, body composition, insulin tolerance, and glucose tolerance were measured to assess the central effects of EPO on metabolic physiology, and muscle strength and histology were analyzed to assess the central effects of EPO on muscle function. In addition, β2-adrenergic receptor knockout bone marrow transplant was employed to determine the potential role of the bone marrow in linking the brain to some of these peripheral functions. Results This study revealed that EPO is expressed in the ventromedial hypothalamus in addition to a few other brain regions and is present in the cerebrospinal fluid. Unlike blood EPO concentration, which increased with aging or dietary obesity, hypothalamic EPO decreased in these disease conditions. Therapeutically, aged mice were chronically treated with EPO in the hypothalamic ventricle, showing an increase in lean mass while body weight and fat mass decreased due to a moderate reduction of food intake. Muscle and metabolic functions were both improved by this central treatment, and mechanistically adrenergic signals to the bone marrow played a role in conveying hypothalamic EPO to these peripheral actions. Dietary obesity was also studied showing that hypothalamic EPO treatment caused a reduction in food intake and obesity leading to improved metabolic functions related to decreased fat as well as increased lean mass. Conclusions Hypothalamic EPO plays a role in the central regulation of muscle and metabolic physiology while its decline contributes to aging and obesity physiology in a manner which is independent of peripheral EPO.

Objective Glucose-dependent insulinotropic polypeptide is an intestinally derived hormone that is essential for normal metabolic regulation. Loss of the GIP receptor (GIPR) through genetic elimination or pharmacological antagonism reduces body weight and adiposity in the context of nutrient excess. Interrupting GIPR signaling also enhances the sensitivity of the receptor for the other incretin peptide, glucagon-like peptide 1 (GLP-1). The role of GLP-1 compensation for loss of GIPR signaling to protect against obesity has not been directly tested. Methods We blocked the GIPR and GLP-1R with specific antibodies, alone and in combination, in healthy and diet-induced obese (DIO) mice. The primary outcome measure of these interventions was the effect on body weight and composition. Results Antagonism of either the GIPR or GLP-1R system reduced food intake and weight gain during high fat feeding, and enhanced sensitivity to the alternative incretin signaling system. Combined antagonism of both GIPR and GLP-1R produced additive effects to mitigate DIO. Acute pharmacological studies using GIPR and GLP-1R agonists demonstrated both peptides reduced food intake, which was prevented by co-administration of the respective antagonists. Conclusions Disruption of either axis of the incretin system protects against diet-induced obesity in mice. However, combined antagonism of both GIPR and GLP-1R produced additional protection against diet-induced obesity, suggesting additional factors beyond compensation by the complementary incretin axis. While antagonizing the GLP-1 system decreases weight gain, GLP-1R agonists are used in clinical to target obesity. Hence, the phenotype arising from loss-of-function of GLP-1R does not implicate GLP-1 as an obesogenic hormone. By extension, caution is warranted in labelling GIP as an obesogenic hormone based on loss-of-function studies.

Background Modern lifestyles, especially high-caloric intake and physical inactivity, contribute to the increased prevalence of non-alcoholic fatty liver disease (NAFLD), which becomes a significant health problem worldwide. Lifestyle changes, however, affect not only parental generation, but also their offspring, reinforcing the need for efficient preventive approaches to deal with this disease. This transgenerational influence of phenotypes dependent on parents (particularly maternal) behaviours may open additional research avenues. Despite persistent attempts to design an effective pharmacological therapy against NAFLD, physical activity, as a non-pharmacological approach, emerges as an exciting strategy. Scope of review Here we briefly review the effect of physical exercise on liver mitochondria adaptations in NAFLD, highlighting the importance of mitochondrial metabolism and transgenerational and epigenetic mechanisms in liver diseases. Major Conclusions A deeper look into cellular mechanisms sheds a light on possible effects of physical activity in the prevention and treatment of NAFLD through modulation of function and structure of particular organelles, namely mitochondria. Additionally, despite of increasing evidence regarding the contribution of epigenetic mechanisms in the pathogenesis of different diseases, the role of microRNAs, DNA methylation, and histone modification in NAFLD pathogenesis still needs to be elucidated.

Background Cancer cells rewire their metabolism to meet the energetic and biosynthetic demands of their high proliferation rates and environment. Metabolic reprogramming of cancer cells may result in strong dependencies on nutrients that could be exploited for therapy. While these dependencies may be in part due to the nutrient environment of tumors, mutations or expression changes in metabolic genes also reprogram metabolic pathways and create addictions on extracellular nutrients. Scope of review This review summarizes the major nutrient dependencies of cancer cells focusing on their discovery and potential mechanisms by which metabolites become limiting for tumor growth. We further detail available therapeutic interventions based on these metabolic features and highlight opportunities for restricting nutrient availability as an anti-cancer strategy. Major conclusions Strategies to limit nutrients required for tumor growth by dietary interventions or the use of nutrient degrading enzymes have recently been suggested for cancer therapy. The best clinical example of exploiting cancer nutrient dependencies is the treatment of leukemia with L-asparaginase, a first line chemotherapeutic that depletes serum asparagine. Despite the success of nutrient starvation in blood cancers, it remains unclear whether this approach could be extended to other solid tumors. Systematic studies to identify nutrient dependencies unique to individual tumor types have the potential to discover targets for therapy.

Objective microRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains a challenge. In this study we aimed to identify miRNAs and their downstream targets involved in regeneration of islet beta cells following partial pancreatectomy in mice. Methods RNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in beta cell regeneration. Altered expression of selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were selected through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA and protein expression levels and signaling upon miR-132 knockdown and/or overexpression in mouse MIN6 and human EndoC-βH1 insulinoma cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132-/- and control mice. Results Partial pancreatectomy significantly increased the number of BrdU+/insulin+ islet cells. Microarray profiling revealed 14 miRNAs, including miR-132 and -141, to be significantly upregulated in LCM islets of partially pancreatectomized compared to LCM islets of control mice. In the same comparison miR-760 was the only miRNA found to be downregulated. Changed expression of these miRNAs in islets of partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we focused our attention on miR-132. Downregulation of miR-132 reduced the proliferation of MIN6 cells while enhancing the levels of pro-apoptotic cleaved caspase-9. Opposite observations were made in miR-132 overexpressing MIN6 cells. Microarray profiling, RT-PCR and immunoblotting of the latter cells revealed their downregulated expression of Pten, with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb as well as inactivation of pro-apoptotic Foxo3a via its phosphorylation. Downregulation of Pten was further confirmed in LCM islets of pancreatectomized mice in comparison to sham operated mice. Moreover, overexpression of miR-132 correlated with increased proliferation of EndoC-βH1 cells. Finally, regeneration of beta cells following partial pancreatectomy was reduced in miR-132/212-/- mice compared to control littermates. Conclusions/Interpretations: Our study provides compelling evidence about the critical role of miR-132 for regeneration of mouse islet beta cells through downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.

Objective Brown adipocyte tissue (BAT) mediated thermogenesis plays a key role in energy homeostasis and the maintenance of body temperature. Previous work suggests that brain derived neurotrophic factor (BDNF) is involved in BAT thermogenesis, but the underlying neural circuits and molecular mechanism remain largely unknown. This is in part due to the difficulties in manipulating BDNF expression in different brain regions through different promoters, and the lack of tools to identify neurons in the brain specifically involved in BAT thermogenesis. Methods We have created several lines of mutant mice in which BDNF transcription from a specific promoter was selectively disrupted by replacing Bdnf with green fluorescent protein (GFP) (Bdnf-e1, e4, and e6-/- mice). As such, cells expressing Bdnf-e1, e4 or e6 were labeled with GFP. To identify brown adipose tissue (BAT)-connected thermogenesis neurons in brain, we applied retrograde PRV labeling method from BAT. We also used chemogenetic tools to manipulate specific neurons coupled with BAT temperature recording. Moreover, we developed a new TrkB agonist antibody to rescue the BAT thermogenesis deficits. Results We show that selective disruption of Bdnf expression from promoter 1 (Bdnf-e1) resulted in severe obesity and deficits of BAT-mediated thermogenesis. Body-temperature response to cold was impaired in Bdnf-e1-/- mice. BAT expression of Ucp1 and Pcg1a, genes known to regulate thermogenesis, was also reduced, accompanying a decrease in sympathetic activity of BAT. Staining of cells expressing Bdnf-e1 transcript, combined with trans-synaptic, retrograde-tracing labeling of BAT-connected neurons, identified a group of excitatory neurons in lateral hypothalamus (LH) critical for thermogenesis regulation. Moreover, adaptive thermogenesis defect in Bdnf-e1-/- mice was rescued by injecting an agonistic antibody for TrkB, the BDNF receptor, into LH. Remarkably, activation of the excitatory neurons (VGLUT2+) in LH through chemogenetic tools resulted in a rise of BAT temperature. Conclusions These results reveal a specific role of BDNF promoter I in thermogenesis regulation, and define a small subset of neurons in LH that contribute to such regulation.

Objectives The incretin hormone glucagon-like peptide-1 (GLP-1) is secreted from intestinal L-cells upon nutrient intake. While recent evidence has shown that GLP-1 is released in a circadian manner in rats, whether this occurs in mice and if this pattern is regulated by the circadian clock remain to be elucidated. Furthermore, although circadian GLP-1 secretion parallels expression of the core clock gene Bmal1, the link between the two remains largely unknown. Secretagogin (Scgn) is an regulatory SNARE exocytotic protein that demonstrates circadian expression and is essential for insulin secretion from ß-cell. The objective of the current study was to establish the necessity of the core clock gene Bmal1 and of the SNARE protein SCGN as essential regulators of circadian GLP-1 secretion. Methods Oral glucose tolerance tests were performed at different times of the day on 4 hour fasted C57BL/6J, Bmal1 wild-type and Bmal1 knockout mice. Mass spectrometry, RNA-seq, qRT-PCR and/or microarray analyses, as well as immunostaining were conducted on murine and human primary L-cells and mGLUTag and human (h) NCI-H716 L-cell lines. At peak and trough GLP-1 secretory time points, mGLUTag cells were co-stained for SCGN and a membrane-marker, ChIP was used to analyze BMAL1 binding sites in the Scgn promoter, protein interaction with SCGN was tested by co-immunoprecipitation, and siRNA was used to knockdown Scgn for GLP-1 secretion assay. Results C57BL/6J mice displayed a circadian rhythm in GLP-1 secretion that peaked at the onset of their feeding period. Rhythmic GLP-1 release was impaired in Bmal1 knockout mice as compared to wild-type controls at the peak (p < 0.05) but not at the trough secretory time point. Microarray identified SNARE and transport vesicle pathways as highly upregulated in mGLUTag L-cells at the peak time point of GLP-1 secretion (p < 0.001). Mass spectrometry revealed that SCGN was also increased at this time (p < 0.001), while RNA-seq, qRT-PCR, and immunostaining demonstrated Scgn expression in all human and murine primary L-cells and cell lines. mGLUTag and hNCI-H716 L-cells exhibited circadian rhythms in Scgn expression (p < 0.001). ChIP analysis demonstrated increased binding of BMAL1, only at the peak of Scgn expression (p < 0.01). Immunocytochemistry showed translocation of SCGN to the cell membrane after stimulation, at the peak time point only (p < 0.05), while CoIP showed that SCGN was pulled down with SNAP25 and β-actin, but that only the latter interaction was time-dependent (p < 0.05). Finally, Scgn siRNA-treated cells demonstrated significantly blunted GLP-1 secretion (p < 0.01) in response to stimulation, at the peak time point only. Conclusions These data demonstrate, for the first time, that mice display a circadian pattern in GLP-1 secretion, which is impaired in Bmal1 knockout mice, and that Bmal1 regulation of Scgn expression plays an essential role in the circadian release of the incretin hormone, GLP-1.

Objectives The carbohydrate-insulin model (CIM) predicts that increases in fasting and postprandial insulin in response to dietary carbohydrate, stimulate energy intake and lower energy expenditure, leading to positive energy balance and weight gain. The objective of the present study was to directly test predictions from the CIM using C57BL/6 mice. Methods Diets were designed by altering dietary carbohydrate with either fixed protein or fat content, and were fed to C57BL/6 mice acutely or chronically for 12 weeks. Body weight, body composition, food intake and energy expenditure of the mice were measured. Fasting and postprandial glucose and insulin levels were also measured. RNAseq was performed on RNA from hypothalamus and subcutaneous white adipose tissue. Pathway analysis was performed using IPA. Results Only postprandial insulin and fasting glucose followed the CIM predictions. Lipolysis and leptin signalling pathways in sWAT were inhibited, in relation to elevated fasting insulin, supporting the predicted impact of high insulin by the CIM. However, because higher fasting insulin was unrelated to carbohydrate intake, the overall pattern did not support the model. Moreover, hypothalamic hunger pathways were inhibited in relation to increased fasting insulin, and energy intake was not increased. The browning pathway in sWAT was inhibited at higher insulin levels, but daily energy expenditure was not altered. Conclusions In conclusion, two of the predictions were partially supported (and hence also partially not supported) and the other three predictions were not supported. We conclude the CIM does not explain the impact of dietary macronutrients on adiposity in mice.

Objective Recently, we observed that specialized pro-resolving mediators (SPMs) entity resolvin D1 activates lipoxin A4/formyl peptide receptor 2 (ALX/FPR2) that facilitates cardiac healing and persistent inflammation is a hallmark of impaired cardiac repair in aging. Splenic leukocyte-directed SPMs are essential for the safe clearance of inflammation and cardiac repair after injury; however, the target of SPMs remains undefined in cardiac healing and repair. Methods To define the mechanistic basis of ALX/FPR2 as resolvin D1 target, FPR2 null mice were examined extensively. The systolic-diastolic heart function was assessed using echocardiography, leukocytes were phenotyped using flow cytometry, and SPMs were quantitated using mass spectrometry. The presence of cardiorenal syndrome was validated using histology and renal markers. Results Lack of ALX/FPR2 led to the development of spontaneous obesity and diastolic dysfunction with reduced survival with aging. After cardiac injury, FPR2-/- mice showed lower lipoxygenases (-5, -12, -15) expression, reducing SPMs in the infarcted left ventricle and spleen, indicating non-resolving inflammation. Reduced SPMs levels in the infarcted heart and spleen is suggestive of impaired cross-talk between the injured heart and splenic leukocytes, which are required for the resolution of inflammation. In contrast, cyclooxygenases (-1 and -2) were over-amplified in the infarcted heart. Together, these results suggest inter-organ signaling in which the spleen acts as both a SPMs biosynthesizer and supplier in acute heart failure. FPR2 dysfunction magnified obesogenic cardiomyopathy and renal inflammation (↑NGAL, ↑TNF-α, ↑CCL2, ↑IL-1β) with elevated plasma creatinine levels in aging mice. At the cellular level, ALX/FPR2-/- mice showed impairment in macrophage phagocytic function ex-vivo with expansion of neutrophils post-MI. Conclusions Lack of ALX/FPR2 induced obesity, reduced the lifespan, amplified leukocyte dysfunction, and facilitated profound inter-organ non-resolving inflammation. Our study shows the integrative and indispensable role of ALX/FPR2 in lipid metabolism, cardiac inflammation-resolution processes, obesogenic aging, and renal homeostasis.

Background Formate is a one-carbon molecule at the crossroad between cellular and whole body metabolism, between host and microbiome metabolism, and between nutrition and toxicology. This centrality confers formate with a key role in human physiology and disease that is currently unappreciated. Scope of review Here we review the scientific literature on formate metabolism, highlighting cellular pathways, whole body metabolism, and interactions with the diet and the gut microbiome. We will discuss the relevance of formate metabolism in the context of embryonic development, cancer, obesity, immunometabolism, and neurodegeneration. Major conclusions We will conclude with an outlook of some open questions bringing formate metabolism into the spotlight.

Background Tumors are highly plastic metabolic entities composed of cancer and host cells that can adopt different metabolic phenotypes. For energy production, cancer cells may use 4 main fuels that are shuttled in 5 different metabolic pathways. Glucose fuels glycolysis that can be coupled to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in oxidative cancer cells or to lactic fermentation in proliferating and in hypoxic cancer cells. Lipids fuel lipolysis, glutamine fuels glutaminolysis, and lactate fuels the oxidative pathway of lactate, all of which are coupled to the TCA cycle and OXPHOS for energy production. This review focuses on the latter metabolic pathway. Scope of review Lactate, which is prominently produced by glycolytic cells in tumors, was only recently recognized as a major fuel for oxidative cancer cells and as a signaling agent. Its exchanges across membranes are gated by monocarboxylate transporters MCT1-4. This review summarizes the current knowledge about MCT structure, regulation and functions in cancer, with a specific focus on lactate metabolism, lactate-induced angiogenesis and MCT-dependent cancer metastasis. It also describes lactate signaling via cell surface lactate receptor GPR81. Major conclusions Lactate and MCTs, especially MCT1 and MCT4, are important contributors to tumor aggressiveness. Analyses of MCT-deficient (MCT+/- and MCT−/-) animals and (MCT-mutated) humans indicate that they are druggable, with MCT1 inhibitors being in advanced development phase and MCT4 inhibitors still in the discovery phase. Imaging lactate fluxes non-invasively using a lactate tracer for positron emission tomography would further help to identify responders to the treatments.

Objectives The present study aims to verify the relationship between glucose consumption and uptake of [18F]-2-deoxy-glucose (FDG) in the skeletal muscle (SM) of experimental models of streptozotocin-induced diabetes mellitus (STZ-DM). Methods The study included 36 Balb/c mice. Two weeks after intraperitoneal administration of saline (n=18 “control”) or 150 mg streptozotocin (n=18 “STZ-DM”), the two cohorts were submitted to oral glucose tolerance test and were further subdivided into three groups (n=6 each) untreated and treated metformin (MTF) at low or high doses (10 or 750 mg/Kg daily, respectively). Two weeks thereafter, all mice were submitted to dynamic micro-PET imaging after prolonged fasting. After sacrifice, enzymatic pathways and response to oxidative stress were evaluated in harvested SM. Results At PET imaging, FDG uptake rate in hindlimb SM was significantly lower in non-diabetic with respect to STZ-DM untreated mice. MTF had no significant effect on SM FDG uptake in untreated mice, however its high dose induced a significant decrease in STZ-DM animals. At conventional analysis, SM standard uptake value (SUV) was higher in STZ-DM while MTF was virtually ineffective in either control or STZ-DM models. This metabolic reprogramming was not explained by any change in cytosolic glucose metabolism. By contrast, it closely agreed with the catalytic function of hexose-6P-dehydrogenase (H6PD), i.e. the trigger of a specific pentose phosphate pathway selectively located within the endoplasmic reticulum. In agreement with this role, H6PD enzymatic response to both STZ-DM and MTF matched the activation of the NADPH-dependent antioxidant responses to the increased ROS generation caused by chronic hyperglycemia. Ex vivo analysis of tracer kinetics confirmed that the enhanced SM avidity for FDG occurred despite a significant reduction in glucose consumption while it was associated with an increased radioactivity transfer to the endoplasmic reticulum. Conclusions These data challenge the current dogma linking FDG uptake to the glycolytic rate. They instead introduce a new model considering a strict link between the uptake of this glucose analog, H6PD reticular activity and oxidative damage in diabetes, at least under fasting condition.

Objective Fasting results in major metabolic changes including a switch from glycogenolysis to gluconeogenesis to maintain glucose homeostasis. However, the relationship between the length of fasting and the relative contribution of gluconeogenic substrates remains unclear. We investigated the relative contribution of glycogen, lactate, and glycerol in glucose production of male C57BL/6J-albino mice after 6, 12, and 18 hours of fasting. Methods We used non-perturbative infusions of 13C3 lactate, 13C3 glycerol, and 13C6 glucose combined with liquid chromatography mass spectrometry and metabolic flux analysis to study the contribution of substrates in gluconeogenesis (GNG). Results During infusion studies, both lactate and glycerol significantly label about 60% and 30-50% glucose carbon, respectively, but glucose labels much more lactate (∼90%) than glycerol carbon (∼10%). Our analyses indicate that lactate, but not glycerol is largely recycled during all fasting periods such that lactate is the largest direct contributor to GNG via the Cori cycle but a minor source of new glucose carbon (overall contribution). In contrast, glycerol is not only a significant direct contributor to GNG but also the largest overall contributor to GNG regardless of fasting length. Prolonged fasting decreases both the whole body turnover rate of glucose and lactate but increases that of glycerol, indicating that the usage of glycerol in GNG become more significant with longer fasting. Conclusion Collectively, these findings suggest that glycerol is the dominant overall contributor of net glucose carbon in GNG during both short and prolonged fasting.

Objective Roux-en-Y gastric surgery (RYGB) promotes a rapid and sustained weight loss and amelioration of glucose control in obese patients. A high number of molecular hypotheses were previously tested using duodenal-jejunal bypass (DJB) performed in various genetic models of mice with knockouts for various hormones or receptors. The data were globally negative or inconsistent. Therefore, the mechanisms remained elusive. Intestinal gluconeogenesis is a gut function that has been suggested to contribute to the metabolic benefits of RYGB in obese patients. Methods We studied the effects of DJB on body weight and glucose control in obese mice fed a high fat-high sucrose diet. Wild type mice and mice with a genetic suppression of intestinal gluconeogenesis were studied in parallel using glucose- and insulin-tolerance tests. Fecal losses, including excretion of lipids, were studied from the feces recovered in metabolic cages. Results DJB induced a dramatic decrease in body weight and improvement in glucose control (glucose- and insulin-tolerance) in obese wild type mice fed a high calorie diet, for 25 days after the surgery. The DJB-induced decrease in food intake was transient and resumed to normal in 7-8 days, suggesting that decreased food intake could not account for the benefits. Total fecal losses were about 5 times and lipid losses 7 times higher in DJB-mice than in control (sham-operated and pair-fed) mice, and could account for the weight loss of mice. The results were comparable in mice with suppression of intestinal gluconeogenesis. There was no effect of DJB on food intake, body weight or fecal loss in lean mice fed a normal chow diet. Conclusions DJB in obese mice fed a high calorie diet promotes dramatic fecal loss, which could account for the dramatic weight loss and metabolic benefits observed. This could dominate the effects of the mouse genotype/phenotype. Thus, fecal energy loss should be considered as an essential process contributing to the metabolic benefits of DJB in obese mice.

Objective Fibroblast growth factor 21 (FGF21) has been shown to rapidly lower body weight in the Siberian hamster, a preclinical model of adiposity. This induced negative energy balance mediated by FGF21 is associated with both lowered caloric intake and increased energy expenditure. Previous research demonstrated that adipose tissue (AT) is one of the primary sites of FGF21 action and may be responsible for its ability to increase the whole-body metabolic rate. The present study sought to determine the relative importance of white (subcutaneous WAT [sWAT], visceral WAT [vWAT]), and brown (interscapular brown AT [iBAT]) in governing FGF21-mediated metabolic improvements using the tissue-specific uptake of glucose and lipids as a proxy for metabolic activity. Methods We used positron emission tomography-computed tomography (PET-CT) imaging in combination with both glucose (18F-fluorodeoxyglucose) and lipid (18F-4-thiapalmitate) tracers to assess the effect of FGF21 on the tissue-specific uptake of these metabolites and compared responses to a control group pair-fed to match the food intake of the FGF21-treated group. In vivo imaging was combined with ex vivo tissue-specific functional, biochemical, and molecular analyses of the nutrient uptake and signaling pathways. Results Consistent with previous findings, FGF21 reduced body weight via reduced caloric intake and increased energy expenditure in the Siberian hamster. PET-CT studies demonstrated that FGF21 increased the uptake of glucose in BAT and WAT independently of reduced food intake and body weight as demonstrated by imaging of the pair-fed group. Furthermore, FGF21 increased glucose uptake in the primary adipocytes, confirming that these in vivo effects may be due to a direct action of FGF21 at the level of the adipocytes. Mechanistically, the effects of FGF21 are associated with activation of the ERK signaling pathway and upregulation of GLUT4 protein content in all fat depots. In response to treatment with FGF21, we observed an increase in the markers of lipolysis and lipogenesis in both the subcutaneous and visceral WAT depots. In contrast, FGF21 was only able to directly increase the uptake of lipid into BAT. Conclusions These data identify brown and white fat depots as primary peripheral sites of action of FGF21 in promoting glucose uptake and also indicate that FGF21 selectively stimulates lipid uptake in brown fat, which may fuel thermogenesis.

Objective Long-term glucocorticoids (GCs) therapy usually causes many metabolic side effects, including fatty liver. However, the molecular mechanisms remain poorly understood. Herein, we explored the molecular basis of GCs in the development of fatty liver. Methods C57BL/6 male mice were injected with Dexamethasone (DEX) while mouse primary hepatocytes (MPHs), HepG2 and Hep1-6 cells were cultured in the presence of DEX. Genes expression in liver tissues and hepatocytes were assessed by quantitative real-time PCR and western blotting, respectively. To explore whether Periostin is involved in the development of GCs-induced fatty liver, wild-type and Periostin knockout mice were treated with DEX or vehicle control. Luciferase reporter and chromatin immunoprecipitation assays were used to determine the regulatory roles of GCs on Periostin expression. Results We show that treatment of dexamethasone (DEX), a synthetic analog of GCs, led to the accumulation of triglycerides in the livers of mice, but not in cultured hepatocytes, suggesting that GCs may promote liver steatosis through integrative organ crosstalk mediated by systemic factors. We further found that DEX upregulated the expression levels of Periostin in white adipose tissues, which in turn promoted liver steatosis. Administration of a Periostin-neutralizing antibody or genetic ablation of Periostin largely attenuated DEX-induced hepatic steatosis in mice. Conclusions Our findings provided a novel insight that GCs could promote liver steatosis through integrative organ crosstalk mediated by white fat-secreted Periostin. These results establish Periostin as an endocrine factor with therapeutic potential for the treatment of GCs-associated fatty liver.

ObjectivePhosphatidylethanolamine methyltransferase (PEMT) generates phosphatidylcholine (PC), the most abundant phospholipid in the mitochondria and an important acyl chain donor for cardiolipin (CL) biosynthesis. Mice lacking PEMT (PEMTKO) are cold-intolerant when fed a high-fat diet (HFD) due to unclear mechanisms. The purpose of this study was to determine whether PEMT-derived phospholipids are important for the function of uncoupling protein 1 (UCP1) and thus for maintenance of core temperature. MethodsTo test whether PEMT-derived phospholipids are important for UCP1 function, we examined cold-tolerance and brown adipose (BAT) mitochondria from PEMTKO mice with or without HFD feeding. We complemented these studies with experiments on mice lacking functional CL due to tafazzin knockdown (TAZKD). We generated several conditional mouse models to study the tissue-specific roles of PEMT, including mice with BAT-specific knockout of PEMT (PEMT-BKO). ResultsChow- and HFD-fed PEMTKO mice completely lacked UCP1 protein in BAT, despite a lack of difference in mRNA levels, and the mice were accordingly cold-intolerant. While HFD-fed PEMTKO mice exhibited reduced mitochondrial CL content, this was not demonstrated in chow-fed PEMTKO mice or TAZKD mice, indicating that the lack of UCP1 was not attributable to CL deficiency. Surprisingly, the PEMT-BKO mice exhibited normal UCP1 protein levels. Knockout of PEMT in the adipose tissue (PEMT-AKO), liver (PEMT-LKO), or skeletal muscle (PEMT-MKO) also did not affect UCP1 protein levels, suggesting that lack of PEMT in other non-UCP1-expressing cells communicates to BAT to suppress UCP1. Instead, we identified an untranslated UCP1 splice variant that was triggered during the perinatal period in the PEMTKO mice. ConclusionsPEMT is required for UCP1 splicing that yields functional protein. This effect is derived by PEMT in nonadipocytes that communicate to BAT during embryonic development. Future research will focus on identifying the non-cell-autonomous PEMT-dependent mechanism of UCP1 splicing.

Objective Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energy dissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Here we evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in the pathophysiology of metabolic syndrome. Methods We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared their metabolic phenotypes. We also carried out ex vivo assays with primary and immortalized mouse brown adipocytes to establish the autonomy of the effect of loss of iRhom2 on thermogenesis and respiration. Results Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improved insulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beige adipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced thermogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak. Conclusion Our data suggest that iRhom2 is a negative regulator of thermogenesis and plays a role in the control of adipose tissue homeostasis during metabolic disease.

BACKGROUND/OBJECTIVES Although the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age- and BMI-matched) donors must exist. SUBJECTS/METHODS Using Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined. RESULTS Dissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity. CONCLUSIONS Collectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose.

OBJECTIVE The inability of leptin to suppress food intake in diet-induced obesity, sometimes referred to as leptin resistance, is associated with several distinct pathological hallmarks. One prevailing theory is that impaired transport of leptin across the blood-brain barrier (BBB) represents a molecular mechanism that triggers this phenomenon. Recent evidence, however, has challenged this notion, suggesting that leptin BBB transport is acquired during leptin resistance. METHODS To resolve this debate, we utilized a novel cerebral Open Flow Microperfusion (cOFM) method to examine leptin BBB transport in male C57BL/6J mice, fed a chow diet or high fat diet (HFD) for 20 days. RESULTS Basal plasma leptin levels were 3.8-fold higher in HFD-fed mice (p < 0.05). Leptin administration (2.5 mg/kg) elicited similar pharmacokinetic profiles of circulating leptin. However, while leptin reduced food intake by 20% over 22 h in chow-fed mice, it did not affect food intake in HFD-fed mice. In spite of this striking functional difference, hypothalamic leptin levels, as measured by cOFM, did not differ between chow-fed mice and HFD-fed mice following leptin administration. CONCLUSIONS These data suggest that leptin transport across the BBB is not impaired in non-obese leptin resistant mice and thus unlikely to play a direct role in the progression of pharmacological leptin resistance.

OBJECTIVE The fatty acid receptor 1 (FFAR1/GPR40) mediates fatty acid-dependent augmentation of glucose-induced insulin secretion (GIIS) in pancreatic β-cells. Genetically engineered Ffar1-knockout/congenic mice univocally displayed impaired fatty acid-mediated insulin secretion, but in vivo experiments delivered controversial results regarding the function of FFAR1 in glucose homeostasis and liver steatosis. This study presents a new coisogenic mouse model carrying a point mutation in Ffar1 with functional consequence. These mice reflect the situations in humans in which point mutations can lead to protein malfunction and disease development. METHODS The Munich N-ethyl-N-nitrosourea (ENU) mutagenesis-derived F1 archive containing over 16,800 sperms and corresponding DNA samples was screened for mutations in the coding region of Ffar1. Two missense mutations (R258W and T146S) in the extracellular domain of the protein were chosen and homozygote mice were generated. The functional consequence of these mutations was examined in vitro in isolated islets and in vivo in chow diet and high fat diet fed mice. RESULTS Palmitate, 50 μM, and the FFAR1 agonist TUG-469, 3 μM, stimulated insulin secretion in islets of Ffar1T146S/T146S mutant mice and of wild-type littermates, while in islets of Ffar1R258W/R258W mutant mice, these stimulatory effects were abolished. Insulin content and mRNA levels of Ffar1, Glp1r, Ins2, Slc2a2, Ppara, and Ppard were not significantly different between wild-type and Ffar1R258W/R258W mouse islets. Palmitate exposure, 600 μM, significantly increased Ppara mRNA levels in wild-type but not in Ffar1R258W/R258W mouse islets. On the contrary, Slc2a2 mRNA levels were significantly reduced in both wild-type and Ffar1R258W/R258W mouse islets after palmitate treatment. HFD feeding induced glucose intolerance in wild-type mice. Ffar1R258W/R258W mutant mice remained glucose tolerant although their body weight gain, liver steatosis, insulin resistance, and plasma insulin levels were not different from those of wild-type littermates. Worth mentioning, fasting plasma insulin levels were lower in Ffar1R258W/R258W mice. CONCLUSION A point mutation in Ffar1 abrogates the stimulatory effect of palmitate on GIIS, an effect that does not necessarily translate to HFD-induced glucose intolerance.

OBJECTIVE Glycogen metabolism has emerged as a mediator in the control of energy homeostasis and studies in murine models reveal that adipose tissue might contain glycogen stores. Here we investigated the physio(patho)logical role of glycogen in human adipose tissue in the context of obesity and insulin resistance. METHODS We studied glucose metabolic flux of hypoxic human adipoctyes by nuclear magnetic resonance and mass spectrometry-based metabolic approaches. Glycogen synthesis and glycogen content in response to hypoxia was analyzed in human adipocytes and macrophages. To explore the metabolic effects of enforced glycogen deposition in adipocytes and macrophages, we overexpressed PTG, the only glycogen-associated regulatory subunit (PP1-GTS) reported in murine adipocytes. Adipose tissue gene expression analysis was performed on wild type and homozygous PTG KO male mice. Finally, glycogen metabolism gene expression and glycogen accumulation was analyzed in adipose tissue, mature adipocytes and resident macrophages from lean and obese subjects with different degrees of insulin resistance in 2 independent cohorts. RESULTS We show that hypoxia modulates glucose metabolic flux in human adipocytes and macrophages and promotes glycogenesis. Enforced glycogen deposition by overexpression of PTG re-orients adipocyte secretion to a pro-inflammatory response linked to insulin resistance and monocyte/lymphocyte migration. Furthermore, glycogen accumulation is associated with inhibition of mTORC1 signaling and increased basal autophagy flux, correlating with greater leptin release in glycogen-loaded adipocytes. PTG-KO mice have reduced expression of key inflammatory genes in adipose tissue and PTG overexpression in M0 macrophages induces a pro-inflammatory and glycolytic M1 phenotype. Increased glycogen synthase expression correlates with glycogen deposition in subcutaneous adipose tissue of obese patients. Glycogen content in subcutaneous mature adipocytes is associated with BMI and leptin expression. CONCLUSION Our data establish glycogen mishandling in adipose tissue as a potential key feature of inflammatory-related metabolic stress in human obesity.

OBJECTIVE Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. METHODS AND RESULTS Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase. CONCLUSIONS These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy.

BACKGROUND Elucidation of the basic molecular mechanism of autophagy was a breakthrough in understanding various physiological events and pathogenesis of diverse diseases. In the fields of diabetes and metabolism, many cellular events associated with the development of disease or its treatment cannot be explained well without taking autophagy into account. While a grand picture of autophagy has been established, detailed aspects of autophagy, particularly that of selective autophagy responsible for homeostasis of specific organelles or metabolic intermediates, are still ambiguous and currently under intensive research. SCOPE OF REVIEW Here, results from previous and current studies on the role of autophagy and its dysregulation in the physiology of metabolism and pathogenesis of diabetes are summarized, with an emphasis on the pancreatic β-cell autophagy. In addition to nonselective (bulk) autophagy, machinery and significance of selective autophagy such as mitophagy of pancreatic β-cells is discussed. Novel findings regarding autophagy types other than macroautophagy are also covered, since several types of autophagy or lysosomal degradation pathways other than macroautophagy coexist in pancreatic β-cells. MAJOR CONCLUSION Autophagy plays a critical role in cellular metabolism, homeostasis of the intracellular environment and function of organelles such as mitochondria and endoplasmic reticulum. Impaired autophagic activity due to aging, obesity or genetic predisposition could be a factor in the development of β-cell dysfunction and diabetes associated with lipid overload or human-type diabetes characterized by islet amyloid deposition. Modulation of autophagy of pancreatic β-cells is likely to be possible in the near future, which would be valuable in the treatment of diabetes associated with lipid overload or accumulation of islet amyloid.

BACKGROUND Insulin is stored within large dense-core granules in pancreatic beta (β)-cells and is released by Ca2+-triggered exocytosis with increasing blood glucose levels. Polarized and targeted secretion of insulin from β-cells in pancreatic islets into the vasculature has been proposed; however, the mechanisms related to cellular and molecular localization remain largely unknown. Within nerve terminals, the Ca2+-dependent release of a polarized transmitter is limited to the active zone, a highly specialized area of the presynaptic membrane. Several active zone-specific proteins have been characterized; among them, the CAST/ELKS protein family members have the ability to form large protein complexes with other active zone proteins to control the structure and function of the active zone for tight regulation of neurotransmitter release. Notably, ELKS but not CAST is also expressed in β-cells, implying that ELKS may be involved in polarized insulin secretion from β-cells. SCOPE OF REVIEW This review provides an overview of the current findings regarding the role(s) of ELKS and other active zone proteins in β-cells and focuses on the molecular mechanism underlying ELKS regulation within polarized insulin secretion from islets. MAJOR CONCLUSIONS ELKS localizes at the vascular-facing plasma membrane of β-cells in mouse pancreatic islets. ELKS forms a potent insulin secretion complex with L-type voltage-dependent Ca2+ channels on the vascular-facing plasma membrane of β-cells, enabling polarized Ca2+ influx and first-phase insulin secretion from islets. This model provides novel insights into the functional polarity observed during insulin secretion from β-cells within islets at the molecular level. This active zone-like region formed by ELKS at the vascular side of the plasma membrane is essential for coordinating physiological insulin secretion and may be disrupted in diabetes.

BACKGROUND A growing body of literature suggests the cell-intrinsic activity of Atf6α during ER stress responses has implications for tissue cell number during growth and development, as well as in adult biology and tumorigenesis [1]. This concept is important, linking the cellular processes of secretory protein synthesis and endoplasmic reticulum stress response with functional tissue capacity and organ size. However, the field contains conflicting observations, especially notable in secretory cell types like the pancreatic beta cell. SCOPE OF REVIEW Here we summarize current knowledge of the basic biology of Atf6α, along with the pleiotropic roles Atf6α plays in cell life and death decisions and possible explanations for conflicting observations. We include studies investigating the roles of Atf6α in cell survival, death and proliferation using well-controlled methodology and specific validated outcome measures, with a focus on endocrine and metabolic tissues when information was available. MAJOR CONCLUSIONS The net outcome of Atf6α on cell survival and cell death depends on cell type and growth conditions, the presence and degree of ER stress, and the duration and intensity of Atf6α activation. It is unquestioned that Atf6α activity influences the cell fate decision between survival and death, although opposite directions of this outcome are reported in different contexts. Atf6α can also trigger cell cycle activity to expand tissue cell number through proliferation. Much work remains to be done to clarify the many gaps in understanding in this important emerging field.

BACKGROUND Human pancreatic β-cells are heterogeneous. This has been known for a long time and is based on various functional and morphological readouts. β-Cell heterogeneity could reflect fixed subpopulations with distinct functions. However, recent pseudotime analysis of large-scale RNA sequencing data suggest that human β-cell subpopulations may rather reflect dynamic interchangeable states characterized by low expression of genes involved in the unfolded protein response (UPR) and low insulin gene expression, low UPR and high insulin expression or high UPR and low insulin expression. SCOPE OF REVIEW This review discusses findings obtained by single-cell RNA sequencing combined with pseudotime analysis that human β-cell heterogeneity represents dynamic interchangeable functional states. The physiological significance and potential implications of β-cell heterogeneity in the development and progression of diabetes is highlighted. MAJOR CONCLUSIONS The existence of dynamic functional states allow β-cells to transition between periods of high insulin production and UPR-mediated stress recovery. The recovery state is important since proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum a stressful event. The transition of β-cells between dynamic states is likely controlled at multiple levels and influenced by the microenvironment within the pancreatic islets. Disturbances in the ability of the β-cells to transition between periods of high insulin biosynthesis and UPR-mediated stress recovery may contribute to diabetes development. Diabetes medications that restore the ability of the β-cells to transition between the functional states should be considered.

BACKGROUND Myriad challenges to the proper folding and structural maturation of secretory pathway client proteins in the endoplasmic reticulum (ER) - a condition referred to as "ER stress" - activate intracellular signaling pathways termed the unfolded protein response (UPR). SCOPE OF REVIEW Through executing transcriptional and translational programs the UPR restores homeostasis in those cells experiencing manageable levels of ER stress. But the UPR also actively triggers cell degeneration and apoptosis in those cells that are encountering ER stress levels that exceed irremediable thresholds. Thus, UPR outputs are "double-edged". In pancreatic islet β-cells, numerous genetic mutations affecting the balance between these opposing UPR functions cause diabetes mellitus in both rodents and humans, amply demonstrating the principle that the UPR is critical for the proper functioning and survival of the cell. MAJOR CONCLUSIONS Specifically, we have found that the UPR master regulator IRE1α kinase/endoribonuclease (RNase) triggers apoptosis, β-cell degeneration, and diabetes, when ER stress reaches critical levels. Based on these mechanistic findings, we find that novel small molecule compounds that inhibit IRE1α during such "terminal" UPR signaling can spare ER stressed β-cells from death, perhaps affording future opportunities to test new drug candidates for disease modification in patients suffering from diabetes.

BACKGROUND Current therapeutic strategies for type 1 (T1DM) and type 2 diabetes mellitus (T2DM) rely on increasing or substituting endogenous insulin secretion in combination with lifestyle changes. β-cell regeneration, a process whereby new β-cells arise from progenitors, self-renewal or transdifferentiation, has the potential to become a viable route to insulin self-sufficiency. Current regeneration strategies capture many of the transcriptomic and protein features of native β-cells, generating cells capable of glucose-dependent insulin secretion in vitro and alleviation of hyperglycemia in vivo. However, whether novel β-cells display appreciable heterogeneity remains poorly understood, with potential consequences for long-term functional robustness. SCOPE OF REVIEW The review brings together crucial discoveries in the β-cell regeneration field with state-of-the-art knowledge regarding β-cell heterogeneity. Aspects that might aid production of longer-lasting and more plastic regenerated β-cells are highlighted and discussed. MAJOR CONCLUSIONS Different β-cell regeneration approaches result in a similar outcome: glucose-sensitive, insulin-positive cells that mimic the native β-cell phenotype but which lack normal plasticity. The β-cell subpopulations identified to date expand our understanding of β-cell survival, proliferation and function, signposting the direction for future regeneration strategies. Therefore, regenerated β-cells should exhibit stimulus-dependent differences in gene and protein expression, as well as establish a functional network with different β-cells, all while coexisting with other cell types on a three-dimensional platform.

BACKGROUND Pancreatic β-cells adapt to high metabolic demand by expanding their β-cell mass and/or enhancing insulin secretion to maintain glucose homeostasis. Type 2 diabetes (T2D) is typically characterized by β-cell decompensation. SCOPE OF THE REVIEW The current review focuses on summarizing the "omics" and "epi-omics" approaches that particularly focus on addressing the β-cell adaptation to insulin resistance and T2D. MAJOR CONCLUSIONS The molecular mechanisms underlying successful versus compromised β-cell adaptation to insulin resistance are not entirely understood. The last decade has seen an exponential increase in the use of "omics" and "epi-omics" approaches to dissect pathophysiology of metabolic diseases. One recent example is the emergence of m6A mRNA methylation as a new layer of regulation of gene expression with the potential to impact diverse physiological processes in metabolic cells.

BACKGROUND Obesity and type 2 diabetes (T2D) are major public health issues worldwide, and put a significant burden on the healthcare system. Genetic variants, along with traditional risk factors such as diet and physical activity, could account for up to approximately a quarter of disease risk. Epigenetic factors have demonstrated potential in accounting for additional phenotypic variation, along with providing insights into the causal relationship linking genetic variants to phenotypes. SCOPE OF REVIEW In this review article, we discuss the epidemiological and functional insights into epigenetic disturbances in obesity and diabetes, along with future research directions and approaches, with a focus on DNA methylation. MAJOR CONCLUSIONS Epigenetic mechanisms have been shown to contribute to obesity and T2D disease development, as well as potential differences in disease risks between ethnic populations. Technology to investigate epigenetic profiles in diseased individuals and tissues has advanced significantly in the last years, and suggests potential in application of epigenetic factors in clinical monitoring and as therapeutic options.