The gastrointestinal tract is a major source of the acute metformin-stimulated rise in GDF15.

The hormone GDF15 is secreted in response to cellular stressors. Metformin elevates circulating levels of GDF15, an action important for the drug's beneficial effects on body weight. Metformin can also inhibit mammalian respiratory complex I, leading to decreases in ATP:AMP ratio, activation of AMP Kinase (AMPK), and increased GDF15 production. We undertook studies using a range of mice with tissue-specific loss of Gdf15 (namely gut, liver and global deletion) to determine the relative contributions of two classical metformin target tissues, the gut and liver, to the elevation of GDF15 seen with metformin. In addition, we performed comparative studies with another pharmacological agent, the AMP kinase pan-activator, MK-8722. Deletion of Gdf15 from the intestinal epithelium significantly reduced the circulating GDF15 response to oral metformin, whereas deletion of Gdf15 from the liver had no effect. In contrast, deletion of Gdf15 from the liver, but not the gut, markedly reduced circulating GDF15 responses to MK-8722. Further, our data show that, while GDF15 restricts high-fat diet-induced weight gain, the intestinal production of GDF15 is not necessary for this effect. These findings add to the body of evidence implicating the intestinal epithelium in key aspects of the pharmacology of metformin action.

Cachexia: A systemic consequence of progressive, unresolved disease.

Cachexia, a systemic wasting condition, is considered a late consequence of diseases, including cancer, organ failure, or infections, and contributes to significant morbidity and mortality. The induction process and mechanistic progression of cachexia are incompletely understood. Refocusing academic efforts away from advanced cachexia to the etiology of cachexia may enable discoveries of new therapeutic approaches. Here, we review drivers, mechanisms, organismal predispositions, evidence for multi-organ interaction, model systems, clinical research, trials, and care provision from early onset to late cachexia. Evidence is emerging that distinct inflammatory, metabolic, and neuro-modulatory drivers can initiate processes that ultimately converge on advanced cachexia.

Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia.

The dependency of cancer cells on glucose can be targeted with high-fat low-carbohydrate ketogenic diet (KD). However, hepatic ketogenesis is suppressed in IL-6 producing cancers, which prevents the utilization of this nutrient source as energy for the organism. In two IL-6 associated murine models of cancer cachexia we describe delayed tumor growth but accelerated onset of cancer cachexia and shortened survival when mice are fed KD. Mechanistically, we find this uncoupling is a consequence of the biochemical interaction of two simultaneously occurring NADPH-dependent pathways. Within the tumor, increased production of lipid peroxidation products (LPPs) and, consequently, saturation of the glutathione (GSH) system leads to ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impairs the biosynthesis of corticosterone, the main regulator of metabolic stress, in the adrenal glands. Administration of dexamethasone, a potent glucocorticoid, improves food intake, normalizes glucose homeostasis and utilization of nutritional substrates, delays onset of cancer cachexia and extends survival of tumor-bearing mice fed KD, while preserving reduced tumor growth. Our study highlights that the outcome of systemic interventions cannot necessarily be extrapolated from the effect on the tumor alone, but that they have to be investigated for anti-cancer and host effects. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer.

HypoMap-a unified single-cell gene expression atlas of the murine hypothalamus.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

Murine neuronatin deficiency is associated with a hypervariable food intake and bimodal obesity.

Neuronatin (Nnat) has previously been reported to be part of a network of imprinted genes downstream of the chromatin regulator Trim28. Disruption of Trim28 or of members of this network, including neuronatin, results in an unusual phenotype of a bimodal body weight. To better characterise this variability, we examined the key contributors to energy balance in Nnat+/-p mice that carry a paternal null allele and do not express Nnat. Consistent with our previous studies, Nnat deficient mice on chow diet displayed a bimodal body weight phenotype with more than 30% of Nnat+/-p mice developing obesity. In response to both a 45% high fat diet and exposure to thermoneutrality (30 °C) Nnat deficient mice maintained the hypervariable body weight phenotype. Within a calorimetry system, food intake in Nnat+/-p mice was hypervariable, with some mice consuming more than twice the intake seen in wild type littermates. A hyperphagic response was also seen in Nnat+/-p mice in a second, non-home cage environment. An expected correlation between body weight and energy expenditure was seen, but corrections for the effects of positive energy balance and body weight greatly diminished the effect of neuronatin deficiency on energy expenditure. Male and female Nnat+/-p mice displayed subtle distinctions in the degree of variance body weight phenotype and food intake and further sexual dimorphism was reflected in different patterns of hypothalamic gene expression in Nnat+/-p mice. Loss of the imprinted gene Nnat is associated with a highly variable food intake, with the impact of this phenotype varying between genetically identical individuals.

Chronic glucocorticoid treatment induces hepatic lipid accumulation and hyperinsulinaemia in part through actions on AgRP neurons.

Glucocorticoids (GCs) are widely prescribed anti-inflammatory medicines, but their use can lead to metabolic side-effects. These may occur through direct actions of GCs on peripheral organs, but could also be mediated by the hypothalamic AgRP neurons, which can increase food intake and modify peripheral metabolism. Therefore, the aim of this study was to examine the metabolic effects of chronic treatment with the GC corticosterone (Cort, 75 µg/ml in drinking water) in mice lacking the glucocorticoid receptor (GR) on AgRP neurons. Female AgRP-GR KO mice had delayed onset of Cort-induced hyperphagia. However, AgRP-GR KO had little impact on the increased body weight or adiposity seen with 3 weeks Cort treatment. Cort caused hepatic steatosis in control mice, but in Cort treated female AgRP-GR KO mice there was a 25% reduction in liver lipid content and lower plasma triglycerides. Additionally, Cort treatment led to hyperinsulinaemia, but compared to controls, Cort-treated AgRP-GR KO mice had both lower fasting insulin levels and lower insulin levels during a glucose tolerance test. In conclusion, these data indicate that GCs do act through AgRP neurons to contribute, at least in part, to the adverse metabolic consequences of chronic GC treatment.

Activation of the hypothalamic-pituitary-adrenal axis by exogenous and endogenous GDF15.

An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15-/- mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies.

A survey of the mouse hindbrain in the fed and fasted states using single-nucleus RNA sequencing.

OBJECTIVE: The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain. METHODS: Using single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of mice in the fed and fasted states. RESULTS: Of these, 8,910 were neurons that group into 30 clusters, with 4,289 from mice fed ad libitum and 4,621 from overnight fasted mice. A total of 7,124 nuclei were from non-neuronal cells, including oligodendrocytes, astrocytes, and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL, and CALCR, which bind GLP1, GIP, GDF15, and amylin, respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC-expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2, or both, implying that melanocortin peptides are likely produced by these neurons. CONCLUSION: We provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds and also prove useful in the continued search for other novel therapeutic targets.

Day-to-day variability of insulin requirements in the inpatient setting: Observations during fully closed-loop insulin delivery.

The aim of this study was to characterize the variability of exogenous insulin requirements during fully closed-loop insulin delivery in hospitalized patients with type 2 diabetes or new-onset hyperglycaemia, and to determine patient-related characteristics associated with higher variability of insulin requirements. We retrospectively analysed data from two fully closed-loop inpatient studies involving adults with type 2 diabetes or new-onset hyperglycaemia requiring insulin therapy. The coefficient of variation quantified day-to-day variability of exogenous insulin requirements during up to 15 days using fully automated closed-loop insulin delivery. Data from 535 days in 67 participants were analysed. The coefficient of variation of day-to-day exogenous insulin requirements was 30% ± 16%, and was higher between nights than between any daytime period (56% ± 29% overnight [11:00 pm to 4:59 am] compared with 41% ± 21% in the morning [5:00 am to 10:59 am], 39% ± 15% in the afternoon [11:00 am to 4:59 pm] and 45% ± 19% during the evening [5:00 pm to 10:59 pm]; all P < 0.01). There is high day-to-day variability of exogenous insulin requirements in inpatients, particularly overnight, and diabetes management approaches should account for this variability.

Inhibition of mitochondrial function by metformin increases glucose uptake, glycolysis and GDF-15 release from intestinal cells.

Even though metformin is widely used to treat type2 diabetes, reducing glycaemia and body weight, the mechanisms of action are still elusive. Recent studies have identified the gastrointestinal tract as an important site of action. Here we used intestinal organoids to explore the effects of metformin on intestinal cell physiology. Bulk RNA-sequencing analysis identified changes in hexose metabolism pathways, particularly glycolytic genes. Metformin increased expression of Slc2a1 (GLUT1), decreased expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1) whilst increasing GLUT-dependent glucose uptake and glycolytic rate as observed by live cell imaging of genetically encoded metabolite sensors and measurement of oxygen consumption and extracellular acidification rates. Metformin caused mitochondrial dysfunction and metformin's effects on 2D-cultures were phenocopied by treatment with rotenone and antimycin-A, including upregulation of GDF15 expression, previously linked to metformin dependent weight loss. Gene expression changes elicited by metformin were replicated in 3D apical-out organoids and distal small intestines of metformin treated mice. We conclude that metformin affects glucose uptake, glycolysis and GDF-15 secretion, likely downstream of the observed mitochondrial dysfunction. This may explain the effects of metformin on intestinal glucose utilisation and food balance.

Trappc9 deficiency causes parent-of-origin dependent microcephaly and obesity.

Some imprinted genes exhibit parental origin specific expression bias rather than being transcribed exclusively from one copy. The physiological relevance of this remains poorly understood. In an analysis of brain-specific allele-biased expression, we identified that Trappc9, a cellular trafficking factor, was expressed predominantly (~70%) from the maternally inherited allele. Loss-of-function mutations in human TRAPPC9 cause a rare neurodevelopmental syndrome characterized by microcephaly and obesity. By studying Trappc9 null mice we discovered that homozygous mutant mice showed a reduction in brain size, exploratory activity and social memory, as well as a marked increase in body weight. A role for Trappc9 in energy balance was further supported by increased ad libitum food intake in a child with TRAPPC9 deficiency. Strikingly, heterozygous mice lacking the maternal allele (70% reduced expression) had pathology similar to homozygous mutants, whereas mice lacking the paternal allele (30% reduction) were phenotypically normal. Taken together, we conclude that Trappc9 deficient mice recapitulate key pathological features of TRAPPC9 mutations in humans and identify a role for Trappc9 and its imprinting in controlling brain development and metabolism.

Publisher Correction: GDF15 mediates the effects of metformin on body weight and energy balance.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

GDF15 mediates the effects of metformin on body weight and energy balance.

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk1,2. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner3. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.

Global transcriptomic analysis of the arcuate nucleus following chronic glucocorticoid treatment.

OBJECTIVE: Glucocorticoids (GCs) are widely prescribed medications that are well recognized to cause adverse metabolic effects including hyperphagia, obesity, and hyperglycemia. These effects have been recapitulated in a murine model of GC excess, and we hypothesize that they are mediated, in part, through central mechanisms. This study aimed to identify genes in the hypothalamic arcuate nucleus (ARC) that are altered with GC treatment and evaluate their contribution to GC-induced metabolic abnormalities. METHODS: Corticosterone (Cort; 75 µg/ml) was administered in the drinking water to male C57Bl/6J mice for 2 days or 4 weeks. Phenotypic analysis of each group was undertaken and central and peripheral tissues were collected for biochemical and mRNA analyses. Arcuate nuclei were isolated by laser capture microdissection and tissue analyzed by RNA-seq. RESULTS: RNA-seq analysis of ARC tissue from 4 week Cort treated mice revealed 21 upregulated and 22 downregulated genes at a time when mice had increased food intake, expansion of adipose tissue mass, and insulin resistance. In comparison, after 2 days Cort treatment, when the main phenotypic change was increased food intake, RNA-seq identified 30 upregulated and 16 downregulated genes. Within the genes altered at 2 days were a range of novel genes but also those known to be regulated by GCs, including Fkbp5, Mt2, Fam107a, as well as some involved in the control of energy balance, such as Agrp, Sepp1, Dio2, and Nmb. Of the candidate genes identified by RNA-seq, type-II iodothyronine deiodinase (Dio2) was chosen for further investigation as it was increased (2-fold) with Cort, and has been implicated in the control of energy balance via the modulation of hypothalamic thyroid hormone availability. Targeted knockdown of Dio2 in the MBH using AAV-mediated CRISPR-Cas9 produced a mild attenuation in GC-induced brown adipose tissue weight gain, as well as a 56% reduction in the GC-induced increase in Agrp. However, this conferred no protection from GC-induced hyperphagia, obesity, or hyperglycemia. CONCLUSIONS: This study identified a comprehensive set of genes altered by GCs in the ARC and enabled the selection of key candidate genes. Targeted knockdown of hypothalamic Dio2 revealed that it did not mediate the chronic GC effects on hyperphagia and hyperglycemia.

Outcome of trans-metatarsal amputations in patients with diabetes mellitus.

BACKGROUND: This retrospective case series reports the reoperation rate, survival rate and mobility status in patients with diabetes mellitus who had undergone a trans-metatarsal amputation (TMA) managed within a diabetic foot care service. METHODS: Forty-one consecutive patients (37 men, 4 women) underwent a TMA with primary wound closure between January 2008 and December 2017. Eighty-eight per cent (36/41) of the patients were followed-up for a mean of 2.3 years. The outcomes were retrospectively reviewed. RESULTS: Four (11%) of the 36 patients required reoperation, including three (8%) major amputations. All of the patients requiring a reoperation had peripheral vascular disease. Eleven patients died giving a four-year survival rate of 69% (25/36). Of the surviving patients who had not required revision to a major amputation 96% (21/22) were fully mobile in bespoke orthoses. A third used a walking cane. CONCLUSION: This study shows that a TMA with primary wound closure in patients with diabetes mellitus, is effective for limb salvage with low reoperation and major amputation rates. A well healed TMA stump provides independent mobility in the majority of patients. The failures occurred in patients with peripheral vascular disease who, even after percutaneous trans-luminal angioplasty, had a 19% major amputation rate. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Fully closed-loop insulin delivery improves glucose control of inpatients with type 2 diabetes receiving hemodialysis.

Inpatient diabetes management of those on hemodialysis poses a major challenge. In a post hoc analysis of a randomized controlled clinical trial, we compared the efficacy of fully automated closed-loop insulin delivery vs. usual care in patients undergoing hemodialysis while in hospital. Compared to control patients receiving conventional subcutaneous insulin therapy, those patients receiving closed-loop insulin delivery significantly increased the proportion of time when a continuous glucose monitor was in the target range of 5.6-10.0 mmol/l by 37.6 percent without increasing the risk of hypoglycemia. Thus, closed-loop insulin delivery offers a novel way to achieve effective and safe glucose control in this vulnerable patient population.

Fully closed-loop insulin delivery in inpatients receiving nutritional support: a two-centre, open-label, randomised controlled trial.

BACKGROUND: Glucose management is challenging in patients who require nutritional support in hospital. We aimed to assess whether fully closed-loop insulin delivery would improve glycaemic control compared with conventional subcutaneous insulin therapy in inpatients receiving enteral or parenteral nutrition or both. METHODS: We did a two-centre (UK and Switzerland), open-label, randomised controlled trial in adult inpatients receiving enteral or parenteral nutrition (or both) who required subcutaneous insulin therapy. Patients recruited from non-critical care surgical and medical wards were randomly assigned (1:1) using a computer-generated minimisation schedule (stratified by type of nutritional support [parenteral nutrition on or off] and pre-study total daily insulin dose [<50 or ≥50 units]) to receive fully closed-loop insulin delivery with faster-acting insulin aspart (closed-loop group) or conventional subcutaneous insulin therapy (control group) given in accordance with local clinical practice. Continuous glucose monitoring in the control group was masked to patients, ward staff, and investigators. Patients were followed up for a maximum of 15 days or until hospital discharge. The primary endpoint was the proportion of time that sensor glucose concentration was in target range (5·6-10·0 mmol/L), assessed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01774565. FINDINGS: Between Feb 8, 2018, and Sept 21, 2018, 90 patients were assessed for eligibility, of whom 43 were enrolled and randomly assigned to the closed-loop group (n=21) or the control group (n=22). The proportion of time that sensor glucose was in the target range was 68·4% [SD 15·5] in the closed-loop group and 36·4% [26·6] in the control group (difference 32·0 percentage points [95% CI 18·5-45·5; p<0·0001]). One serious adverse event occurred in each group (one cardiac arrest in the control group and one episode of acute respiratory failure in the closed-loop group), both of which were unrelated to study interventions. There were no adverse events related to study interventions in either group. No episodes of severe hypoglycaemia or hyperglycaemia with ketonaemia occurred in either study group. INTERPRETATION: Closed-loop insulin delivery is an effective treatment option to improve glycaemic control in patients receiving nutritional support in hospital. FUNDING: Diabetes UK, Swiss National Science Foundation, National Institute for Health Research Cambridge Biomedical Research Centre, Wellcome Trust, and European Foundation for the Study of Diabetes.

Metabolic Abnormalities of Chronic High-Dose Glucocorticoids Are Not Mediated by Hypothalamic AgRP in Male Mice.

Glucocorticoids are potent and widely used medicines but often cause metabolic side effects. A murine model of corticosterone treatment resulted in increased hypothalamic expression of the melanocortin antagonist AgRP in parallel with obesity and hyperglycemia. We investigated how these adverse effects develop over time, with particular emphasis on hypothalamic involvement. Wild-type and Agrp-/- male mice were treated with corticosterone for 3 weeks. Phenotypic, biochemical, protein, and mRNA analyses were undertaken on central and peripheral tissues, including white and brown adipose tissue, liver, and muscle, to determine the metabolic consequences. Corticosterone treatment induced hyperphagia within 1 day in wild-type mice, which persisted for 3 weeks. Despite this early increase in food intake, the body weight only started to increase after 10 days. Hyperinsulinemia occurred at day 1. Also, although after 2 days, alterations were present in the genes often associated with insulin resistance in several peripheral tissues, hyperglycemia only developed at 3 weeks. Throughout, sustained elevation in hypothalamic Agrp expression was present. Mice with Agrp deleted [using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, Agrp-/-] were partially protected against corticosterone-induced hyperphagia. However, Agrp-/- mice still had corticosterone-induced increases in body weight and adiposity similar to those of the Agrp+/+ mice. Loss of Agrp did not diminish corticosterone-induced hyperinsulinemia or correct changes in hepatic gluconeogenic genes. Chronic glucocorticoid treatment in mice mimics many of the metabolic side effects seen in patients and leads to a robust increase in Agrp. However, AgRP does not appear to be responsible for most of the glucocorticoid-induced adverse metabolic effects.

GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression. Circulating GDF15 levels manifest very modest changes in response to moderate caloric surpluses or deficits in mice or humans, differentiating it from classical intestinally derived satiety hormones and leptin. However, GDF15 levels do increase following sustained high-fat feeding or dietary amino acid imbalance in mice. We demonstrate that GDF15 expression is regulated by the integrated stress response and is induced in selected tissues in mice in these settings. Finally, we show that pharmacological GDF15 administration to mice can trigger conditioned taste aversion, suggesting that GDF15 may induce an aversive response to nutritional stress.