Background

Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming a global health problem. Cardiovascular diseases (CVD) are the most common cause of mortality in NAFLD patients. NAFLD and CVD share several common risk factors including obesity, insulin resistance and type 2 diabetes (T2D). Atherogenic dyslipidemia, characterized by plasma hypertriglyceridemia, increased small dense low-density lipoprotein (LDL) particles and decreased high-density lipoprotein-cholesterol (HDL-C) levels, is often observed in NAFLD patients.

Scope of review

In the present review, we highlight recent epidemiological studies evaluating the link between NAFLD and CVD risk. We further focus on recent mechanistic insights into the links between NAFLD and altered lipoprotein metabolism. Finally, we discuss current therapeutic strategies for NAFLD and their potential impact on NAFLD-associated CVD risk.

Major conclusions

Alterations in hepatic lipid and lipoprotein metabolism are major contributing factors for the increased CVD risk in NAFLD patients and many promising NASH therapies in development also improve dyslipidemia in clinical trials.

Objective

Tumor cells experience hypoxia, acidosis, and hypoglycemia. Metabolic adaptation to a glucose shortage is essential to maintain tumor cell survival because of their high glucose requirement. This study aimed to study the hypothesis that acidosis might promote tumor survival during a glucose shortage and if so, to explore a novel drug targeting metabolic vulnerability to glucose shortage.

Methods

Cell survival and bioenergetics metabolism were assessed in lung cancer cell lines. Our in-house small-molecule compounds were screened to identify those that kill cancer cells under low-glucose conditions. Cytotoxicity against non-cancerous cells was also assessed. Tumor growth was evaluated in vivo using a mouse engraft model.

Results

Acidosis limited the cellular consumption of glucose and ATP, causing tumor cells to enter a metabolically dormant but energetically economic state, which promoted tumor cell survival during glucose deficiency. We identified ESI-09, a previously known exchange protein directly activated by cAMP (EAPC) inhibitor, as an anticancer compound that inhibited cancer cells under low-glucose conditions, even when associated with acidosis. Bioenergetic studies showed that independent of EPAC inhibition, ESI-09 was a safer mitochondrial uncoupler than a classical uncoupler and created a futile cycling of mitochondrial respiration leading to decreased ATP production, increased ATP dissipation, and fuel scavenging. Accordingly, ESI-09 exhibited more cytotoxic effects under low-glucose conditions than under normal glucose conditions. ESI-09 was also more effective than actively proliferating cells on quiescent glucose-restricted cells. Cisplatin showed opposite effects. ESI-09 inhibited tumor growth in lung-cancer engraft mice.

Conclusions

This study highlights the acidosis-induced promotion of tumor survival during glucose shortage and demonstrates that ESI-09 is a novel potent anticancer mitochondrial uncoupler that targets a metabolic vulnerability to glucose shortage even when associated with acidosis. The higher cytotoxicity under lower than normal glucose conditions suggests that ESI-09 is safer than conventional chemotherapy, can target the metabolic vulnerability of tumor cells to low-glucose stress, and is applicable to many cancer cell types.

Objective

Transformation of white into brown fat (“browning”) reduces obesity in many preclinical models and holds great promise as therapeutic concept in metabolic disease. Vitamin A metabolites (retinoids) have been linked to thermogenic programming of adipose tissue, however the physiologic importance of systemic retinoid transport for adipose tissue browning and adaptive thermogenesis is unknown.

Methods

We performed cold exposure studies in mice and humans and used a genetic model of defective vitamin A transport, the retinol binding protein deficient (Rbp^-/-) mouse, to study the effects of cooling on systemic vitamin A and the relevance of intact retinoid transport on cold-induced adipose tissue browning.

Results

We show that cold stimulation in mice and humans leads to an increase in circulating retinol and its plasma transporter Rbp. In Rbp^-/- mice thermogenic programming of adipocytes and oxidative mitochondrial function are dramatically impaired in subcutaneous white fat, which renders Rbp^-/- mice more cold sensitive. In contrast, retinol stimulation in primary human adipocytes promotes thermogenic gene expression and mitochondrial respiration. In humans, cold-mediated retinol increase is associated with a shift in oxidative substrate metabolism suggestive of higher lipid utilization.

Conclusions

Systemic vitamin A levels are regulated by cold exposure in mice and humans and intact retinoid transport is essential for cold-induced adipose tissue browning and adaptive thermogenesis.

Objective

The human adaptive fasting response enables survival during periods of caloric deprivation. A crucial component of the fasting response is the shift from glucose metabolism to utilization of lipids, underscoring the importance of adipose tissue as the central lipid-storing organ. The objective of this study was to investigate the response of adipose tissue to a prolonged fast in humans.

Methods

We performed RNA sequencing of subcutaneous adipose tissue samples longitudinally collected during a 10-day, 0-calorie fast in humans. We further investigated observed transcriptional signatures utilizing cultured human monocytes and Thp1 cells. We examined the cellularity of adipose tissue biopsies with transmission electron microscopy and tested for associated changes in relevant inflammatory mediators in the systemic circulation by ELISA assays of longitudinally collected blood samples.

Results

Coincident with the expected shift away from glucose utilization and lipid storage, we demonstrated downregulation of pathways related to glycolysis, oxidative phosphorylation, and lipogenesis. The canonical lipolysis pathway was also downregulated, whereas fasting drove alternative lysosomal paths to lipid digestion. Unexpectedly, the dominant induced pathways were associated with immunity and inflammation, although this only became evident at the 10-day time point. Among the most augmented transcripts were those associated with macrophage identity and function, such as members of the erythroblast transformation-specific (ETS) transcription factor family. Key components of the macrophage transcriptional signal in fasting adipose tissue were recapitulated with induced expression of two of the ETS transcription factors via cultured macrophages, SPIC and SPI1. The inflammatory signal was further reflected by an increase in systemic inflammatory mediators.

Conclusions

Collectively, this study demonstrates an unexpected role of metabolic inflammation in the human adaptive fasting response.

Objective

Obesity due to overnutrition causes adipose tissue dysfunction, serving as a critical pathological step on the road to Type 2 diabetes (T2D) and other metabolic disorders. Here, we performed an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity.

Methods

We used NuTRAP (Nuclear tagging and Translating Ribosome Affinity Purification) reporter mice crossed with Adipoq-Cre to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), and PPARγ cistrome (ChIP-seq) profiles in mice fed chow or high fat diet (HFD) for 10 weeks. We also assessed the impact of the PPARγ agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from HFD-fed mice. We integrated these data to determine what are the transcription factors underlying adipocyte responses to HFD and performed functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes.

Results

Adipocytes from HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes while showing enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARγ binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGFβ1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and to be associated with myofibroblast signature genes. TGFβ1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFβ1.

Conclusions

Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPARγ activity and elevated TGFβ-SMAD signaling. This cellular identity crisis may serve as a fundamental mechanism that drives functional decline of adipose tissues during obesity.

Objective

The endocannabinoid (eCB) system is increasingly recognized as being of crucial importance in obesity-related hepatic steatosis. eCBs, via activation of the hepatic cannabinoid-1 receptor (CB₁R), modulate lipogenesis and fatty acid oxidation. However, the underlying molecular mechanisms are largely unknown.

Methods

We combined unbiased bioinformatics techniques, mouse genetic manipulations, multiple pharmacological, molecular and cellular biology approaches, and genomic sequencing to systematically decipher the role of hepatic CB₁R in modulating fat utilization in the liver, and explored the downstream molecular mechanisms.

Results

Using an unbiased normalized phylogenetic profiling analysis, we found that CB₁R evolutionarily coevolves with peroxisome proliferator-activated receptor-alpha (PPARα), a key regulator of hepatic lipid metabolism. In diet-induced obese (DIO) mice, peripheral CB₁R blockade (using AM6545) induced the reversal of hepatic steatosis and improved liver injury in WT, but not in PPARα^-/- mice. The antisteatotic effect, mediated by AM6545 in WT DIO mice, was accompanied by increased hepatic expression and activity of PPARα as well as elevated hepatic levels of the PPARα-activating eCB-like molecules, oleoylethanolamine and palmitoylethanolamide. Moreover, AM6545 was unable to rescue hepatic steatosis in DIO mice lacking liver sirtuin 1 (SIRT1), an upstream regulator of PPARα. Both of these signaling molecules were modulated by CB₁R, as measured in hepatocytes exposed to lipotoxic conditions or treated with CB₁R agonists in the absence/presence of AM6545. Furthermore, using microRNA transcriptomic profiling, we found that CB₁R regulated the hepatic expression, acetylation, and transcriptional activity of p53, resulting in the enhanced expression of miR-22, which was found to specifically target SIRT1 and PPARα.

Conclusions

We provide a strong evidence for a functional role of the p53/miR-22/SIRT1/PPARα signaling pathway in potentially mediating the antisteatotic effect of peripherally restricted CB₁R blockade.

Objective

Diabetic nephropathy (DN) is one of the most common complications of diabetes and a critical risk factor for developing end-stage renal disease. Activation of purinergic receptors including P2Y2R has been associated to the pathogenesis of renal diseases such as polycystic kidney and glomerulonephritis. However, the role of P2Y2R and its precise mechanisms in DN remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in DN by modulating autophagy signaling pathway.

Methods

We used a mouse model of DN by combining a treatment of high fat diet and streptozotocin after unilateral nephrectomy in wild type or P2Y2R knockout mice. We measured renal functional parameter in plasma, examined renal histology, and analysed expression of autophagy regulatory proteins.

Results

Hyperglycemia and ATP release were induced in wild type DN mice and positively correlated with renal dysfunction. Conversely, P2Y2R knockout markedly attenuates albuminuria, podocyte loss, development of glomerulopathy, renal tubular injury, apoptosis and interstitial fibrosis induced by DN. These protective effects were associated with inhibition of AKT-mediated FOXO3a phosphorylation and induction of FOXO3a-induced autophagy gene transcription. Furthermore, inhibitory phosphorylation of ULK-1 was decreased, and the downstream Beclin-1 autophagy signaling was activated in P2Y2R deficiency. An increased SIRT-1 and FOXO3a expression in P2Y2R deficiency also enhanced autophagy response, thereby ameliorating renal dysfunction in DN.

Conclusions

P2Y2R contributes to the pathogenesis of DN by impairing autophagy and serves as a therapeutic target for treating DN.

Background

Glucagon-like peptide-1 receptor (GLP-1R) agonists are approved for the treatment of type 2 diabetes and obesity and elicit robust improvements in glycemic control and weight loss, coupled with cardioprotection in individuals at risk for or with pre-existing cardiovascular disease. These attributes make GLP-1 a preferred partner for next generation therapies exhibiting improved efficacy yet retained safety for the treatment of diabetes, obesity, non-alcoholic steatohepatitis and related cardiometabolic disorders. The available clinical data demonstrates the best GLP-1R agonists are not yet competitive with bariatric surgery, emphasizing the need to further improve the efficacy of current medical therapy.

Scope of the Review

Here we discuss data highlighting the physiological and pharmacological attributes of potential peptide and non-peptide partners, exemplified by amylin, glucose-dependent insulinotropic polypeptide (GIP), and steroid hormones. We review progress, limitations and future considerations for translating findings from preclinical experiments to competitive efficacy and safety in humans with type 2 diabetes and obesity.

Conclusions

Multiple co-agonist combinations exhibit promising efficacy in the clinic, notably tirzepatide, and investigational amylin combinations. Simultaneously, increasing doses of GLP-1R agonists such as semaglutide produces substantial weight loss, raising the bar for development of new unimolecular co-agonists. Collectively, the available data suggests that new co-agonists with robust efficacy should prove superior to GLP-1R agonists alone for the treatment of metabolic disorders.

Objective

Although the hypothalamus is crucial for peripheral metabolism control, the signals in specific neurons involved remain poorly understood. The aim of our current study was to explore the role of the hypothalamic gene mothers against decapentaplegic homolog 7 (Smad7) in peripheral glucose disorders.

Methods

We studied glucose metabolism in mice under chow diet, high fat diet (HFD) or middle-aged conditions with Cre-mediated recombination causing 1) over-expression of Smad7 in hypothalamic POMC neurons, 2) deletion of Smad7 in POMC neurons, and 3) over-expression of AKT in arcuate nucleus (ARC) in Smad7 over-expressed mice. Intracerebroventricular (ICV) cannulation of insulin was used to test the hypothalamic insulin sensitivity in the above mice. Hypothalamic primary neurons were used to investigate the mechanism of Smad7 regulating hypothalamic insulin signaling.

Results

We found that Smad7 expression was increased in POMC neurons in the hypothalamic ARC of HFD-fed or middle-aged mice. Furthermore, over-expression of Smad7 in POMC neurons disrupted the glucose balance, and deletion of Smad7 in POMC neurons prevented diet- or age-induced glucose disorders which was likely to be independent of changes in body weight or food intake. Moreover, the effect of Smad7 was reversed by over-expression of AKT in the ARC. Finally, Smad7 reduced AKT phosphorylation by activating protein phosphatase 1c in hypothalamic primary neurons.

Conclusions

Our results demonstrated that excess of central Smad7 in POMC neurons disrupts glucose balance by attenuating hypothalamic insulin signaling. In addition, we found that this regulation was mediated by the activity of protein phosphatase 1c.

Objective

Single nucleotide polymorphisms in the FTO gene encoding an m⁶Am and an m⁶A demethylase are associated with obesity. Moreover, recent studies have linked a dysregulation of m⁶A modifications and its machinery, including FTO, to the development of several forms of cancers. However, the functional role of hepatic FTO in metabolism and the development and progression of hepatocellular carcinoma (HCC), a proteotypic obesity-associated cancer, remains unclear. Thus, we aimed at uncovering the role of hepatic FTO in metabolism and in the initiation and progression of HCC in vivo.

Methods

We have generated mice with hepatic FTO deficiency (FTO^L-KO). The effect of hepatic FTO on metabolism was investigated by extensive metabolic phenotyping. To determine the impact of hepatic FTO on HCC development, FTO^L-KO and Ctrl mice were subjected to long-term Diethylnitrosamine (DEN)-induced HCC-development and the tumor initiation phase was examined via a short-term DEN protocol.

Results

In long-term DEN experiments, FTO^L-KO mice exhibit increased HCC burden compared to Ctrl mice. In the tumor initiation phase, Ctrl mice display a dynamic regulation of FTO upon induction of liver damage, while this response is abrogated in FTO-deficient mice. Proteomic analyses revealed that liver damage-induced increases in FTO expression reduces CUL4A protein abundance. Functionally, simultaneous knockdown of Cul4a reverses the increased hepatocyte proliferation observed upon loss of FTO.

Conclusion

Collectively, our study demonstrates that hepatic FTO is dispensable for the control of energy homeostasis and glucose metabolism. However, we show a protective function of FTO in liver carcinogenesis and suggest the FTO-dependent dynamic mRNA demethylation of Cul4a in the initiation of HCC development contributes to this effect.

Introduction

Individuals born with intrauterine growth retardation (IUGR) are more prone to cardio-metabolic diseases as adults. The prevalence of IUGR (5 to 7% of births in developed countries) may herald a worsening of the current worldwide epidemic of obesity and related comorbidities.

Methods

We used a preclinical mouse model of induced IUGR, in which we modulated the nutrition of the pups during the suckling period, to modify their susceptibility to cardio-metabolic diseases in adulthood. Mice born with IUGR that were overfed (IUGR-O) during lactation rapidly developed obesity, hepatic steatosis and insulin resistance, by three months of age, whereas those subjected to nutrition restriction during lactation (IUGR-R) remained permanently thin and highly sensitive to insulin. Mice born with IUGR and fed normally during lactation (IUGR-N) presented an intermediate phenotype and developed insulin resistance by 12 months of age. Molecular alterations to the insulin signaling pathway with an early onset were observed in the livers of adult IUGR-N mice, nine months before the appearance of insulin resistance. The implication of epigenetic changes was revealed by ChIP sequencing, with both posttranslational H3K4me3 histone modifications and microRNAs involved.

Conclusion

These two changes lead to the coherent regulation of insulin signaling, with a decrease in Akt gene transcription associated with an increase in the translation of its inhibitor, Pten. Moreover, we found that the levels of the implicated miRNA19a-3p also decreased in the blood of young adult IUGR mice nine months before the appearance of insulin resistance, suggesting a possible role for this miRNA as an early circulating biomarker of metabolic fate of potential use for precision medicine.

Introduction

Obesity results in lymphatic dysfunction, but the cellular mechanisms that mediate this effect remain largely unknown. Previous studies in obese mice have shown that inducible nitric oxide synthase-expressing (iNOS⁺) inflammatory cells accumulate around lymphatic vessels. In the current study, we therefore tested the hypothesis that increased expression of iNOS results in nitrosative stress and injury to the lymphatic endothelial cells (LECs). In addition, we tested the hypothesis that lymphatic injury, independent of obesity, can modulate glucose and lipid metabolism.

Methods

We compared the metabolic changes and lymphatic function of wild-type and iNOS knockout mice fed a normal chow or high-fat diet for 16 weeks. To corroborate our in vivo findings, we analyzed the effects of reactive nitrogen species on isolated LECs. Finally, using a genetically engineered mouse model that allows partial ablation of the lymphatic system, we studied the effects of acute lymphatic injury on glucose and lipid metabolism in lean mice.

Results

The mesenteric lymphatic vessels of obese wild-type animals were dilated, leaky, and surrounded by iNOS⁺ inflammatory cells with resulting increased accumulation of reactive nitrogen species when compared with lean wild-type or obese iNOS knockout animals. These changes in obese wild-type mice were associated with systemic glucose and lipid abnormalities, as well as decreased mesenteric LEC expression of lymphatic-specific genes, including vascular endothelial growth factor receptor 3 (VEGFR-3) and antioxidant genes as compared with lean wild-type or obese iNOS knockout animals. In vitro experiments demonstrated that isolated LECs were more sensitive to reactive nitrogen species than blood endothelial cells, and that this sensitivity was ameliorated by antioxidant therapies. Finally, by using mice in which the lymphatics were specifically ablated using diphtheria toxin, we found that the interaction between metabolic abnormalities caused by obesity and lymphatic dysfunction is bidirectional. Targeted partial ablation of mesenteric lymphatic channels of lean mice resulted in increased accumulation of iNOS⁺inflammatory cells and increased reactive nitrogen species. Lymphatic ablation also caused marked abnormalities in insulin sensitivity, serum glucose and insulin concentrations, expression of insulin-sensitive genes, lipid metabolism, and significantly increased systemic and mesenteric white adipose tissue (M-WAT) inflammatory responses.

Conclusions

Our studies suggest that increased iNOS production in obese animals plays a key role in regulating lymphatic injury by increasing nitrosative stress. In addition, our studies suggest that obesity-induced lymphatic injury may amplify metabolic abnormalities by increasing systemic and local inflammatory responses and regulating insulin sensitivity. These findings suggest that manipulation of the lymphatic system may represent a novel means of treating metabolic abnormalities associated with obesity.

Objective

Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s) while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms behind the cycle and the possible impact of hepatic steatosis are unclear.

Methods

We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD).

Results

Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis).

Conclusions

Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.

Objective

Canonical Wnt/β-catenin signaling is a well-studied endogenous regulator of mesenchymal cell fate determination, promoting osteoblastogenesis and inhibiting adipogenesis. However, emerging genetic evidence in humans links a number of Wnt pathway members to body fat distribution, obesity, and metabolic dysfunction, suggesting that this pathway also functions in adipocytes. Recent studies in mice have uncovered compelling evidence that the Wnt signaling pathway plays important roles in adipocyte metabolism, particularly under obesogenic conditions. However, complexities in Wnt signaling and differences in experimental models and approaches have thus far limited our understanding of its specific roles in this context.

Methods

To investigate the roles of the canonical Wnt pathway in the regulation of adipocyte metabolism, we generated adipocyte-specific β-catenin (β-cat) knockout mouse and culture cell models. We used RNA sequencing, ChIP sequencing, and molecular approaches to assess expression of Wnt targets and lipogenic genes. We then used functional assays to evaluate effects of β-catenin deficiency on adipocyte metabolism, including lipid and carbohydrate handling. In mice maintained on normal chow and high-fat diets, we assessed the cellular and functional consequences of adipocyte-specific β-catenin deletion on adipose tissues and systemic metabolism.

Results

We report that in adipocytes, the canonical Wnt/β-catenin pathway regulates de novolipogenesis (DNL) and fatty acid monounsaturation. Further, β-catenin mediates effects of Wnt signaling on lipid metabolism in part by transcriptional regulation of Mlxipl and Srebf1. Intriguingly, adipocyte-specific loss of β-catenin is sensed and defended by CD45^-/CD31^- stromal cells to maintain tissue-wide Wnt signaling homeostasis in chow-fed mice. With a long-term high-fat diet, this compensatory mechanism is overridden, revealing that β-catenin deletion promotes resistance to diet-induced obesity and adipocyte hypertrophy and subsequent protection from metabolic dysfunction.

Conclusions

Taken together, studies demonstrate that Wnt signaling in adipocytes is required for lipogenic gene expression, de novo lipogenesis, and lipid desaturation. In addition, adipose tissues rigorously defend Wnt signaling homeostasis under standard nutritional conditions, such that stromal-vascular cells sense and compensate for adipocyte-specific loss. These findings underscore the critical importance of this pathway in adipocyte lipid metabolism and adipose tissue function.

Objective

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor that plays major roles in the central control of energy balance. Loss-of-function mutations of MC4R constitute the most common monogenic cause of early-onset extreme obesity in humans, whereas gain-of-function mutations appear to be protective. In particular, two relatively frequent alleles carrying the non-synonymous coding mutations V103I or I251L are associated with lower risks of obesity and type-2 diabetes. Although V103I and I251L MC4Rs showed more efficient signalling in transfected cells, their specific effects in live animals remain unexplored. Here, we investigated whether the introduction of V103I and I251L mutations into the mouse MC4R leads to a lean phenotype and provides protection against an obesogenic diet.

Methods

Using CRISPR/Cas9, we generated two novel strains of mice carrying single-nucleotide mutations into the mouse Mc4r which are identical to those present in V103I and I251L MCR4 human alleles, and studied their phenotypic outcomes in mice fed with normal chow or a high-fat diet. In particular, we measured body weight progression, food intake and adiposity. In addition, we analysed glucose homeostasis through glucose and insulin tolerance tests.

Results

We found that homozygous V103I females displayed shorter longitudinal length and decreased abdominal white fat, whereas homozygous I251L females were also shorter and leaner due to decreased weight in all white fat pads examined. Homozygous Mc4r^V103I/V103I and Mc4r^I251L/I251L mice of both sexes showed improved glucose homeostasis when challenged in a glucose tolerance test, whereas Mc4r^I251L/I251L females showed improved responses to insulin. Despite being leaner and metabolically more efficient, V103I and I251L mutants fed with a hypercaloric diet increased their fasting glucose levels and adiposity similar to their wild-type littermates.

Conclusions

Our results demonstrate that mice carrying V103I and I251L MC4R mutations displayed gain-of-function phenotypes that were more evident in females. However, hypermorphic MC4R mutants were as susceptible as their control littermates to the obesogenic and diabetogenic effects elicited by a long-term hypercaloric diet, highlighting the importance of healthy feeding habits even under favourable genetic conditions.

Objective

Perinatal exposure to maternal obesity results in predisposition of offspring to develop obesity later in life. Increased weight gain in offspring exposed to maternal obesity is usually associated with hyperphagia, implicating altered central regulation of food intake as a cause. We aimed to define how maternal obesity impacts early development of the hypothalamus to program lasting dysfunction in feeding regulatory pathways.

Methods

Mice offspring of diet-induced obese mothers were compared to the offspring of lean control mothers. We analysed gene expression in the fetal hypothalamus, alongside neurosphere assays to investigate the effects of maternal obesity on neural progenitor cell proliferation in vitro. Western blotting was used to investigate the insulin signalling pathway in the fetal hypothalamus. Characterisation of cell type and neuropeptide profile in adulthood was linked with analyses of feeding behaviour.

Results

There was a reduction in the expression of proliferative genes in the fetal hypothalamus of offspring exposed to maternal obesity. This reduction in proliferation was maintained in vitro when hypothalamic neural progenitor cells were grown as neurospheres. Hypothalamic fetal gene expression and neurosphere growth correlated with maternal body weight and insulin levels. Foetuses of obese mothers showed hypothalamic insulin resistance, which may be causative of reduced proliferation. Furthermore, maternal obesity activated the Notch signalling pathway in neonatal offspring hypothalamus, resulting in decreased neurogenesis. Adult offspring of obese mothers displayed an altered ratio of anorexigenic and orexigenic signals in the arcuate nucleus, associated with an inability to maintain energy homeostasis when metabolically challenged.

Conclusions

These findings show that maternal obesity alters the molecular signature in the developing hypothalamus, which is associated with disrupted growth and development of hypothalamic precursor cells and defective feeding regulation in adulthood. This is the first report of fetal hypothalamic insulin resistance in an obese pregnancy and suggests a mechanism by which maternal obesity causes permanent changes to hypothalamic structure and function.

Objectives

There is increasing evidence that intestinal microbiota play a role in diverse metabolic processes via intestinal butyrate production. Based on bariatric surgery data in humans it has been suggested that the gut-brain axis is also involved in this process, however the underlying mechanisms are still unknown.

Design

We therefore compared the effect of fecal microbiota transfer (FMT) from post-Roux-en-Y gastric bypass (RYGB) donors versus oral butyrate supplementation on (¹²³I-FP-CIT determined) brain dopamine transporter (DAT) and serotonin transporter (SERT) binding as well as stable isotope determined insulin sensitivity at baseline and after 4 weeks in 24 male and female treatment-naïve metabolic syndrome subjects. Also, plasma metabolites and fecal microbiota were determined at these timepoints.

Results

We observed an increase in brain DAT upon donor FMT compared to oral butyrate that reduced this binding. However, no effect on body weight and insulin sensitivity was seen upon post-RYGB donor feces transfer in either humans with metabolic syndrome. In line, increases in fecal levels of Bacteroides uniformis were significantly associated with an increase in DAT whereas increases in Prevotella spp. showed an inverse association. Furthermore, changes in the plasma metabolites glycine, betaine, methionine and lysine (associated with the S-adenosylmethionine cycle) were also associated with altered striatal DAT expression.

Conclusion

Although more and larger studies are needed, our data suggest a potential gut microbiota-driven modulation of brain dopamine and serotonin transporters in human obese metabolic syndrome subjects. Moreover, these data suggest the presence of a gut-brain axis in humans,that can be modulated. NTR registration: 4488

Objectives

In this study, we used NTS nutrient sensing as an entry point to characterize stimulus-defined neuronal ensembles engaged by the NTS to produce physiological satiety.

Methods

We combined histological analysis, neuroanatomical assessment using inducible viral tracing tools, and functional tests to characterize hindbrain-forebrain circuits engaged by NTS leucine sensing to suppress hunger.

Results

We found that NTS detection of leucine engages NTS prolactin-releasing peptide (PrRP) neurons to inhibit AgRP neurons via a population of leptin receptor-expressing neurons in the dorsomedial hypothalamus. This circuit is necessary for the anorectic response to NTS leucine, the appetite-suppressive effect of high-protein diets, and the long-term control of energy balance.

Conclusions

These results extend the integrative capability of AgRP neurons to include brainstem nutrient sensing inputs.

Objective

Sodium-glucose cotransporter 2 (SGLT2) inhibitors (SGLT2i), or gliflozins, are anti-diabetic drugs that lower glycemia by promoting glucosuria. They stimulate endogenous glucose and ketone body production. The likely causes of these metabolic responses are increased blood glucagon levels and decreased blood insulin levels, but the mechanisms involved are hotly debated. This study verified whether or not SGLT2i affect glucagon and insulin secretion by direct action on islet cells in three species using multiple approaches.

Methods and results

SGLT2 inhibition in mice increased the plasma glucagon/insulin ratio in the fasted state, an effect correlated with a decline in glycemia. Gene expression analyses and immunodetections showed no SGLT2 mRNA or protein expression in rodent and human islet cells, but moderate SGLT1 mRNA expression in human α-cells. However, functional experiments on rat, mouse, and human (29 donors) islets and in situ perfused mouse pancreas did not reveal any direct effect of selective SGLT2i (dapagliflozin and empagliflozin) and SGLT1/2i (sotagliflozin) on glucagon and insulin secretion. SGLT2i did not affect glucagon gene expression in rat and human islets.

Conclusions

The data indicate that the SGLT2i-induced increase in the plasma glucagon/insulin ratio in vivo does not result from a direct action of the gliflozins on islet cells.

Background

The prevalence of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NAFLD/NASH) is increasing. NAFLD/NASH may progress to cirrhosis and hepatocellular carcinoma. However, most patients with NAFLD/NASH will die from a vascular cause. There are no currently approved pharmacological treatments for NASH/NAFLD. A large number of clinical trials have been, or are being, undertaken; however, the challenge is the assessment of the clinical endpoint.

Scope of review

The main objective of this narrative review was to evaluate the efficacy of drugs used in clinical trials for the treatment of NAFLD/NASH that included a liver biopsy as the gold standard. A literature search was carried out using 3 databases (PubMed, Scopus, and Google Scholar) to identify the clinical trials that included liver biopsy assessment before and after treatment. Interventional clinical trials (n=33) involving 18 different agents, alone and in combination, were identified.

Major conclusions

Pioglitazone is the only agent that has shown consistent benefit and efficacy in a number of clinical trials. Pentoxifylline, rosiglitazone and ursodeoxycholic acid had both positive and negative results from clinical trials. There is also evidence for vitamin E and metformin. Other drugs including bicyclol, cysteamine bitartrate, L-carnitine, liraglutide, obeticholic acid, oligofructose, selonsertib, silymarin, and statins each had a single clinical study. There is an urgent need for further research in this field.