Food perception promotes phosphorylation of MFFS131 and mitochondrial fragmentation in liver.

Liver mitochondria play a central role in metabolic adaptations to changing nutritional states, yet their dynamic regulation upon anticipated changes in nutrient availability has remained unaddressed. Here, we found that sensory food perception rapidly induced mitochondrial fragmentation in the liver through protein kinase B/AKT (AKT)-dependent phosphorylation of serine 131 of the mitochondrial fission factor (MFFS131). This response was mediated by activation of hypothalamic pro-opiomelanocortin (POMC)-expressing neurons. A nonphosphorylatable MFFS131G knock-in mutation abrogated AKT-induced mitochondrial fragmentation in vitro. In vivo, MFFS131G knock-in mice displayed altered liver mitochondrial dynamics and impaired insulin-stimulated suppression of hepatic glucose production. Thus, rapid activation of a hypothalamus-liver axis can adapt mitochondrial function to anticipated changes of nutritional state in control of hepatic glucose metabolism.

An Approach to Intersectionally Target Mature Enteroendocrine Cells in the Small Intestine of Mice.

Enteroendocrine cells (EECs) constitute only a small proportion of Villin-1 (Vil1)-expressing intestinal epithelial cells (IECs) of the gastrointestinal tract; yet, in sum, they build the largest endocrine organ of the body, with each of them storing and releasing a distinct set of peptides for the control of feeding behavior, glucose metabolism, and gastrointestinal motility. Like all IEC types, EECs are continuously renewed from intestinal stem cells in the crypt base and terminally differentiate into mature subtypes while moving up the crypt-villus axis. Interestingly, EECs adjust their hormonal secretion according to their migration state as EECs receive altering differentiation signals along the crypt-villus axis and thus undergo functional readaptation. Cell-specific targeting of mature EEC subtypes by specific promoters is challenging because the expression of EEC-derived peptides and their precursors is not limited to EECs but are also found in other organs, such as the brain (e.g., Cck and Sst) as well as in the pancreas (e.g., Sst and Gcg). Here, we describe an intersectional genetic approach that enables cell type-specific targeting of functionally distinct EEC subtypes by combining a newly generated Dre-recombinase expressing mouse line (Vil1-2A-DD-Dre) with multiple existing Cre-recombinase mice and mouse strains with rox and loxP sites flanked stop cassettes for transgene expression. We found that transgene expression in triple-transgenic mice is highly specific in I but not D and L cells in the terminal villi of the small intestine. The targeting of EECs only in terminal villi is due to the integration of a defective 2A separating peptide that, combined with low EEC intrinsic Vil1 expression, restricts our Vil1-2A-DD-Dre mouse line and the intersectional genetic approach described here only applicable for the investigation of mature EEC subpopulations.

NIK/MAP3K14 in hepatocytes orchestrates NASH to hepatocellular carcinoma progression via JAK2/STAT5 inhibition.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis (NASH), which often progresses to hepatocellular carcinoma (HCC) through a largely undefined mechanism. NASH and HCC depend on inflammatory signaling, whose master regulator is the NFκB transcription factor family, activated by canonical and non-canonical pathways. METHODS: Here, we investigated non-canonical NFκB-inducing kinase (NIK/MAP3K14) in metabolic NASH, NASH to HCC transition, and DEN-induced HCC. To this end, we performed dietary and chemical interventions in mice that were analyzed via single nucleus sequencing, gene expression and histochemical methods. Ultimately, we verified our mouse results in human patient samples. RESULTS: We revealed that hepatocyte-specific NIK deficiency (NIKLKO) ameliorated metabolic NASH complications and reduced hepatocarcinogenesis, independent of its role in the NFκB pathway. Instead, hepatic NIK attenuated hepatoprotective JAK2/STAT5 signaling that is a prerequisite for NASH and NASH to HCC progression in mice and humans. CONCLUSIONS: Our data suggest NIK-mediated inhibitory JAK2 phosphorylation at serine 633 that might be amenable for future therapeutic interventions in patients.

Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing.

Wound healing is a coordinated process that initially relies on pro-inflammatory macrophages, followed by a pro-resolution function of these cells. Changes in cellular metabolism likely dictate these distinct activities, but the nature of these changes has been unclear. Here, we profiled early- versus late-stage skin wound macrophages in mice at both the transcriptional and functional levels. We found that glycolytic metabolism in the early phase is not sufficient to ensure productive repair. Instead, by combining conditional disruption of the electron transport chain with deletion of mitochondrial aspartyl-tRNA synthetase, followed by single-cell sequencing analysis, we found that a subpopulation of early-stage wound macrophages are marked by mitochondrial ROS (mtROS) production and HIF1α stabilization, which ultimately drives a pro-angiogenic program essential for timely healing. In contrast, late-phase, pro-resolving wound macrophages are marked by IL-4Rα-mediated mitochondrial respiration and mitohormesis. Collectively, we identify changes in mitochondrial metabolism as a critical control mechanism for macrophage effector functions during wound healing.

Gut-brain communication by distinct sensory neurons differently controls feeding and glucose metabolism.

Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

GLP-1 and hunger modulate incentive motivation depending on insulin sensitivity in humans.

OBJECTIVE: To regulate food intake, our brain constantly integrates external cues, such as the incentive value of a potential food reward, with internal state signals, such as hunger feelings. Incentive motivation refers to the processes that translate an expected reward into the effort spent to obtain the reward; the magnitude and probability of a reward involved in prompting motivated behaviour are encoded by the dopaminergic (DA) midbrain and its mesoaccumbens DA projections. This type of reward circuity is particularly sensitive to the metabolic state signalled by peripheral mediators, such as insulin or glucagon-like peptide 1 (GLP-1). While in rodents the modulatory effect of metabolic state signals on motivated behaviour is well documented, evidence of state-dependent modulation and the role of incentive motivation underlying overeating in humans is lacking. METHODS: In a randomised, placebo-controlled, crossover design, 21 lean (body mass index [BMI] < 25 kg/m2) and 16 obese (BMI³ 30 kg/m2) volunteer participants received either liraglutide as a GLP-1 analogue or placebo on two separate testing days. Incentive motivation was measured using a behavioural task in which participants were required to exert physical effort using a handgrip to win different amounts of food and monetary rewards. Hunger levels were measured using visual analogue scales; insulin, glucose, and systemic insulin resistance as assessed by the homeostasis model assessment of insulin resistance (HOMA-IR) were quantified at baseline. RESULTS: In this report, we demonstrate that incentive motivation increases with hunger in lean humans (F(1,42) = 5.31, p = 0.026, ß = 0.19) independently of incentive type (food and non-food reward). This effect of hunger is not evident in obese humans (F(1,62) = 1.93, p = 0.17, ß = -0.12). Motivational drive related to hunger is affected by peripheral insulin sensitivity (two-way interaction, F(1, 35) = 6.23, p = 0.017, ß = -0.281). In humans with higher insulin sensitivity, hunger increases motivation, while poorer insulin sensitivity dampens the motivational effect of hunger. The GLP-1 analogue application blunts the interaction effect of hunger on motivation depending on insulin sensitivity (three-way interaction, F(1, 127) = 5.11, p = 0.026); no difference in motivated behaviour could be found between humans with normal or impaired insulin sensitivity under GLP-1 administration. CONCLUSION: We report a differential effect of hunger on motivation depending on insulin sensitivity. We further revealed the modulatory role of GLP-1 in adaptive, motivated behaviour in humans and its interaction with peripheral insulin sensitivity and hunger. Our results suggest that GLP-1 might restore dysregulated processes of midbrain DA function and hence motivational behaviour in insulin-resistant humans.

Hepatic FTO is dispensable for the regulation of metabolism but counteracts HCC development in vivo.

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 to reveal the role of hepatic FTO in metabolism and in the initiation and progression of HCC in vivo. METHODS: We generated mice with hepatic FTO deficiency (FTOL-KO). The effect of hepatic FTO on metabolism was investigated by extensive metabolic phenotyping. To determine the impact of hepatic FTO on HCC development, FTOL-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, FTOL-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 reduce 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.

MCH Neurons Regulate Permeability of the Median Eminence Barrier.

Melanin-concentrating hormone (MCH)-expressing neurons are key regulators of energy and glucose homeostasis. Here, we demonstrate that they provide dense projections to the median eminence (ME) in close proximity to tanycytes and fenestrated vessels. Chemogenetic activation of MCH neurons as well as optogenetic stimulation of their projections in the ME enhance permeability of the ME by increasing fenestrated vascular loops and enhance leptin action in the arcuate nucleus of the hypothalamus (ARC). Unbiased phosphoRiboTrap-based assessment of cell activation upon chemogenetic MCH neuron activation reveals MCH-neuron-dependent regulation of endothelial cells. MCH neurons express the vascular endothelial growth factor A (VEGFA), and blocking VEGF-R signaling attenuates the leptin-sensitizing effect of MCH neuron activation. Our experiments reveal that MCH neurons directly regulate permeability of the ME barrier, linking the activity of energy state and sleep regulatory neurons to the regulation of hormone accessibility to the ARC.

PNOCARC Neurons Promote Hyperphagia and Obesity upon High-Fat-Diet Feeding.

Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia.

Lipid signalling drives proteolytic rewiring of mitochondria by YME1L.

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis1-3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1-LIPIN1-YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics.

m6A RNA Methylation Maintains Hematopoietic Stem Cell Identity and Symmetric Commitment.

Stem cells balance cellular fates through asymmetric and symmetric divisions in order to self-renew or to generate downstream progenitors. Symmetric commitment divisions in stem cells are required for rapid regeneration during tissue damage and stress. The control of symmetric commitment remains poorly defined. Using single-cell RNA sequencing (scRNA-seq) in combination with transcriptomic profiling of HSPCs (hematopoietic stem and progenitor cells) from control and m6A methyltransferase Mettl3 conditional knockout mice, we found that m6A-deficient hematopoietic stem cells (HSCs) fail to symmetrically differentiate. Dividing HSCs are expanded and are blocked in an intermediate state that molecularly and functionally resembles multipotent progenitors. Mechanistically, RNA methylation controls Myc mRNA abundance in differentiating HSCs. We identified MYC as a marker for HSC asymmetric and symmetric commitment. Overall, our results indicate that RNA methylation controls symmetric commitment and cell identity of HSCs and may provide a general mechanism for how stem cells regulate differentiation fate choice.

The role of C16:0 ceramide in the development of obesity and type 2 diabetes: CerS6 inhibition as a novel therapeutic approach.

OBJECTIVE: Ectopic fat deposition is associated with increased tissue production of ceramides. Recent genetic mouse studies suggest that specific sphingolipid C16:0 ceramide produced by ceramide synthase 6 (CerS6) plays an important role in the development of insulin resistance. However, the therapeutic potential of CerS6 inhibition not been demonstrated. Therefore, we pharmacologically investigated the selective ablation of CerS6 using antisense oligonucleotides (ASO) in obese insulin resistance animal models. METHODS: We utilized ASO as therapeutic modality, CerS6 ASO molecules designed and synthesized were initially screened for in-vitro knock-down (KD) potency and cytotoxicity. ASOs with >85% inhibition of CerS6 mRNA were selected for further investigations. Most promising ASOs verified for in-vivo KD efficacy in healthy mice. CerS6 ASO (AAGATGAGCCGCACC) was found most active with hepatic reduction of CerS6 mRNA expression. Prior to longitudinal metabolic studies, we performed a dose titration target engagement analysis with CerS6 ASO in healthy mice to select the optimal dose. Next, we utilized leptin deficiency ob/ob and high fat diet (HFD) induced obese mouse models for pharmacological efficacy study. RESULTS: CerS6 expression were significantly elevated in the liver and brown adipose, this was correlated with significantly elevated C16:0 ceramide concentrations in plasma and liver. Treatment with CerS6 ASO selectively reduced CerS6 expression by ∼90% predominantly in the liver and this CerS6 KD resulted in a significant reduction of C16:0 ceramide by about 50% in both liver and plasma. CerS6 KD resulted in lower body weight gain and accompanied by a significant reduction in whole body fat and fed/fasted blood glucose levels (1% reduction in HbA1c). Moreover, ASO-mediated CerS6 KD significantly improved oral glucose tolerance (during oGTT) and mice displayed improved insulin sensitivity. Thus, CerS6 appear to play an important role in the development of obesity and insulin resistance. CONCLUSIONS: Our investigations identified specific and selective therapeutic valid ASO for CerS6 ablation in in-vivo. CerS6 should specifically be targeted for the reduction of C16:0 ceramides, that results in amelioration of insulin resistance, hyperglycemia and obesity. CerS6 mediated C16:0 ceramide reduction could be a potentially attractive target for the treatment of insulin resistance, obesity and type 2 diabetes.

Hepatic leptin receptor expression can partially compensate for IL-6Rα deficiency in DEN-induced hepatocellular carcinoma.

OBJECTIVE: The current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice. METHODS: We generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations. RESULTS: We demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL-KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes. CONCLUSIONS: Our findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.

p53 in AgRP neurons is required for protection against diet-induced obesity via JNK1.

p53 is a well-known tumor suppressor that has emerged as an important player in energy balance. However, its metabolic role in the hypothalamus remains unknown. Herein, we show that mice lacking p53 in agouti-related peptide (AgRP), but not proopiomelanocortin (POMC) or steroidogenic factor-1 (SF1) neurons, are more prone to develop diet-induced obesity and show reduced brown adipose tissue (BAT) thermogenic activity. AgRP-specific ablation of p53 resulted in increased hypothalamic c-Jun N-terminal kinase (JNK) activity before the mice developed obesity, and central inhibition of JNK reversed the obese phenotype of these mice. The overexpression of p53 in the ARC or specifically in AgRP neurons of obese mice decreased body weight and stimulated BAT thermogenesis, resulting in body weight loss. Finally, p53 in AgRP neurons regulates the ghrelin-induced food intake and body weight. Overall, our findings provide evidence that p53 in AgRP neurons is required for normal adaptations against diet-induced obesity.

Targeted deletion of the AAA-ATPase Ruvbl1 in mice disrupts ciliary integrity and causes renal disease and hydrocephalus.

Ciliopathies comprise a large number of hereditary human diseases and syndromes caused by mutations resulting in dysfunction of either primary or motile cilia. Both types of cilia share a similar architecture. While primary cilia are present on most cell types, expression of motile cilia is limited to specialized tissues utilizing ciliary motility. We characterized protein complexes of ciliopathy proteins and identified the conserved AAA-ATPase Ruvbl1 as a common novel component. Here, we demonstrate that Ruvbl1 is crucial for the development and maintenance of renal tubular epithelium in mice: both constitutive and inducible deletion in tubular epithelial cells result in renal failure with tubular dilatations and fewer ciliated cells. Moreover, inducible deletion of Ruvbl1 in cells carrying motile cilia results in hydrocephalus, suggesting functional relevance in both primary and motile cilia. Cilia of Ruvbl1-negative cells lack crucial proteins, consistent with the concept of Ruvbl1-dependent cytoplasmic pre-assembly of ciliary protein complexes.

Diet-Induced Growth Is Regulated via Acquired Leptin Resistance and Engages a Pomc-Somatostatin-Growth Hormone Circuit.

Anorexigenic pro-opiomelanocortin (Pomc)/alpha-melanocyte stimulating hormone (αMSH) neurons of the hypothalamic melanocortin system function as key regulators of energy homeostasis, also controlling somatic growth across different species. However, the mechanisms of melanocortin-dependent growth control still remain ill-defined. Here, we reveal a thus-far-unrecognized structural and functional connection between Pomc neurons and the somatotropic hypothalamo-pituitary axis. Excessive feeding of larval zebrafish causes leptin resistance and reduced levels of the hypothalamic satiety mediator pomca. In turn, this leads to reduced activation of hypophysiotropic somatostatin (Sst)-neurons that express the melanocortin receptor Mc4r, elevated growth hormone (GH) expression in the pituitary, and enhanced somatic growth. Mc4r expression and αMSH responsiveness are conserved in Sst-expressing hypothalamic neurons of mice. Thus, acquired leptin resistance and attenuation of pomca transcription in response to excessive caloric intake may represent an ancient mechanism to promote somatic growth when food resources are plentiful.

VEGF and GLUT1 are highly heritable, inversely correlated and affected by dietary fat intake: Consequences for cognitive function in humans.

OBJECTIVE: Reduction of brain glucose transporter GLUT1 results in severe neurological dysfunction. VEGF is required to restore and maintain brain glucose uptake across the blood brain barrier via GLUT1, which was shown to be acutely diminished in response to a high fat diet (HFD) in mice. The genetic and HFD-related regulation and association of VEGF and GLUT1 (SLC2A1) in humans was investigated in the NUtriGenomic Analysis in Twins (NUGAT) study. METHODS: 92 healthy and non-obese twins were standardized to a high-carbohydrate low-fat diet for 6 weeks before switched to a 6-week HFD under isocaloric conditions. Three clinical investigation days were conducted: after 6 weeks of low-fat diet and after 1 and 6 weeks of HFD. Serum VEGF and other cytokine levels were measured using ELISA. Gene expression in subcutaneous adipose tissue was assessed by quantitative Real-Time PCR. Genotyping was performed using microarray. The Auditory Verbal Learning Task was conducted to measure cognitive performance. RESULTS: In this human study, we showed that the environmental regulation of SLC2A1 expression and serum VEGF by HFD was inversely correlated and both factors showed strong heritability (>90%). In response to the HFD containing 45% fat, serum VEGF levels increased (P = 0.002) while SLC2A1 mRNA expression in adipose tissue decreased (P = 0.001). Higher BMI was additionally associated with lower SLC2A1 expression. AA-genotypes of the rs9472159 polymorphism, which explained ∼39% of the variation in circulating VEGF concentrations, showed significantly reduced serum VEGF levels (P = 6.4 × 10-11) but higher SLC2A1 expression (P = 0.009) in adipose tissue compared to CC/CA-genotypes after 6 weeks of HFD. Memory performance in AA-genotypes declined in response to the HFD compared to CC- and CA-genotypes. CONCLUSIONS: The results provide evidence to suggest the translatability of the dietary regulation of VEGF and GLUT1 from mouse models to humans. Our data demonstrate that HFD induces a genetically determined and correlated decrease of GLUT1 and increase of VEGF which may affect memory performance. CLINICAL TRIAL REGISTRATION NUMBER: NCT01631123.

N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response.

The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2α. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2α signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). While depleting m6A demethylases represses ATF4 reinitiation, knocking down m6A methyltransferases promotes ATF4 translation. We demonstrate that m6A in the 5' UTR controls ribosome scanning and subsequent start codon selection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by dynamic mRNA methylation. Consistently, Fto transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of m6A in translational regulation of ISR at cellular and organismal levels.

A microRNA screen reveals that elevated hepatic ectodysplasin A expression contributes to obesity-induced insulin resistance in skeletal muscle.

Over 40% of microRNAs (miRNAs) are located in introns of protein-coding genes, and many of these intronic miRNAs are co-regulated with their host genes. In such cases of co-regulation, the products of host genes and their intronic miRNAs can cooperate to coordinately regulate biologically important pathways. Therefore, we screened intronic miRNAs dysregulated in the livers of mouse models of obesity to identify previously uncharacterized protein-coding host genes that may contribute to the pathogenesis of obesity-associated insulin resistance and type 2 diabetes mellitus. Our approach revealed that expression of both the gene encoding ectodysplasin A (Eda), the causal gene in X-linked hypohidrotic ectodermal dysplasia (XLHED), and its intronic miRNA, miR-676, was increased in the livers of obese mice. Moreover, hepatic EDA expression is increased in obese human subjects and reduced upon weight loss, and its hepatic expression correlates with systemic insulin resistance. We also found that reducing miR-676 expression in db/db mice increases the expression of proteins involved in fatty acid oxidation and reduces the expression of inflammatory signaling components in the liver. Further, we found that Eda expression in mouse liver is controlled via PPARγ and RXR-α, increases in circulation under conditions of obesity, and promotes JNK activation and inhibitory serine phosphorylation of IRS1 in skeletal muscle. In accordance with these findings, gain- and loss-of-function approaches reveal that liver-derived EDA regulates systemic glucose metabolism, suggesting that EDA is a hepatokine that can contribute to impaired skeletal muscle insulin sensitivity in obesity.