Molecular Metabolism

The GLP-1-based obesity drug semaglutide lowers bodyweight primarily by increasing satiation and satiety, whilst also reducing food reward and commonly causing nausea. The brainstem dorsal vagal complex (DVC) has been identified as a key site of action for these phenotypic components of semaglutides anorectic effect. However, which GLP-1 receptor (GLP-1R) populations within the DVC are recruited to mediate these phenotypic components, and whether they are dissociable, are translationally important but unresolved questions. We addressed these using metabolic and behavioural phenotyping, combined with activity-dependent genetic labelling ( Sema-TRAP) and chemogenetic manipulation of semaglutide-recruited brainstem circuits. Semaglutide potentiated satiation and satiety, caused behavioural proxies of nausea, and suppressed motivation for Western diet, in a largely sex-independent manner. It activated a substantial proportion of GLP-1R-expressing neurons in the brainstem area postrema (AP), but surprisingly most semaglutide-activated neurons in the nucleus tractus solitarius (NTS) did not express GLP-1R. Chemogenetic reactivation of Sema-TRAP neurons in the NTS alone was sufficient to recapitulate the acute effects of semaglutide on satiation, nausea, food reward, and bodyweight. Knockdown of GLP-1R expression in the AP before Sema-TRAPing abolished the recruitment of Sema-TRAPNTS neurons which elicited all these effects, while leaving the effects of semaglutide on satiety and bodyweight intact. These data demonstrate that semaglutide recruits dissociable anorectic circuits to suppress eating via distinct behavioural mechanisms, with non-GLP-1R NTS neurons downstream of GLP-1RAP representing potential therapeutic targets to tune GLP-1-based obesity drugs towards a better-tolerated effect profile.

Angiotensinogen (AGT) deletion in hepatocytes reduces Western diet-induced adiposity and hepatic steatosis in mice maintained under conventional room-temperature (RT) housing. Given the high metabolic activity of mice, this temperature imposes adaptive metabolic responses in this species. Whether this metabolic protection persists independent of increased thermogenic demand remains unclear. In this study, we first determined whether thermoneutral housing (TN, 30 {degrees}C) alters Western diet-induced metabolic phenotypes compared with RT housing (20 {degrees}C) in wild-type mice. Although body weight did not differ significantly between housing conditions, Western diet-fed mice housed at TN exhibited brown adipose tissue whitening and more pronounced hepatic steatosis than mice housed at RT, confirming that thermoneutrality exacerbated diet-induced metabolic dysfunction. We then housed hepatocyte Agt deficient (hepAGT-/-) mice and wild-type (hepAGT+/+) littermates at TN and fed them Western diet for 12 weeks. Despite enhanced metabolic dysfunction under TN, hepatocyte AGT deletion resulted in reductions in diet-induced body weight gain, fat mass, liver weight, and hepatic triglyceride accumulation. Bulk RNA sequencing of liver revealed hepatocyte AGT deficiency-dependent alterations in lipid-metabolic pathways. Cross-temperature analysis of RT and TN housing identified 35 shared differentially expressed genes, including 27 concordantly downregulated genes enriched in lipid metabolism and transport. Extended Western diet feeding for 24 weeks confirmed sustained reductions in body weight gain, liver weight, and hepatic lipid accumulation in hepAGT-/- mice. These findings demonstrate that hepatocyte AGT deletion provides sustained protection against Western diet-induced metabolic dysfunction under thermoneutral housing, a condition that more closely recapitulates human basal metabolism. NEW & NOTEWORTHYThis study investigated hepatocyte angiotensinogen (AGT) biology during Western diet feeding in mice under thermoneutral housing, a condition relevant to human metabolism. By minimizing adaptive thermogenesis induced by standard room temperature housing, thermoneutrality more closely recapitulates human basal metabolic conditions. Under this condition, hepatocyte AGT deletion remains protective against adipo and hepatic lipid accumulation, despite exacerbated Western diet-induced metabolic dysfunction in wild-type mice, demonstrating that this protection persists in a human-relevant thermal environment. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/742617v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1ac7094org.highwire.dtl.DTLVardef@131cfforg.highwire.dtl.DTLVardef@d4dba6org.highwire.dtl.DTLVardef@a09acc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Obesity is a global epidemic characterized by metabolic dysfunction, with white adipose tissue playing a pivotal role in these processes. Noncoding RNAs, such as long non-coding RNAs (lncRNAs) and short non-coding RNAs (e.g., microRNAs), have been identified as an emerging class of regulatory molecules that can influence metabolic function. Here, the Dleu2/miR-15a/16-1 cluster (known as 13q14-Minimal Deleted Region, i.e., MDR), which encodes the lncRNA Dleu2 and miR-15a/16-1, a previously unrecognized player in metabolic function, is shown to contribute to obesity and insulin resistance. Using a combination of phenotypic and molecular approaches, this study establishes that MDR governs metabolic regulation for the first time. In a nutshell, this study identifies a new role of a lncRNA-miRNA cluster, previously implicated exclusively in cancer, in the regulation of obesity, thereby extending its biological significance beyond oncology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/745519v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@424a1borg.highwire.dtl.DTLVardef@f6e3eorg.highwire.dtl.DTLVardef@10ebf0borg.highwire.dtl.DTLVardef@120803c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeletion of MDR contributes to obesity, insulin resistance, and impaired energy metabolism C_LIO_LILoss of MDR reduces circulating adiponectin levels, indicating metabolic dysfunction C_LIO_LIMDR regulates satiety signaling in visceral adipose tissue and increases serum leptin levels C_LIO_LIMDR modulates several unrecognized new transcriptional regulators in obesity C_LIO_LIFirst evidence to establish the metabolic role of MDR beyond cancer biology C_LI

To address the long-standing question of the respective physiological contributions of classical brown versus beige adipocytes to adaptive nonshivering thermogenesis, we generated mice with lineage-specific ablation of UCP1 in thermogenic adipocytes of myogenic origin. This selectively targeted the major classical brown adipocyte lineage while preserving UCP1 expression in the remaining thermogenic adipocytes, reducing total UCP1 content by approximately 80 %. Unexpectedly, despite this profound reduction in UCP1 abundance, cold acclimation-recruited thermogenic capacity, assessed by adrenergic stimulation, remained largely preserved. In contrast, complete UCP1 deficiency abolished the adrenergically induced thermogenic response, demonstrating that UCP1 is indispensable for adaptive nonshivering thermogenesis. These findings indicate that in cold-acclimated mice only a fraction of the UCP1 normally present is required to sustain maximal thermogenic capacity. We further establish that the capacity to support UCP1-dependent oxidative metabolism, rather than UCP1 abundance, is the principal constraint on maximal thermogenic output under these conditions.

Uncoupling protein 1 (UCP1) mediates thermogenesis in brown adipose tissue. Whether human-UCP1 shares the bioenergetic properties established for rodent UCP1 (innate uncoupling, GDP sensitivity, fatty acid (re)activation) is not known. Therefore, we expressed human and mouse UCP1 in mouse liver, using adeno-associated viral vectors, and characterized their properties in isolated liver mitochondria. Both UCP1s induced marked innate uncoupling, characterized by increased substrate-supported respiration and decreased membrane potential, in the absence of exogenous fatty acids. Mouse-UCP1 in liver retained the classical regulatory properties of native brown-fat UCP1, including potent inhibition by GDP and reactivation by oleate. In contrast, human-UCP1 was only weakly inhibited by GDP but was strongly responsive to fatty acids. However, ATP potently inhibited human-UCP1, with an apparent IC of {approx}0.4 mM compared with {approx}1.4 mM for GDP, and ATP markedly decreased the sensitivity of human-UCP1 to oleate (re)activation. Despite substantial UCP1-mediated uncoupling, oxidative phosphorylation capacity and mitochondrial OXPHOS protein levels were preserved. Molecular dynamics simulations suggested a structural basis for the species difference. GDP formed persistent interactions with F88 in mouse-UCP1, an interaction absent at the corresponding S88 residue in human-UCP1. In-silico substitution of F88 by serine reduced GDP interaction at this site. Thus, human and mouse UCP1 share innate thermogenic activity but differ fundamentally in nucleotide regulation. The F88/S88 difference may contribute to the preferential GDP sensitivity of mouse-UCP1, whereas ATP provides effective nucleotide control of human-UCP1.

Understanding the feeding-induced neuropeptidome cascade requires analytical strategies capable of quantifying low-abundance, highly modified peptides across multiple tissues and time points. Herein, we apply 12-plex N,N-dimethyl leucine (DiLeu) isobaric labeling to perform the first multiplexed, tissue-wide, temporal quantitation of the Cancer borealis feeding neuropeptidome. This approach enabled sensitive measurement of neuropeptides across five tissues over six timepoints, revealing distinct regulatory patterns. The pericardial organ (PO) showed rapid early upregulation followed by suppression aligned with foregut emptying, whereas the thoracic ganglion (TG) displayed inverse and strongly condition-dependent responses, indicating previously unrecognized neuromodulatory roles. Single-residue variants and post-translational modifications, including pyro-Glu formation and amidation, produced markedly different temporal profiles, underscoring the functional specificity of closely related isoforms. We further identify differential regulation of proctolin and its amidated form, suggesting modified variants may contribute uniquely to feeding physiology. Collectively, these results establish multiplexed DiLeu labeling as a powerful platform for quantitative neuropeptidomics and reveal new dimensions of peptide-mediated feeding regulation.

Pancreatic beta cells exhibit marked resistance to proliferation, posing a barrier to therapeutic strategies aimed at restoring beta-cell mass in diabetes. Here, we present a transient, non-integrative approach to stimulate beta-cell proliferation using in vitro transcribed (IVT) mRNAs encoding cell cycle regulators. In rodent beta cells and human-beta cell derived EndoC-BH5 cells, chemically modified IVT mRNAs activated cell cycle entry and subsequent mitosis. A single dose of cyclin D1 and CDK4 IVT mRNAs nearly doubled the number of rat beta cells. However, achieving cell division in human beta cells required co-delivery of MYC IVT mRNA. The mitogenic response of beta cells peaked within 36-60 hours, and declined thereafter, reflecting the transient nature of IVT mRNA. Transcriptomic profiling revealed temporary activation of proliferative pathways and reversible downregulation of beta-cell maturation markers. Importantly, we detected no evidence of sustained proliferation. Our findings demonstrate that mRNA-based delivery of cell cycle regulators can overcome the intrinsic cell cycle block in beta cells and may provide a controllable approach for beta-cell regeneration.

Dysregulated hormone secretion and erosion of endocrine cell identity are features of type 1 and type 2 diabetes, but the transcriptional programs maintaining adult human islet identity and function remain poorly defined. The large MAF transcription factor MAFB is expressed in human - and {beta}-cells, marks their most functionally mature subpopulations, and is downregulated in diabetes, but its role in adult human islets has not been tested directly. Using shRNA-mediated MAFB knockdown (KD) in whole and CD26+ -cell-enriched human pseudoislets, we found that whole pseudoislet MAFB KD impaired glucagon synthesis and secretion while only modestly reducing insulin content and cAMP-potentiated insulin release. Single-cell profiling detected no {beta}-cell transcriptional response beyond MAFB KD itself, consistent with buffering by the related {beta}-cell-enriched MAFA transcription factor. In contrast, -cell-restricted MAFB KD unmasked a cell-autonomous requirement for MAFB in stimulus-secretion coupling. MAFB deficiency also destabilized -cell identity, downregulating canonical -cell and neuroendocrine secretory genes while ectopically inducing mesenchymal and extracellular matrix remodeling programs. In addition, MAFB-dependent downregulation of electron transport chain genes was confined to a large -cell subcluster, manifesting as impaired islet-wide mitochondrial respiration within the broader -cell population. Together, these findings identify MAFB as an essential adult human -cell maintenance factor that links diabetes-associated downregulation to impaired glucagon secretion, -cell identity erosion, and mitochondrial dysfunction. RESEARCH IN CONTEXTO_LIWhat is already known about this subject? O_LIMAFB is expressed in adult human - and {beta}-cells, marks their most functionally mature subpopulations, and is downregulated in type 1 and type 2 diabetes C_LIO_LIIn human stem cell models, MAFB is essential for generating insulin-producing {beta}-like cells, whereas glucagon-producing -like cells are reduced but still formed C_LIO_LINeither model addresses adult human islets: rodent MafB becomes -cell restricted after birth, and stem cell models capture differentiation, not maintenance C_LI C_LIO_LIWhat is the key question? O_LIIs MAFB required to maintain identity and secretory function in adult human islet cells? C_LI C_LIO_LIWhat are the new findings? O_LIMAFB knockdown in primary human pseudoislets impaired glucagon synthesis and secretion but minimally affected {beta}-cells, consistent with buffering by MAFA C_LIO_LIKnockdown in CD26+ -cell-enriched pseudoislets revealed a cell-autonomous requirement for MAFB in stimulus-secretion coupling, and destabilized -cell identity by inducing mesenchymal and extracellular matrix programs C_LIO_LIMAFB loss downregulated electron transport chain genes in the largest -cell subcluster and reduced mitochondrial respiration C_LI C_LIO_LIHow might this impact on clinical practice in the foreseeable future? O_LIPreserving MAFB activity in adult human -cells may represent a strategy to limit -cell dysfunction in diabetes C_LI C_LI

The release of glucagon from pancreatic alpha cells is a core component of hypoglycaemic counter regulation. Several mechanisms regulate glucagon release including paracrine control by neighbouring cell types, and changes in extracellular glucose. While the inhibitory effect of glucose on glucagon secretion is well established, the exact way in which glucose metabolism contributes to alpha cell function remains unclear. Here, we use live-cell imaging of the redox potential in alpha cells within intact islets to investigate whether non-mitochondrial glucose metabolism contributes to the potentiation of glucagon secretion at low glucose. Our findings show that increased glucose metabolism through the pentose phosphate pathway elevates the cytosolic redox potential in alpha cells. Using a combination of antioxidant treatment and pre-incubation in 5 mM glucose, we find that the cytosolic redox potential affects PKA activity in alpha cells and that changes in whole body redox state affects the counterregulatory response in mice. These findings indicate that prior glucose-driven redox potential charging is essential for maintaining glucagon secretion at low glucose.

Non-typhoidal Salmonella is a major cause of bacterial foodborne illness leading to acute gastroenteritis. In individuals with type 2 diabetes (T2D), Salmonella infection is more likely to cause life-threatening extraintestinal infections. The mechanism underlying this susceptibility remains unclear. In this study, 8-week-old TALLYHO mice were fed either a chow or high-fat diet (HFD, 45% fat) for 8 weeks to induce the T2D. As expected, HFD-fed mice gained more weight and developed diabetic-range blood glucose levels by 16 weeks of age. Next, mice from each diet group were orally infected with a fully virulent bioluminescent Salmonella Typhimurium to monitor infection spread by in-vivo imaging. Although both groups developed clinical signs of salmonellosis, Salmonella spread was accelerated and followed an unusual pattern in T2D mice compared with healthy animals. Additionally, hyperglycemia increased gut-derived lipopolysaccharide leakage into the bloodstream. Based on the link between T2D and altered levels of butyrate-producing bacteria in the gut, we analyzed the intestinal short-chain fatty acid (SCFA) profiles in the TALLYHO mice. As expected, intestinal SCFA concentrations, including butyrate, were lower in HFD mice than in chow-fed animals. Given butyrate?s role in gut health and its ability to downregulate Salmonella invasion genes, mice received oral butyrate supplementation. We found that butyrate supplementation reduced the extraintestinal spread of Salmonella in normoglycemic chow-fed animals. Unexpectedly, although butyrate improved intestinal health in hyperglycemic mice, it failed to decrease Salmonella spread in diabetic animals. Taken together, these findings provide novel insights into the pathogenesis of enteric salmonellosis in the context of T2D.

Brown adipose tissue (BAT) is a mitochondria-rich thermogenic organ whose function depends on high oxidative capacity, yet how primary mitochondrial dysfunction remodels BAT identity and metabolism remains poorly defined. Using the Deletor mouse model of progressive mtDNA deletion disease, we identify a pseudohypoxiairon-NAD+ axis as a central organiser of BAT pathology. Deletor BAT underwent profound structural, transcriptional and metabolic remodelling, characterised by mitochondrial ultrastructural damage, loss of thermogenic identity, PHD3/HIF associated pseudohypoxic signalling, iron dysregulation and NAD+/NADH redox imbalance. Indirect calorimetry confirmed that this molecular disease program translates to functional thermogenic failure under physiological demand. Deletor mice showed significantly reduced heat production under acute cold challenge and failed to switch to fatty acid oxidation Metabolomic profiling revealed altered TCA cycle intermediates, glycolytic rewiring and selective amino acid accumulation. Pharmacological perturbation showed that the PHD inhibitor roxadustat worsened disease-associated features, whereas HIF-1 suppression with PX-478 attenuated the integrated stress response, indicating that pseudohypoxic signalling is maladaptive in this setting. Nicotinamide riboside broadly attenuated the disease metabolome and transcriptome, restoring NAD+/NADH balance, suppressing ISRmt, iron-stress and pseudohypoxic gene programs, and correcting selective carnitine and acylcarnitine abnormalities consistent with impaired fatty-acid handling. These findings define a therapeutically tractable pseudohypoxia-iron-NAD+ axis as a core determinant of BAT dysfunction in mitochondrial disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major cause of chronic liver disease, with weight loss as the pivotal therapeutic strategy. However, the metabolic and molecular adaptations underlying rapid weight loss remain incompletely defined. In this pilot study, women with obesity and MASLD but without diabetes consumed a very low-calorie diet (VLCD) for 8 weeks. Clinical parameters, hepatic steatosis measured by controlled attenuation parameter (CAP), circulating metabolites, and microRNAs (miRs) were assessed before and after the dietary intervention. Integrated correlation and hierarchical clustering analyses were performed to identify molecular networks associated with clinical improvement. VLCD was well tolerated, resulting in significant weight loss (~11%) with ~80% adherence. Significant improvements in metabolic parameters were observed, including fat mass, waist circumference, blood pressure, insulinemia, HOMA-IR, HbA1c, and triglycerides, with unchanged liver enzymes. Hepatic steatosis decreased markedly, as indicated by a reduction in CAP, while stiffness remained unchanged. Metabolomic profiling revealed elevated ketone bodies and broad reductions in amino acid levels, consistent with enhanced fatty acid oxidation and a catabolic metabolic state. Correlation analysis identified distinct metabolite signatures associated with hepatic steatosis, with changes in CAP positively associated with changes in amino acids and inversely associated with changes in ketone bodies and tricarboxylic acid cycle intermediates. Circulating miRs underwent selective rather than global remodeling, with only a limited subset showing strong associations with clinical parameters, including CAP and HOMA-IR. Specifically, VLCD altered the circulating levels of miR-148a-3p, miR-140-3p, miR-10b-5p, and miR-345-5p. Integration of metabolomic and miR datasets identified coordinated metabolite-miR modules involving glucose metabolism, branched-chain amino acid catabolism, mitochondrial metabolism, purine metabolism, microbial metabolites, and cellular redox pathways. These findings demonstrate that improvement in hepatic steatosis during VLCD-induced weight loss is accompanied by coordinated remodeling of circulating metabolite-miR networks. Integrated multi-omics analysis identifies candidate molecular signatures associated with metabolic adaptation and highlights circulating miR-metabolite modules as potential biomarkers of therapeutic response in MASLD.

High-protein diets increase hepatic sulfur amino acid metabolism, but the underlying transcriptional mechanisms remain unclear. This study investigated whether Kruppel-like factor 15 (KLF15) directly regulates cystathionine {gamma}-lyase (CTH), a key enzyme linking methionine transsulfuration to hydrogen sulfide (H2S) and taurine production. Promoter-reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation identified two functional KLF15-binding elements, designated 1-1 and 2-2, within the proximal Cth promoter. Mutation of either element attenuated KLF15-dependent promoter activation, whereas mutation of both largely abolished it. In vivo luciferase imaging further demonstrated that these elements were required for the hepatic transcriptional response to a high-protein diet. KLF15 loss of function reduced high-protein-diet-induced Cth expression and altered the hepatic sulfur amino acid profile. Methionine, cystathionine, and cystine accumulated, whereas taurine production and the high-protein-diet-induced increase in hepatic H2S were attenuated. Gene expression analyses further indicated that KLF15 selectively regulates components of methionine, taurine, and H2S metabolism rather than controlling the entire sulfur metabolic program. Collectively, these findings establish the high-protein diet-KLF15-CTH axis as a physiologically relevant transcriptional pathway that amplifies hepatic sulfur amino acid disposal and directs sulfur toward H2S and taurine production.

Elephant seals are remarkable breath-hold divers, capable of remaining submerged for up to two hours during diving bouts. These dives entail repeated, extreme hypoxia/reoxygenation events that would induce severe lipid peroxidation and tissue dysfunction in most mammals. Here, we show that primary vascular endothelial cells derived from elephant seals possess an intrinsic resistance to lipid peroxidation. Comparative transcriptomic and lipidomic profiling across seal, human, and sheep cells identified ferroptosis - an iron-dependent, lipid peroxidation-driven cell death pathway - as uniquely regulated in seal cells following hydroperoxide exposure. Mechanistically, seal cells exhibit robust baseline expression of acyl-CoA synthetase long-chain family member 3 (ACSL3), alongside rapid, seal-specific induction of the sole mammalian iron exporter, ferroportin (SLC40A1). Functional validation using genetic and pharmacological approaches revealed that seal cells are naturally enriched in monounsaturated fatty acids and triglycerides and utilize lipid droplet biogenesis and active iron export as dual protective axes to evade lipid peroxidation. Together, these findings show that elephant seal cells employ a coordinated cytoprotective network of lipid remodeling and iron handling to withstand the severe challenges of deep diving. SIGNIFICANCE STATEMENTDeep-diving marine mammals repeatedly experience extreme hypoxia-reoxygenation events that would induce severe oxidative damage in most terrestrial mammals. However, vascular cells derived from seals naturally resist lipid peroxidation, a major driver of ischemia-reperfusion injury. Here, we show that elephant seal endothelial cells evade lipid peroxidation through two complementary mechanisms: lipid droplets that sequester peroxidation-prone phospholipids, and rapid iron export that limits lipid peroxide formation. These findings reveal naturally evolved cellular strategies that protect against vascular oxidative stress, offering new insights into physiological resilience against ischemia-reperfusion injury.

Background Lifestyle interventions are central to obesity prevention and management, yet interindividual variability in response remains incompletely understood. Here, we leveraged genetically defined, distinct obesity endotypes to examine lifestyle-body mass index (BMI) associations across biological pathways. Methods In the UK Biobank, we analyzed 305,713 participants with partitioned polygenic scores (pPSs) representing 10 obesity endotypes. We evaluated interactions between endotype-specific genetic susceptibility and physical activity, diet, sedentary behavior, and sleep on BMI using multivariable linear regression. Primary findings were externally evaluated in the All of Us Research Program using Fitbit-derived lifestyle measures. Results Favorable lifestyle behaviors were associated with lower BMI for all obesity endotypes, but the magnitude of these associations varied significantly across endotypes. Higher endotype-specific pPSs strengthened the benefits of physical activity (7 endotypes), healthy diet (3 endotypes), nonsedentary behavior (5 endotypes), and adequate sleep (7 endotypes) on BMI. Distinct endotypes demonstrated the greatest responsiveness to different lifestyle domains, with the metabolically unhealthy endotype showing the strongest interaction with physical activity, metabolically healthy endotype with sedentary behavior, hypothalamic dysregulation endotype with diet, and hypoinsulin 2 endotype with sleep, corresponding to differences in BMI of 0.22-0.49 kg/m2 between the highest and lowest pPS deciles. These interaction patterns were consistent in the All of Us cohort. Conclusions Obesity endotypes modify the association between lifestyle behaviors and BMI, demonstrating that responsiveness to lifestyle behaviors is heterogeneous and pathway dependent. These findings provide a framework for precision obesity prevention by identifying individuals who may derive greater benefit from specific lifestyle interventions.

AMP-activated protein kinase (AMPK) regulates metabolism in response to metabolic stress that includes stimulating glucose uptake in skeletal muscle independently of the canonical insulin signalling pathway, positioning it as an attractive therapeutic target for insulin resistance and type 2 diabetes mellitus (T2DM). AMPK is an {beta}{gamma} heterotrimer, with multiple isoforms for each subunit enabling the formation of 12 different complexes with distinct tissue expression profiles. Among these, the 2{beta}2{gamma}3 complex is predominantly expressed in skeletal muscle, the major site of glucose disposal and a highly desirable therapeutic target for T2DM. Here, we characterise the functional role of a unique, 182 residue N-terminal extension (NTE) within {gamma}3 subunit. Deletion of the {gamma}3-NTE from 2{beta}2{gamma}3 complex increases basal AMPK activity without affecting activation by AMP or pharmacological AMPK activators, demonstrating the {gamma}3-NTE performs an autoinhibitory function. Using complementary biophysical techniques, including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, chemical crosslinking and co-pulldowns, we identified a 39-residue sequence in the {gamma}3-NTE (residues 129-168), that directly interacts with the C-helix of the AMPK kinase domain small lobe, a key regulatory element in many protein kinases. Using AlphaFold3, we probe the interaction predicted to take place between a {gamma}3-NTE -helix ({gamma}3-iHelix; [~]T142-E154) and the C-helix in the 2{beta}2{gamma}3 complex. These findings provide the groundwork for developing novel T2DM therapies that target AMPK activation selectively in skeletal muscle involving reversal of the {gamma}3 autoinhibition.

Background/Objectives: Childhood appetitive traits are heritable behavioural phenotypes hypothesized to link genetic susceptibility to obesity risk. Yet their genetic architecture and role in mediating polygenic adiposity risk remain poorly understood. Methods: We conducted the largest survey of childhood eating behaviour to date, allowing us to perform genome-wide association studies of six appetitive domains derived from 18 items of the parent-reported Children's Eating Behaviour Questionnaire in up to 31,018 eight-year-old children from the Norwegian Mother, Father and Child Cohort Study (MoBa). A trio-based design enabled decomposition of direct and indirect genetic effects on appetite and BMI. Results: We identified ten independent genome-wide significant loci for childhood eating behaviour, primarily across Food Responsiveness, Satiety Responsiveness, and Food Fussiness, eight of which lie at established childhood or adult BMI loci. Food Responsiveness and Satiety Responsiveness showed both phenotypic and genetic correlations with BMI trajectories from early childhood through adolescence. Statistical mediation analyses indicated that 22.1% and 10.4% of the aggregated genetic association with BMI at age 8 could be decomposed through these traits, respectively. Locus-specific patterns further suggested mechanistic pathways, with the FTO locus acting predominantly via Food Responsiveness, and the ADCY3 locus via Satiety Responsiveness. Trio analyses demonstrated that both BMI and eating behaviour associations were predominantly explained by children's inherited alleles, with minimal contribution from indirect effect from parental adiposity, although parental genetic liability influenced reporting of Satiety Responsiveness. Conclusions: Childhood appetitive traits capture a substantial proportion of genetic susceptibility to adiposity through distinct eating behaviour pathways (under standard mediation assumptions). These effects are primarily driven by the child's own genotype rather than indirect parental influences, positioning appetite as a plausible, biologically grounded target for early obesity prevention.

Background and AimsPregnancy requires dynamic, stage-specific adaptations in maternal liver metabolism and growth to sustain fetal development while preserving systemic homeostasis. Estrogen signaling, which significantly increases during pregnancy, is primarily mediated in hepatocytes by estrogen receptor (ER). Although hepatic ER regulates female liver metabolism under non-pregnant conditions, its role in pregnancy-induced hepatic remodeling remains unclear. MethodsWe studied non-pregnant and pregnant control and liver-specific ER knockout (LERKO) mice across gestational stages using longitudinal physiological measurements, liver transcriptomics, targeted metabolomics, histological assessment of cell proliferation, and metabolic phenotyping. ResultsIn control mice, pregnancy elicited sequential hepatic remodeling characterized by early induction of cell-cycle programs, a mid-gestational peak in hepatocyte proliferation with transient suppression of selected metabolic pathways, and late reactivation of specific metabolic programs. Chronic hepatic ER deficiency alters this temporal pattern. LERKO livers showed premature activation of proliferative and anabolic transcriptional programs, changes in amino acid- and fatty acid-related metabolic pathways, and altered temporal regulation of AKT-mTORC1-related signaling. At mid-gestation, LERKO mice displayed reduced hepatocyte proliferation, altered expression of metabolic and insulin-related genes, blunted gestational glucose adaptation without overt evidence of systemic insulin resistance, and changes in the light/dark-phase metabolic patterns. ConclusionsThese findings suggest that hepatic ER is required for the appropriate stage-specific coupling of liver growth, metabolic remodeling, and insulin-responsive signaling during pregnancy. Its loss is associated with gestational hepatic maladaptation and systemic metabolic phenotypes, providing a framework for investigating estrogen-dependent mechanisms underlying pregnancy-associated metabolic and liver disorders. HighlightsHepatic ER is required for stage-specific liver remodeling during pregnancy. Loss of hepatic ER alters temporal coupling of liver growth and metabolism. LERKO mice show early changes in amino acid- and fatty acid-related pathways. Hepatic ER loss reduces proliferation and alters gestational glucose adaptation. Hepatic ER loss is associated with altered light/dark-phase metabolic organization. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/743939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d52bborg.highwire.dtl.DTLVardef@b27511org.highwire.dtl.DTLVardef@23b286org.highwire.dtl.DTLVardef@19d9314_HPS_FORMAT_FIGEXP M_FIG C_FIG

GPR27/SREB1 is a highly conserved orphan class A G-protein coupled receptor implicated in insulin production, metabolic regulation, tumour biology, neurodegeneration and L-lactate homeostasis, but its immediate second-messenger signalling remains poorly defined. We used single-cell Forster resonance energy transfer nanosensors to monitor cytosolic Ca2+ and cAMP in wild-type 3T3 MEF cells, CRISPR-Cas9 GPR27-knockout cells (GPR27KO) and GPR27-knockout cells transiently re-expressing FLAG-tagged GPR27 (GPR27-rescued). The GPR27 surrogate agonist 8535n (1 {micro}M) increased intracellular Ca2+ in wild-type and rescued cells but not in GPR27-knockout cells and produced no significant cAMP response in wild-type cells. Basal Ca2+ and cAMP levels were unaffected by GPR27 deletion. Extracellular L-lactate (2 mM) induced a GPR27-dependent increase in Ca2+ and cAMP in wild-type and rescued cells, but not in knockout cells, raising the possibility that L-lactate acts as an endogenous ligand or modulator of GPR27. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/743761v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@2af45dorg.highwire.dtl.DTLVardef@113e2corg.highwire.dtl.DTLVardef@8dea4org.highwire.dtl.DTLVardef@50ec49_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGPR27 surrogate agonist 8535n increases intracellular Ca2+ but not cAMP in 3T3 cells. C_LIO_LIExtracellular L-lactate induces GPR27-dependent intracellular Ca2+ and cAMP increases in 3T3 cells. C_LIO_LIThese findings identify GPR27 as a putative candidate lactate sensor. C_LI

Lateral hypothalamic GABAergic (LHAGABA) neurons regulate arousal, feeding, and reward-related behaviors, but how their downstream projections coordinate motivated behaviors across domains remains incompletely defined. Our histological analyses revealed that LHAGABA fibers were distributed across the dorsal pons (DP) subregions, including the peri-locus coeruleus, laterodorsal tegmentum, and Barringtons nucleus, and extend throughout the lateral preoptic area (LPO), thereby refining existing anatomical descriptions. We then used optogenetics to systematically compare the effects of activating LHAGABA somata and their projections to the DP and LPO across assays of feeding, non-food-directed gnawing, predatory behavior, real-time place preference, and operant self-stimulation. In sated mice, optogenetic activation of LHAGABA somata or their terminals in the DP or LPO increased caloric food intake, whereas non-caloric cellulose intake was minimally affected during terminal stimulation. Across conditions, activation increased gnawing and shredding of non-food objects while reducing inactivity. In cricket hunting, stimulation increased cricket killing and consumption relative to controls. Similarly, all stimulation conditions supported positive-valence and reinforcement-related responding, as indicated by real-time place preference and operant self-stimulation. Together, these results provide new functional evidence that activation of LHAGABA somata and projections to both the DP and LPO recruit largely overlapping behavioral responses across feeding, non-food behavior, predatory hunting, and reinforcement-related assays. These findings support a distributed hypothalamic output architecture in which major ascending and descending LHAGABA pathways contribute to a shared motivational repertoire rather than wholly discrete behavioral functions.