Molecular Metabolism

Fructose ingestion increases circulating GLP-1 and insulin, yet the specific contributions of these hormonal responses to glycaemic control remain incompletely defined. We hypothesised that fructose metabolism in intestinal L-cells triggers GLP-1 secretion, which then potentiates insulin secretion and counteracts fructose-induced hyperglycaemia. To test this hypothesis, we systematically characterised metabolic responses across multiple mouse strains after 24 h ad libitum fructose ingestion. In both lean (NSY.B6-a/a) and obese diabetic (NSY.B6-Ay/a) mice, fructose elevated plasma insulin, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP). The insulin response was preserved in GIP receptor-deficient mice (Gipr-/-) but was abolished in proglucagon-deficient mice (Gcg-/-) by pharmacological GLP-1 receptor antagonism, indicating a requirement for GLP-1, but not GIP. Across strains, fructose-induced insulin response correlated with attenuation of post-fructose glycaemia, consistent with insulin being essential for suppressing fructose-induced hyperglycaemia. To explore the mechanism underlying fructose-induced GLP-1 secretion, we combined ATP-sensitive potassium channel-deficient mice (Kcnj11-/-), GLUTag L-cell line, and metabolic tracing of 13C-labelled fructose in freshly isolated intestinal crypts. These complementary approaches support a model in which fructolysis increases the ATP/ADP ratio in L-cells, closes KATP channels, and stimulates GLP-1 secretion. In obese diabetic mice, increased fructolytic flux and a higher ATP/ADP ratio were associated with elevated GLP-1 levels, further corroborating this model. Collectively, our findings indicate that intestinal fructose metabolism drives GLP-1 secretion required to potentiate insulin secretion, thereby establishing a gut-pancreas axis that counter-regulates fructose-induced hyperglycaemia. KEY POINTS SUMMARYO_LIFructose ingestion acutely increases plasma insulin levels, but the underlying mechanisms and physiological significance remain elusive. C_LIO_LIOur study demonstrates that short-term (24h) fructose ingestion in mice elevates both insulin and glucagon-like peptide 1 (GLP-1) levels in the blood, with the plasma insulin response being GLP-1-dependent. C_LIO_LIWe found that fructose metabolism in intestinal L-cells triggered GLP-1 secretion by increasing the ATP/ADP ratio and closing ATP-sensitive K+ channels (KATP channels). C_LIO_LIThis intestinal fructose metabolism/GLP-1/{beta}-cell axis plays a crucial role in preventing fructose-induced hyperglycaemia, an effect that is compromised in obese diabetic mice. C_LIO_LIThese insights highlight the previously unclear metabolic responses following short-term fructose ingestion and their importance in glucose homeostasis. C_LI 1. ABSTRACT FIGURE LEGENDThis study investigated the hormonal effects of short-term fructose consumption in mice, allowing them ad-lib access to fructose solution for 24 h. Fructose metabolism in intestinal L-cells increases the intracellular ATP/ADP ratio, leading to GLP-1 secretion via KATP channel closure and Ca2+ influx. GLP-1 promotes insulin secretion from pancreatic {beta}-cells. The fructose metabolism/GLP-1/insulin pathway is essential for mitigation of fructose-induced hyperglycaemia. Figures were drawn using BioRender.com.

Consumption of diets high in sugar and fat are well-established risk factors for the development of obesity and its metabolic complications, including non-alcoholic fatty liver disease. Metabolic dysfunction associated with sugar intake is dependent on fructose metabolism via ketohexokinase (KHK). Here, we compared the effects of systemic, small molecule inhibition of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. Both modalities led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown profoundly decreased lipogenesis, while the inhibitor increased the fatty acid oxidation pathway. Moreover, hepatocyte-specific KHK knockdown completely prevented hepatic fructose metabolism and improved glucose tolerance. Conversely, KHK inhibitor only partially reduced fructose metabolism, but it also decreased downstream triokinase. This led to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. In summary, KHK profoundly impacts hepatic metabolism, likely via both kinase-dependent and independent mechanisms. HIGHLIGHTSO_LIKHK knockdown or inhibition of its kinase activity differently target hepatic metabolism. C_LIO_LIKHK inhibitor increases F1P and glycogen accumulation as it also lowers triokinase. C_LIO_LIKHK knockdown completely prevents hepatic fructose metabolism and lipogenesis. C_LIO_LIE of wild type, but not mutant, kinase dead KHK-C increases glycogen accumulation. C_LI

Background & AimsPNPLA3(I148M) is the strongest genetic risk factor for steatotic liver disease (SLD), but its functional role and tissue-specific regulation remain unclear. In mice, PNPLA3 is abundant in liver, yet undetectable in adipose depots. Here, we characterize the molecular mechanisms underlying these tissue-specific differences in PNPLA3 expression in mice to clarify its functional role and link to SLD risk. MethodsPnpla3 mRNA and PNPLA3 protein levels were quantified in liver and adipose depots of fasted and refed mice at 30{degrees}C and 6{degrees}C. Signaling pathways regulating PNPLA3 expression in adipocytes were examined using adrenergic agonists and pathway-specific modulators. Translation and proteasomal inhibitors were used during adrenergic stimulation to investigate the discordance between Pnpla3 mRNA and protein levels. Relationship between PNPLA3 levels and triglyceride (TG) fatty acid composition was also assessed. ResultsAt thermoneutrality, feeding strongly increased PNPLA3 levels in liver but it remained undetectable in adipose tissue of mice. Conversely, cold exposure or {beta}3-adrenergic stimulation had no effect on hepatic PNPLA3, but increased PNPLA3 >19-fold in brown adipose tissue (BAT), despite causing a >75% reduction in Pnpla3 mRNA, indicating robust post-translational regulation. In BAT, adrenergic signaling via cAMP/PKA and PI3K/AKT elevated PNPLA3 by reducing proteasomal degradation. PNPLA3 expression correlated with depletion of TG-long-chain polyunsaturated fatty acids (TG-LCPUFAs) in both liver and BAT, consistent with a role in lipid remodeling. ConclusionsThese findings reveal striking tissue- and context-specific regulation of PNPLA3, but a conserved association between its expression and TG-LCPUFAs levels, suggesting that PNPLA3 modulates lipid remodeling in response to metabolic stress and that disrupting this function may contribute to SLD susceptibility. Impact and implicationsDespite being the strongest genetic risk factor for SLD, PNPLA3s physiological role remains unclear. Using mouse models, this study reveals that PNPLA3 is regulated in a tissue-specific manner in response to feeding and cold exposure, thereby promoting remodeling of cellular lipids to adapt to dietary and environmental challenges. The localization of PNPLA3 action and its tissue-specific regulation are directly relevant to hepatologists and metabolic researchers aiming to understand its influence on intracellular lipid composition and its effects on disease susceptibility. Moreover, modulation of PNPLA3 turnover--and its impact on LCPUFAs remodeling--emerges as a potential therapeutic strategy for regulating lipid homeostasis in SLD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=191 SRC="FIGDIR/small/684800v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@ee6ac7org.highwire.dtl.DTLVardef@a45d74org.highwire.dtl.DTLVardef@f39929org.highwire.dtl.DTLVardef@cc826b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPNPLA3 is regulated in a highly tissue-specific manner in mice. C_LIO_LIIn liver, feeding-but not cold exposure-induces PNPLA3 primarily through transcriptional mechanisms. C_LIO_LIIn adipose tissue, cold exposure-but not feeding-induces PNPLA3 through post-transcriptional mechanisms. C_LIO_LIIn adipose tissue, cold exposure increases PNPLA3 despite a reduction in Pnpla3 mRNA. C_LIO_LIPNPLA3 remodels lipids in liver and adipose tissue to maintain lipid homeostasis, a process disrupted in SLD. C_LI

AbstractThe progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) to metabolic-dysfunction-associated steatohepatitis (MASH) involves complex alterations in both liver-autonomous and systemic metabolism that influence the livers balance of fat accretion and disposal. Here, we quantify the relative contribution of hepatic oxidative pathways to liver injury in MASLD-MASH. Using NMR spectroscopy, UHPLC-MS, and GC-MS, we performed stable-isotope tracing and formal flux modeling to quantify hepatic oxidative fluxes in humans across the spectrum of MASLD-MASH, and in mouse models of impaired ketogenesis. We found in humans with MASH, that liver injury correlated positively with ketogenesis and total fat oxidation, but not with turnover of the tricarboxylic acid cycle. The use of loss-of-function mouse models demonstrated that disruption of mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting step of ketogenesis, impairs overall hepatic fat oxidation and induces a MASLD-MASH-like phenotype. Disruption of mitochondrial {beta}-hydroxybutyrate dehydrogenase (BDH1), the terminal step of ketogenesis, also impaired fat oxidation, but surprisingly did not exacerbate steatotic liver injury. Taken together, these findings suggest that quantifiable variations in overall hepatic fat oxidation may not be a primary determinant of MASLD-to-MASH progression, but rather, that maintenance of hepatic ketogenesis could serve a protective role through additional mechanisms that extend beyond quantified overall rates of fat oxidation.

Carrying a germline mutation in BRCA1 is associated with an increased risk of several cancers, including breast and ovarian. Our recent work has demonstrated that obesity is associated with elevated levels of DNA damage in breast glands in this high-risk population. BRCA1 is a canonical tumor suppressor gene primarily recognized for its role in DNA damage repair, yet emerging evidence suggests broader functions in metabolic regulation. To determine whether heterozygous loss of Brca1, as seen in individuals who carry a germline mutation, modifies susceptibility to diet-induced metabolic dysfunction in a sex-dependent manner, we subjected wild-type (WT) and Brca1+/- mice of both sexes to a high-fat diet (HFD) and performed longitudinal metabolic phenotyping. Female Brca1+/- mice exhibited pronounced obesity, increased adiposity, hyperinsulinemia, and impaired glucose tolerance. In contrast, male Brca1+/- mice showed modest resistance to HFD-induced weight gain and displayed improved glucose tolerance compared to WT controls. Notably, Brca1 heterozygosity led to more severe hepatic steatosis with HFD, indicating a shared susceptibility to liver lipid accumulation despite divergent systemic outcomes. In females, steatosis was associated with reduced mitochondrial respiratory complex IV activity and transcriptional remodeling that favored lipid storage. Treatment with the dual GLP1/GIP receptor agonist tirzepatide ameliorated systemic metabolic dysfunction and hepatic steatosis in HFD-fed female Brca1+/- mice. These findings identify Brca1 heterozygosity as a modifier of metabolic disease risk, expanding BRCA1 biology beyond tumor suppression. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/708005v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@17f9592org.highwire.dtl.DTLVardef@134c2c2org.highwire.dtl.DTLVardef@de69d8org.highwire.dtl.DTLVardef@1f6f1af_HPS_FORMAT_FIGEXP M_FIG C_FIG

Adipose tissues exhibit a remarkable capacity to expand, regress, and remodel in response to energy status. The cellular mechanisms underlying adipose remodelling are central to metabolic health. Hypertrophic remodelling - characterised by the enlargement of existing adipocytes - is associated with insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, hyperplastic remodelling - in which new adipocytes are generated - is linked to improved metabolic outcomes. Despite its clinical importance, the regulation of hypertrophic and hyperplastic adipose morphology remains poorly understood. Here, we integrate human transcriptomic data with a quantitative CRISPR-imaging platform in zebrafish to identify regulators of adipose morphology. We developed an image-based phenotyping pipeline that captures lipid droplet size, number, and spatial patterning, and applied generalised additive modelling to quantify hyperplastic versus hypertrophic morphology signatures. Using this platform, we conducted an F0 CRISPR screen targeting 25 candidate genes and identified three that induced hypertrophic morphology (txnipa, mmp14b and foxp1b) and an additional candidate that altered total adiposity (kazna). For functional validation, we generated stable loss-of-function alleles for both zebrafish foxp1 paralogues. Spatial analysis along the anterior-posterior axis revealed that foxp1b mutants display developmental hypertrophy but profoundly blunted adaptive responses to high-fat diet ([~]68% reduction across all spatial zones), while foxp1a mutants show normal baseline morphology but disrupted spatial patterning of diet-induced hypertrophy. Together, these findings establish a scalable CRISPR-imaging platform for in vivo genetic screening of adipose morphology, and reveal distinct roles for Foxp1 paralogues in developmental patterning and adaptive responses to dietary challenge in adipose tissue.

Glucagon-like peptide-1 receptor (GLP-1R) agonists and fibroblast growth factor 21 (FGF21) confer similar metabolic benefits. Studies report that GLP-1RA induce FGF21. Here, we investigated the mechanisms engaged by the GLP-1R agonist liraglutide to increase FGF21 levels and the metabolic relevance of liraglutide-induced FGF21. We show that liraglutide increases FGF21 levels via neuronal GLP-1R activation. We also demonstrate that lack of liver Fgf21 expression confers partial resistance to liraglutide-induced weight loss. Since FGF21 reduces carbohydrate intake, we tested whether the contribution of FGF21 to liraglutide-induced weight loss is dependent on dietary carbohydrate content. In control and liver Fgf21 knockout (LivFgf21-/-) mice fed calorically matched diets with low- (LC) or high-carbohydrate (HC) content, we found that only HC-fed LivFgf21-/- mice were resistant to liraglutide-induced weight loss. Similarly, liraglutide-induced weight loss was partially impaired in LivFgf21-/- mice fed a high-fat, high-sugar (HFHS) diet. Lastly, we show that loss of neuronal {beta}-klotho expression also diminishes liraglutide-induced weight loss in mice fed a HC or HFHS diet, indicating that FGF21 mediates liraglutide-induced weight loss via neuronal FGF21 action. Our findings support a novel role for a GLP-1R-FGF21 axis in regulating body weight in the presence of high dietary carbohydrate content.

ObjectiveDelineating the nodal control points that maintain whole-body energy homeostasis is critical for understanding potential treatments of obesity and cardiometabolic diseases. The nutrient-sensing transcription factor MondoA is a regulator of skeletal muscle fuel storage, where muscle-specific inhibition improves glucose tolerance and insulin sensitivity. However, the role of MondoA in whole body energy metabolic homeostasis is not understood. MethodsGeneralized MondoA knockout (gKO) mice were generated and assessed for glucose tolerance and insulin sensitivity, body composition, energy expenditure, cold tolerance, and tissue specific transcriptional changes in response to high fat diet. Complementary studies in cultured human adipocytes assessed the impact of MondoA deficiency on substrate utilization and lipolysis. ResultsgKO mice are protected from diet-induced obesity and insulin resistance, through increased whole body energy expenditure. gKO mice exhibit reduced brown and inguinal white adipose tissue mass, without evidence of beiging. The gKO mice are hyperlactatemic and isolated MondoA-deficient adipocytes have increased 2-deoxyglucose uptake and glycolytic function. Lastly, gKO mice and KO adipocytes display increased circulating glycerol relative to free fatty acids in response to adrenergic stimulus consistent with elevated re-esterification. However, this phenotype is not recapitulated in adipocyte-specific KO mice. ConclusionsMondoA deficiency alters cellular sensing of nutrient availability and storage/utilization mechanisms. In the whole-body setting, this results in increased energy expenditure, potentially related to increased glucose uptake and glycolytic flux driving glycerol synthesis to supply high rates of lipolysis and lipid re-esterification. These results suggest that MondoA functions to maintain fuel storage and when lost, inter-organ futile cycling ensues. O_FIG O_LINKSMALLFIG WIDTH=140 HEIGHT=200 SRC="FIGDIR/small/680559v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1ccf42dorg.highwire.dtl.DTLVardef@b2da28org.highwire.dtl.DTLVardef@1081aforg.highwire.dtl.DTLVardef@1b2168d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO 1) Global MondoA deficiency drives 2) tissue glucose uptake which in skeletal muscle is 3) converted and excreted as lactate, while in adipose tissue 4) triglyceride re-esterification requires 5) de novo glycerol synthesis to feed into the futile cycle. C_FIG

Emerging evidence that circulating levels of key metabolic intermediates are sensed by a range of G-Protein Coupled receptors (GPCRs) is providing critical new insights into the control of systemic metabolic homeostasis, and how disturbances in such sensing may contribute to metabolic disease. The hydroxycarboxylic acid receptors for lactate (HCAR1), {beta}-hydroxybutyrate (HCAR2), and octanoate (HCAR3) are encoded by three closely homologous GPCR genes co-located in a region where common genetic variation has been reportedly associated with lipid levels and body fat distribution. By resolving sequence homology in this region, we were able to refine this signal to a coding variant (R311C) in HCAR2. Using corrected genotypes from [~]500K participants from UK Biobank and direct genotyping of four other studies, we found that carriage of the HCAR2 p.R311C variant was significantly associated with type 2 diabetes risk, reduced gynoid fat mass, increased waist-hip ratio, higher circulating triglycerides, glucose and alanine aminotransferase levels, lower levels of HDL cholesterol and adiponectin and impaired suppression of circulating levels of non-esterified fatty acids after oral glucose. Adipose tissue explants from mice engineered to express the equivalent mutation variant (p.R308C) in the mouse ortholog showed increased lipolytic activity, basally and after {beta}-hydroxybutyrate (BHB) treatment. In vivo, the mice were insulin resistant and had increased liver fat and impaired post-prandial suppression of NEFAs. The variant alters an amino acid located in the intracellular C-terminal tail of HCAR2, increasing recruitment of {beta}-arrestin and resulting in enhanced internalisation and reduced cell surface expression. In conclusion, a common variant in the human ketone body receptor results in impaired control of adipocyte lipolysis and adversely impacts systemic lipid and glucose metabolism. These findings highlight the importance of anti-lipolytic ketone body signalling in adipocytes for the maintenance of metabolic health Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=182 HEIGHT=200 SRC="FIGDIR/small/25336995v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1dd7e54org.highwire.dtl.DTLVardef@90ca10org.highwire.dtl.DTLVardef@1c1ef16org.highwire.dtl.DTLVardef@137c18b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Obesity is a major public health crisis, affecting billions worldwide and increasing the risk of metabolic and cardiovascular diseases. While lifestyle factors play a role, genetic variation is a key determinant of both obesity susceptibility and the efficacy of treatment strategies. Recent studies have implicated the Semaphorin 3 signalling pathway in obesity; however, specific roles for pathway components remain largely unexplored. Here, we focus on Class A Plexins and their potential contributions to body weight regulation. Using large-scale genetic association data, we identified that rare, predicted loss-of-function mutations in PLXNA4 were associated with body mass index (BMI) in females. Furthermore, common variant analysis revealed that genetic variation at PLXNA4 was linked to BMI, height, and various neuropsychiatric disorders. To investigate the biological role of Plxna4, we generated zebrafish plxna4 loss-of-function mutants, which exhibited an 85-92% reduction in Plxna4 protein. Despite appearing morphologically normal, mutant zebrafish at juvenile stages were shorter, had increased body fat levels relative to size-matched wild-type siblings, and displayed hypertrophic subcutaneous adipose tissue. Feeding assays revealed that plxna4 mutants consumed more food than wild-type siblings and exhibited food-stimulated hyperactivity, characterised by increased swimming speed, higher speed variability, and frequent high-speed bursts. Together, these findings demonstrate a conserved role for Plxna4 in regulating feeding behaviour and body fat levels, providing new insights into the genetic basis of obesity and warranting further studies to elucidate the molecular mechanisms underlying these effects.

Intracellular fatty acids (FAs) activate and fuel non-shivering thermogenesis (NST) via uncoupling protein 1 (UCP1). Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) control FA availability. Since mice lacking ATGL in brown adipose tissue (BAT) exhibit intact recruitable adrenergic thermogenesis, we hypothesized that HSL-mediated FA release is sufficient to activate UCP1-dependent NST. We demonstrate that mice with inducible brown adipocyte-specific loss of ATGL and HSL (iBDKO) exhibit normal recruitable adrenergic thermogenesis upon prolonged cold exposure. Mechanistically, we show that BAT thermogenic capacity is impaired in cold-adapted iBDKO mice due to diminished mitochondrial numbers. Increased browning of white adipose tissue (WAT) in iBDKO mice indicates a shift in thermogenesis from BAT to WAT. Consistently, the loss of ATGL and HSL in BAT and WAT disrupts thermogenesis in both depots, resulting in blunted UCP1-dependent NST. Our study highlights the metabolic adaptability of adipose tissue and the critical role of intracellular lipolysis in regulating thermogenesis.

The Staub-Traugott effect describes the improved glycemic response observed after consuming a second identical meal. We previously showed that morning (AM) hyperinsulinemia primes the liver for enhanced afternoon (PM) meal-associated net hepatic glucose uptake (NHGU) and glycogen storage. However, depending on the meal composition, both insulin and glucagon may rise. Therefore, we investigated whether AM hyperglucagonemia alters the priming effect of AM hyperinsulinemia on subsequent hepatic glucose metabolism. Dogs underwent a 4h AM hyperinsulinemic-euglycemic clamp paired with either basal (AM INS, n=8) or elevated glucagon (AM INS+GCG, n=8). After a 1.5h rest, dogs underwent a 2.5h PM hyperinsulinemic-hyperglycemic clamp designed to mimic postprandial conditions. AM hyperglucagonemia reduced PM NHGU through additive shifts in both hepatic glucose uptake and production, leading to lower direct glycogen synthesis and less glycolytic flux. Mechanistically, hepatic glucokinase protein was reduced in the AM INS+GCG vs. AM INS group, suggesting diminished capacity for glucose phosphorylation and lower intracellular G6P, a central node regulating downstream hepatic glucose metabolism. Thus, morning hepatic glucagon exposure diminishes insulins ability to prime the liver, limiting net hepatic glucose uptake during a later meal. These results underscore how antecedent hormonal signals govern subsequent postprandial hepatic glucose metabolism.

IntroductionAbout 1 billion people are living with obesity worldwide. GLP-1-based drugs have massively transformed care, but long-term consequences are unclear in part due to reductions in energy expenditure with ongoing use. Diet-induced thermogenesis (DIT) and cold exposure (CE) raise EE via brown adipose tissue (BAT) activation and beiging of white adipose tissue (WAT). Methionine restriction (MetR) is a candidate DIT stimulus, but its EE effect has not been benchmarked against CE, nor have their tissue-level interactions been defined. Objective & MethodsIn a 2x2 design (Control vs. MetR; room temperature, RT: 22 {degrees}C vs. CE: 4 {degrees}C for 24 h), we used male C57BL/6N mice to benchmark MetR-induced thermogenesis against CE and mapped how diet and temperature interact across tissues. Bulk RNA-seq profiled liver, iBAT, iWAT, and eWAT. Differential expression was modeled with main effects and a dietxtemperature interaction. KEGG GSEA was used to assess pathway-level enrichment. ResultsMetR increased EE at RT and shifted fuel use towards lipid oxidation, supporting MetR as a bona fide DIT stimulus. CE elevated EE across diets and blunted diet differences. Transcriptomic responses were tissue-specific: in liver, CE dominated gene induction while MetR and CE cooperatively repressed genes. The combination enriched glucagon/AMPK-linked and core metabolic pathways. In iBAT, CE dominated thermogenic and lipid-oxidation programs with minimal MetR contribution. In iWAT, MetR and CE acted largely additively with high concordance, enhancing fatty-acid degradation, PPAR signaling, thermogenesis, and TCA cycle pathways. In eWAT, robust co-dependent and synergistic differential expression emerged only with MetR+CE. ConclusionMetR is a genuine DIT stimulus that remodels metabolism in a tissue-specific manner. Our study provides a tissue-resolved transcriptomic resource that benchmarks diet-induced (MetR) against cold-induced thermogenesis and maps their interactions across liver, iBAT, iWAT, and eWAT.

Pharmacological administration of Fibroblast growth factor 21 (FGF21) alters food choice, including that it decreases the consumption of sucrose and other sweet tastants. Conversely, endogenous secretion of FGF21 by the liver is modulated by diet, such that plasma FGF21 is increased after eating foods that have a low dietary protein: total energy (P: E) ratio. Together, these findings suggest a strategy to promote healthy eating, in which the macronutrient content of a pre-load meal could reduce the later consumption of sweet desserts. Here, we tested the prediction that individuals eating a low P: E pre-load meal, and next offered a highly palatable sweet dessert, would eat less of the sugary snack compared to controls, due to increased FGF21 signaling. In addition to decreasing sweet intake, FGF21 increases the consumption of dietary protein. Thus, we predicted that individuals eating a low protein pre-load meal, and subsequently offered a very high-protein pellet as dessert or snack, would eat more of the high protein pellet compared to controls, and that this depends on FGF21. We tested this in C57Bl/6J, and liver-specific FGF21-null (FGF21{Delta}L) null male and female mice and littermate controls. Contrary to expectation, eating a low protein pre-load did not reduce the later consumption of a sweet solution in either males or females, despite robustly increasing plasma FGF21. Rather, eating the low protein pre-load increased later consumption of a high protein pellet. This was more apparent among males and was abrogated in the FGF21{Delta}L mice. We conclude that physiologic induction of hepatic FGF21 by a low protein pre-load is not sufficient to reduce later consumption of sweet dessert, though it effectively increases the subsequent intake of dietary protein in male mice.

To curb the obesity epidemic, it is imperative that we improve our understanding of the mechanisms controlling fat mass and body weight regulation. While great progress has been made in mapping the biological feedback forces opposing weight loss, the mechanisms countering weight gain remain less well defined. Here, we integrate a mouse model of intragastric overfeeding with a comprehensive evaluation of the regulatory aspects of energy balance, encompassing food intake, energy expenditure, and fecal energy excretion. To evaluate the role of adipose tissue thermogenesis in the homeostatic protection against overfeeding-induced weight gain, we exposed uncoupling protein 1 (UCP1) knockout (KO) mice to overfeeding. Our results confirm that 7 days of 150% overfeeding induces [~]11% weight gain and triggers a potent and prolonged reduction in voluntary food intake that drives body weight back to baseline following overfeeding. Overfeeding has no effects on energy expenditure, consistent with the observation that mice lacking UCP1 are not compromised in their ability to defend against overfeeding-induced weight gain. These data emphasize that whole-body energy expenditure and adipose thermogenesis are not key contributors to protection against overfeeding in mice. Lastly, we show that fecal energy excretion decreases in response to overfeeding, primarily driven by a reduction in fecal output rather than in fecal caloric content. In conclusion, these results challenge the prevailing notion that adaptive thermogenesis contributes to the defense against weight gain induced by overfeeding. Instead, the protection against enforced weight gain in mice is primarily linked to a profound reduction in food intake.

Whole-body estrogen receptor (ER) knockout mice develop hepatic steatosis; however, liver-specific ER knockout (LERKO) mice fail to recapitulate this susceptibility and maintain normal hepatic mitochondrial function. However, estrogen-mediated protection against hepatic steatosis is lost in LERKO mice following ovariectomy (OVX). Here, we tested whether loss of hepatic ER blunts estrogen modulation of hepatic mitochondrial respiratory capacity and mitochondrial proteome following ovariectomy (OVX). Sham or ovariectomy (OVX) surgery was performed in middle-aged female mice (36-40 weeks), followed by AAV injection to generate Control (Con; GFP) or LERKO mice (Cre). All mice were placed on a high-fat diet (HFD) for 10 weeks following surgery. Half of the OVX mice received 17-beta estradiol (E2) replacement (OVX+E2) for the last 4 weeks of HFD. OVX mice had greater body mass and adiposity, which was reversed by E2 replacement in both Con and LERKO mice. While E2 replacement reduced steatosis in both Con and LERKO OVX mice, the LERKO OVX mice maintained greater hepatic triglyceride content. E2 replacement promoted greater basal and ADP-stimulated (State 3) mitochondrial respiration in Con OVX but not in LERKO OVX mice under palmitate-supported conditions. Changes in mitochondrial respiration could not be attributed to altered responses to changes in energy demand (GATP) or to alterations in mitochondrial H2O2 production. Conversely, maximal coupled branched-chain amino acid-supported respiration was universally suppressed by E2 replacement. Proteomics analysis revealed E2-mediated reductions in hepatic mitochondrial energy transduction, with relatively minimal differences between Con and LERKO mice. In conclusion, post-ovariectomy estrogen treatment reduces steatosis in the absence of hepatic ER; however, triglyceride levels remain higher, and mitochondrial respiratory deficits persist despite similar proteomic signatures, suggesting that ER signaling is required for optimal estrogen hepatic responsiveness.

Fatty liver is characterised by the expansion of lipid droplets and is associated with the development of many metabolic diseases, including insulin resistance, dyslipidaemia and cardiovascular disease. We assessed the morphology of hepatic lipid droplets and performed quantitative proteomics in lean, glucose-tolerant mice compared to high-fat diet (HFD) fed mice that displayed hepatic steatosis and glucose intolerance as well as high-starch diet (HStD) fed mice who exhibited similar levels of hepatic steatosis but remained glucose tolerant. Both HFD and HStD-fed mice had more and larger lipid droplets than Chow-fed animals. We observed striking differences in liver lipid droplet proteomes of HFD and HStD-fed mice compared to Chow-fed mice, with fewer differences between HFD and HStD. Taking advantage of our diet strategy, we identified a fatty liver lipid droplet proteome consisting of proteins common in HFD- and HStD-fed mice. Likewise, a proteome associated with glucose tolerance that included proteins common in Chow and HStD but not HFD-fed mice was identified. Notably, glucose intolerance was associated with changes in the ratio of adipose triglyceride lipase (ATGL) to perilipin 5 (PLIN5) in the lipid droplet proteome, suggesting dysregulation of neutral lipid homeostasis in glucose-intolerant fatty liver, which supports bioactive lipid synthesis and impairs hepatic insulin action. We conclude that our novel dietary approach uncouples ectopic lipid burden from insulin resistance-associated changes in the hepatic lipid droplet proteome.

The mechanisms by which exercise modulate liver metabolism, a central regulator of systemic metabolism, are poorly understood. Leveraging data from MoTrPAC, we analyzed liver adaptations across 1, 2, 4, and 8 weeks of exercise in male and female rats using multi-omic approaches. Female livers displayed a progressive increase in oxidative phosphorylation (OXPHOS) complexes (at the protein level), while male livers showed an increase in acetylation of OXPHOS, TCA cycle, and fatty acid oxidation enzymes. Exercise also enhanced liver cholesterol and bile acid synthesis, reducing liver lipid metabolites in males after 8 weeks of exercise. Male rats had higher fecal cholesterol and cholic acid levels, indicating a sex-specific mechanism of lipid excretion with exercise. Moreover, 8 weeks of training reduced markers related to hepatic stellate cell activation and fibrosis in both sexes. This study highlights the sexual dimorphic and temporal molecular signatures by which exercise modulates liver metabolism to provide hepatoprotective effects.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homolog ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

Skeletal muscle mitochondrial fatty acid (FA) overload in response to chronic overnutrition is a prominent pathophysiological mechanism in obesity-induced metabolic disease. Increased disposal of FAs is therefore an attractive strategy for intervening in obesity and related disorders. Skeletal muscle uncoupling protein 3 (UCP3) activity is associated with increased FA oxidation and antagonizes weight gain in mice on obesogenic diets, but the mechanisms involved are not clear. Here, we show that UCP3 forms a direct, FA-stimulated, mitochondrial matrix-localized complex with the auxiliary unsaturated FA-metabolizing enzyme, {Delta}3,5-{Delta}2,4dienoyl-CoA-isomerase (ECH1). Expression studies in C2C12 myoblasts that functionally augments state 4 (uncoupled) respiration and FA oxidation in skeletal myocytes.\n\nMechanistic studies indicate that ECH1:UCP3 complex formation is likely stimulated by FA import into the mitochondria to enhance uncoupled respiration and unsaturated FA oxidation in mouse skeletal myocytes. In order to characterize the contribution of ECH1-dependent FA metabolism in NST, we generated an ECH1 knockout mouse and found that these mice were severely cold intolerant, despite an up-regulation of UCP3 expression in SKM. These findings illuminate a novel mechanism that links unsaturated FA metabolism with mitochondrial uncoupling and non-shivering thermogenesis in SKM.