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High-sugar diets cause human metabolic diseases, yet several bird lineages convergently adapted to feeding on sugar-rich nectar or fruits. We investigated the underlying molecular mechanisms in hummingbirds, parrots, honeyeaters, and sunbirds by generating nine new genomes and 90 tissue-specific transcriptomes. Comparative screens revealed an excess of repeated selection in both protein-coding and regulatory sequences in sugar-feeding birds, suggesting reuse of genetic elements. Sequence or expression changes in sugar-feeders affect genes involved in blood pressure regulation and lipid, amino acid, and carbohydrate metabolism, with experiments showing functional changes in honeyeater hexokinase 3. Show less

Neuropathic pain (NP), a chronic disorder caused by somatosensory nervous system lesions, severely impairs the quality of life. Microglial metabolic reprogramming and neuroinflammation drive NP progression. Although ChREBP (key metabolic regulator) protects against NP, its specific mechanisms remain unclear. NP rat model was established via spared nerve injury (SNI) surgery, and mechanical allodynia was evaluated using Von Frey tests. ChREBP expression in microglia was detected through immunofluorescence, RT-qPCR, and western blot. Functional studies involved ChREBP knockdown/overexpression to assess effects on microglial polarization, neuroinflammation, neuronal excitability, pain behaviors, and fatty acid metabolism. Mechanisms were explored via dual-luciferase reporter and chromatin immunoprecipitation assays. Mechanical pain thresholds were significantly decreased on the ipsilateral side after SNI. ChREBP was upregulated in SDH microglia after SNI and in LPS-stimulated microglia in vitro. ChREBP knockdown inhibited anti-inflammatory microglial polarization, exacerbated neuroinflammation, and aggravated pain. Conversely, ChREBP overexpression promoted the anti-inflammatory phenotype, suppressed neuroinflammation, and alleviated pain. ChREBP enhanced microglial fatty acid oxidation and energy metabolism. Mechanistically, ChREBP bound to the TFBS1 site on the PGC-1α promoter to activate its transcription. PGC-1α overexpression rescued the impairments caused by ChREBP knockdown, including reduced fatty acid oxidation, suppressed anti-inflammatory polarization, elevated inflammatory factors, and increased neuronal excitability. The protective effects of ChREBP were attenuated by the fatty acid oxidation inhibitor Etomoxir. ChREBP alleviates NP by enhancing microglial fatty acid oxidation and anti-inflammatory phenotype via PGC-1α transcriptional activation, revealing a novel metabolic-immune axis for potential NP therapy. Show less

Glyphosate, one of the most widely used herbicides worldwide, has raised significant concerns regarding its potential involvement in hepatotoxicity and molecular changes associated with liver cancer biology. These concerns highlight the need to better understand its underlying molecular mechanisms in hepatoma cells. Emerging evidence suggests that glyphosate exposure may increase the risk of liver cancer and chronic liver disease. However, the precise molecular alterations and promising biomarkers associated with glyphosate-induced hepatic toxicity and disease remain largely unexplored. In this study, an RNA-Seq-based in silico systems biology approach was employed to elucidate glyphosate-induced differential transcriptional profiling in hepatoma cells. This analysis revealed significant transcriptional profiling characterized by the upregulated hub genes Show less

Excessive fructose intake is strongly associated with metabolic diseases, with the carbohydrate response element-binding protein (ChREBP) playing a key role in its metabolism, particularly in renal tubules. However, the role of its active form, ChREBP-β, was previously unclear. In this study, ChREBP-β overexpression and ChREBP knockout mouse models were utilized to investigate the effects of excessive fructose intake in vivo. In addition, primary renal tubular epithelial cells from mice and human kidney-2 (HK2) cells were applied for further validation in vitro. We found that ChREBP-β leads to increased transcription to mediate endoplasmic reticulum stress and mitochondrial dysfunction, which ultimately impairs renal function. Our findings underscore the critical role of ChREBP-β in fructose-related renal disorders. Show less

Metabolic dysfunction-associated steatotic liver disease (MASLD) has become highly prevalent worldwide, largely as a consequence of the global obesity epidemic. This research endeavors to elucidate the role and molecular mechanisms of hepatic glycogen synthase (GS) in MASLD progression. Published transcriptomic data reveal a downward trend in GYS2 gene expression in patients with obesity, MASLD, and metabolic dysfunction-associated steatohepatitis. In mouse models of MASLD, GYS2 gene or protein expression was downregulated, consistent with the human data. Here, GS-deficient mice fed with a normal diet displayed hepatic lipid accumulation and liver injury, whereas hepatic steatosis progression and inflammation were aggravated in mice fed with a high-fat diet. Loss of hepatic GS stimulated fatty acid de novo synthesis through carbohydrate-response element-binding protein and AKT-mTOR1-sterol regulatory element-binding protein 1 axis pathways. In GS-deficient mice, lipid accumulation in the hepatocytes significantly decreased when carbohydrate-response element-binding protein and sterol regulatory element-binding protein 1 levels were suppressed to levels comparable to those of cytotoxic T lymphocyte hepatocytes. Forced expression of hepatic GS by adeno-associated virus in db/db mice ameliorated lipid accumulation in male mice. Our findings provide proof of concept whereby targeting glycogen metabolism in hepatocytes may offer potential therapeutic avenues to treat MASLD. Show less

Overactivation of hepatic de novo lipogenesis (DNL) contributes to fatty liver disease. Although glucose and fructose strongly promote DNL, diary-rich galactose is only weakly lipogenic. However, whether and how it regulates hepatic DNL remains unclear. In this study, we investigated whether low-dose galactose supplementation attenuates glucose- or fructose-induced DNL activation and protects against fatty liver diseases driven by DNL overactivation, such as alcohol-associated liver disease (ALD). In this study, we used integrated hepatocyte and mouse models to assess hepatic DNL and related signaling under high-glucose or high-fructose conditions, with or without low-dose galactose. Pharmacological and genetic interventions targeting the Leloir and hexosamine biosynthetic pathways (HBP) defined underlying mechanisms. For in vivo validation, male C57BL/6 mice were fed an isocaloric control or ethanol-containing diet for 4 wk. We found that glucose engages the HBP-mTORC1-SREBP-1c axis to stimulate hepatic DNL, whereas fructose acts predominantly through carbohydrate-responsive element-binding protein (ChREBP). Low-dose galactose selectively suppressed glucose-induced hepatic fat accumulation, concomitant with the inhibition of the HBP-mTORC1-SERBP-1c pathway. These effects required an intact Leloir pathway for galactose metabolism and were not observed with fructose. In alcohol-fed mice, hepatic HBP-mTORC1-SREBP-1c signaling was markedly upregulated, contributing to steatosis and liver injury. Replacing even a small fraction of dietary glucose with galactose normalized these alterations, attenuating hepatic lipid accumulation and injury without altering systemic glucose levels. In conclusion, glucose-induced hepatic lipogenesis involves the HBP-mTORC1-SREBP-1c pathway, which is also activated during chronic alcohol exposure. Low-dose galactose, obtainable from dairy sources, attenuates this pathway, thereby limiting excessive lipogenesis and protecting against early-stage ALD. Show less

Williams-Beuren Syndrome (WBS), a neurodevelopmental disorder caused by a heterozygous microdeletion at chromosome 7q11.23, is characterized by hypersociability and enhanced affective empathy. However, the specific genetic and neural mechanisms within the WBS locus underlying this elevated empathic response remain unknown. Here, we investigated empathy-related behaviors, including observational fear and allogrooming, in WBS mouse models harboring a deletion within the conserved syntenic region on mouse chromosome 5. We demonstrate that WBS mice exhibited emotional contagion and prosocial consolation behaviors comparable to their wild-type controls. Furthermore, WBS mice with single-gene deletions of the cortex-enriched genes Abhd11, Limk1, Mlxipl, and Stx1a also showed unaffected empathic freezing behavior. Collectively, our findings suggest that the enhanced empathic responsiveness reported in individuals with WBS may be influenced by reduced social inhibition toward others, while acknowledging that limitations of current rodent behavioral assays preclude definitive conclusions regarding primary neural mechanisms of empathy. Show less

Genetic variants near LYPLAL1 are associated with Metabolic dysfunction-Associated Steatotic Liver Disease (MASLD) in humans, but their impact on LYPLAL1 function is unknown. We identified LYPLAL1 loss-of-function variants from UK BioBank (UKBB) whole-exome sequencing data that had AlphaMissense or GPN-MSA scores in the top 20% of LYPLAL1 variants for being disruptive. We aggregated these variants and carried out burden analysis for effects on MRI Proton Density-Fat Fraction (MRI-PDFF) and ICD-based MASLD in UKBB. Rare loss-of-function LYPLAL1 variants were associated with reduced MRI-PDFF and ICD diagnosed MASLD across sexes. We used CRISPR to knockout and overexpress LYPLAL1 in human hepatoma cells (HuH-7), measuring lipid content, lipid uptake/export, and changes in de novo lipogenesis and mitochondrial β-oxidation. LYPLAL1 subcellular localization was determined by overexpressing LYPLAL1-HA tagged protein. We purified GST tagged human LYPLAL1 protein and conducted in vitro tests for esterase and depalmitoylase activity. Knocking out LYPLAL1 reduced triglycerides biochemically as well as lipid intensity after oleic (18:1, n-9) acid treatment. LYPLAL1 KO cells had increased expression of PPARα and MLXIPL, increased mitochondrial β-oxidation, and reduced capacity to both import fatty acids (FAs) and export lipoproteins. Overexpression of LYPLAL1 increased lipid droplet accumulation and decreased PPARα and MLXIPL. LYPLAL1-HA is partly localized to mitochondria when treated with oleic acid. Biochemical analyses showed that LYPLAL1 has strong esterase activity but lacks depalmitoylase activity. Reduction of LYPLAL1 esterase function likely increases β-oxidation of FAs in mitochondria through PPARα and MLXIPL and decreases FA import to protect against lipid accumulation in human liver cancer cells. Together, our results indicate that LYPLAL1 loss-of-function protects against MASLD in Europeans and in vitro, LYPLAL1 is an esterase for short-chain substrates which is involved in the regulation of mitochondrial β-oxidation and uptake of fatty acids, influencing lipid accumulation in the liver. Show less

Renal tubulointerstitial fibrosis (TIF) is a hallmark pathological feature of diabetic kidney disease (DKD). This study investigates the role and molecular mechanisms of retinol saturase (RetSat) in DKD-associated TIF. RetSat expression was assessed in renal tissues from DKD patients and mice and correlated with the severity of TIF. Functional experiments were conducted RetSat expression was significantly up regulated in the renal tissues of both DKD patients and mice, correlating with the deterioration of TIF. These findings indicate that RetSat promotes TIF in DKD by disrupting the Smurf2-ChREBP ubiquitination axis, highlighting RetSat as a promising therapeutic target for DKD. Show less

Plin4 is transcriptionally regulated by peroxisome proliferator-activated receptor gamma and is primarily expressed in white adipose tissue (WAT). We found that expression of Plin4 is elevated in the liver upon prolonged feeding with an obesogenic diet containing saturated fat, fructose, and cholesterol (Western diet). To investigate the functional role of Plin4 in energy metabolism, we generated Plin4 Show less

Diabetes is an increasingly prevalent global disease and is often accompanied by sarcopenia, particularly in older adults. While insulin resistance is a well-known contributor to muscle loss in diabetes, the role of glucose signaling in diabetic skeletal muscle atrophy, particularly under insulin-deficient conditions, remains poorly understood. This study aimed to elucidate the pathophysiological role of the carbohydrate-responsive element-binding protein (ChREBP), a glucose-sensing transcription factor encoded by the Chrebp gene in mice, in diabetic sarcopenia by generating Chrebp-deficient, insulin-deficient Ins2Akita/+ mice. We evaluated Chrebp +/+, Chrebp -/-, Ins2Akita/+ /Chrebp +/+, and Ins2Akita/+ /Chrebp -/- mice for muscle strength, endurance, survival, body composition, and muscle histology. Skeletal muscles were analyzed for gene expressions related to anabolic and catabolic pathways. We found that Ins2Akita/+ /Chrebp -/- mice exhibited significant reductions in body weight, grip strength, survival, and skeletal muscle mass - particularly in the tibialis anterior, soleus, gastrocnemius, and quadriceps - compared to Ins2Akita/+ controls, despite similar hyperglycemia. Histological analysis revealed a smaller mean muscle fiber size and reduced cross-sectional area of type 2A and 2B fibers, without changes in fiber-type composition. Furthermore, Igf-1 expression was suppressed, while the atrophy marker Fbxo32/Atrogin-1 was upregulated. These findings demonstrate that Chrebp deletion exacerbates muscle atrophy and frailty in insulin-deficient mice, underscoring a key role for ChREBP-mediated glucose signaling in maintaining muscle mass under diabetic conditions. The Ins2Akita/+ /Chrebp -/- model provides a valuable platform for exploring diabetic sarcopenia mechanisms and potential therapeutic targets. Show less

Metabolically Dysfunctional-Associated Steatotic Liver Disease (MASLD) affects both metabolically healthy obese (MHO) individuals and metabolically unhealthy lean (MUL) individuals. Key genes linked to liver dysfunction, such as Show less

Tumor metabolic reprogramming has been recognized as a critical determinant in tumor development and cancer immunotherapy response. Aberrant choline metabolism is emerging as a defining hallmark of cancer. In this study, we found that carbohydrate-responsive element-binding protein (ChREBP)-mediated choline deprivation induced tumor-associated macrophage (TAM) reprogramming and maintained an immunosuppressive tumor microenvironment. Mechanistically, ChREBP interacted with SP1 to increase the expression of immunosuppressive chemokines CCL2 and CCL7 and choline transporter SLC44A1. As such, high CCL2 and CCL7 expression promoted recruitment of TAMs. Tumor cells with high SLC44A1 levels competed with M1-like TAMs for choline, inhibiting cGAS/STING signaling and promoting the repolarization of M1-like to M2-like macrophages. Clinically, ChREBP-SP1-choline metabolism axis expression was associated with poor clinical outcome in colorectal cancer. Thus, the study identified the interplay between tumors and TAMs via choline competition as a previously unknown immune evasion mechanism in the tumor microenvironment and proposes ChREBP as a potential immunotherapeutic target in cancer. ChREBP induces a choline-deprived tumor microenvironment and promotes chemokine secretion to facilitate immune evasion, suggesting targeting ChREBP as a therapeutic approach to improve the efficacy of immunotherapy. Show less

Type 1 diabetes mellitus is a major risk factor for both sarcopenia and osteoporosis, primarily due to the body's inability to utilize glucose as a result of insulin deficiency. Impairments in insulin and glucose signaling can accelerate the decline in muscle and bone health. To investigate this interaction, we examined whether insulin deficiency exacerbates muscle and bone deterioration in Show less

Alzheimer's disease (AD) is a common neurodegenerative condition involving a complex blend of disturbances in synaptic development and maintenance, neurovascular cross-talk, ionic and nutrient transport, and mitochondrial metabolism. The precise molecular profile of AD onset with insight for major pathological contributors remains unclear with corresponding impedances in therapeutic development. The current study sought two objectives, as (i) to resolve the molecular pathogenesis from cognitive impairment to the onset of AD-like neuropathology and (ii) whether the novel agent cannabidiol (CBD), noted for its neuroprotective effects, influences the molecular transition associated with AD onset. Dietary CBD was administered daily (80-100 mg/kg/day) in male There were >1,000 differentially expressed markers of AD onset, whereby >75% were either eliminated or reversed in the direction of expression in response to CBD. Signaling pathways encompassed synaptic development and plasticity (e.g., Foxp2), neurovascular interactions (Smad9, Angptl6), receptors and ion channels (Gria4, Chrna2, Rgs7/Rgs7bp), mitochondrial genes (Ndufa7, Cox7a2), immunity (Ncr1), oxidation-reduction (Esr1), lipid synthesis (Fasn, ApoE), and carbohydrate metabolism (Mafa, Mlxipl). As potentially addressable with CBD treatment, AD onset represents molecular integration of neurovascular interactions, channelopathies, metabolic disturbances, and aberrations in developmental genes with involvement of major pathological contributors such as inflammation, oxidative signaling, dyslipidemia, and insulin resistance. Show less

To determine whether insulin controls hepatic de novo lipogenesis (DNL) through an HCF-1-dependent modulation of ChREBP that is distinct from the canonical SREBP1c pathway. AML-12 mouse hepatocytes were subjected to 10 μg/mL insulin and 25 mM glucose for 6 h. IRβ or HCF-1 was knocked down with lentiviral shRNA (≈80 % efficiency). Lipid droplets were quantified by Nile-Red staining; mRNA and protein levels were measured by RT-qPCR, Western blot, immunofluorescence and RNA-seq. Co-immunoprecipitation was used to test complex formation. Insulin reduced lipid accumulation and suppressed ChREBP protein and its nuclear localization in AML-12 hepatocytes without altering SREBP1c. Knock-down of IRβ or HCF-1 abolished insulin-mediated ChREBP suppression, increased lipid droplets and up-regulated lipogenic genes. HCF-1 co-immunoprecipitated with IRβ, indicating formation of an insulin-responsive IRβ/HCF-1 complex that restrains ChREBP-driven lipogenesis. We identify an IRβ/HCF-1/ChREBP regulatory node in hepatocytes that can repress lipogenic genes independently of SREBP1c. The axis constitutes a testable target for understanding selective insulin action on hepatic lipid metabolism and for future in-vivo studies of fatty-liver disease. Show less

Carbohydrate-responsive element binding protein (ChREBP) and Max-like protein X (MLX) are key Mondo Family Proteins (MFPs) acting as transcription factors. They are known to couple intracellular sugar levels with carbohydrate and lipid metabolism by regulating glucose-responsive gene expression. MondoA regulates lipid metabolism and insulin signaling in addition to controlling glucose uptake, whereas ChREBP primarily regulates Show less

The Carbohydrate-Responsive Element-Binding Protein (ChREBP) is a glucose-sensitive transcription factor that regulates the carbohydrate and lipid metabolism. We investigated its cell-type-specific role in hepatocarcinogenesis using a chemically induced mouse model. Additionally, we examined the functions of its isoforms, ChREBPα and ChREBPβ. After the diethylnitrosamine (DEN) administration, we analyzed hepatocellular adenomas and carcinomas in systemic ChREBP-knockout (KO), hepatocyte-specific ChREBP-KO (L-KO), and wildtype (WT) mice at 4, 12, and 36 weeks using histology, morphometry, proliferation measurements, immunohistochemistry, a Western blot, and a quantitative PCR. Tumors developed 36 weeks after the DEN administration in 27% of WT mice but less frequently in KO (18%) and L-KO (9%) mice. However, preneoplastic foci were less common in KO mice but not in L-KO mice (39% vs. 9%; Show less

HNF1A-MODY, the most prevalent form of monogenic diabetes, displays incomplete penetrance, indicating the involvement of other environmental and genetic factors in the disease etiology. Currently, it is largely unknown what the influence of environmental factors, such as toxins or diet, is on HNF1A-MODY onset and progression. Here we address this issue by exploring the impact of diet on islet and insulin-secreting beta-cells in the context of HNF1A mutation. Transgenic mice allowing the specific Hnf1a mutation in insulin-secreting beta-cells were exposed to four distinct dietary regimens including combinations of high-fat diet and caloric restriction. In vitro stem cell islets bearing the HNF1A Hnf1a-deficient beta-cells exhibited high sensitivity to dietary cues. Exposure to a high-fat diet exacerbated the glucose regulation defects, while caloric restriction significantly improved blood glucose levels in vivo, without perturbing islet architecture. The high-throughput methods identified changes in the Hnf1a-deficient beta-cells proteome landscape, involving conserved critical regulators of metabolic and growth processes, such as the Carbohydrate Response Element Binding Protein (Chrebp/Mlxipl) and ATP citrate lyase (Acly) among others. This study hallmarks the important impact of diet on Hnf1a-deficient beta-cells, stemming new therapeutic perspectives, such as future diet management approaches. Show less

White adipocyte differentiation or adipogenesis requires coordination of metabolic sensing and transcriptional modifications to orchestrate lipid storage. Creatine and its kinases are implicated in adipose energy buffering, but the roles of cytosolic (CKB) and mitochondrial (CKMT2) creatine kinases in adipogenesis are unclear. We find that both CKB and CKMT2 are progressively upregulated during differentiation. Functional studies show that CKB restrains de novo lipogenesis (DNL) by limiting activation of carbohydrate-responsive element-binding protein (ChREBP), a key regulator of lipogenic genes. Mechanistically, CKB interacts with AKT and regulates its activation in response to insulin. Loss of CKB causes persistent AKT-mTORC1 signaling, increases glycolytic flux, and enhances ChREBP activation, thereby promoting glucose-derived lipid synthesis. Thus, CKB acts as a metabolic rheostat linking creatine-kinase activity to insulin signaling and nutrient-responsive transcription. We propose a CKB-AKT-ChREBP regulatory axis that contributes to metabolic remodeling and lipid homeostasis during adipocyte differentiation. Show less

Citrin deficiency (CD) is caused by the inactivation of SLC25A13, a mitochondrial membrane protein required to move electrons from cytosolic NADH to the mitochondrial matrix in hepatocytes. People with CD do not like sweets. Here we show that SLC25A13 loss causes the accumulation of glycerol-3-phosphate (G3P), which activates the carbohydrate response element-binding protein (ChREBP) to transcribe FGF21, which acts in the brain to restrain intake of sweets and alcohol and to transcribe key genes driving lipogenesis. Mouse and human data suggest that G3P-ChREBP is a mechanistic component of the Randle Cycle that contributes to metabolic-dysfunction-associated steatotic liver disease and forms part of a system that communicates metabolic states from the liver to the brain in a manner that alters food and alcohol choices. The data provide a framework for understanding FGF21 induction in varied conditions, suggest ways to develop FGF21-inducing drugs and suggest potential drug candidates for lean metabolic-dysfunction-associated steatotic liver disease and support of urea cycle function in CD. Show less

The association between obesity and cholelithiasis has been identified. However, the causal relationship between age-specific childhood obesity and adult cholelithiasis remains unclear. In addition, the biological basis for the association between childhood obesity and adult cholelithiasis is poorly understood, which poses a challenge for preventing adult cholelithiasis in specific biological pathways. Summary statistics of genome-wide association studies (GWASs) of childhood age-specific body mass index (BMI) at 12 time points and adult cholelithiasis derived from FinnGen were used in this study, with the former covering data from birth to 8 years. Linkage disequilibrium score regression (LDSC) analyses were used to assess the genetic correlations of age-specific childhood BMI to cholelithiasis. Two-sample Mendelian randomization (MR) and multivariable Mendelian randomization (MVMR) analyses were utilized to explore the causal associations. As downstream analyses, summary-based Mendelian randomization (SMR) analyses, transcriptome-wide association studies (TWAS), and Bayesian colocalization were conducted to discover the shared transcriptomic signals. The GWAS summary statistics of cholelithiasis from the UK Biobank were used for sensitivity analyses. LDSC analyses revealed significant genetic correlations between 11 age-specific childhood BMIs and adult cholelithiasis (except for birth BMI). Two-sample MR and MVMR analyses indicated causal relationships between birth BMI and BMI at 8 months, 1.5 years, 7 years, and 8 years after birth and adult cholelithiasis. SMR, TWAS, and colocalization analyses identified MLXIPL as the strongest overlapping signal between age-specific BMI and adult cholelithiasis. This study provides new evidence on the relationships between childhood obesity and adult cholelithiasis, highlighting the role of early intervention for obesity in childhood at key time points. MLXIPL gene expression was identified as a potential biological pathway, suggesting potential therapeutic targets and precise intervention strategies for childhood obesity and adult cholelithiasis. Show less

Brown adipose tissue (BAT) comprises a heterogeneous population of adipocytes and non-adipocyte cell types. To characterize these cellular subpopulations and their adaptation to cold, we performed single-nucleus mRNA-sequencing (snRNA-seq) on interscapular BAT from mice maintained at room temperature or exposed to acute (24h) or chronic (10 days) cold (6 °C). To investigate the role of the de novo lipogenesis (DNL)-regulating transcription factor carbohydrate response element-binding protein (ChREBP), we analyzed control and brown adipocyte-specific ChREBP knockout mice. We identified different cell populations, including seven brown adipocyte subtypes with distinct metabolic profiles. One of them highly expressed ChREBP and DNL enzymes. Notably, these lipogenic adipocytes were highly sensitive to acute cold exposure, showing a marked depletion in BAT of control mice that was compensated by other brown adipocyte subtypes maintaining DNL. Chronic cold exposure resulted in an expansion of basal brown adipocytes and adipocytes putatively derived from stromal and endothelial precursors. In ChREBP-deficient mice, lipogenic adipocytes were almost absent under all conditions, identifying the transcription factor as a key determinant of this adipocyte subtype. Detailed expression analyses revealed Ttc25 as a specific marker of lipogenic brown adipocytes and as a downstream target of ChREBP. Furthermore, pathway and cell-cell interaction analyses implicated a Wnt-ChREBP axis in the maintenance of lipogenic adipocytes, with Wnt ligands from stromal and muscle cells providing instructive cues. Our findings provide a comprehensive atlas of BAT cellular heterogeneity and reveal a critical role for ChREBP in lipogenic adipocyte identity, with implications for BAT plasticity and metabolic function. Show less

Recent advancements in transcriptomic analysis, combined with single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, have deepened our understanding of the tumor microenvironment and cellular heterogeneity, laying the groundwork for personalized therapies. The aim of this research is to explore the heterogeneity of tumor cells in colorectal cancer (CRC) and evaluate their prognostic value in different therapeutic contexts, emphasizing the impact of tumor cell heterogeneity on disease progression. scRNA-seq alongside spatial transcriptomics was employed to analyze the heterogeneity of tumor cells in CRC, the spatial distribution of tumor cells, and their interactions with the microenvironment. We identified nine distinct tumor cell subtypes, with MLXIPL + neoplasm prevalent in advanced CRC, while ADH1C + and MUC2 + neoplasms were more common in early-stage CRC. MLXIPL + neoplasm was significantly associated with chemotherapy and targeted therapy efficacy. Analysis of spatial transcriptomics indicated that MLXIPL + neoplasm is located in the core area of the tumor cells. We developed a 13-gene prognostic signature (PS) using machine learning algorithm (StepCox backward), which predicts the prognosis of CRC patients. Furthermore, the patients with low PS score demonstrated higher immune cell infiltration and immune regulatory factors, suggesting enhanced immune surveillance and treatment response. The findings highlight the critical role of tumor cell heterogeneity in CRC progression and treatment response, suggesting the need for personalized therapeutic strategies targeting different subpopulations. The constructed PS demonstrates significant clinical application value in predicting patient prognosis. Show less

This study aims to investigate the in vitro mechanisms underlying the beneficial effects of puerarin on hepatic insulin resistance(IR) based on the carbohydrate response element-binding protein(ChREBP)/peroxisome proliferator-activated receptor(PPAR)α/PPARγ axis involved in glucose and lipid metabolism. An IR-HepG2 cell model was established by treating cells with dexamethasone for 48 h, and the cells were then treated with 10, 20, and 40 μmol·L~(-1) puerarin for 24 h. Glucose levels and output in the extracellular fluid were measured by the glucose oxidase method, while cell viability was assessed by the cell counting kit-8(CCK-8) assay. The adenosine triphosphate(ATP) content and glycogen synthesis were evaluated through chemiluminescence and periodic acid-Schiff staining, respectively. Western blot was employed to quantify the protein levels of forkhead box protein O1(FoxO1), phosphorylated forkhead box protein O1 [p-FoxO1(Ser256)], glucagon, phosphofructokinase, liver type(PFKL), pyruvate kinase L-R(PKLR), pyruvate dehydrogenase complex 1(PDHA1), insulin receptor substrate 2(IRS2), phosphatidylinositol 3-kinase p85(PI3KR1), phosphorylated protein kinase B [p-Akt(Thr308)], glycogen synthase(GYS), glycogen phosphorylase, liver type(PYGL), adiponectin(ADPN), ChREBP, PPARα, and PPARγ. Additionally, the protein levels of acetyl-CoA carboxylase 1(ACC1), phosphorylated ATP citrate lyase [p-ACLY(Ser455)], sterol regulatory element binding protein 1c(SREBP-1c), peroxisome proliferator-activated receptor gamma coactivator 1α(PGC1α), carnitine palmitoyltransferase 1α(CPT1α), and glucagon receptor(GCGR) were also determined. Immunofluorescence was employed to visualize the expression and nuclear location of ChREBP/PPARα/PPARγ. Furthermore, quantitative PCR with the antagonists GW6471 and GW9662 was employed to assess Pparα, Pparγ, and Chrebp. The findings indicated that puerarin effectively reduced both the glucose level and glucose output in the extracellular fluid of IR-HepG2 cells without obvious effect on the cell viability, and it increased intracellular glycogen and ATP levels. Puerarin down-regulated the protein levels of FoxO1 and glucagon while up-regulating the protein levels of p-FoxO1(Ser256), PFKL, PKLR, PDHA1, IRS2, PI3KR1, p-Akt(Thr308), GYS, PYGL, ADPN, ACC1, SREBP-1c, p-ACLY(Ser455), PGC1α, CPT1α, and GCGR in IR-HepG2 cells. Furthermore, puerarin up-regulated both the mRNA and protein levels of ChREBP, PPARα, and PPARγ and promoted the translocation into the nucleus. GW6471 was observed to down-regulate the expression of Pparα while up-regulating the expression of Chrebp and Pparγ. GW9662 down-regulated the expression of Pparγ while up-regulating the expression of Pparα, with no significant effect on Chrebp. In summary, puerarin activated the hepatic ChREBP/PPARα/PPARγ axis, thereby coordinating the glucose and lipid metabolism, promoting the conversion of glucose to lipids to exert the blood glucose-lowering effect. Show less

We recently reported that the long non-coding RNA TRIBAL/TRIB1AL was required to sustain key hepatocyte functions. Here, we identify HepaRG cells as a model for studying TRIBAL and provide additional validation and functional insights. In contrast to HepG2 and HuH-7 cells, differentiated HepaRG cells showed similarities to primary hepatocytes in response to TRIBAL suppression. TRIBAL suppression was associated with reduced HNF4A and MLXIPL abundance in hepatocytes and HepaRG cells. TRIBAL targeting using a panel of cognate antisense oligonucleotides confirmed specificity. A comparison of TRIBAL-suppressed hepatocyte and HepaRG transcriptomics identified extensive functional overlap. Biological ontologies associated with key hepatic metabolic functions were predicted to be inhibited in both models. Comparative analyses with TRIB1-suppressed HepaRG cells, a central metabolic regulator vicinal to TRIBAL, also revealed extensive functional congruence with TRIBAL. Interestingly, TRIBAL transduction failed to restore function in TRIBAL-suppressed cells, which may be linked to structural differences, as supported by contrasting RNAse R sensitivities between the endogenous and transduced forms. In summary, these findings support the use of HepaRG cells as an experimental model to study TRIBAL and underscore its importance in regulating key hepatocyte genes essential for metabolic function. Show less

Brown adipose tissue (BAT), a thermogenic tissue that plays an important role in systemic energy expenditure, has histological and functional sex differences. BAT thermogenic activity is higher in female mice than in male mice. However, the molecular mechanism underlying this functional sex difference has not been fully elucidated. Herein, we demonstrate the role and mechanism of PGC-1α in this sex difference. Inducible adipocyte-specific PGC-1α knockout (KO) mice display mitochondrial morphological defects and decreased BAT thermogenesis only in females. Expression of carbohydrate response-element binding protein beta (Chrebpβ) and its downstream de novo lipogenesis (DNL)-related genes are both reduced only in female KO mice. BAT-specific knockdown of ChREBPβ displays decreased DNL-related gene expression and mitochondrial morphological defects followed by reduced BAT thermogenesis in female wild-type mice. Lipidomics reveals that, PGC-1α increases ether-linked phosphatidylethanolamine (PE) and cardiolipin(18:2) Show less