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The relation between hepatic ChREBP level and insulin sensitivity remains equivocal. Our study, however, provides compelling evidence that hepatic ChREBP depletion can significantly enhance insulin sensitivity in high-fat and sucrose-fed mice. We have identified that transcriptional induction of hepatic PTEN is driven by ChREBP. Mechanistically, two critical stimuli are elicited in the hepatic ChREBP knockdown condition. The PTEN level is reduced for one stimulus, thereby promoting hepatic insulin sensitivity. The second stimulus, where reduced hepatic PTEN leads to the enhanced release of FGF21, spreads systemic insulin sensitivity. These findings identify hepatic ChREBP as a critical modulator of systemic insulin signaling and suggest that ChREBP downregulation may lead to protection against insulin resistance. Building on this, our molecular dynamics simulation analysis has led to the discovery of a small molecule, Quercetin, that sequesters ChREBP in the cytosol. We report that Quercetin treatment can sequester ChREBP in the cytosol and abrogate high-fat and sucrose-fed-mediated ChREBP nuclear translocation, thereby mimicking the insulin-sensitizing abilities of the hepatic ChREBP knockdown condition. These findings have significant therapeutic implications, suggesting that liver-selective downregulation of ChREBP could protect against systemic insulin resistance that frequently develops early in the pathogenesis of NAFLD and T2DM. 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

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

Ethanol rapidly stimulates the liver to synthesize the hormone fibroblast growth factor 21 (FGF21), which then acts on the brain to elicit a multifaceted protective response. We show that in mice, this induction of FGF21 occurs at the level of gene transcription and is regulated by two byproducts of ethanol metabolism, glycerol-3-phosphate (G3P) and acetyl-CoA. Using cell-based reporter and thermal shift binding assays, we show that G3P binds to a conserved domain and activates the transcription factor carbohydrate-responsive element-binding protein (ChREBP), which regulates the Show less

Glucocorticoids are potent anti-inflammatory agents that are frequently used to treat inflammatory and autoimmune diseases. Chronic glucocorticoid treatment, however, causes unwanted adverse effects such as hypertriglyceridemia and hepatic steatosis. Here we showed that reducing the expression of sphingosine-1-phosphate receptor 2 (S1PR2) in mice liver reduced chronic glucocorticoid exposure induced triglyceride accumulation in the liver and the plasma. Chronic glucocorticoid treatment increased the recruitment of sterol regulatory element-binding protein 1c (Srebp1c) to the sterol regulatory element of mouse fatty acid synthase (Fasn) gene. This response was attenuated in hepatic S1PR2 knockdown mice. Chronic glucocorticoid treatment also increased the recruitment of carbohydrate response element binding protein (ChREBP) to the carbohydrate response elements (ChoREs) of lipogenic and glycolytic genes. This response was partially reduced in hepatic S1PR2 knockdown mice. Reducing hepatic ChREBP expression reduced the expression of Pklr, Me1, and Fasn. However, long-term glucocorticoid induced triglyceride accumulation in the liver and the plasma were not affected whereas the hepatic lactate levels were decreased. Thus, ChREBP plays a major role in chronic glucocorticoid induced glycolysis whereas its role in hypertriglyceridemia and hepatic steatosis was modest. Overall, this study demonstrated that hepatic S1PR2 signaling plays a partial but significant role in chronic glucocorticoid exposure-activated Srebp1c and ChREBP which promote lipogenesis and glycolysis, respectively. Show less

Sustained activation of hepatic stellate cells (HSCs) drives liver fibrosis in response to chronic liver injury and inflammation. It is reported that profibrogenic signals released from stressed/injured hepatocytes evoke fibrogenic responses in HSCs. However, intrahepatocyte players that modulate such cell-to-cell communications remain poorly defined. In this study, hepatic ChREBPα is found to be reduced in mouse models of chemical-induced liver fibrosis as well as in three groups of human patients with liver fibrosis. Chrebpα-LKO mice are highly sensitive to both chemical (CCL4 and TAA) and bile duct ligation (BDL)-induced liver injury and developed more advanced liver fibrosis without affecting liver lipid content. Hepatocyte ChREBPα overexpression suppressed the activation of HSCs in an in vitro medium transfer experiment in part via inhibiting the expression of profibrogenic factors THBS1 and CTGF. RNA-Seq analysis revealed that E2F1, a novel effector of TGFβ-mediated fibrogenic pathway, is highly induced in the liver of Chrebpα-LKO mice. Hepatic knockdown of E2F1 ameliorated the increased liver fibrosis in mice with hepatic Chrebpα deficiency while reducing the expression of hepatic THBS1 and CTGF. Show less

Non-alcoholic steatohepatitis (NASH) is characterized by hepatic lipid accumulation and fibrosis, yet the molecular mechanisms linking metabolic dysfunction to extracellular matrix (ECM) remodeling remain poorly defined. This study investigated the role of laminin γ2 (LAMC2, encoded by Lamc2), a basement membrane component, in NASH pathogenesis. Using a high-fat choline-deficient l-amino acid-defined (HF-CDAA) diet-induced murine NASH model and AML12 hepatocytes, we assessed LAMC2 expression via qPCR, immunohistochemistry, and lipidomics. Functional studies included LAMC2 overexpression (adenovirus) and TGFβ pathway inhibition (SB431542). LAMC2 was markedly upregulated in steatotic hepatocytes and localized adjacent to lipid droplets. Laminin-332 (Ln-332), which contains the γ2 chain (LAMC2), directly amplified lipogenesis by increasing Srebf1 and Mlxipl gene expression. TGFβ1 signaling via TGFBR1/Fra2 drove LAMC2 expression. Crucially, LAMC2 amplified both lipogenesis and fibrogenesis, thereby forming a feedforward loop that exacerbated hepatic fibrosis in this predominantly fibrotic NASH model. In vivo, LAMC2 overexpression exacerbated hepatic lipid accumulation and collagen deposition in the HF-CDAA diet-induced NASH model compared to controls. Fra2 silencing via AAV-shRNA attenuated NASH progression. LAMC2 bridges metabolic and fibrotic reprogramming in NASH through TGFβ1/Fra2-dependent mechanisms. Targeting this ECM-metabolism axis, particularly LAMC2 or Fra2, offers novel therapeutic strategies for fibrosis-dominant NASH, addressing a critical unmet clinical need. Show less

Carbohydrate response element-binding protein (ChREBP) is a transcription factor activated by glucose metabolites that orchestrates the expression of genes involved in glycolysis, de novo lipogenesis, and ATP homeostasis. Inadequate ChREBP activity impairs the cellular adaptations to glucose exposure and in humans associates with dyslipidemia, fatty liver disease, and type 2 diabetes. ChREBP activity is regulated by cytosolic-nuclear translocation involving its low-glucose inhibitory domain (LID). Whether this domain is targeted by post-translational lysine acetylation is unknown. Here we report a novel LID acetylation site that controls activity and protein interactions of ChREBP. Mutation of this residue increased glucose-induced activity and target gene expression of ChREBP. Mechanistically, mutant ChREBP protein showed more nuclear localization and enhanced genomic binding to a target promoter. Interactions with proteins that exhibit differential binding upon glucose exposure were attenuated by the mutation, demonstrating the importance of the LID in the formation of the protein interactome. Particularly interactions with 14-3-3 proteins, factors that regulate cytosolic/nuclear trafficking of ChREBP, were reduced, whereas interactions with proteins of the nucleosome remodeling deacetylase complex (NuRD) were increased. These molecular insights may shape new therapeutic strategies to target ChREBP activity and counteract metabolic diseases. 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

Many traditional treatments include honey owing to its magnificent health beneficiary effects. Recent studies have demonstrated the potent anti-diabetic activity of honey. However, its actual mechanism of action remains elusive. Moreover, being rich in sugar (75%-80%), its role in maintaining glucose homeostasis remains questionable. Although the polyphenol content of honey aids its hypoglycaemic activity, the small quantity of bioactive compounds in honey (0.5%-1.0%) may not be solely responsible for this. In the current study, an attempt was made to understand the role of Indian lychee honey (LyH) in regulating blood glucose levels under diabetic conditions. This study investigated whether LyH, although rich in sugars, can be used as an alternative to regulate glucose and lipid homeostasis under insulin-resistant conditions by regulating the ChREBP/Glut4 signalling pathway. This study was first performed 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

Diabetic kidney disease (DKD), characterized by mesangial fibrosis and renal dysfunction, is a major microvascular complication of diabetes. Studies have shown that miRNAs are closely related to the progression of DKD. Therefore, in this study, we aimed to explore whether miR-1225-3p can regulate Smad ubiquitin regulatory factor 2 (SMURF2)-mediated carbohydrate response element binding protein (ChREBP) ubiquitination through Rho GTPase-activating protein 5 (ARHGAP5) to affect fibrosis in DKD. DKD mice were established by intraperitoneally injecting streptozocin (STZ), and a DKD cell model was generated by culturing in media supplemented with 25 mmol/L glucose (high glucose, HG). StarBase was used to predict the target binding sites between miR-1225-3p and ARHGAP5, and a dual-luciferase reporter gene assay was used to verify this relationship. Western blotting, RT-qPCR, flow cytometry, immunoprecipitation, ELISAs, HE staining, and Masson staining were used to detect relevant indicators. ARHGAP5 and SMURF2 expression was decreased, but ChREBP was highly expressed in the renal tissue of DKD mice and HG-induced mouse mesangial cells (MMCs). miR-1225-3p could target and regulate the transcription of ARHGAP5, and an association between ARHGAP5 and SMURF2 was revealed. miR-1225-3p facilitated fibrosis and oxidative stress in MCCs by inhibiting ARHGAP5. In addition, SMURF2 promoted the ubiquitination of HA-ChREBP, and miR-1225-3p facilitated fibrosis and oxidative stress by mediating the ARHGAP5/SMURF2-mediated ubiquitination of ChREBP in MCCs. Furthermore, the miR-1225-3p inhibitor inhibited fibrosis and inflammation in the renal tissues of DKD mice. miR-1225-3p facilitates fibrosis and oxidative stress by mediating ARHGAP5/SMURF2-mediated ubiquitination of ChREBP. 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

The transcription factor carbohydrate response element binding protein (ChREBP) has emerged as a crucial regulator of hepatic glucose and lipid metabolism. The increased ChREBP activity involves the pro-oncogenic PI3K/AKT/mTOR signaling pathway that induces aberrant lipogenesis, thereby promoting hepatocellular carcinomas (HCC). However, the molecular pathogenesis of ChREBP-related hepatocarcinogenesis remains unexplored in the high-fat diet (HFD)-induced mouse model. Male C57BL/6J (WT) and liver-specific (L)-ChREBP-KO mice were maintained on either a HFD or a control diet for 12, 24, and 48 weeks, starting at the age of 4 weeks. At the end of the feeding period, mice were perfused, and liver tissues were formalin-fixed, paraffin-embedded, sectioned, and stained for histological and immunohistochemical analysis. Biochemical and gene expression analysis were conducted using serum and frozen liver tissue. Mice fed with HFD showed a significant increase ( Show less

The deficiency of fructose-1,6-bisphosphatase 1 (FBP1), a key enzyme of gluconeogenesis, causes fatty liver. However, its underlying mechanism and physiological significance are not fully understood. Here we demonstrate that carbohydrate response element-binding protein (ChREBP) mediates lipid metabolic remodeling and promotes progressive triglyceride accumulation against metabolic injury in adult FBP1-deficient liver. Inducible liver-specific deletion of Fbp1 gene caused progressive hepatomegaly and hepatic steatosis, with a marked increase in hepatic de novo lipogenesis (DNL) as well as a decrease in plasma β-hydroxybutyrate levels. Notably, FBP1 deficiency resulted in a persistent activation of ChREBP and its target genes involved in glycolysis, lipogenesis, and fatty acid oxidation, even under fasting conditions. Furthermore, liver-specific ChREBP disruption could markedly restore the phenotypes of enhanced DNL and triglyceride accumulation in FBP1-deficient liver but exacerbated its hepatomegaly and liver injury, which was associated with remarkable energy deficit, impaired mammalian target of rapamycin (mTOR) activation, and increased oxidative stress. Furthermore, metabolomics analysis revealed a robust elevation of phosphoenolpyruvate, phosphoglycerates, phospholipids, and ceramides caused by ChREBP deletion in FBP1-deficient liver. Put together, these results suggest that overactivation of ChREBP pathway mediates liver metabolic remodeling in the absence of FBP1, which contributes to the pathogenesis of progressive hepatic steatosis and provides a protection against liver injury. Thus, our findings point to a beneficial role of ChREBP in metabolic remodeling in the context of excessive gluconeogenic intermediates. Show less

The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) with two major splice isoforms (α and β). In chronic hyperglycemia and glucolipotoxicity, ChREBPα-mediated ChREBPβ expression surges, leading to insulin-secreting β-cell dedifferentiation and death. 14-3-3 binding to ChREBPα results in cytoplasmic retention and suppression of transcriptional activity. Thus, small molecule-mediated stabilization of this protein-protein interaction (PPI) may be of therapeutic value. Here, we show that structure-based optimizations of a 'molecular glue' compound led to potent ChREBPα/14-3-3 PPI stabilizers with cellular activity. In primary human β-cells, the most active compound retained ChREBPα in the cytoplasm, and efficiently protected β-cells from glucolipotoxicity while maintaining β-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative 'molecular glue' approach for achieving small molecule control of notoriously difficult to target TFs. 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

Glucose-sensing ChREBP and MondoA are transcriptional factors involved in the lipogenic, inflammatory, and insulin signaling pathways implicated in metabolic disorders; however, limited ocular studies have been conducted on these proteins. We aimed to investigate the potential role of ChREBP in the pathogenesis of diabetic retinopathy (DR). We used diabetic human and mouse retinal cryosections analyzed by immunohistochemistry. qRT-PCR was performed to quantify gene expression. To explore the role of ChREBP in rods, we generated caChREBP Show less

Suicide is a manner of death resulting from complex environmental and genetic risks that affect millions of people globally. Both structural and functional studies identified the hippocampus as one of the vulnerable brain regions contributing to suicide risk. We have identified the hippocampal tissue transcriptomes, gene ontology, cell type proportions, and dendritic spine morphology in controls (n = 28) and suicide decedents (n = 22). In addition, the transcriptomic signature in iPSC-derived neuronal precursor cells (NPCs) and neurons were also investigated in controls (n = 2) and suicide decedents (n = 2). The hippocampal tissue transcriptomic data revealed that NPAS4 gene expression was downregulated while ALDH1A2, NAAA, and MLXIPL gene expressions were upregulated in hippocampal tissue of suicide decedents. The gene ontology identified 29 significant pathways including NPAS4-associated gene ontology terms "excitatory post-synaptic potential", "regulation of postsynaptic membrane potential" and "long-term memory" indicating alteration of glutamatergic synapses in the hippocampus of suicide decedents. The cell type deconvolution identified decreased excitatory neuron proportion and an increased inhibitory neuron proportion providing evidence of excitation/inhibition imbalance in the hippocampus of suicide decedents. In addition, suicide decedents had increased dendric spine density in the hippocampus, due to an increase of thin (relatively unstable) dendritic spines, compared to controls. The transcriptomes of iPSC-derived hippocampal-like NPCs and neurons revealed 31 and 33 differentially expressed genes in NPC and neurons, respectively, of suicide decedents. Our findings will provide new insights into the hippocampal neuropathology of suicide. Show less

The relationship between type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) has long been extensively recognized, but their crosstalk mechanisms based on gene regulation remain elusive. In our study, for the first time, bulk RNA-seq and single-cell RNA-seq data were used to explore the shared molecular mechanisms between T2DM and CRC. Moreover, Connectivity Map and molecular docking were employed to determine potential drugs targeting the candidate targets. Eight genes ( Show less

Coffee and tea consumption account for most caffeine intake and 2-3 billion cups are taken daily around the world. Caffeine dependence is a widespread but under recognized problem. To conduct a systematic review on the genetic susceptibility factors affecting caffeine metabolism and caffeine reward and their association with caffeine intake. We conducted PubMed and Embase searches using the terms "caffeine", "reward", "gene", "polymorphism", "addiction", "dependence" and "habit" from inception till 2024. The demographics, genetic and clinical data from included studies were extracted and analyzed. Only case-control studies on habitual caffeine drinkers with at least 100 in each arm were included. A total of 2552 studies were screened and 26 studies involving 1,851,428 individuals were included. Several genes that were involved with caffeine metabolism such as CYP1A2, ADORA2A, AHR, POR, ABCG2, CYP2A6, PDSS2 and HECTD4 rs2074356 (A allele specific to East Asians and monomorphic in Europeans, Africans and Americans) were associated with habitual caffeine consumption with effect size difference of 3% to 32% in number of cups of caffeinated drink per day per effect allele. In addition, ALDH2 was linked to the Japanese population. Genes associated with caffeine reward included BDNF, SLC6A4, GCKR, MLXIPL and dopaminergic genes such as DRD2 and DAT1 which had around 2-5% effect size difference in number of cups of caffeinated drink for each allele per day. Several genes that were involved in caffeine metabolism and reward were associated with up to 30% effect size difference in number of cups of caffeinated drink per day, and some associations were specific to certain ethnicities. Identification of at-risk caffeine dependence individuals can lead to early diagnosis and stratification of at-risk vulnerable individuals such as pregnant women and children, and can potentially lead to development of drug targets for dependence to caffeine. Show less

Intrahepatic lipid accumulation (IHL), a hallmark of metabolic disorders, is closely associated with de novo lipogenesis (DNL). Notably, fructose feeding increased the DNL. Lifestyle modifications resulting from dietary changes and increased physical activity/exercise can decrease the IHL content. We examined the effects of vitamin D Forty male rats were assigned to 5 groups (n = 8): CS (the control group had a standard diet); CF (the control group had HFrD (10% (w/v) fructose solution in tap water)); and FT (HFrD + HIIT: 10 bouts of 4 min of high-intensity running, corresponding to 85-90% of the maximal speed with 2 min active rest periods of 50% maximal speed, 5 days per week); FD (HFrD + intervention of intraperitoneal injection of 10000 IU/kg/week VDS); FTD (HFrD + HIIT + VDS) that were maintained for 12 weeks. ELISA, the GOD-POD assay, folch, western blotting, and oil red O staining were used to determine insulin, fasting blood glucose (FBG), hepatic triglyceride (TG) and cholesterol levels; SREBP1c, ChREBP-β, ACC1, FASN, p-ACC1, AMPK, p-AMPK, and PKA protein expression; and IHL content, respectively. Both HIIT and VDS led to significant increases in the levels of PKA, AMPK, p-AMPK, and p-ACC1, as well as significant decreases in the levels of SREBP1c, ChREBP-β, ACC1, FASN, insulin, FBG, liver TG, liver cholesterol, and IHL. HIIT exhibited superior efficacy over VDS in reducing ChREBP-β, ACC1, insulin, FBG, liver TG and cholesterol, as well as increasing p-ACC1 and PKA. Notably, the combined intervention of HIIT and VDS yielded the most substantial improvements across all the parameters. HFrD causes IHL accumulation and the onset of diabetes, whereas VDS and HIIT, along with their combined effects, prevent the consequences of HFrD. Show less

Glycogen-storing so-called clear cell kidney tubules (CCTs), precursor lesions of renal cell carcinoma, have been described in diabetic rats and in humans. The lesions show upregulation of the Akt/mTOR-pathway and the related transcription factor carbohydrate responsive element binding protein (ChREBP), which is supposedly pro-oncogenic. We investigated the effect of ChREBP-knockout on nephrocarcinogenesis in streptozotocin-induced diabetic and normoglycemic mice. Diabetic, but not non-diabetic mice, showed CCTs at 3, 6 and 12 months of age. Glycogenosis was confirmed by periodic acid schiff reaction and transmission electron microscopy. CCTs in ChREBP-knockout mice consisted of larger cells and occurred more frequently compared to wildtype mice. Progression towards kidney tumors was observed in both diabetic groups but occurred earlier in ChREBP-knockout mice. Proliferative activity assessed by BrdU-labeling was lower in 1-week-old but higher in 12-month-old diabetic ChREBP-knockout mice. Surprisingly, renal neoplasms occurred spontaneously in non-diabetic ChREBP-knockout, but not non-diabetic wildtype mice, indicating an unexpected tumor-suppressive function of ChREBP. Immunohistochemistry showed upregulated glycolysis and lipogenesis, along with activated Akt/mTOR-signaling in tumors of ChREBP-knockout groups. Immunohistochemistry of human clear cell renal cell carcinomas revealed reduced ChREBP expression compared to normal kidney tissue. However, the molecular mechanisms by which loss of ChREBP might facilitate tumorigenesis require further investigation. Show less

Diabetes, a global epidemic, is the leading cause of mortality globally. The aim of this study is to get better understanding of pathophysiology of diabetes. Palmitic acid (PA)-treated β-cells, db/db mice and high fat diet (HFD)-fed mouse model of type 2 diabetes were established. H&E was used to assess the histological changes of pancreas. IHC, FISH, western blot or qRT-PCR was employed to detect the expression of key molecules in primary islets or lipotoxic β-cells. Cell behaviors were detected by MTT, EdU incorporation assay, TUNEL assay and glucose-induced insulin secretion (GSIS). The associations among circMlxipl, Mbnl1 and Rbbp6 were validated by RIP and RNA pull-down assays, and the direct binding between Hdac3 and Mbnl1 promoter was examined by ChIP and luciferase assays. Co-IP was employed to assess the interaction between ChREBP and Rbbp6, as well as the ubiquitination of ChREBP. Hdac3 and ChREBP were upregulated, but Mbnl1 and circMlxipl were downregulated in islets from diabetic mice and lipotoxic β-cells. Mbnl1 overexpression protected against PA-induced impairments in lipotoxic β-cells through modulating back-splicing of circMlxipl and suppressing ChREBP. Hdac3 served as a transcriptional repressor of Mbnl1, and it was implicated in circMlxipl-mediated protection via regulating ChREBP expression in lipotoxic β-cells. Lack of circMlxipl inhibited Rbbp6-mediated ubiquitin-proteasomal degradation of ChREBP in lipotoxic β-cells. In vivo studies revealed that Hdac3 knockdown or Mbnl1 overexpression alleviated diabetes symptoms through circMlxipl-regulated ChREBP in diabetic mice. Mbnl1-mediated alternative splicing of circMlxipl regulates Rbbp6-involved ChREBP turnover to inhibit lipotoxicity-induced β-cell damage. Show less

Activation of thermogenic brown adipose tissue (BAT) and inducible beige adipose tissue (BeAT) is triggered by environmental or metabolic stimuli, including cold ambient temperatures and nutrient stress. Thioesterase superfamily member 1 (Them1), a long-chain fatty acyl-CoA thioesterase that is enriched in BAT, suppresses acute cold-induced thermogenesis. Here, we demonstrate that Show less

Stable isotope studies have shown that hepatic de novo lipogenesis (DNL) plays an important role in the pathogenesis of intrahepatic lipid (IHL) deposition. Furthermore, previous research has demonstrated that fructose 1-phosphate (F1P) not only serves as a substrate for DNL, but also acts as a signalling metabolite that stimulates DNL from glucose. The aim of this study was to elucidate the mediators of F1P-stimulated DNL, with special focus on two key regulators of intrahepatic glucose metabolism, i.e., glucokinase regulatory protein (GKRP) and carbohydrate response element binding protein (ChREBP). Aldolase B deficient mice (Aldob Aldob Both GKRP and ChREBP mediate F1P-stimulated DNL in aldolase B deficient mice. Further studies are needed to unravel the role of GKRP and hepatic ChREBP in regulating IHL accumulation in aldolase B deficiency. Show less

Dendrobine (DDB), an alkaloid isolated from the Chinese herb Dendrobium, has antioxidant and anti-inflammatory effects; however, whether DDB reduces oleic acid (OA)-induced lipid accumulation remains unclear. OA-induced lipid accumulation model of HepG2 cells were treated with DDB. Cellular lipid deposition was assessed by Oil Red O (ORO) staining and triglyceride and total cholesterol detection. RNA-Sequencing (RNA-seq), biological function analysis, and transcription factor (TFs) prediction were combined to identify key TF in the DDB-treated OA model. Finally, the roles of FOS and METTL14 were examined using a DDB-induced lipid accumulation model. DDB inhibited OA-induced lipid accumulation. We identified 895 differentially expressed genes (DEGs) that were mainly enriched in various biological processes of lipid synthesis and transport. Four transcription factors (SOX9, MLXIPL, FOS, and JUN) associated with lipid metabolism and FOS levels in the OA-induced lipid accumulation model after DDB treatment had the greatest changes in expression change. Overexpression of FOS alleviates the inhibitory effect of DDB on OA-induced lipid accumulation. METTL14 is a target gene of FOS, and simultaneous interference with METTL14 in cells with high FOS expression restored the alleviating effect of DDB on lipid accumulation. DDB alleviated OA-induced lipid accumulation by inhibiting the FOS/METTL14 pathway. Show less

SNAP25 plays an essential role in the glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells. Carbohydrate response element-binding protein (ChREBP) is an important transcription factor in β-cells and, in this study, we aimed to explore whether ChREBP regulates SNAP25 expression in β-cells. We show that diabetic Goto-Kakizaki (GK) rats exhibited impaired insulin secretion and hyperglycemia, along with decreased SNAP25 expression and ChREBP phosphorylation in islets. SNAP25 knockdown decreased GSIS in β-cells, while SNAP25 overexpression increased GSIS in β-cells. Activation or overexpression of ChREBP led to reduced SNAP25 expression and subsequent suppression of GSIS. Conversely, ChREBP knockdown mitigated the reduction in SNAP25 expression caused by high glucose. Mechanistically, the activation of ChREBP by high glucose increased its occupancy and decreased the level of H3K4me3 at the Snap25 promoter. Our findings reveal the novel regulatory mechanisms of SNAP25 expression in β-cells and suggest that SNAP25 may be involved in the regulation of β-cell secretory function controlled by ChREBP. Show less

Recombinant adeno-associated virus (rAAV) vectors are powerful tools for the sustained expression of proteins in vivo and have been successfully used for mechanistic studies in mice. A major challenge associated with this method is to obtain tissue specificity and high expression levels without need of local virus administration. To achieve this goal for brown adipose tissue (BAT), we developed a rAAV vector for intravenous bolus injection, which includes an expression cassette comprising an uncoupling protein-1 enhancer-promoter for transcription in brown adipocytes and miR122 target sequences for suppression of expression in the liver, combined with packaging in serotype Rec2 capsid protein. To test tissue specificity, we used a version of this vector expressing Cre recombinase to transduce mice with floxed alleles to knock out MLXIPL (ChREBP) or tdTomato-Cre reporter mice. We demonstrated efficient Cre-dependent recombination in interscapular BAT and variable effects in minor BAT depots, but little or no efficacy in white adipose tissues, liver and other organs. Direct overexpression of glucose transporter SLC2A1 (GLUT1) using the rAAV vector in wild type mice resulted in increased glucose uptake and glucose-dependent gene expression in BAT, indicating usefulness of this vector to increase the function even of abundant proteins. Taken together, we describe a novel brown adipocyte-specific rAAV method to express proteins for loss-of-function and gain-of-function metabolic studies. The approach will enable researchers to access brown fat swiftly, reduce animal breeding time and costs, as well as enable the creation of new transgenic mouse models combining multiple transgenes. Show less

Carbohydrate-responsive element-binding protein (ChREBP) is a transcription factor that regulates several metabolic genes, including the lipogenic enzymes necessary for the metabolic conversion of carbohydrates into lipids. Although the crucial role of ChREBP in the liver, the primary site of de novo lipogenesis, has been studied, its functional role in adipose tissues, particularly brown adipose tissue (BAT), remains unclear. In this study, we investigated the role of ChREBP in BAT under conditions of a high-carbohydrate diet (HCD) and ketogenic diet (KD), represented by extremely low carbohydrate intake. Using an adeno-associated virus and Cas9 knock-in mice, we rapidly generated Chrebp brown adipocyte-specific knock-out (B-KO) mice, bypassing the necessity for prolonged breeding by using the Cre-Lox system. We demonstrated that ChREBP is essential for glucose metabolism and lipogenic gene expression in BAT under HCD conditions in Chrebp B-KO mice. After nutrient intake, Chrebp B-KO attenuated the KD-induced expression of several inflammatory genes in BAT. Our results indicated that ChREBP, a nutrient-sensing regulator, is indispensable for expressing a diverse range of metabolic genes in BAT. Show less