Objectives

Glucagon is essential for maintaining glucose homeostasis, yet the molecular mechanisms governing α-cell function remain incompletely understood. Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed ion channel with an intrinsic kinase domain, which regulates the mammalian target of rapamycin (mTOR) signaling in various cell types. Given the central role of mTOR in α-cell regulation, this study investigates how TRPM7 influences α-cell biology and examines whether its function is modulated through interaction with the mTOR signaling pathway.

Methods

Islets were isolated from wild-type (WT) mice and mice lacking TRPM7 kinase activity (Trpm7^R/R). Functional analyses included Bio-Plex assays, RNA sequencing, glucagon ELISA, qRT-PCR, Western blotting, immunocytochemistry, and patch-clamp recordings. αTC1c9 cells were used as a murine α-cell model. NS8593, a small synthetic compound, was used as a potent TRPM7 inhibitor.

Results

Ex vivo analysis revealed impaired mTOR signaling in Trpm7^R/R islets. Trpm7^R/R islets secreted less glucagon in response to various secretagogues compared to WT controls. This reduction was partially caused by diminished glucagon content due to downregulation of key transcriptional regulators of glucagon biosynthesis, including Gcg and Mafb. Morphological analysis identified reduced proliferation and enhanced apoptosis of Trpm7^R/R α-cells. Similarly, pharmacological inhibition of TRPM7 impaired mTOR signaling, suppressed α -cell identity, and α-cell proliferation in both WT islets and αTC1c9 cells.

Conclusions

Loss of TRPM7 kinase function impairs mTOR signaling, leading to reduced α-cell proliferation and glucagon secretion. Our findings show that the TRPM7 kinase/mTOR signaling pathway axis is a critical regulator of α-cell function in mice.

Objectives

Global but not liver-specific deletion of protein kinase C epsilon (PKCε) improves glucose tolerance in fat-fed mice, suggesting that extra-hepatic tissues are involved. AgRP neurons within the arcuate nucleus (ARC) of the hypothalamus can affect glucose homeostasis acutely, in addition to their role in energy homeostasis. We therefore deleted PKCε specifically in AgRP neurons to examine its effects at this site.

Methods

Fat-fed AgRP-PKCε^−/− mice were subjected to glucose tolerance tests and euglycaemic-hyperinsulinaemic clamps. c-Fos and tyrosine hydroxylase were used as markers to map neuronal activity in serial brain sections. Transcriptional changes in liver and adipose tissue were examined by qRT-PCR while alterations in protein levels and phosphorylation were determined by immunoblotting and mass spectrometry.

Results

Fat-fed AgRP-PKCε^−/− mice exhibited improved glucose tolerance but not insulin sensitivity determined by clamp. c-Fos mapping demonstrated that glucose challenge resulted in greater activation of neurons in the paraventricular nucleus (PVN) in AgRP-PKCε^−/− mice, but reduced expression of tyrosine hydroxylase in the PVN, suggestive of reduced sympathetic outflow. This was associated with a reduction in hormone sensitive lipase phosphorylation and plasma fatty acid levels. Proteomic analysis indicated overlapping alterations in proteins and protein phosphorylation in adipose tissue and liver, consistent with changes in a common, potentially neuronal, cell type.

Conclusions

Ablation of PKCε in AgRP neurons improves glucose homeostasis in fat-fed mice. This appears to be mediated through glucose sensing mechanisms, potentially reducing sympathetic outflow from the hypothalamus to tissues such as adipose, reducing lipolysis to indirectly lower hepatic glucose production.

Atherosclerosis is a long-term complication of obesity and diabetes and as such a key driver of vascular dysfunction and eventually mortality in affected patients. Both aberrant lipid metabolism and inflammatory reactions promote atherosclerotic plaque development in the vessel wall by triggering a cascade of cellular events involving multiple cell types, including smooth muscle cells, monocytic macrophages, and lymphocytes. Despite its eminent impact on human health, molecular drivers of cellular dysfunction in atherosclerosis remain poorly defined and therapeutic options are scarce.

Here we show by single-cell RNA sequencing that the expression of the nuclear receptor co-factors, TBL1X and TBL1XR1, was particularly prominent in the CD4⁺ T cell population of human carotid artery plaques. Indeed, genetic double deletion of TBL1X/TBL1XR1 in CD4⁺ T cells led to a substantial shift from naïve CD44^lowCD62L^hi cells to CD44^hiCD62L^low effector and Foxp3⁺ Tregs. CD4⁺ TBL1X/TBL1XR1 KO cells exhibited enhanced cytokine production capacity upon ionomycin/PMA stimulation, correlating with the induction of pro-inflammatory and cytokine-producing transcriptional pathways in these cells. Consistently, transplantation of bone marrow from CD4⁺-specific TBL1X/TBL1XR1 knock out mice into LDLR KO recipients doubled the development of atherosclerotic plaques in the aortic arch compared with wild-type bone marrow transplanted littermates. As TBL1X/TBL1XR1 expression levels were diminished in carotid arteries from patients with advanced unstable plaques compared to stable plaques or healthy controls, these data suggest that aberrant inhibition of TBL1X/TBL1XR1 in CD4⁺ T cells may contribute to the development of atherosclerosis in humans. Restoration of TBL1X/TBL1XR1 functionality may thus serve as a novel, druggable strategy for preventing or limiting atherosclerosis progression.

Sterile inflammation is associated with a broad range of metabolic stressors including both dietary excess and prolonged fasting. In a 10-day human fasting study, we previously identified a surge in the circulating inflammatory biomarker, C-reactive protein (CRP), which we leveraged in the current study to identify novel metabolic inflammatory correlates. With a variety of longitudinal metabolic variables as input, including metabolomics, we identified branched chain amino acids (BCAA) as the top candidate inflammatory correlate. We then used in vitro myeloid/macrophage culture and in vivo murine models to test BCAA as a determinant of inflammatory signaling. Short-term exposure to BCAA alone had modest effects on a variety of immune readouts; however, when coupled with a second stimulus, such as exposure to endotoxin or when administered to diet-induced obese mice, members of the JAK/STAT/cytokine signaling pathways were augmented on the transcriptional level by concurrent BCAA administration in multiple tissues, including visceral adipose and liver. The modifying effect of BCAA on inflammatory stressors translated into increased levels of circulating inflammatory cytokines. Collectively, these data position BCAA as an immune priming factor, a potential mechanism underlying the well-established association between circulating BCAA and diverse diseases of aging.

Objectives

The gut microbiota plays a key role in maintaining brain health and homeostasis. Previous studies have demonstrated that metabolites in the brain respond to alterations in gut microbial composition. In this study we aimed to explore how depletion of the gut microbiota is associated with alterations in the diurnal rhythmicity of metabolites in the brain.

Methods

We used antibiotic-induced microbial depletion in mice to examine the impact of the gut microbiota on the rhythmicity of metabolites in the prefrontal cortex. Metabolite profiles were assessed across multiple timepoints using untargeted metabolomics.

Results

Microbial depletion was associated with alterations in the rhythmic profile of metabolites in the prefrontal cortex, with amino acids showing a robust inversion of their normal rhythm. These alterations were specific to the prefrontal cortex, with hippocampus and amygdala showing minimal changes. This altered gut microbial environment was associated with potential consequences for neurotransmitter production, including glutamate and serotonin.

Conclusions

These findings provide further evidence that the gut microbiota shapes rhythmic diurnal processes in the brain. Future studies are warranted to investigate how such microbial effects influence actual neurotransmitter levels and behavioral phenotypes associated with the prefrontal cortex.

Understanding tissue-specific mechanisms of protein regulation gives crucial insights into cardiometabolic disease and informs drug discovery. Most proteomic studies have primarily concentrated on plasma, overlooking tissue-specific effects. Utilizing Olink technology, we assessed relative protein levels across plasma and tissue (aortic wall, mammary artery, liver, and skeletal muscle) from the STARNET cohort: 284 individuals with a high prevalence of coronary artery disease (CAD). We identified 608 cis protein quantitative trait loci (pQTLs), primarily in plasma, reflecting greater protein variability. Of 190 proteins with cis-pQTLs in non-plasma tissues, 50% also had plasma pQTLs, validating Olink technology in these tissues while reinforcing the relevance of plasma data for understanding protein regulation. To identify potential mechanistic pathways linking genetic variants to clinical traits, we performed Bayesian colocalization and Mendelian randomization. These analyses revealed shared genetic regulation between tissues at the gene expression and protein level, and key cardiometabolic traits including low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides. Notably, analyses provide further support to SORT1 and PSRC1 gene and protein expression having liver-specific influences on CAD risk and lipid profiles. We also observed distinct genetic regulation of gene expression and protein within the same tissues, underscoring the value of tissue proteomics for therapeutic insights.

Background

Controlling brown adipose tissue (BAT) plasticity offers potential for novel obesity therapies. DNA methylation is closely linked to thermogenic and metabolic pathways and thereby influences BAT function. How metabolic state and cold exposure interact to shape methylation-dependent BAT gene regulation was investigated.

Methods

Five-week-old mice were fed either chow for 11 weeks (lean) or high-fat diet for 22 weeks to induce obesity (DIO), after which cold exposure was applied for seven days. BAT transcriptomes (RNAseq) and methylomes (RRBS) were generated, and differentially methylated and expressed genes (DMEGs) showing metabolic state–dependent cold responses were identified. Pathway enrichment, epigenetic regulator screening, and transcription factor (TF) motif analyses were performed. DNA methylation was experimentally modulated in vitro to validate selected gene expression responses.

Results

A total of 1,364 differentially expressed genes (DEGs) were uniquely affected by the interaction of metabolic state and cold, with most downregulated in DIO mice. Sixty-five DMEGs (4 % of DEGs) showed metabolic state–specific responses to cold. In DIO mice, DMEGs were enriched in pathways associated with mitochondrial dysfunction, altered lipid metabolism, neuroendocrine signaling, and stress responses. Several epigenetic regulators, including Tet2, Dnmt3a, and Apobec1, exhibited metabolic state- and cold-dependent expression, and TF-motif analyses highlighted roles for AhrArnt and Foxn1. In vitro assays confirmed that DNA methylation influences expression of thermogenic genes.

Conclusion

These findings provide the first evidence that the epigenetic cold response of BAT differs by metabolic condition. BAT remodeling is shaped by coordinated transcriptional and epigenetic mechanisms integrating environmental and metabolic cues.

Graphical abstract

Mice were housed under cold exposure (8 °C) or thermoneutrality (30 °C) and fed either chow (lean) or high-fat diet (HFD; diet-induced obese (DIO)). RNA sequencing (RNAseq) and reduced representation bisulfite sequencing (RRBS) was performed on brown adipose tissue (BAT) which provide the fundament for the identification of differentially methylated positions (DMP) and regions (DMR) as well as differentially expressed genes (DEG) in three models: COLD_lean, comparing 8° versus 30 °C mice on chow diet; COLD_DIO, comparing 8° versus 30 °C mice on HFD diet and ΔCOLD, comparing COLD_lean versus COLD_DIO. Differentially methylated and expressed genes (DMEGs) were identified for all comparisons, based on DMPs within DMRs that significantly correlated with differentially expressed genes (DEGs; the heatmap shows a representative example). DMEGs were further characterized. First, using a published single cell dataset (Shamsi et al., 2023, https://doi.org/10.1038/s42003-023-05140-2), the distribution of DMEGs across cell types was analyzed. For further characterization, DMEGS were categorized into similarly (among COLD_lean and COLD_DIO) and uniquely (COLD_lean, COLD_DIO and ΔCOLD) regulated. Pathway enrichment analyses were performed and to identify differentially regulated epigenetic regulators among the DMEGs, the EpiFactor Database (https://epifactors.autosome.org) was used. A TF-binding site motive enrichment analyses (https://jaspar.elixir.no)was applied to add information of most enriched Transcription Factors (TF). Finally, to gain functional insights of DNA methylation changes on selected DMEG candidates, cell cultures of immortalized brown adipocytes and primary brown adipocytes were treated with 5′aza-2′-deoxycytidine (demethylation) or S-adenosylmethionine (upregulation of methylation), following gene expression analyses. The figure was created with BioRender.

Abstract/objective

Metabolic associated steatotic liver disease (MASLD) is the most prevalent liver disorder and a major risk factor for hepatic fibrosis. Activated hepatic stellate cells (HSCs) are the primary source of collagen production in the liver, contributing to fibrosis. However, the mechanisms by which HSCs reprogram their metabolism to support sustained collagen production, particularly in a lipid-rich environment such as MASLD, remain inadequately understood. In this study, we investigated the effect of extracellular fatty acids on HSC substrate metabolism, HSC activation, and collagen synthesis.

Methods

Immortalized human HSCs (LX-2 cells) were cultured with or without transforming growth factor-beta 1 (TGF-β1) and varying concentrations of palmitate or oleate. Cellular lipid composition was assessed by mass spectrometry lipidomics. Fatty acid metabolism was assessed using radiometric techniques and isotopic labelling experiments using ¹³C-glucose or ¹³C-palmitate. HSC activation was assessed by measuring ACTA2, TGFB1, and COL1A1 mRNA levels and collagen secretion by ELISA.

Results

TGF-β1 reduced the abundance of many lipid types in LX-2 cells. Exogenous palmitate did not increase HSC activation, as determined by ACTA2, TGFB1, COL1A1 mRNA levels. Palmitate potentiated TGF-β1 induced collagen secretion but not in the presence of oleate. Palmitate reduced glucose incorporation into glycine in activated HSCs and induced a reciprocal increase in palmitate incorporation into glycine, most likely via carbons derived from TCA cycle intermediates. Pharmacological inhibition of fatty acid uptake reduced TGF-β1-mediated collagen secretion.

Conclusions

These results suggest that in activated HSCs, palmitate oxidation is reduced and that TCA cycle intermediates derived from palmitate are used as carbon sources for amino acid production that supports collagen synthesis and secretion.

Animals adaptively adjust nutrient intake based on internal physiological need. Although protein deficiency elicits robust behavioral and endocrine responses, the sensory mechanisms that detect dietary protein and guide selective feeding remain incompletely understood. Here, we identify a population of vagal sensory neurons that respond selectively to intragastric protein and are required for adaptive regulation of protein intake. Using activity-dependent genetic labeling and in vivo calcium imaging, we show that these neurons are activated by dietary protein, exhibit enhanced responses in protein-restricted states, and are distinct from previously characterized calorie-sensing populations. Selective ablation of protein-responsive vagal sensory neurons disrupts the ability to adapt eating behavior to internal protein need, blunts motivation to work for protein rewards, and prevents behavioral updating following protein repletion. These neurons also mediate protein-specific satiety, limiting further protein intake without affecting carbohydrate consumption. Notably, protein preference is suppressed under mild caloric restriction, indicating that caloric and amino acid needs are hierarchically organized and likely monitored by separate interoceptive systems. Our findings reveal a novel vagal circuit that integrates internal protein status with nutrient-specific cues to guide adaptive protein appetite and maintain amino acid homeostasis.

Objectives

Hereditary fructose intolerance (HFI), caused by Aldolase B deficiency, is a rare genetic disorder where fructose exposure leads to severe metabolic pathologies including Type-2 diabetes and liver steatosis. Despite adhering to fructose-free diets, some individuals still present with disease. Using a rat model of HFI we demonstrate that fructose independent pathologies exist and identify the molecular pathways driving disease.

Methods

Aldob was deleted in Sprague Dawley rats using CRIPSR/Cas9 (AldoB-KO). Phenotypic, metabolomic and transcriptomic studies were conducted to identify mechanisms promoting fructose-independent pathologies. Potential molecular causes were tested using pharmacologic inhibitors and ASOs.

Results

Deletion of Aldob caused hepatic steatosis, fibrosis and stunted growth in rats weaned on low fructose chow recapitulating human HFI. On fructose-free chow, AldoB-KO rats were phenotypically normal. However, upon fasting, male and female AldoB-KO rats developed hepatic steatosis and hyperlipidemia due to impaired fatty acid oxidation (FAOx) and elevated de novo lipogenesis (DNL). Transcriptional and metabolomic profiling revealed increased hepatic Carbohydrate Response Element Binding Protein (ChREBP) activation in AldoB-KO rats due to glycolytic metabolite accumulation caused by impaired gluconeogenesis. Treatment with Acetyl-CoA Carboxylase (ACC) and Diacylglycerol Acyl Transferase 2 (DGAT2) inhibitors reduced hepatic lipids and plasma triglycerides in AldoB-KO rats. Finally, using electronic health records we observed increased metabolic dysfunction-associated steatohepatitis (MASH) diagnosis in individuals with HFI.

Conclusions

Aldob deletion caused fructose-independent hyperlipidemia and steatosis upon fasting in rats. Individuals with HFI may have risk for hepatic disease and hyperlipidemia even upon fructose abstinence suggesting additional therapies may be needed to mitigate disease.

Following recurrence, the cornerstone clinical therapy to treat prostate cancer (PCa) is to inhibit the androgen receptor (AR) signaling. While AR inhibition is initially successful, tumors will eventually develop treatment resistance and evolve into lethal castration-resistant PCa. To discover new anti-metabolic treatments for PCa, a high-throughput anti-metabolic drug screening was performed in PC3 cells, an AR-negative PCa cell line. This screening identified the dihydroorotate dehydrogenase (DHODH) enzyme as a metabolic vulnerability, using both AR-positive and AR-negative models, including the neuroendocrine cell line LASCPC-01 and patient-derived organoids. DHODH is required for de novo pyrimidine synthesis and is the sole mitochondrial enzyme of this pathway. Using extracellular flux assays and targeted metabolomics, DHODH inhibition was shown to impair the pyrimidine synthesis pathway, as expected, along with a significant reprogramming of mitochondrial metabolism, with a massive increase in fumarate (>10-fold). Using ¹³C₆-glucose, it was shown that following DHODH inhibition, PCa cells redirect carbons from glucose toward biosynthetic pathways rather than the TCA cycle. In parallel, using ¹³C₅-glutamine, it was shown that PCa cells use this amino acid to fuel a reverse TCA cycle. Finally, ¹³C₁-aspartate and ¹⁵N₁-glutamine highlighted the connection between pyrimidine synthesis and the urea cycle, redirecting pyrimidine synthesis intermediates toward the urea cycle as a stress response mechanism upon DHODH inhibition. Consequently, combination therapies targeting DHODH and glutamine metabolism were synergistic in impairing PCa cell proliferation. Altogether, these results highlight DHODH as a metabolic vulnerability of AR-positive and AR-negative PCa cells by regulating central carbon and nitrogen metabolism.