Objective

Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency.

Methods

We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation.

Results

Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect.

Conclusions

Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency.

Objectives

Experimental and genetic studies suggest that fibroblast growth factor 21 (FGF21) modulates macronutrient and alcohol preferences, but evidence of such regulation in humans remains scarce. To address this gap in translation, we aimed to map the relationships between plasma FGF21 levels, FGF21 genetic variation and habitual macronutrient intake in a large human population.

Methods

We fine-mapped and performed colocalization of the FGF21 genetic region in GWAS summary statistics of plasma FGF21 levels and macronutrient intake. UK Biobank data were used to investigate the associations between FGF21 genetic variants, plasma FGF21 protein levels, and macronutrient intake (including alcohol) assessed with repeated 24-hour recalls. One- and two-sample mendelian randomization were performed to estimate the effects of plasma FGF21 on macronutrient intake.

Results

We show that the main macronutrient-associated variant rs838133 and the FGF21 cis-pQTL rs838131, both in the FGF21 gene, are distinct genetic signals. Effect directions also suggest that the influence of FGF21 variation on macronutrient intake appear more complex than by direct mediation through plasma FGF21. Only when considering this complexity at FGF21, is plasma FGF21 estimated to reduce alcohol and increase protein and fat intake using mendelian randomization. Importantly, plasma FGF21 levels also appear markedly elevated by primarily high alcohol and low protein intake.

Conclusions

These findings support the feedback diet-regulatory mechanism of FGF21 in humans, but highlights the need for mechanistic characterization of the complex FGF21 genetic region.

Background

AMP-activated protein kinase (AMPK) is an evolutionarily conserved regulator of energy metabolism. AMPK is sensitive to acute perturbations to cellular energy status and leverages fundamental bioenergetic pathways to maintain cellular homeostasis. AMPK is a heterotrimer comprised of αβγ-subunits that in humans are encoded by seven individual genes (isoforms α1, α2, β1, β2, γ1, γ2 and γ3), permitting formation of at least 12 different complexes with personalised biochemical fingerprints and tissue expression patterns. While the canonical activation mechanisms of AMPK are well-defined, delineation of subtle, as well as substantial, differences in the regulation of heterogenous AMPK complexes remain poorly defined.

Scope of review

Here, taking advantage of multidisciplinary findings, we dissect the many aspects of isoform-specific AMPK function and links to health and disease. These include, but are not limited to, allosteric activation by adenine nucleotides and small molecules, co-translational myristoylation and post-translational modifications (particularly phosphorylation), governance of subcellular localisation, and control of transcriptional networks. Finally, we delve into current debate over whether AMPK can form novel protein complexes (e.g., dimers lacking the α-subunit), altogether highlighting opportunities for future and impactful research.

Major conclusions

Baseline activity of α1-AMPK is higher than its α2 counterpart and is more sensitive to synergistic allosteric activation by metabolites and small molecules. α2 complexes however, show a greater response to energy stress (i.e., AMP production) and appear to be better substrates for LKB1 and mTORC1 upstream. These differences may explain to some extent why in certain cancers α1 is a tumour promoter and α2 a suppressor. β1-AMPK activity is toggled by a ‘myristoyl-switch’ mechanism that likely precedes a series of signalling events culminating in phosphorylation by ULK1 and sensitisation to small molecules or endogenous ligands like fatty acids. β2-AMPK, not entirely beholden to this myristoyl-switch, has a greater propensity to infiltrate the nucleus, which we suspect contributes to its oncogenicity in some cancers. Last, the unique N-terminal extensions of the γ2 and γ3 isoforms are major regulatory domains of AMPK. mTORC1 may directly phosphorylate this region in γ2, although whether this is inhibitory, especially in disease states, is unclear. Conversely, γ3 complexes might be preferentially regulated by mTORC1 in response to physical exercise.

Cancer is a disease characterized by the acquisition of a multitude of unique traits. It has long been understood that cancer cells divert significantly from normal cell metabolism. The most obvious of metabolic changes is that cancer cells strongly rely on glucose conversion by aerobic glycolysis. In addition, they also regularly develop mechanisms to use lipids and fatty acids for their energy needs. Peroxisomes lie central to these adaptive changes of lipid metabolism.

Peroxisomes are metabolic organelles that take part in over 50 enzymatic reactions crucial for cellular functioning. Thus, they are essential for an effective and comprehensive use of lipids’ energy supplied to cells. Cancer cells display a substantial increase in the biogenesis of peroxisomes and an increased expression of proteins necessary for the enzymatic functions provided by peroxisomes. Moreover, the enzymatic conversion of FAs in peroxisomes is a significant source of reactive oxygen and nitrogen species (ROS/RNS) that strongly impact cancer malignancy. Important regulators in peroxisomal FA oxidation and ROS/RNS generation are the transcription factors of the peroxisome proliferator-activated receptor (PPAR) family. This review describes the metabolic changes in tumorigenesis and cancer progression influenced by peroxisomes. We will highlight the ambivalent role that peroxisomes and PPARs play in the different stages of tumor development and summarize our current understanding of how to capitalize on the comprehension of peroxisomal biology for cancer treatment.

Background

Sodium-glucose cotransporters (SGLTs) play a crucial role in glucose regulation and are essential therapeutic targets for diabetes management. Recent advancements have leveraged SGLT-targeted PET imaging to examine these transporters' roles in both health and disease.

Scope of Review

This review highlights recent innovations in PET imaging targeting SGLTs, with a particular focus on SGLT-specific radiotracers, such as alpha-methyl-4-deoxy-4-¹⁸F-fluoro-d-glucopyranoside (Me-4FDG). It emphasizes the advantages of these radiotracers over conventional ¹⁸F-2-fluoro-2-deoxy-d-glucose (2-FDG) imaging, especially in assessing SGLT activity. Additionally, the review addresses their potential in evaluating the pharmacodynamics of SGLT inhibitors, investigating metabolic changes in diabetes, and staging cancers.

Major Conclusions

SGLT-targeted PET imaging offers promising improvements in diagnostic accuracy and therapeutic planning. The findings underscore the physiological and pathological significance of SGLTs, indicating that this imaging approach could shape future diagnostic and therapeutic strategies in metabolic and oncologic fields.

With age, metabolic perturbations accumulate to elevate our obesity burden. While age-onset obesity is mostly driven by a sedentary lifestyle and high calorie intake, genetic and epigenetic factors also play a role. Among these, members of the large histone deacetylase (HDAC) family are of particular importance as key metabolic determinants for healthy ageing, or metabolic dysfunction. Here, we aimed to interrogate the role of class 2 family member HDAC5 in controlling systemic metabolism and age-related obesity under non-obesogenic conditions. Starting at 6 months of age, we observed adult-onset obesity in chow-fed male global HDAC5-KO mice, that was accompanied by marked reductions in adrenergic-stimulated ATP-consuming futile cycles, including BAT activity and UCP1 levels, WAT-lipolysis, skeletal muscle, WAT and liver futile creatine and calcium cycles, and ultimately energy expenditure. Female mice did not differ between genotypes. The lower peripheral sympathetic nervous system (SNS) activity in mature male KO mice was linked to higher dopaminergic neuronal activity within the dorsomedial arcuate nucleus (dmARC) and elevated hypothalamic dopamine levels. Mechanistically, we reveal that hypothalamic HDAC5 acts as co-repressor of STAT5b over the control of Tyrosine hydroxylase (TH) gene transactivation, which ultimately orchestrates the activity of dmARH dopaminergic neurons and energy metabolism in male mice under non-obesogenic conditions.

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system – comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

Objective

Dysregulation of hepatic cholesterol metabolism can contribute to elevated circulating cholesterol levels, which is a significant risk factor for cardiovascular disease. Cholesterol homeostasis in mammalian cells is tightly regulated by an integrated network of transcriptional and post-transcriptional signalling pathways. Whilst prior studies have identified many of the central regulators of these pathways, the extended supporting networks remain to be fully elucidated.

Methods

Here, we leveraged an integrated discovery platform, combining multi-omics data from 107 strains of mice to investigate these supporting networks. We identified retinol dehydrogenase 11 (RDH11; also known as SCALD) as a novel protein associated with cholesterol metabolism. Prior studies have suggested that RDH11 may be regulated by alterations in cellular cholesterol status, but its specific roles in this pathway are mostly unknown.

Results

Here, we show that mice fed a Western diet (high fat, high cholesterol) exhibited a significant reduction in hepatic Rdh11 mRNA expression. Conversely, mice treated with a statin (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) inhibitor) exhibited a 2-fold increase in hepatic Rdh11 mRNA expression. Studies in human and mouse hepatocytes demonstrated that RDH11 expression was regulated by altered cellular cholesterol conditions in a manner consistent with SREBP2 target genes HMGCR and LDLR. Modulation of RDH11 in vitro and in vivo demonstrated modulation of pathways associated with cholesterol metabolism, inflammation and cellular stress. Finally, RDH11 silencing in mouse liver was associated with a reduction in hepatic cardiolipin abundance and a concomitant reduction in the abundance of proteins of the mitochondrial electron transport chain.

Conclusion

Taken together, these findings suggest that RDH11 likely plays a role in protecting cells against the cellular toxicity that can arise as a by-product of endogenous cellular cholesterol synthesis.

Objective

Adipose tissue remodeling plays a critical role in obesity-induced metabolic dysfunction, but the underlying molecular mechanisms remain incompletely understood. This study investigates the role of miR-10a-5p in adipose tissue inflammation and metabolic dysfunction induced by a high-fat diet (HFD).

Methods

Male miR-10a knockout (KO) mice were fed a HFD to induce obesity for up to 16 weeks. RNA sequencing (RNA-seq) analysis was performed to profile mRNA expression and assess the effects of miR-10a-5p KO in gonadal white adipose tissue (gWAT). Additional analyses included immunoblotting, qPCR, histological examination, and validation of the miR-10a-5p target sequence using a dual-luciferase reporter assay.

Results

miR-10a-5p was highly expressed in gWAT but decreased after 8 weeks of HFD feeding. Over the 16-week HFD period, miR-10a KO mice exhibited greater weight gain and reduced energy expenditure compared to wild-type (WT) controls. gWAT of miR-10a KO mice on a HFD showed an increased population of proinflammatory macrophages, elevated inflammation, and increased cell death, characterized by upregulated apoptosis and necrosis markers. This was also associated with increased triglyceride accumulation in liver. Mechanistically, the proapoptotic gene Bcl2l11 was identified as a direct target of miR-10a-5p. Loss of miR-10a-5p led to BIM-mediated adipocyte death and inflammation, contributing to mitochondrial metabolic dysregulation, increased fibrosis marker expression, and the onset of inflammation in adipose tissue.

Conclusions

This study demonstrates the significant role of miR-10a-5p and its downstream target BIM in regulating adipocyte death during diet-induced obesity. This signaling pathway presents a potential therapeutic target for modulating obesity-induced inflammation and cell death in adipose tissue.

Background

D-amino acids are being recognized as important molecules in mammals with function. This is a first identification of endogenous D-cysteine in mammalian pancreas.

Methods

Using a novel stereospecific bioluminescent assay, chiral chromatography, enzyme kinetics and a transgenic mouse model we identify endogenous D-cysteine. We elucidate its function in two mice models of type 1 diabetes (STZ and NOD), and in tests of Glucose Stimulated Insulin Secretion in isolated mouse and human islets and INS-1 832/13 cell line.

Results and Discussion

D-cysteine is synthesized by serine racemase (SR) and SR^−/− mice produce 6–10 fold higher levels of insulin in the pancreas and plasma including higher glycogen and ketone bodies in the liver. The excess insulin is stored as amyloid in secretory vesicles and exosomes. In glucose stimulated insulin secretion in mouse and human islets, equimolar amount of D-cysteine showed higher inhibition of insulin secretion compared to D-serine, another closely related stereoisomer synthesized by SR. In mouse models of diabetes (Streptozotocin (STZ) and Non Obese Diabetes (NOD) and human pancreas, the diabetic state showed increased expression of D-cysteine compared to D-serine followed by increased expression of SR. SR^−/− mice show decreased cAMP in the pancreas, lower DNA methyltransferase enzymatic and promoter activities followed by reduced phosphorylation of CREB (S133), resulting in decreased methylation of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1. Dietary supplementation with methyl donors restored the high insulin levels and low DNMT enzymatic activity in SR^−/− mice.

Conclusions

Our data show that endogenous D-cysteine in the mammalian pancreas is a regulator of insulin secretion.

Objectives

Protein kinase D (PKD) family is emerging as relevant regulator of metabolic homeostasis. However, the precise role of PKD2 in modulating hepatic insulin signaling has not been fully elucidated and it is the aim of this study.

Methods

PKD inhibition was analyzed for insulin signaling in mouse and human hepatocytes. PKD2 was overexpressed in Huh7 hepatocytes and mouse liver, and insulin responses were evaluated. Mice with hepatocyte-specific PKD2 depletion (PKD2^ΔHep) and PKD2^fl/fl mice were fed a chow (CHD) or high fat diet (HFD) and glucose homeostasis and lipid metabolism were investigated.

Results

PKD2 silencing enhanced insulin signaling in hepatocytes, an effect also found in primary hepatocytes from PKD2^ΔHep mice. Conversely, a constitutively active PKD2 mutant reduced insulin-stimulated AKT phosphorylation. A more in-depth analysis revealed reduced IRS1 serine phosphorylation under basal conditions and increased IRS1 tyrosine phosphorylation in PKD2^ΔHep primary hepatocytes upon insulin stimulation and, importantly PKD co-immunoprecipitates with IRS1. In vivo constitutively active PKD2 overexpression resulted in a moderate impairment of glucose homeostasis and reduced insulin signaling in the liver. On the contrary, HFD-fed PKD2^ΔHep male mice displayed improved glucose and pyruvate tolerance, as well as higher peripheral insulin tolerance and enhanced hepatic insulin signaling compared to control PKD2^fl/fl mice. Despite of a remodeling of hepatic lipid metabolism in HFD-fed PKD2^ΔHep mice, similar steatosis grade was found in both genotypes.

Conclusions

Results herein have unveiled an unknown role of PKD2 in the control of insulin signaling in the liver at the level of IRS1 and point PKD2 as a therapeutic target for hepatic insulin resistance.

Objective

The peptide hormone ghrelin exerts potent effects in the brain, where its receptor is highly expressed. Here, we investigated the role of hypothalamic tanycytes in transporting ghrelin across the blood-cerebrospinal fluid (CSF) interface.

Methods

We investigated the internalization and transport of fluorescent ghrelin (Fr-ghrelin) in primary cultures of rat hypothalamic tanycytes, mouse hypothalamic explants, and mice. We also tested the impact of inhibiting clathrin-mediated endocytosis of ghrelin in the brain ventricular system on the orexigenic and locomotor effects of the hormone.

Results

In vitro, we found that Fr-ghrelin is selectively and rapidly internalized at the soma of tanycytes, via a GHSR-independent and clathrin-dependent mechanism, and then transported to the endfoot. In hypothalamic explants, we also found that Fr-ghrelin is internalized at the apical pole of tanycytes. In mice, Fr-ghrelin present in the CSF was rapidly internalized by hypothalamic β-type tanycytes in a clathrin-dependent manner, and pharmacological inhibition of clathrin-mediated endocytosis in the brain ventricular system prolonged the ghrelin-induced locomotor effects.

Conclusions

We propose that tanycyte-mediated transport of ghrelin is functionally relevant, as it may contribute to reduce the concentration of this peptide hormone in the CSF and consequently shortens the duration of its central effects.

Background

Long non-coding RNAs (lncRNAs) can perform tasks of key relevance in fat cells, contributing, when defective, to the burden of obesity and its sequelae. Here, scrutiny of adipose tissue transcriptomes before and after bariatric surgery (GSE53378) granted identification of 496 lncRNAs linked to the obese phenotype. Only expression of linc-GALNTL6-4 displayed an average recovery over 2-fold and FDR-adjusted p-value <0.0001 after weight loss. The aim of the present study was to investigate the impact on adipocyte function and potential clinical value of impaired adipose linc-GALNTL6-4 in obese subjects.

Methods

We employed transcriptomic analysis of public dataset GSE199063, and cross validations in two large transversal cohorts to report evidence of a previously unknown association of adipose linc-GALNTL6-4 with obesity. We then performed functional analyses in human adipocyte cultures, genome-wide transcriptomics, and untargeted lipidomics in cell models of loss and gain of function to explore the molecular implications of its associations with obesity and weight loss.

Results

The expression of linc-GALNTL6-4 in human adipose tissue is adipocyte-specific and co-segregates with obesity, being normalized upon weight loss. This co-segregation is demonstrated in two longitudinal weight loss studies and two cross-sectional samples. While compromised expression of linc-GALNTL6-4 in obese subjects is primarily due to the inflammatory component in the context of obesity, adipogenesis requires the transcriptional upregulation of linc-GALNTL6-4, the expression of which reaches an apex in terminally differentiated adipocytes. Functionally, we demonstrated that the knockdown of linc-GALNTL6-4 impairs adipogenesis, induces alterations in the lipidome, and leads to the downregulation of genes related to cell cycle, while propelling in adipocytes inflammation, impaired fatty acid metabolism, and altered gene expression patterns, including that of apolipoprotein C1 (APOC1). Conversely, the genetic gain of linc-GALNTL6-4 ameliorated differentiation and adipocyte phenotype, putatively by constraining APOC1, also contributing to the metabolism of triglycerides in adipose.

Conclusions

Current data unveil the unforeseen connection of adipocyte-specific linc-GALNTL6-4 as a modulator of lipid homeostasis challenged by excessive body weight and meta-inflammation.

Objective

Triglycerides (TGs) associate with apolipoprotein B100 (apoB100) to form very low density lipoproteins (VLDLs) in the liver. The repertoire of factors that facilitate this association is incompletely understood. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytosolic lipid droplet (LD) biogenesis in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen.

Methods

Experiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, stimulated Raman scattering (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy.

Main findings

1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to low density lipoprotein (LDL) density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress.

Conclusion

The results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be an important factor in the partitioning of TG between cytosolic LDs and VLDL particles.

Objectives

Adipose tissue depots vary markedly in their ability to store and metabolize triglycerides, undergo beige adipogenesis and susceptibility to metabolic disease. The molecular mechanisms that underlie such heterogeneity are not entirely clear. Previously, we showed that TGF-β signaling suppresses beige adipogenesis via repressing the recruitment of dedicated beige progenitors. Here, we find that TGF-β signals dynamically regulate the balance between adipose tissue fibrosis and beige adipogenesis.

Methods

We investigated adipose tissue depot-specific differences in activation of TGF-β signaling in response to dietary challenge. RNA-seq and fluorescence activated cell sorting was performed to identify and characterize cells responding to changes in TGF-β signaling status. Mouse models, pharmacological strategies and human adipose tissue analyses were performed to further define the influence of TGF-β signaling on fibrosis and functional beige adipogenesis.

Results

Elevated basal and high-fat diet inducible activation of TGF-β/Smad3 signaling was observed in the visceral adipose tissue depot. Activation of TGF-β/Smad3 signaling was associated with increased adipose tissue fibrosis. RNA-seq combined with fluorescence-activated cell sorting of stromal vascular fraction of epididymal white adipose tissue depot resulted in identification of TGF-β/Smad3 regulated ITGA5+ fibrogenic progenitors. TGF-β/Smad3 signal inhibition, genetically or pharmacologically, reduced fibrosis and increased functional beige adipogenesis. TGF-β/Smad3 antagonized the beneficial effects of PPARγ whereas TGF-β receptor 1 inhibition synergized with actions of rosiglitazone, a PPARγ agonist, to dampen fibrosis and promote beige adipogenesis. Positive correlation between TGF-β activation and ITGA5 was observed in human adipose tissue, with visceral adipose tissue depots exhibiting higher fibrosis potential than subcutaneous or brown adipose tissue depots.

Conclusions

Basal and high-fat diet inducible activation of TGF-β underlies the heterogeneity of adipose tissue depots. TGF-β/Smad3 activation promotes adipose tissue fibrosis and suppresses beige progenitors. Together, these dual mechanisms preclude functional beige adipogenesis. Controlled inhibition of TβRI signaling and concomitant PPARγ stimulation can suppress adipose tissue fibrosis and promote beige adipogenesis to improve metabolism.

Objective

Activating brown adipose tissue (BAT) thermogenesis is a promising approach to combat obesity and metabolic disorders. The post-transcriptional regulation of BAT thermogenesis mediated by RNA-binding proteins (RBPs) is still not fully understood. This study explores the physiological role of novel RBPs in BAT differentiation and thermogenesis.

Methods

We used multiple public datasets to screen out novel RBPs responsible for BAT differentiation and thermogenesis. In vitro loss- and gain-of-function experiments were performed in both C3H10T1/2 preadipocytes and mature brown adipocytes to determine the role of Y-box binding protein 3 (YBX3) in brown adipocyte differentiation and thermogenesis. Adeno-associated virus (AAV)-mediated BAT-specific knockdown or overexpression of Ybx3 was applied to investigate the function of YBX3 in vivo.

Results

YBX3 is a brown adipocyte-enriched RBP induced by cold stimulation and β-adrenergic signaling. Both in vitro loss- and gain-of-function experiments demonstrate that YBX3 is essential for brown adipocyte differentiation and thermogenesis. BAT-specific loss of Ybx3 dampens thermogenesis and exacerbates diet-induced obesity in mice, while overexpression of Ybx3 promotes thermogenesis and confers protection against diet-induced metabolic dysfunction. Transcriptome analysis and mitochondrial stress test indicate that Ybx3 deficiency compromises the mitochondrial oxidative phosphorylation, leading to thermogenic failure. Mechanistically, YBX3 stabilizes the mRNA of Slc3a2 and Pparg, which facilitates branched-chain amino acid (BCAA) influx and catabolism and fuels brown adipocyte differentiation and thermogenesis.

Conclusions

YBX3 facilitates BAT fueling BCAA to boost thermogenesis and energy expenditure, which protects against obesity and metabolic dysfunction. Thus, YBX3 could be a promising therapeutic target for obesity.

Objective

The uncoupling protein 1 (UCP1) is induced in brown or “beige” adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPARγ agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously.

Methods

Adipocytes generated from primary human progenitor cells were differentiated without exposure to PPARγ agonists, and treated for 3, 6 or 78 h to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses.

Results

UCP1 mRNA expression was induced within 3 h of exposure to either Rsg or Fsk, indicating that Rsg’s effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2, the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPARγ agonists.

Conclusions

UCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPARγ-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPARγ in response to cAMP may result from the production of natural PPARγ activating ligands through ATGL-mediated lipolysis.

Objective

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates.

Method

To address the pleotropic nature of O-GlcNAc, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics.

Results

We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways.

Conclusion

These data show how a multi-Omics platform can disentangle the pleotropic nature of O-GlcNAc to discern how OGT fine-tunes multiple cellular pathways involved in aneuploidy.

Objectives

Elevated circulating branched-chain amino acids (BCAAs) have been associated with obesity, insulin resistance, and MASLD. Nonetheless, BCAA supplementation has been shown to provide protective outcomes towards the intervention of MASLD. Currently, there is a lack of study towards the contribution of the BCAA: valine on MASLD. Herein, the effect of low-dose valine supplementation was investigated for its role in the progression of MASLD.

Methods

C57BL/6J mice were fed a high-fat/high-cholesterol diet (HFD) to induce MASLD. Upon the establishment of MASLD, valine was supplemented via voluntary oral administration. Clinical and biochemical parameters associated with MASLD were measured, and molecular mechanism and gut microbiota modulation from the effect of valine were investigated.

Results

Low-dose valine was found to attenuate the progression of MASLD, significantly reducing the gain in body weight, liver weight, and epididymal white adipose tissue (eWAT) weight, while also attenuating hyperglycemia and hyperleptinemia, and improving serum lipid profiles. Mechanistically, in the liver, genes related to hepatic lipogenesis and cholesterol biosynthesis were downregulated, while those associated with fatty acid oxidation, autophagy, and antioxidant capacity were upregulated, and AMPK pathway activity was enhanced. Liver and hypothalamic leptin resistance and inflammation were also attenuated, allowing better appetite control in mice fed a HFD and leading to reduced food intake. Additionally, metabolic flexibility in the eWAT was improved, and the gut microbiome was modulated by low-dose valine supplementation.

Conclusion

Low-dose valine supplementation attenuates MASLD by enhancing systemic leptin sensitivity and modulating the gut microbiome.

Objective

The gene encoding TOMM40 (Transporter of Outer Mitochondrial Membrane 40) is adjacent to that encoding APOE, which has a central role in lipid and lipoprotein metabolism. While human genetic variants near APOE and TOMM40 have been shown to be strongly associated with plasma lipid levels, a specific role for TOMM40 in lipid metabolism has not been established, and the present study was aimed at assessing this possibility.

Methods

TOMM40 was knocked down by siRNA in human hepatoma HepG2 cells, and effects on mitochondrial function, lipid phenotypes, and crosstalk between mitochondria, ER, and lipid droplets were examined. Additionally, hepatic and plasma lipid levels were measured in mice following shRNA-induced knockdown of Tomm40 shRNA.

Results

In HepG2 cells, TOMM40 knockdown upregulated expression of APOE and LDLR in part via activation of LXRB (NR1H2) by oxysterols, with consequent increased uptake of VLDL and LDL. This is in part due to disruption of mitochondria-endoplasmic reticulum contact sites, with resulting accrual of reactive oxygen species and non-enzymatically derived oxysterols. With TOMM40 knockdown, cellular triglyceride and lipid droplet content were increased, effects attributable in part to receptor-mediated VLDL uptake, since lipid staining was significantly reduced by concomitant suppression of either LDLR or APOE. In contrast, cellular cholesterol content was reduced due to LXRB-mediated upregulation of the ABCA1 transporter as well as increased production and secretion of oxysterol-derived cholic acid. Consistent with the findings in hepatoma cells, in vivo knockdown of TOMM40 in mice resulted in significant reductions of plasma triglyceride and cholesterol concentrations, reduced hepatic cholesterol and increased triglyceride content, and accumulation of lipid droplets leading to development of steatosis.

Conclusions

These findings demonstrate a role for TOMM40 in regulating hepatic lipid and plasma lipoprotein levels and identify mechanisms linking mitochondrial function with lipid metabolism.

Objective

Exercise plays a crucial role in maintaining and improving human health. However, the precise molecular mechanisms that govern the body’s response to exercise or/compared to periods of inactivity remain elusive. Current evidence appears to suggest that exercise exerts a seemingly dual influence on macrophage polarization states, inducing both pro-immune response M1 activation and cell-repair-focused M2 activation. To reconcile this apparent paradox, we leveraged a comprehensive meta-analysis of 75 diverse exercise and immobilization published datasets (7000+ samples), encompassing various exercise modalities, sampling techniques, and species.

Methods

75 exercise and immobilization expression datasets were identified and processed for analysis. The data was analyzed using boolean relationships which uses binary gene expression relationships in order to increase the signal to noise achieved from the data, allowing for the use of comparison across such a diverse set of datasets. We utilized a boolean relationship-aided macrophage gene model [1], to model the macrophage polarization state in pre and post exercise samples in both immediate exercise and long term training.

Results

Our modeling uncovered a key temporal dynamic: exercise triggers an immediate M1 surge, while long term training transitions to sustained M2 activation. These patterns were consistent across different species (human vs mouse), sampling methods (blood vs muscle biopsy), and exercise type (resistance vs endurance), and routinely showed statistically significant results. Immobilization was shown to have the opposite effect of exercise by triggering an immediate M2 activation. Individual characteristics like gender, exercise intensity and age were found to impact the degree of polarization without changing the overall patterns. To model macrophages within the specific context of muscle tissue, we identified a focused gene set signature of muscle resident macrophage polarization, allowing for the precise measurement of macrophage activity in response to exercise within the muscle.

Conclusions

These consistent patterns across all 75 examined studies suggest that the long term health benefits of exercise stem from its ability to orchestrate a balanced and temporally-regulated interplay between pro-immune response (M1) and reparative macrophage activity (M2). Similarly, it suggests that an imbalance between pro-immune and cell repair responses could facilitate disease development. Our findings shed light on the intricate molecular choreography behind exercise-induced health benefits with a particular insight on its effect on the macrophages within the muscle.

Objective

The early consumption of calorie-rich diet disrupts circadian rhythms and has adverse effects on memory, yet the effects of time-restricted feeding (TRF) and the underlying molecular mechanisms are unknown. Here, we set out to identify the behavioral and molecular circadian rhythms disruptions generated by juvenile obesogenic diet consumption and their restoration by TRF in male mice.

Methods

Metabolic rhythms were measured by indirect calorimetry and memory performances by behavioral tasks. Hippocampal translatome (pS6_TRAP), enrichment and co-regulated gene network analyses were conducted to identify the molecular pathways involved in memory impairments and their restoration by TRF. Differential exon usage analyses, mass spectrometry and pharmacological intervention were used to confirm thyroid hormone signaling involvement.

Results

We show that four weeks of TRF restore the rhythmicity of metabolic parameters and prevents memory impairments in mice fed a high fat-high sucrose (HFS) diet since weaning, independently of body fat levels. Hippocampal translatome and differential exon usage analyses indicate that impaired memory of mice under ad libitum HFS diet is accompanied by reduced thyroid hormone signaling and altered expression of astrocytic genes regulating glutamate neurotransmission. TRF restored the diurnal expression variation of part of these genes and intra-hippocampal infusion of T3, the active form of thyroid hormone, rescues memory performances and astrocytic gene expression of ad libitum HFS diet-fed mice.

Conclusions

Thus, thyroid hormones contribute to the TRF positive effects on both metabolism and memory in mice fed an obesogenic diet, highlighting this nutritional approach as a powerful tool in addressing obesity brain comorbidities and paving the way for further mechanistic studies on hippocampal thyroid signaling.

Objectives

Deficiency in the transcription factor (TF) GLI-Similar 3 (GLIS3) in humans and mice leads to the development of polycystic kidney disease (PKD). In this study, we investigate the role of GLIS3 in the regulation of energy metabolism and mitochondrial functions in relation to its role in normal kidney and metabolic reprogramming in PKD pathogenesis.

Methods

Transcriptomics, cistromics, and metabolomics were used to obtain insights into the role of GLIS3 in the regulation of energy homeostasis and mitochondrial metabolism in normal kidney and PKD pathogenesis using GLIS3-deficient mice.

Results

Transcriptome analysis showed that many genes critical for mitochondrial biogenesis, oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and the tricarboxylic acid (TCA) cycle, including Tfam, Tfb1m, Tfb2m, Ppargc1a, Ppargc1b, Atp5j2, Hadha, and Sdha, are significantly suppressed in kidneys from both ubiquitous and tissue-specific Glis3-deficient mice. ChIP-Seq analysis demonstrated that GLIS3 is associated with the regulatory region of many of these genes, indicating that their transcription is directly regulated by GLIS3. Cistrome analyses revealed that GLIS3 binding loci frequently located near those of hepatocyte nuclear factor 1-Beta (HNF1B) and nuclear respiratory factor 1 (NRF1) suggesting GLIS3 regulates transcription of many metabolic and mitochondrial function-related genes in coordination with these TFs. Seahorse analysis and untargeted metabolomics corroborated that mitochondrial OXPHOS utilization is suppressed in GLIS3-deficient kidneys and showed that key metabolites in glycolysis, TCA cycle, and glutamine pathways were altered indicating increased reliance on aerobic glycolysis and glutamine anaplerosis. These features of metabolic reprogramming may contribute to a bioenergetic environment that supports renal cyst formation and progression in Glis3-deficient mice kidneys.

Conclusions

We identify GLIS3 as a novel positive regulator of the transition from aerobic glycolysis to OXPHOS in normal early postnatal kidney development by directly regulating the transcription of mitochondrial metabolic genes. Loss of GLIS3 induces several features of renal cell metabolic reprogramming. Our study identifies GLIS3 as a new participant in an interconnected transcription regulatory network, that includes HNF1B and NRF1, critical in the regulation of mitochondrial-related gene expression and energy metabolism in normal postnatal kidneys and PKD pathogenesis in Glis3-deficient mice.

Objective

Thyroid hormone (TH) action is mediated by thyroid hormone receptor (THR) isoforms. While THRβ1 is likely the main isoform expressed in liver, its role in human hepatocytes is not fully understood.

Methods

To elucidate the role of THRβ1 action in human hepatocytes we used CRISPR/Cas9 editing to knock out THRβ1 in induced pluripotent stem cells (iPSC). Following directed differentiation to the hepatic lineage, iPSC-derived hepatocytes were then interrogated to determine the role of THRβ1 in ligand-independent and -dependent functions.

Results

We found that the loss of THRβ1 promoted alterations in proliferation rate and metabolic pathways regulated by T3, including gluconeogenesis, lipid oxidation, fatty acid synthesis, and fatty acid uptake. We observed that key genes involved in liver metabolism are regulated through both T3 ligand-dependent and -independent THRβ1 signaling mechanisms. Finally, we demonstrate that following THRβ1 knockout, several key metabolic genes remain T3 responsive suggesting they are THRα targets.

Conclusions

These results highlight that iPSC-derived hepatocytes are an effective platform to study mechanisms regulating TH signaling in human hepatocytes.

Objective

The study aimed to investigate the effects of glucagon on metabolic pathways in mouse models of obesity, fatty liver disease, and type 2 diabetes (T2D) to determine the extent and variability of hepatic glucagon resistance in these conditions.

Methods

We investigated glucagon's effects in mouse models of fatty liver disease, obesity, and type 2 diabetes (T2D), including male BKS-db/db, high-fat diet-fed, and western diet-fed C57Bl/6 mice. Glucagon tolerance tests were performed using the selective glucagon receptor agonist acyl-glucagon (IUB288). Blood glucose, serum and liver metabolites include lipids and amino acids were measured. Additionally, liver protein expression related to glucagon signalling and a comprehensive liver metabolomics were performed.

Results

Western diet-fed mice displayed impaired glucagon response, with reduced blood glucose and PKA activation. In contrast, high-fat diet-fed and db/db mice maintained normal glucagon sensitivity, showing significant elevations in blood glucose and phospho-PKA motif protein expression. Acyl-glucagon treatment also lowered liver alanine and histidine levels in high-fat diet-fed mice, but not in western diet-fed mice. Additionally, some amino acids, such as methionine, were increased by acyl-glucagon only in chow diet control mice. Despite normal glucagon sensitivity in PKA signalling, db/db mice had a distinct metabolomic response, with acyl-glucagon significantly altering 90 metabolites in db/+ mice but only 42 in db/db mice, and classic glucagon-regulated metabolites, such as cyclic adenosine monophosphate (cAMP), being less responsive in db/db mice.

Conclusions

The study reveals that hepatic glucagon resistance in obesity and T2D is complex and not uniform across metabolic pathways, underscoring the complexity of glucagon action in these conditions.

Objectives

Obesity, diabetes and high-calorie diets are associated with defective sperm function and lowered male fertility. Mature spermatozoa primarily use fructose and glucose, and glucose and glycerol metabolism are important for sperm function. We recently discovered a novel mammalian enzyme, glycerol-3-phosphate (Gro3P) phosphatase (G3PP), and showed that it operates the glycerol shunt by hydrolyzing Gro3P to glycerol, and regulates glucose, lipid and energy metabolism in pancreatic β-cells and liver. We now observed that G3PP expression is the highest in the testis and spermatozoa, and investigated its role in male fertility.

Methods

We examined G3PP expression during spermatogenesis in mouse and assessed male fertility and spermatozoon function in conditional germ cell specific G3PP-KO (cG3PP-KO) mice and tamoxifen-inducible conditional germ cell G3PP-KO (icG3PP-KO) mice. We also determined the structural and metabolic parameters and oxidative stress in the spermatozoa from icG3PP-KO and control mice.

Results

G3PP expression in mouse spermatocytes and spermatids markedly increases during spermatogenesis. Male cG3PP-KO mice, in which germ cell G3PP is deleted from embryonic stage, are infertile due to dysfunctional sperm with reduced motility and capacitation, and elevated spontaneous acrosomal reaction and oxidative stress. However, icG3PP-KO male mice do not have altered fertility, due to the presence of ∼10% normal spermatozoa. icG3PP-KO spermatozoa display significantly reduced functionality and morphological and ultrastructural alterations. The icG3PP-KO spermatozoa show reduced glycerol production, elevated levels of Gro3P and reactive oxygen species (ROS), and oxidative stress that is associated with increased mitochondrial membrane potential.

Conclusions

Germ cell G3PP deletion leads to the generation of spermatozoa that are functionally and structurally abnormal, likely due to the build-up of Gro3P that increases mitochondrial membrane potential, ROS, and oxidative stress and alters spermatozoa function. Overall, the results indicate that G3PP and the glycerol shunt are essential for normal spermatozoa function and male fertility.

Objective

Dysregulated glucagon secretion and inadequate functional beta cell mass are hallmark features of diabetes. While glucagon receptor (GCGR) antagonism ameliorates hyperglycemia and elicits beta cell regeneration in pre-clinical models of diabetes, it also promotes alpha and delta cell hyperplasia. We sought to investigate the mechanism by which loss of glucagon action impacts pancreatic islet non-alpha cells, and the relevance of these observations in a human islet context.

Methods

We used zebrafish, rodents, and transplanted human islets comprising six different models of interrupted glucagon signaling to examine their impact on delta and beta cell proliferation and mass. We also used models with global deficiency of the cationic amino acid transporter, SLC7A2, and mTORC1 inhibition via rapamycin, to determine whether amino acid-dependent nutrient sensing was required for islet non-alpha cell growth.

Results

Inhibition of glucagon signaling stimulated delta cell proliferation in mouse and transplanted human islets, and in mouse islets. This was rapamycin-sensitive and required SLC7A2. Likewise, gcgr deficiency augmented beta cell proliferation via SLC7A2- and mTORC1-dependent mechanisms in zebrafish and promoted cell cycle engagement in rodent beta cells but was insufficient to drive a significant increase in beta cell mass in mice.

Conclusions

Our findings demonstrate that interruption of glucagon signaling augments islet non-alpha cell proliferation in zebrafish, rodents, and transplanted human islets in a manner requiring SLC7A2 and mTORC1 activation. An increase in delta cell mass may be leveraged for future beta cell regeneration therapies relying upon delta cell reprogramming.

Objectives

Histone acetylation is an important epigenetic modification that regulates various biological processes and cell homeostasis. Acetyl-CoA, a hub molecule of metabolism, is the substrate for histone acetylation, thus linking metabolism with epigenetic regulation. However, still relatively little is known about the dynamics of histone acetylation and its dependence on metabolic processes, due to the lack of integrated methods that can capture site-specific histone acetylation and deacetylation reactions together with the dynamics of acetyl-CoA synthesis.

Methods

In this study, we present a novel proteo-metabo-flux approach that combines mass spectrometry-based metabolic flux analysis of acetyl-CoA and histone acetylation with computational modelling. We developed a mathematical model to describe metabolic label incorporation into acetyl-CoA and histone acetylation based on experimentally measured relative abundances.

Results

We demonstrate that our approach is able to determine acetyl-CoA synthesis dynamics and site-specific histone acetylation and deacetylation reaction rate constants, and that consideration of the metabolically labelled acetyl-CoA fraction is essential for accurate determination of histone acetylation dynamics. Furthermore, we show that without correction, changes in metabolic fluxes would be misinterpreted as changes in histone acetylation dynamics, whereas our proteo-metabo-flux approach allows to distinguish between the two processes.

Conclusions

Our proteo-metabo-flux approach expands the repertoire of metabolic flux analysis and cross-omics and represents a valuable approach to study the regulatory interplay between metabolism and epigenetic regulation by histone acetylation.