Background

Cellular metabolism plays a pivotal role in the development and progression of pancreatic ductal adenocarcinoma (PDAC), with dysregulated metabolic pathways contributing to tumorigenesis and therapeutic resistance. Distinct metabolic heterogeneity in pancreatic cancer significantly impacts patient prognosis, as variations in metabolic profiles influence tumor behavior and treatment responses.

Scope of the Review: This review explores the intricate interplay between mitochondrial dynamics, mitophagy, and cellular metabolism in PDAC. We discuss the significance of mitophagy dysregulation in PDAC pathogenesis, emphasizing its influence on treatment responses and prognosis. Furthermore, we analyze the impact of mitochondrial dynamics alterations, including fission and fusion processes, on PDAC progression and tumorigenesis.

Major Conclusion

Targeting mitochondrial metabolism holds promise for advancing PDAC therapeutics. Ongoing clinical trials underscore the therapeutic potential of modulating key regulators of mitochondrial dynamics and mitophagy. Despite inherent challenges, these approaches offer diverse strategies to enhance treatment efficacy and improve patient outcomes.

Objectives

Dysregulation of lipid homeostasis pathway causes many liver diseases, including hepatic steatosis. One of the primary factors contributing to lipid accumulation is fatty acid uptake by the liver. Transmembrane protein 135 (TMEM135), which exists in mitochondria and peroxisomes, participates in intracellular lipid metabolism. This study aims to investigate the role of TMEM135 on regulating cellular lipid import in the liver.

Methods

We used in vivo, ex vivo, and in vitro models of steatosis. TMEM135 knockout (TMEM135KO) and wild type (WT) mice were fed a high-fat diet (HFD) to induce hepatic steatosis. Primary mouse hepatocytes and AML12 cells were treated with free fatty acid (FFA). Additionally, TMEM135-deficient stable cells and overexpressed cells were established using AML12 cells.

Results

TMEM135 deficiency mitigated lipid accumulation in the liver of HFD-fed TMEM135KO mice. TMEM135-depleted primary hepatocytes and AML12 cells exhibited less lipid accumulation when treated with FFA compared to control cells, as shown as lipid droplets. Consistently, the effect of TMEM135 depletion on lipid accumulation was completely reversed under TMEM135 overexpression conditions. CD36 expression was markedly induced by HFD or FFA, which was reduced by TMEM135 depletion. Among the SIRT family proteins, only SIRT1 expression definitely increased in the liver of HFD-fed TMEM135KO mice along with a significant increase in NAD⁺/NADH ratio. However, inhibition of SIRT1 in TMEM135-depleted cells using siSIRT1 or the SIRT1 inhibitor EX-527 resulted in an increase of CD36 expression and consequent TG levels.

Conclusions

TMEM135 depletion attenuates CD36 expression in a SIRT1-dependent manner, thereby reducing cellular lipid uptake and hepatic steatosis.

In white adipose tissue, disturbed creatine metabolism through reduced creatine kinase B (CKB) transcription contributes to obesity-related inflammation. However, the mechanisms regulating CKB expression in human white adipocytes remain unclear. By screening conditions perturbed in obesity, we identified endoplasmic reticulum (ER) stress as a key suppressor of CKB transcription across multiple cell types. Through follow-up studies, we found that ER stress through the IRE1–XBP1s pathway, promotes CKB promoter methylation via the methyltransferase DNMT3A. This epigenetic change represses CKB transcription, shifting metabolism towards glycolysis and increasing the production of the pro-inflammatory chemokine CCL2. We validated our findings in vivo, demonstrating that individuals living with obesity show an inverse relationship between CKB expression and promoter methylation in white adipocytes, along with elevated CCL2 secretion. Overall, our study uncovers a regulatory axis where ER stress drives inflammation in obesity by reducing CKB abundance, and consequently altering the bioenergetic state of the cell.

Objective

Antagonism of the muscarinic acetylcholine type 1 receptor (M₁R) promotes sensory axon repair and is protective in peripheral neuropathy, however, the mechanism remains elusive. We investigated the role of the heat-sensing transient receptor potential melastatin-3 (TRPM3) cation channel in M₁R antagonism-mediated nerve regeneration and explored the potential of TRPM3 activation to facilitate axonal plasticity.

Methods

Dorsal root ganglion (DRG) neurons from adult control or diabetic rats were cultured and treated with TRPM3 agonists (CIM0216, pregnenolone sulfate) and M₁R antagonists pirenzepine (PZ) or muscarinic toxin 7 (MT7). Ca²⁺ transients, mitochondrial respiration, AMP-activated protein kinase (AMPK) expression, and mitochondrial inner membrane potential were analyzed. The effect of M₁R activation or blockade on TRPM3 activity mediated by phosphatidylinositol 4,5-bisphosphate (PIP₂) was studied. Metabolic profiling of DRG neurons and human neuroblastoma SH-SY5Y cells was conducted.

Results

M₁R antagonism induced by PZ or MT7 increased Ca²⁺ influx in DRG neurons and was inhibited by TRPM3 antagonists or in the absence of extracellular Ca²⁺. TRPM3 agonists elevated Ca²⁺ levels, augmented mitochondrial respiration, AMPK activation and neurite outgrowth. M₁R antagonism stimulated TRPM3 channel activity through inhibition of PIP₂ hydrolysis to activate Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMPK, leading to augmented mitochondrial function and neuronal metabolism. DRG neurons with AAV-mediated shRNA knockdown of TRPM3 exhibited suppressed antimuscarinic drug-induced neurite outgrowth. TRPM3 agonists increased glycolysis and TCA cycle metabolites, indicating enhanced metabolism in DRG neurons and SH-SY5Y cells.

Conclusions

Activation of the TRPM3/CaMKKβ/AMPK pathway promoted collateral sprouting of sensory axons, positioning TRPM3 as a promising therapeutic target for peripheral neuropathy.

Objectives

Environmental factors such as physical activity induce epigenetic modifications, with exercise-responsive DNA methylation changes occurring in skeletal muscle. To determine the skeletal muscle DNA methylation signature of endurance swim training, we used whole-genome methylated DNA immunoprecipitation (MeDIP) sequencing.

Methods

We utilized endurance-trained rats, cultured L6 myotubes, and human skeletal muscle cells, employing MeDIP sequencing, gene silencing, and palmitate oxidation assays. Additional methods included promoter luciferase assays, fluorescence microscopy, and RNA/DNA analysis to investigate exercise-induced molecular changes.

Results

Gene set enrichment analysis (GSEA) of differentially methylated promoter regions identified an enrichment of four gene sets, including those linked to lipid metabolic processes, with hypermethylated or hypomethylated promoter regions in skeletal muscle of exercise-trained rats. Bisulfite sequencing confirmed hypomethylation of CpGs in the Serhl2 (Serine Hydrolase Like 2) transcription start site in exercise-trained rats. Serhl2 gene expression was upregulated in both exercise-trained rats and an "exercise-in-a-dish" model of L6 myotubes subjected to electrical pulse stimulation (EPS). Serhl2 promoter activity was regulated by methylation and EPS. A Nr4a binding motif in the Serhl2 promoter, when deleted, reduced promoter activity and sensitivity to methylation in L6 myotubes. Silencing Serhl2 in L6 myotubes reduced intracellular lipid oxidation and triacylglycerol synthesis in response to EPS.

Conclusions

Exercise-training enhances intracellular lipid metabolism and phenotypic changes in skeletal muscle through epigenomic modifications on Serhl2. Hypomethylation of the Serhl2 promoter influences Nr4a transcription factor binding, promoter activity, and gene expression, linking exercise-induced epigenomic regulation of Serhl2 to lipid oxidation and triacylglycerol synthesis.

Objective

The aim of the present study was to investigate the effects of targeting the mevalonate pathway (MVP) in rhabdomyosarcoma (RMS), a soft tissue tumor with a prevalence in young people.

Methods

In silico analyses of RNA datasets were performed to correlate MVP with RMS patient survival. The sensitivity of RMS cell lines to MVP inhibitors was assessed in vitro by analysis of cell growth (crystal violet and clonogenic assays), cell migration (wound healing assay), cell survival (neutral red assay), and oxidative stress (ROS assay). The effects of MVP inhibitors were tested in vivo by analyzing RMS xenografts grown in NOD/SCID mice. Quantification of protein targets was performed using immunoblotting or immunohistochemistry analyses.

Results

In silico analysis showed upregulation of sterol regulatory element-binding protein 2 (SREBP2) and MVP genes, including 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), farnesyl-diphosphate synthase (FDPS), squalene epoxidase (SQLE), which correlated with worse overall patient survival. Targeting of MVP in human RD and RH30 lines by inhibitors of SREBP2 (fatostatin), HMGCR (lovastatin and simvastatin), and FDPS (zoledronic acid) resulted in impaired cell growth, migration, and viability, and increased oxidative cell death in combination with actinomycin D. Conversely, cholesterol (CHO) supplementation enhanced cell growth and migration. Fatostatin and lovastatin produced rapid attenuation of Erk1/2 and Akt1 signaling in RMS lines, and oral administration of lovastatin reduced tumor mass growth of xenografted RD cells in NOD/SCID mice. Finally, we found that forced Akt1 activation in RD cells was sufficient to drive SREBP2, HMGCR and SQLE protein expression, promoting increased susceptibility to MVP inhibitors.

Conclusions

These data suggest that the Akt1, SREBP2 and MVP axis is critical for RMS tumor growth, migration, and oxidative stress protection primarily through maintaining adequate CHO levels that enable proper intracellular signaling. Therefore, stimulating CHO depletion via SREBP2 and MVP inhibition may represent a viable option to improve the combination therapy protocol, especially in pAkt1-positive RMS.

Objective

Dynorphin, an endogenous opioid peptide predominantly expressed in the central nervous system and involved in stress response, pain, and addiction, has intrigued researchers due to its expression in pancreatic β-cells. In this study, we aimed to characterize dynorphin expression in mouse and human islets and explore the mechanisms regulating its expression.

Methods

We used primary mouse and human islets with unbiased published datasets to examine how glucose and other nutrients regulate dynorphin expression and secretion in islets.

Results

The prodynorphin gene is significantly upregulated in β-cells under hyperglycemic conditions. In vitro studies revealed that increased glucose concentrations correlate with increased dynorphin expression, indicating a critical interplay involving Ca²⁺, CamKII, and CREB pathways in β-cells. Perifusion studies allowed us to measure the dynamic secretion of dynorphin in response to glucose from mouse and human islets for the first time. Furthermore, we confirmed that increased dynorphin content within the β-cells directly correlates with enhanced dynorphin secretion. Finally, our findings demonstrate a synergistic effect of palmitate in conjunction with high glucose, further amplifying dynorphin levels and secretion in pancreatic islets.

Conclusions

This study demonstrates that the opioid peptide prodynorphin is expressed in mouse and human β-cells. Prodynorphin levels are regulated in parallel with insulin in response to glucose, palmitate, and amino acids. Our findings elucidate the signaling pathways involved, with CamKII playing a key role in regulating prodynorphin levels in β-cells. Finally, our findings are the first to demonstrate active dynorphin secretion from mouse and human islets in response to glucose.

Objective

Although DNA methylation has been suggested to be a potential predictor of the progression of obesity and obesity-related diseases, little is known about its potential role as predictive marker of successful weight loss after bariatric surgery.

Methods

20 patients who underwent sleeve gastrectomy were classified according to the percentage of excess weight loss (%EWL) 1 year after bariatric surgery, using 60% as the cut-off point. Blood DNA methylation was analyzed prior to surgery using the Infinium Methylation EPIC Bead Chip array-based platform.

Results

A total number of 76,559 differentially methylated positions (DMPs) (p < 0.05) were found between <60% EWL and >60% EWL groups. Of them, 59,308 DMPs were annotated to genes. KEGG enrichment analysis showed that pathways involved in the signalling of MAPK, Wnt, mTor, FoxO and AMPK, among others, were involved in weight loss trajectory.

A stepwise logistic regression using the DMPs with an absolute Δβ >0.2 showed that higher methylation levels in the CpG sites cg02405213 (mapping to JAK2) (OR: 1.20098, [0.9586, 1.5044]) and cg01702330 (OR: 2.4426, [0.5761, 10.3567]), were shown to be associated with a higher probability of achieving >60 %EWL after sleeve gastrectomy, whereas higher methylation levels in the CpG site cg04863892 (mapping to HOXA5) were associated with lower probability of achieving >60 %EWL after sleeve gastrectomy (OR: 0.7966, [0.5637, 1.1259]).

Conclusions

Our results show a different pre-surgery methylation pattern according to %EWL. We identified three CpG sites (cg04863892, cg02405213, cg01702330) with potential value as predictor markers of weight loss response to bariatric surgery.

Objective

Several groups of neurons in the NTS suppress food intake, including Prlh-expressing neurons (NTS^Prlh cells). Not only does the artificial activation of NTS^Prlh cells decrease feeding, but also the expression of Prlh (which encodes the neuropeptide PrRP) and neurotransmission by NTS^Prlh neurons contributes to the restraint of food intake and body weight, especially in animals fed a high fat diet (HFD). We set out to determine roles for putative PrRP receptors in the response to NTS PrRP and exogenous PrRP-related peptides.

Methods

We used animals lacking PrRP receptors GPR10 and/or GPR74 (encoded by Prlhr and Npffr2, respectively) to determine roles for each in the restraint of food intake and body weight by the increased expression of Prlh in NTS^Prlh neurons (NTS^PrlhOX mice) and in response to the anorectic PrRP analog, p52.

Results

Although Prlhr played a crucial role in the restraint of food intake and body weight in HFD-fed control animals, the combined absence of Prlhr and Npffr2 was required to abrogate the restraint of food intake in NTS^PrlhOX mice. p52 suppressed feeding independently of both receptors, however.

Conclusions

Hence, each receptor can participate in the NTS^Prlh-mediated suppression of food intake and body weight gain, while PrRP analog treatment can mediate its effects via distinct systems. While Prlhr plays a crucial role in the physiologic restraint of weight gain, the action of either receptor is capable of ameliorating obesity in response to enhanced NTS^Prlh signaling.

Objective

The peroxisome proliferator-activated receptor-alpha (PPARα) plays a central role in lipid metabolism in the liver by stimulating the expression of hundreds of genes. Accordingly, regulation by PPARα could be a screening tool to identify novel genes involved in hepatic lipid metabolism. Previously, the mitochondrial transporter SLC25A47 was suggested to play a role in energy metabolism and liver-specific uncoupling, but further research is lacking.

Methods

We explored the potential role of SLC25A47 through in vitro studies and using mice overexpressing and lacking SLC25A47.

Results

SLC25A47 was identified as a PPARα-regulated and fasting-induced gene in human and mouse hepatocytes. Adenoviral-mediated overexpression of SLC25A47 minimally impacted metabolic parameters during fasting and high-fat feeding. During high-fat feeding, SLC25A47 ablation also did not influence any metabolic parameters, apart from a minor improvement in glucose tolerance. In fasted mice, SLC25A47 ablation was associated with modest, reproducible, and likely indirect reductions in plasma triglycerides and glycerol. SLC25A47 ablation did not influence energy expenditure. Depending on the nutritional status, metabolomic analysis showed modest alterations in plasma, liver, and hepatic mitochondrial levels of various metabolites related to amino acid metabolism, TCA cycle, and fatty acid metabolism. No major and consistent alterations in levels of specific metabolites were found that establish the substrate for and function of SLC25A47.

Conclusion

Collectively, our results hint at a role of SLC25A47 in amino acid and fatty acid metabolism, yet suggest that SLC25A47 is dispensable for hepatic lipid homeostasis during fasting and high-fat feeding.

Objective

AMP-activated protein kinase (AMPK) is a heterotrimer complex consisting of a catalytic α subunit (α1, α2) with a serine/threonine kinase domain, and two regulatory subunits, β (β1, β2) and γ (γ1, γ2, γ3), encoded by different genes. In the hypothalamus, AMPK plays a crucial role in regulating energy balance, including feeding, energy expenditure, peripheral glucose and lipid metabolism. However, most research on hypothalamic AMPK has concentrated on the catalytic subunits AMPKα1 and AMPKα2, with little focus on the regulatory subunits.

Methods

To fill this gap of knowledge, we investigated the effects of selectively deleting the regulatory isoform AMPKγ2, which is a primary “energy sensor”, in steroidogenic factor 1 (SF1) neurons of the ventromedial hypothalamic nucleus (VMH). Complete metabolic phenotyping and molecular analyses in brown adipose tissue (BAT), white adipose tissue (WAT) and liver were carried out.

Results

Our findings reveal that, in contrast to the obesity-protective effect of the genetic deletion of AMPKα subunits, the loss of AMPKγ2 in SF1 neurons leads to a sex-independent and feeding-independent obesity-prone phenotype due to decreased thermogenesis in brown adipose tissue (BAT) and reduced browning of WAT, resulting in lower energy expenditure. Additionally, SF1-Cre AMPKγ2 mice exhibit hepatic lipid accumulation, but surprisingly maintain normal glucose homeostasis.

Conclusions

Overall, these results highlight the distinct roles of AMPK subunits within the hypothalamus.

Objective

Loss of functional β-cell mass is a major cause of diabetes. Thus, identifying regulators of β-cell health is crucial for treating this disease. The Leucine-rich repeat-containing G-protein-coupled receptor (GPCR) 4 (LGR4) is expressed in β-cells and is the fourth most abundant GPCR in human islets. Although LGR4 has regenerative, anti-inflammatory, and anti-apoptotic effects in other tissues, its functional significance in β-cells remains unknown. We have previously identified Receptor Activator of Nuclear Factor Kappa B (NFκB) (RANK) as a negative regulator of β-cell health. In this study, we assessed the regulation of Lgr4 in islets, and the role of LGR4 and LGR4/RANK stoichiometry in β-cell health under basal and stress-induced conditions, in vitro and in vivo.

Methods

We evaluated Lgr4 expression in mouse and human islets in response to acute (proinflammatory cytokines), or chronic (high fat fed mice, db/db mice, and aging) stress. To determine the role of LGR4 we employed in vitro Lgr4 loss and gain of function in primary rodent and human β-cells and examined its mechanism of action in the rodent INS1 cell line. Using Lgr4^fl/fl and Lgr4^fl/fl/Rank^fl/fl × Ins1-Cre mice we generated _{β-cell-specific} conditional knockout (cko) mice to test the role of LGR4 and its interaction with RANK in vivo under basal and stress-induced conditions.

Results

Lgr4 expression in rodent and human islets was reduced by multiple stressors. In vitro, Lgr4 knockdown decreased proliferation and survival in rodent β-cells, while overexpression protected against cytokine-induced cell death in rodent and human β-cells. Mechanistically, LGR4 protects β-cells by suppressing RANK- Tumor necrosis factor receptor associated factor 6 (TRAF6) interaction and subsequent activation of NFκB. Lgr4cko mice exhibit normal glucose homeostasis but increased β-cell death in both sexes and decreased β-cell proliferation and maturation only in females. Male Lgr4cko mice under stress displayed reduced β-cell proliferation and a further increase in β-cell death. The impaired β-cell phenotype in Lgr4cko mice was rescued in Lgr4/Rank double ko (dko) mice. Upon aging, both male and female Lgr4cko mice displayed impaired β-cell homeostasis, however, only female mice became glucose intolerant with decreased plasma insulin.

Conclusions

These data demonstrate a novel role for LGR4 as a positive regulator of β-cell health under basal and stress-induced conditions, through suppressing the negative effects of RANK.

Objectives

Overconsumption of palatable food and energy accumulation are evolutionary mechanisms of survival when food is scarce. These innate mechanisms becom detrimental in obesogenic environment promoting obesity and related comorbidities, including mood disorders. This study aims at elucidating the role of the endocannabinoid system in energy accumulation and hedonic feeding.

Methods

We applied a genetic strategy to reconstitute cannabinoid type-1 receptor (CB1) expression at functional levels specifically in CaMKII+ neurons (CaMKII-CB1-RS) and adipocytes (Ati-CB1-RS), respectively, in a CB1 deficient background.

Results

Rescued CB1 expression in CaMKII+ neurons, but not in adipocytes, promotes feeding behavior, leading to fasting-induced hyperphagia, increased motivation, and impulsivity to palatable food seeking. In a diet-induced obesity model, CB1 re-expression in CaMKII+ neurons, but not in adipocytes, compared to complete CB1 deficiency, was sufficient to largely restore weight gain, food intake without any effect on glucose intolerance associated with high-fat diet consumption. In a model of glucocorticoid-mediated metabolic syndrome, CaMKII-CB1-RS mice showed all metabolic alterations linked to the human metabolic syndrome except of glucose intolerance. In a binge-eating model mimicking human pathological feeding, CaMKII-CB1-RS mice showed increased seeking and compulsive behavior to palatable food, suggesting crucial roles in foraging and an enhanced susceptibility to addictive-like eating behaviors. Importantly, other contingent behaviors, including increased cognitive flexibility and reduced anxiety-like behaviors, but not depressive-like behaviors, were also observed.

Conclusions

CB1 in CaMKII+ neurons is instrumental in feeding behavior and energy storage under physiological conditions. The exposure to risk factors (hypercaloric diet, glucocorticoid dysregulation) leads to obesity, metabolic syndrome, binge-eating and food addiction.

Objective

Obesity and overweight are associated with low-grade inflammation induced by adipose tissue expansion and perpetuated by altered intestinal homeostasis, including increased epithelial permeability. Intestinal epithelium functions are supported by intestinal epithelial cells (IEC) mitochondria function. However, diet-induced obesity (DIO) may impair mitochondrial activity of IEC and consequently, intestinal homeostasis. The aim of the project was to determine whether DIO alters the mitochondrial function of IEC, and what are the consequences on intestinal homeostasis.

Methods

C57Bl/6J mice were fed a control diet for 22 weeks or a high fat diet (58 kcal% fat). Bioenergetic adaptations of IEC were evaluated on isolated crypts and villi from mouse jejunum. To determine the link between mitochondrial function and alterations of intestinal homeostasis in response to lipid overload, we used the jejunal epithelial cell line IPEC-J2 in vitro and mouse jejunum organoids.

Results

Here, we report that DIO in mice induced lipid metabolism adaptations favoring lipid storage in IEC together with reduced number, altered dynamics and diminished oxidative phosphorylation activity of IEC mitochondria. Using the IPEC-J2 cell line, we showed that IEC lipid metabolism and oxidative stress machinery adaptations preceded mitochondrial bioenergetic ones. Moreover, we unraveled the intricate link between IEC energetic status and proliferation / differentiation balance since enhancing mitochondrial function with the AMPK activator AICAR in jejunal organoids reduced proliferation and initiated IEC differentiation and conversely. We confirmed that the reduced IEC mitochondrial function observed in DIO mice was associated with increased proliferation and reduced differentiation, promoting expression of the permissive Cldn2 in the jejunal epithelium of DIO mice.

Conclusions

Our study provides new insights into metabolic adaptations of IEC in obesity by revealing that excess lipid intake diminishes mitochondrial number in IEC, reducing IEC differentiation that contribute to increased epithelial permeability.

Aim

The aim of the present study was to define whether cardiac myosin contributes to energy conservation in the heart of hibernating mammals.

Methods

Thin cardiac strips were isolated from the left ventricles of active and hibernating grizzly bears; and subjected to loaded Mant-ATP chase assays, X-ray diffraction and proteomics.

Main findings

Hibernating grizzly bears displayed an unusually high proportion of ATP-conserving super-relaxed cardiac myosin molecules that are likely due to altered levels of phosphorylation and rod region stability.

Conclusions

Cardiac myosin depresses the heart's energetic demand during hibernation by modulating its function.

Objective

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterised by lipid accumulation in the liver and is often associated with obesity and type 2 diabetes. The gut microbiome recently emerged as a significant player in liver metabolism and health. Hippurate, a host-microbial co-metabolite has been associated with human gut microbial gene richness and with metabolic health. However, its role on liver metabolism and homeostasis is poorly understood.

Methods

We characterised liver biospies from 318 patients with obesity using RNAseq and metabolomics in liver and plasma to derive associations among hepatic hippurate, hepatic gene expression and MASLD and phenotypes. To test a potential beneficial role for hippurate in hepatic insulin resistance, we profile the metabolome of (IHH) using ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-MS/MS), and characterised intracellular triglyceride accumulation and glucose internalisation after a 24 h insulin exposure.

Results

We first report significant associations among MASLD traits, plasma and hepatic hippurate. Further analysis of the hepatic transcriptome shows that liver and plasma hippurate are inversely associated with MASLD, implicating lipid metabolism and regulation of inflammatory responses pathways. Hippurate treatment inhibits lipid accumulation and rescues insulin resistance induced by 24-hour chronic insulin in IHH. Hippurate also improves hepatocyte metabolic profiles by increasing the abundance of metabolites involved in energy homeostasis that are depleted by chronic insulin treatment while decreasing those involved in inflammation.

Conclusions

Altogether, our results further highlight hippurate as a mechanistic marker of metabolic health, by its ability to improve metabolic homeostasis as a postbiotic candidate.

Objectives

There is renewed interest in targeting the glucose-dependent insulinotropic polypeptide receptor (GIPR) for treatment of obesity and type 2 diabetes. G-protein coupled receptor desensitisation is suggested to reduce the long-term efficacy of glucagon-like-peptide 1 receptor (GLP-1R) agonists and may similarly affect the efficacy of GIPR agonists. We explored the extent of pancreatic GIPR functional desensitisation with sustained agonist exposure.

Methods

A long-acting GIPR agonist, GIP108, was used to probe the effect of sustained agonist exposure on cAMP responses in dispersed pancreatic islets using live cell imaging, with rechallenge cAMP responses after prior agonist treatment used to quantify functional desensitisation. Receptor internalisation and β-arrestin-2 activation were investigated in vitro using imaging-based assays. Pancreatic mouse GIPR desensitisation was assessed in vivo via intraperitoneal glucose tolerance testing.

Results

GIP108 treatment led to weight loss and improved glucose homeostasis in mice. Prolonged exposure to GIPR agonists produced homologous functional GIPR desensitisation in isolated islets. GIP108 pre-treatment in vivo also reduced the subsequent anti-hyperglycaemic response to GIP re-challenge. GIPR showed minimal agonist-induced internalisation or β-arrestin-2 activation.

Conclusions

Although GIP108 chronic treatment improved glucose tolerance, it also resulted in partial desensitisation of the pancreatic islet GIPR. This suggests that ligands with reduced desensitisation tendency might lead to improved in vivo efficacy. Understanding whether pancreatic GIPR desensitisation affects the long-term benefits of GIPR agonists in humans is vital to design effective metabolic pharmacotherapies.

Agouti-related peptide (AgRP) is a well-established potent orexigenic peptide primarily expressed in hypothalamic neurons. Nevertheless, the expression and functional significance of extrahypothalamic AgRP remain poorly understood. In this study, utilizing histological and molecular biology techniques, we have identified a significant expression of Agrp mRNA and AgRP peptide production in glomus type I cells within the mouse carotid body (CB). Furthermore, we have uncovered evidence supporting the expression of the AgRP receptor melanocortin receptor 3 (Mc3r) in adjacent sympathetic neurons, suggesting a potential local paracrine role for AgRP within the CB. Importantly, AgRP immunoreactivity was also identified in glomus type I cells of the human CB. Given the unexpected abundance of AgRP in glomus type I cells, a chemoreceptor cell specialized in oxygen sensing, we proceeded to investigate whether Agrp expression in the CB is regulated by hypoxemia and associated oxygen-sensing molecular mechanisms. In vitro luciferase assays reveal that hypoxia stimulates the human and mouse Agrp promoters in a Hypoxia Inducible Factor (HIF1/2)-dependent manner. Our in vivo experiments further demonstrate that exposure to environmental hypoxia (10%) robustly induces Agrp expression in type I glomus cells of mice. Furthermore, these findings collectively highlight the hitherto unknown source of AgRP in murine and human type I glomus cells and underscore the direct control of Agrp transcription by HIF signaling.

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.