Insulin resistance is often characterized as the factor that contributes to the emergence of metabolic diseases. Hepatic microRNAs (miRNAs) played critical roles in the development of metabolic-associated steatotic liver disease (MASLD) and insulin resistance. To investigate the effects of hepatic miR-411-5p in regulating insulin resistance, the present study utilized primary mouse hepatocytes and mice with MASLD. Suppression of miR-411-5p decreased hepatocyte glycogen production and phosphorylation of AKT, but miR-411-5p mimic improved insulin sensitivity. Mechanistically, 3'-UTR of transcription factor Sp2 was one of the binding sites of miR-411-5p. Treatment of miR-411-5p mimic suppressed the Sp2 mRNA and protein levels, enhancing the insulin signaling activity in the primary mouse hepatocytes. Hepatocyte-specific overexpression of Sp2 induced hepatic lipid accumulation and activation of related metabolic pathways. In contrast, inhibition of miR-411-5p reversely upregulated the expression of Sp2 and exaggerated insulin resistance in primary hepatocytes and the mouse model. Similarly, miR-411-5p mimic decreased obesity-induced hyperinsulinemia, glucose intolerance, insulin intolerance, and pyruvate intolerance. Furthermore, the parameters of MASLD, including lipid deposits, inflammation, and fibrosis, were improved after miR-411-5p replenishment, but co-administration with adeno-associated virus (AAV)-Sp2 abolished these benefits in the obese mouse model. Taken together, these findings demonstrated that Sp2-dependent miR-411-5p action regulates insulin resistance and MASLD, which provides a therapeutic approach toward resolving insulin resistance.

Prenatally androgenised (PA) sheep are a clinically realistic model of polycystic ovary syndrome (PCOS). They have dysfunctional subcutaneous adipose tissue (SAT) with reduced adipogenesis in adolescence and enlarged adipocytes with increased inflammation in adulthood. We hypothesised that analysis of SAT in young adults, after adipogenesis is complete but before inflammation is apparent, would give insights into the evolution of adipose tissue dysfunction. Pregnant sheep were treated intramuscularly with 100 mg testosterone propionate or vehicle control (C) twice weekly from day 62-102 of gestation. Weight-matched female offspring (PA = 10; C = 10) were studied up to 22 months of age. Glucose tolerance testing was performed, and at sacrifice SAT was fixed for histological analysis and frozen for RNA sequencing (RNAseq) and gene expression analysis. There was no difference in the average size of SAT adipocytes between PA and C young adults. There were no differences in the expression of the adipogenesis markers PPARG, CEBPA and CEBPB, or the inflammatory markers TNF and IL6, although PA sheep were already hyperinsulinaemic. RNAseq identified 792 potentially differentially expressed (P < 0.05) genes in PA sheep SAT (406 upregulated; 386 downregulated). Ingenuity Pathway Analysis highlighted upregulation of fibrotic pathways in the SAT of PA sheep. POSTN, associated with tissue fibrosis, and COL1A1, COL1A2 and COL3A1 were significantly elevated, and histochemistry showed significantly increased SAT fibrosis in PA sheep. Early fibrotic changes in SAT occur before inflammatory gene expression in PA sheep. A fibrotic barrier to healthy adipocyte expansion may have a mechanistic role in the development of inflammation in PCOS.

Maturity-onset diabetes of the young (MODY) is a form of monogenic diabetes caused by single-gene mutations. MODY3, the most common subtype, results from mutations in the hepatocyte nuclear factor 1-alpha (HNF1α) gene. HNF1α is a transcription factor essential for pancreatic β-cell function and insulin production. Clinically, β-cells in MODY3 patients generally retain intact sulfonylurea receptor function, making sulfonylureas the preferred treatment. However, a novel loss-of-function variant, HNF1α-Q125ter, has been shown to induce sulfonylurea insensitivity in MODY3 patients. This study aimed to investigate the role and mechanism of HNF1α-Q125ter-mediated mitochondrial dysfunction and impaired mitophagy in new variant-induced β-cell dysfunction. Mitophagy-related protein and transcription levels were analysed by Western blotting and reverse transcription-quantitative PCR (RT-qPCR). Mitochondrial morphology was examined by transmission electron microscopy. Ins-1 cells were transfected with overexpression constructs for HNF1α-Q125ter or short hairpin RNA targeting HNF1a (shHNF1α) to assess its effects on mitochondrial function and mitophagy. Ins-1 cells expressing HNF1α-Q125ter showed decreased mitochondrial number, oxygen consumption, and energy metabolism. Correspondingly, mitochondrial morphology was damaged in an hnf1a+/- zebrafish model. HNF1α-Q125ter also inhibited mitophagy by suppressing the mRNA expression of PTEN-induced kinase 1 (PINK1), pyruvate dehydrogenase E1 subunit α1 (PDHA1), and Parkin RBR E3 ubiquitin-protein ligase (Parkin). Mechanistically, HNF1α-Q125ter impaired autophagy by downregulating phosphorylated mammalian target of rapamycin (p-mTOR) (Ser2448) and phosphorylated-70 kDa ribosomal protein S6 kinase (p-p70S6K) (Thr389). In conclusion, our findings suggest that HNF1α-Q125ter induces mitophagy dysfunction by suppressing the p-mTOR(ser2448)/p-p70S6K(Thr389) signalling pathway, providing novel insights into the mechanisms underlying sulfonylurea insensitivity in patients with this variant.

Type 2 diabetes mellitus (DM) is closely associated with cellular senescence (SnC), a state of irreversible cell cycle arrest marked by functional decline. Preventing cellular senescence in already diagnosed DM patients is crucial for limiting disease progression and the onset of co-morbidities. The relationship between oxidative stress, DNA damage, and telomere shortening provides a mechanistic framework for elucidating the role of cellular senescence in the pathogenesis and progression of type 2 DM. This senescence-driven model of metabolic dysfunction not only accounts for impaired β-cell function and insulin resistance but also for the systemic complications observed in DM patients. The accumulation of senescent cells, particularly in metabolically active tissues such as adipose tissue, is increasingly recognised as both a cause and a consequence of the chronic inflammatory environment that characterises diabetes. Evidence from in vitro and preclinical studies highlights the detrimental effects of the senescence-associated secretory phenotype, reinforcing tissue damage through paracrine and autocrine signalling mechanisms. Despite its complexity, approaches targeting the senescent phenotype offer a promising avenue for adjunct therapies. Senotherapeutics, such as senomorphic agents that protect cells from cytotoxic damage and mitigate oxidative stress, can potentially protect against disease onset, whereas senolytic agents have the potential to eliminate senescent cells to limit metabolic disease progression, mitigate complications, and ultimately improve patient outcomes. There is, however, an urgent need to translate the preclinical findings into clinical trials to assess the safety, efficacy, and long-term effects of senotherapeutic agents.

Extracellular vesicle (EV) miRNAs play pivotal roles in metabolic disorders. This study aimed to describe the plasma EV miRNA profiling of type 2 diabetes mellitus (T2DM) and evaluate the association between differentially expressed miRNAs and T2DM. The subjects were from the Henan Rural Cohort. The miRNA profiling of plasma EVs was quantified by the next-generation sequencing of RNA in the discovery sets to identify differentially expressed miRNAs. The association between differentially expressed miR-3120-5p and T2DM was validated in 75 pairs of newly diagnosed T2DM and controls using logistic regression and a generalized linear model. In vitro experiments were performed in HepG2 cells to explore the mRNA and protein expression levels of glucose-related transcription factors and glucose consumption by transfecting miR-3120-5p mimic or inhibitor. We found that in the discovery set, the first phase identified 73 upregulated and 44 downregulated miRNAs, followed by 41 upregulated and 23 downregulated miRNAs in the second phase. miR-3120-5p showed upregulation in the two phases. In the validation set, the miR-3120-5p level in plasma EVs was positively associated with the risk of T2DM (OR: 1.22, 95% CI: 1.05, 1.44). In vitro experiments demonstrated that glucose consumption was reduced in HepG2 cells overexpressing miR-3120-5p compared to mimic negative controls, and that expression of the glucose uptake factor GLUT2 protein was also decreased. We conclude that plasma EV miR-3120-5p was associated with T2DM in the rural populations with limited resources, and might contribute to the pathological process by directly or indirectly inhibiting hepatocyte GLUT2 expression and glucose consumption.

The glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are important incretin receptors that are therapeutic targets for the treatment of type 2 diabetes and obesity. This study extensively characterised the metabolic phenotype of mice with global deletion of either the GLP-1R or GIPR side by side under identical conditions. Age-matched male wild-type (WT) C57Bl6NTac, GLP-1RKO or GIPRKO mice were placed on a high-fat or chow diet for 12 weeks, and a range of in vivo (weight gain, food intake, glucose tolerance, insulin tolerance, and whole-body energy metabolism) and ex vivo (white adipocyte lipolysis, brown adipose tissue and liver mitochondrial function, adipocyte and islet size, and hepatic steatosis) parameters were measured. While both WT and GLP-1RKO mice gained weight similarly on a HFD, obese high-fat-fed GLP-1RKO mice had altered glucose and insulin tolerance, and exhibited hepatic steatosis, highlighting the physiological importance of the GLP-1R in the regulation of blood glucose and lipid homoeostasis. In contrast, GIPRKO mice were partially resistant to diet-induced obesity compared to the WT mice, which was associated with a small reduction in food intake and intact epididymal and subcutaneous white adipocyte β-adrenoceptor-mediated lipolysis. Similarly, WT mice treated with a GIPR antagonist prevented weight gain due to a reduction in food intake on a HFD. These findings provide further support that the GLP-1R is important for normal glycaemic control, whereas the GIPR may play a role in the regulation of body weight.

While pregnancy is known to be an inflammatory condition, preeclampsia (PE) is associated with higher chemokines and pro-inflammatory cytokines and higher Th1/Th2 and Th17/Treg ratios. Since the uteroplacental space can secrete cytokines, including TNF and IL1B, a common assumption is that the proinflammatory immune cell profile of Th1 and Th17 cells dominating over Th2 and Treg cells begins in that space. To date, a possible role for endothelium in this initiation process has not been considered. Nonetheless, recent publications show that endothelium can become immunomodulatory on exposure to TNF and IL1B, and in systemic hypertension, endothelium has been shown to exist as multiple cell subtypes. We have recently shown that uterine artery endothelial cells from late-pregnant sheep (P-UAEC) treated with TNF alone secrete many of the chemokines and cytokines further elevated in PE subjects. Herein, we show that P-UAEC also exist in multiple subtypes with distinct chemokine and cytokine secretory and immunomodulatory properties. The five subtypes are differentially regulated by TNF-alpha (TNF) and IL1-beta (IL1B), which may favor subtype-specific binding and interaction with distinct classes of Th cells, and an altered ability to respond to Th-secreted cytokines (such as IL17 and IL10). Thus, our data demonstrate the possibility that certain endothelial cell subtypes can be pushed to express immunomodulatory proteins by early exposure to increases in TNF or IL1B of immune cell, trophoblast, and decidual origin. This, in turn, begs the question of whether such endothelial changes could contribute to subsequent immune disturbances seen at the time of clinical presentation.

Orbital fibroblast proliferation and activation contribute to the development of thyroid-associated ophthalmopathy (TAO). In this study, nuclear receptor subfamily 4 group A member 3 (NR4A3) was predicted to play a role in TAO based on bioinformatics analysis. Validation of NR4A3 expression in human TAO orbital samples confirmed its elevated levels compared to normal controls. In vitro studies demonstrated that transforming growth factor beta 1 (TGF-β1)-induced NR4A3 expression in human TAO orbital fibroblasts (OFs) enhanced cell viability, DNA synthesis, and fibrotic marker expression. Conversely, NR4A3 knockdown inhibited these fibrotic responses, suggesting a pro-fibrotic role for NR4A3 in TAO. In vivo experiments further validated these findings, with NR4A3 knockdown in a TAO mouse model leading to reduced pathological injury and fibrosis in orbital tissues. In addition, NR4A3 knockdown decreased the expression of fibrotic markers in the orbital tissues of TAO mice, corroborating the in vitro results. Finally, NR4A3 was shown to modulate the nuclear factor kappa B (NF-κB) pathway, which is activated in TAO. NR4A3 overexpression enhanced, while its knockdown suppressed, NF-κB activation in both human TAO OFs and orbital tissues from TAO mice. These findings suggest that NR4A3 promotes TAO progression through its pro-fibrotic effects and activation of NF-κB signaling, highlighting its potential as a therapeutic target for TAO. Collectively, NR4A3 plays a pivotal regulatory role in both fibroblast proliferation and the fibrotic response in TAO, acting through mechanisms involving the NF-κB signaling pathway. Its ability to enhance TGF-β1-induced changes and activate NF-κB underscores its potential as a key therapeutic target for addressing the complex pathophysiology of TAO.

Approximately 10% of children born small for gestational age (SGA) fail to achieve catch-up growth, resulting in persistent short stature and eligibility for growth hormone (GH) therapy under established guidelines. Pathogenic variants in insulin-like growth factor 1 receptor (IGF1R) are associated with SGA, syndromic short stature, neurocognitive impairment, and variable responsiveness to GH therapy. This study aimed to characterize the clinical phenotype and elucidate the molecular mechanism underlying a rare intronic variant in IGF1R identified in an affected family. Here, we performed whole-exome sequencing (WES) on a single individual, followed by segregation studies in the family and Sanger sequencing. cDNA studies were pursued to evaluate mis-spliced transcripts. WES of the proband's affected mother revealed a rare heterozygous variant in IGF1R (NM_000875.5): c.3722+5G>A. Sanger sequencing confirmed segregation of the variant with the affected status in available family members. cDNA analysis showed that the variant results in intronic retention of 134 nucleotides immediately following the penultimate exon of IGF1R. This leads to a frameshift and introduction of a premature truncation codon, supporting the classification of the variant as likely pathogenic. Our study highlights the utility of genetic testing in SGA children with persistent short stature. By characterizing a novel IGF1R intronic variant causing aberrant splicing, we expand the understanding of its clinical spectrum and molecular underpinning. The findings underscore the importance of molecular diagnostics in unexplained short stature and neurodevelopmental disorders and may inform future therapeutic strategies targeting the IGF1R signaling.

Estrogens are steroid hormones that regulate antioxidant and mitochondrial bioenergetic metabolism in addition to activating nuclear genomic pathways. Concentrating these effects within the mitochondria is a novel strategy for ameliorating mitochondrial dysfunction, which is characteristic of cancer, metabolic, and neurodegenerative diseases. The use of synthetic mitochondria-targeted estrogens containing a triphenylphosphonium group may provide a basis for improving mitochondrial function in these conditions. Here, we evaluate the effects of two compounds, one derived from 17β-estradiol (mitoE2) and the other from 17α-ethinylestradiol (mitoEE2), on cell viability in MCF-7 and CCD-1112Sk cells. We further examine their influence on the activities of superoxide dismutase (MnSOD), citrate synthase (CS), cytochrome c oxidase (COX), and ATP synthase, as well as on the glycolytic reserve and cellular respiration. In both cellular models, cell viability assays indicated that mitoE2 was well tolerated below 500 nM, while mitoEE2 allowed treatments up to 100 nM for up to 24 h. We found that the molecules act differently on enzymatic targets. Exposure of MCF-7 cells to mitoE2 resulted in reduced MnSOD activity. Pretreatment with mitoE2 or mitoEE2 restored the viability of MCF-7 cells exposed to H2O2-induced oxidative damage to levels comparable to untreated controls. In addition, mitoEE2 increased the activities of CS and COX. Both mitochondria-targeted estrogens increased glycolytic reserve and mitochondrial respiration, as determined by extracellular flux assays. Overall, these findings suggest that the antioxidant and bioenergetic effects observed encourage further investigation into their potential as therapeutic strategies for conditions linked to mitochondrial dysfunction.

Aldosterone is synthesized by the CYP11B2 enzyme, primarily in the zona glomerulosa of the adrenal gland. It exerts its classical effects on sodium and water balance in the renal distal nephron through binding to the mineralocorticoid receptor (MR). Excess aldosterone production or overactivation of the MR outside the distal nephron leads to cardiac, renal, and vascular injury by increasing oxidative stress and activating the inflammatory and fibrotic pathways. MR antagonists (MRAs) have proved effective at decreasing organ damage and the deleterious effects of excess aldosterone/MR activation. However, MRAs do not fully block the non-genomic effects of aldosterone, which may contribute to residual risks. CYP11B2 inhibition has emerged as an additional therapeutic approach to decreasing the deleterious genomic and non-genomic effects of aldosterone. The development of specific aldosterone synthase inhibitors (ASi) has proved challenging due to the considerable similarity between aldosterone synthase and 11β-hydroxylase, an enzyme encoded by the CYP11B1 gene that catalyzes cortisol synthesis. In this review, we summarize the latest developments on preclinical evidence and clinical trials for ASi and explore the potential clinical advantages of ASi.

Bone marrow stromal cells (BMSCs) play an important role in bone regeneration, but their functional activity is affected by oxidative stress, which is a key pathological feature of osteoporosis. The aim of this study was to investigate the effects of capsaicin on the proliferation and differentiation of BMSCs under oxidative stress. We assessed cell viability and osteogenic potential of capsaicin in promoting BMSC survival and enhancing osteogenic capacity under oxidative stress by cell counting kit-8 (CCK-8), reactive oxygen species fluorescence staining, alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, Western blot (WB), and real-time PCR (RT-PCR). Our results indicate that capsaicin improves cell viability, antioxidant capacity, and osteogenic differentiation in rat BMSCs treated with hydrogen peroxide (H2O2). In addition, immunohistochemistry (IHC) analysis revealed that the surface of BMSCs expressed the capsaicin receptor transient receptor potential vanilloid protein 1 (TRPV1). More importantly, capsaicin increased Ca2+ influx and autophagy and inhibited phosphorylation of the PI3K/AKT/mTOR signaling pathway. In conclusion, capsaicin protects BMSC function during oxidative stress, possibly through inducing TRPV1-mediated Ca2+ influx and PI3K/AKT/mTOR-activated autophagy. The results suggest the potential of capsaicin as a therapeutic agent for osteoporosis.

Classical activation of the hypothalamic-pituitary-adrenal axis is exerted by the stimulation of pituitary POMC gene transcription and ACTH release by the hypothalamic hormone CRH. In parallel, inflammatory cytokines such as IL6 and LIF also stimulate ACTH release and POMC transcription through the JAK/STAT pathway. In recent years, a particular interest in the role of the EGF pathway for POMC activation was sparked by the identification of causative mutations in the USP8 gene that have been implicated in the formation of pituitary corticotroph adenomas that are the hallmark of Cushing's disease. These mutations were associated with the persistent upregulation of the EGF/EGFR pathway and its putative role in ACTH hypersecretion. In the present work, we reassessed the signaling pathways that are activated in response to EGF in pituitary corticotroph cells using the AtT20 cell model. We confirmed the activation of the MAP kinase pathway by EGF and also showed the activation of the AKT/mTOR and JAK/STAT pathways. Whereas activation of all three pathways appears essential for the stimulation of cell proliferation, only the JAK/STAT pathway, and more specifically STAT3, enhances POMC gene transcription. This action is mapped to a single STAT-binding element of the POMC promoter in contrast to the activation by the other STAT-activating cytokines LIF and IL6. Furthermore, EGF signaling is specifically enhanced by STAT3 but not STAT1 in contrast to LIF-dependent activation. All together, the data identified a unique STAT3-dependent target on the POMC promoter that mediates EGF activation of POMC gene transcription.

G protein-coupled receptors (GPCRs) represent a diverse and vital family of membrane proteins that mediate intracellular signaling in response to extracellular stimuli, playing critical roles in physiology and disease. Traditionally recognized as chemical signal transducers, GPCRs have recently been implicated in mechanotransduction, the process of converting mechanical stimuli into cellular responses. This review explores the emerging role of GPCRs in sensing and responding to mechanical forces, with a particular focus on the cardiovascular system. Cardiovascular homeostasis is heavily influenced by mechanical forces such as shear stress, cyclic stretch, and pressure, which are central to both normal physiology and the pathogenesis of diseases such as hypertension and atherosclerosis. GPCRs, including the angiotensin II type 1 receptor (AT1R) and the β2-adrenergic receptor (β2-AR), have demonstrated the ability to integrate mechanical and chemical signals, potentially through conformational changes and/or modulation of lipid interactions, leading to biased signaling. Recent studies highlight the dual activation mechanisms of GPCRs, with β2-AR now serving as a key example of how mechanical and ligand-dependent pathways contribute to cardiovascular regulation. This review synthesizes current knowledge of GPCR mechanosensitivity, emphasizing its implications for cardiovascular health and disease, and explores advancements in methodologies poised to further unravel the mechanistic intricacies of these receptors.

The signalling of kisspeptin-1 through the kisspeptin-1 receptor (KISS1R) is central to mammalian reproduction. Naturally occurring heterozygous KISS1R mutations and Kiss1r +/- knockout mice are less affected than their homozygous counterparts, suggesting that the mutant receptors possibly form oligomers with the wild-type (WT) KISS1R, rescuing the receptor function to some extent. To test this hypothesis, the heterozygous KISS1R mutations R38P, P46Q, S125L and R198G, reported in the literature in cases of delayed puberty, were characterised. In silico analysis predicted that all four mutations affected the receptor function to varying extents, which was substantiated by in vitro studies. Determination of cell surface receptor expression and kisspeptin-stimulated signalling response was carried out post transient transfection of the receptor constructs in CHO cells. Results revealed that these mutations (homozygous condition) impaired the cell surface receptor expression, as quantified by flow cytometry, with a concomitant attenuation of inositol phosphate production. Co-transfection of the WT KISS1R with equal amounts of the mutant receptors, to mimic the heterozygous condition of the mutation in the patients, restored the receptor function and, with increasing amounts of mutant receptors, resulted in attenuation of receptor function. As a direct proof of receptor oligomerisation, co-expression of epitope-tagged KISS1R constructs was carried out. Co-immunoprecipitation and imaging FRET studies revealed that KISS1R forms homo-oligomers in a constitutive manner and that the transmembrane domain 7 contributed to the oligomerisation interface, as demonstrated by the impairment in oligomerisation upon deletion of this domain. Thus, characterisation of heterozygous KISS1R mutations corroborated the oligomerisation status of the KISS1 receptor and helped in establishing a genotype-phenotype association.

Hepatic thyroid hormone action plays an important role in preventing the development and progression of metabolic liver diseases, as evidenced by the recent success of the receptor-specific agonist resmetirom. The liver enzyme deiodinase type I (DIO1) is important for controlling the local availability of thyroid hormone and is upregulated in metabolically associated steatotic liver disease (MASLD), which is thought to be a compensatory mechanism to enhance local hormone action. However, it remains unclear whether this increase is maintained in later stages of MASLD and whether an induction of Dio1 can provide beneficial metabolic effects. Studying mouse models with different stages of MASLD, we show here that Dio1 mRNA expression and activity are rapidly induced within 1 week by high-caloric dietary intervention. In later stages, this increase was less pronounced. Surprisingly, altered Dio1 mRNA concentration became progressively less well associated with altered DIO1 enzyme activity, suggesting uncoupling of mRNA and protein biosynthesis. In order to enhance DIO1 activity in MASLD development, a transgenic strategy was applied by using an adeno-associated virus-based liver-specific gene therapy with either the Dio1 or Socs3 gene. In either model, DIO1 activity was increased, but neither thyroid hormone target genes nor metabolic parameters were positively affected in the time frame of the experiment. We conclude that hepatic DIO1 biosynthesis becomes progressively disturbed with disease progression in MASLD by a decoupling of its transcript and protein levels, highlighting the key importance of translational processes controlling DIO1 in hepatocytes, which are likely affected by local inflammatory mechanisms.

While pregnancy is known to be an inflammatory condition, preeclampsia (PE) is a more extreme state associated with higher cytokines and/or altered growth factors. It is generally assumed these PE-elevated factors come from stimulation of immune cells and/or hypoxic uterine tissue, but several studies have shown that endothelial cells may also be a source. The goal of this study was to determine to what extent TNF, a factor overproduced by uteroplacental tissue in PE pregnancy, may influence uterine artery endothelial cells to contribute to these other PE-specific factors in the maternal circulation. Herein, we use multiple analytical methods to show that uterine artery endothelial cells from pregnant sheep (P-UAEC) on exposure to cytokines can secrete multiple cytokines and chemokines seen in PE women, which may contribute to production of Th17 cells and attraction of these and other cells to the vessel surface. Furthermore, the factors not significantly increased by TNF include those known to be specifically secreted by proinflammatory T cells. This begs the question if endothelium itself is the initial primary orchestrator of chemokine and cytokine imbalance, acting directly and indirectly to promote the symptoms of impaired vasodilation and reduced uteroplacental blood flow. If so, future preventive therapies for PE should be targeted at endothelium as well as immune cells.

The aim of this study was to investigate the mechanism by which chronic stress (CS) induces non-alcoholic fatty liver disease (NAFLD)-like changes, and the role of oxidative stress and the NLRP3 inflammasome in this mechanism. Transcriptomic data extracted from the Sequence Read Archive (SRA) at the NCBI were employed to identify the molecular targets of CS-induced NAFLD. Fifty 8-week-old healthy male Wistar rats were divided into five groups (n = 10 each) as follows: control, CS, CS + mifepristone (CS + Mif), CS + metyrapone (CS + Met), and corticosterone (Cort). The CS, CS + Mif, and CS + Met groups underwent restraint stress training. Rats in the CS + Mif, CS + Met, and Cort groups were administered mifepristone, metyrapone, and corticosterone for 8 weeks. Data showed that CS induced NAFLD-like liver damage via increased glucocorticoids (GCs). Moreover, CS increased malonaldehyde (MDA) levels and decreased superoxide dismutase (SOD) activity in liver and serum samples, suggesting the occurrence of oxidative stress. Furthermore, CS activated various inflammatory pathways via the NLRP3 inflammasome (NLRP3, ASC, caspase-1), which enhanced cytokine levels (IL-1β, IL-6, TNF-α) in liver tissue. Notably, treatment with metyrapone or mifepristone alleviated liver lesions induced by CS, which implies that the GC signalling pathway may be an important mediator of stress-induced liver inflammation. We conclude that GC mediates the development of oxidative stress and inflammation in the liver, and inhibition of GC signalling may be a new therapeutic strategy in NAFLD.

Hypoxia has been implicated as a causal factor in mediating adipocyte dysfunction in obesity. Moreover, protein kinase D 1 (PKD1), a serine/threonine protein kinase, has been shown to contribute to diet-induced adiposity. Therefore, we investigated if PKD isoforms mediate hypoxia-induced dysfunction in 3T3-L1 adipocytes. Hypoxia increased phosphorylation of PKD1 at serine 916 (S916), the autophosphorylation site linked to PKD1 activation, indicating hypoxia-induced activation of PKD1 in adipocytes. Inhibition or depletion of PKD isoforms mitigated hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), the master transcription factor mediating hypoxia-induced gene expression, confirming that PKDs modulate the hypoxia-induced mechanism in adipocytes. Surprisingly, depletion of PKD1 and PKD2, but not PKD3, attenuated hypoxia-induced HIF1α target gene expression. Unlike PKD3, PKD1 and PKD2 possess a unique PDZ-binding motif at their C-terminus. Indeed, hypoxia upregulated a PDZ-containing scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) and its interaction with PKD1, whereas NHERF1 depletion attenuated hypoxia-induced PKD1 phosphorylation, HIF1α protein accumulation, and gene expression. Mechanistically, hypoxia induced nuclear import of active PKD1, which phosphorylated histone deacetylase 5 (HDAC5) at S498, promoting cytoplasmic localization of HDAC5. HDAC5 deacetylated heat shock protein 70 (HSP70) at lysine 77, which dissociated HSP70 from HIF1α, allowing HSP90 association that stabilized HIF1α. Interestingly, PKD inhibition reversed hypoxia effects on subcellular localization of PKD1/HDAC5, HSP70 acetylation, and HIF1α/HSP90 association. In summary, our findings reveal an NHERF1-PKD1-HDAC5 mechanism modulating hypoxia-induced gene expression in adipocytes.

Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic β-cells. The insulin B-chain 9-23 (insB9-23) peptide is a critical epitope in triggering T1D. In our previous study, we showed that Parabacteroides distasonis, a human gut commensal, contains an insB9-23 mimic in its hprt protein (residues 4-18). This mimic (hprt4-18) peptide activates insB9-23-specific T cells, and P. distasonis colonization enhanced diabetes in NOD mice. However, the impact of the P. distasonis colonization on inflammation, gut microbiome, intestinal immune cells, gut permeability, cytokine, and serum metabolome profiles remained unknown. Here, we investigated these effects using specific pathogen-free (SPF) and germ-free (GF) female NOD mice. P. distasonis colonization minimally impacted gut microbiome composition, altering only 28 ASVs. In P. distasonis-colonized mice, there was a reduction in T-helper, T-effector, and B-cell populations in the intraepithelial lymphocytes, indicating a potential decrease in immune activation. Furthermore, P. distasonis colonization did not alter serum metabolome and circulating cytokine profiles (except for a decrease in IL-15) and gut permeability gene expressions. P. distasonis colonization in GF NOD mice induced severe insulitis without affecting gut permeability. Interestingly, mice gavaged with heat-inactivated (HI) P. distasonis did not affect insulitis scores or immune cell composition. These findings support our hypothesis that P. distasonis functions as a gut commensal, exerting no effect on the gut microbiome, metabolome, gut permeability, intestinal immune cell composition, or nonspecific immune activation. Instead, P. distasonis appears to trigger an insB9-23-specific immune response, potentially accelerating T1D onset in NOD mice through molecular mimicry.