Journal of Cellular Physiology

During viral infection, viral replication perturbs endoplasmic reticulum (ER) homeostasis and triggers the unfolded protein response (UPR). XBP1s, a transcription factor generated by one branch of the UPR, is known to potentiate both innate and adaptive immunity, but its role in antiviral responses remains incompletely understood beyond its ability to augment type I interferon (IFN) mRNA induction. Here, we show that XBP1s positively regulates the RIG-I-like receptors (RLRs), ribonuclease L (RNase L), and protein kinase R (PKR) pathways, indicating that it enhances all three major antiviral response pathways. We further show that RNase L activation rapidly decreases XBP1 mRNA levels in an RNase activity-dependent manner, leading to a prompt reduction in XBP1s expression. Consistent with this, RNase L deletion significantly increased both thapsigargin-mediated XBP1s induction and XBP1s expression following Japan encephalitis virus infection. Poly(I:C)-induced IFNB mRNA expression was significantly enhanced in RNase L-knockout cells. This enhancement was completely abolished by RNase L reconstitution. XBP1 knockdown also significantly attenuated IFNB mRNA expression in RNase L-knockout cells. These findings suggest a negative-feedback loop in which RNase L suppresses XBP1s, thereby fine-tuning antiviral responsiveness during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/713401v1_ufig1.gif" ALT="Figure 1000"> View larger version (19K): org.highwire.dtl.DTLVardef@112d312org.highwire.dtl.DTLVardef@df79a9org.highwire.dtl.DTLVardef@1ac571borg.highwire.dtl.DTLVardef@18ac610_HPS_FORMAT_FIGEXP M_FIG C_FIG

The secretion of glucagon from the pancreatic alpha () cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the TC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in TC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.

Acute kidney injury (AKI) is a sudden episode of kidney failure linked to a wide range of health conditions. High mortality in AKI highlights the need to identify new therapeutic approaches. Homeostasis in multicellular organisms is exquisitely regulated by phagocytosis of apoptotic cells, also known as efferocytosis. Apoptotic cells are frequently observed at sites of inflammation, including in AKI. Engulfment and cell motility protein-1 (ELMO1) is a regulator of the actin cytoskeleton that promotes apoptotic cell removal by phagocytes during efferocytosis. Mutations in the human ELMO1 gene are linked with diabetic nephropathy and, in animal models of this disease, high ELMO1 levels promote renal dysfunction. However, the role of ELMO1 in AKI was not known. Here, we describe the links between ELMO1 and kidney pathology and test global and tissue-specific ELMO1-deficient mice in models of AKI. While global loss of Elmo1 expression did not impact the immediate loss of renal function after ischemia-reperfusion elicited AKI, ELMO1 deficiency resulted in increased tissue injury in AKI caused by cisplatin injection. Cisplatin induced robust renal cell apoptosis that was significantly elevated in mice with the global loss of ELMO1, but not in mice with the macrophage-specific Elmo1 deletion. Using primary cell culture and immunofluorescence approaches, we highlight the role of ELMO1 in efferocytosis by several renal cell types, suggesting possible additive effects during nephrotoxic injury.

Pathologic cardiac hypertrophy requires increased protein synthesis, but the mechanosensors that link membrane stretch to translational control remain poorly understood. Polycystin-1 (PC1), encoded by PKD1, has been proposed as a cardiac mechanosensor, with its C-terminal tail (PC1-CT) promoting hypertrophy in rodent cardiomyocytes. However, its subcellular localization and downstream signaling remain incompletely defined, especially in human cardiomyocytes. Here, we examined endogenous PC1 C-terminus localization and the effects of adenoviral PC1-CT overexpression in human iPSC-derived ventricular cardiomyocytes (hiPSC-CMs) and adult mouse ventricular myocytes. Immunofluorescence revealed a striking striated pattern for both endogenous PC1 C-terminus (detected with a PC1-CT antibody) and the overexpressed PC1-CT fragment. In hiPSC-CMs, the PC1 C-terminus localized between the -actinin bands. In contrast, in adult cardiomyocytes, the overexpressed protein colocalized with -actinin and desmin, suggesting that PC1-CT sarcomeric distribution depends on cardiomyocyte maturation. We performed RNA-seq to assess transcriptional responses downstream of PC1-CT overexpression in hiPSC-CMs relative to LacZ controls. Gene Set Enrichment Analysis (GSEA) revealed enrichment of gene sets related to ribosome biogenesis, RNA processing, and protein synthesis, while classical hypertrophic markers remained unchanged. Pathway analysis suggested increased PI3K activity. PC1-CT overexpression increased phosphorylation of Akt, ERK, S6K1, and ribosomal protein S6 without altering 4EBP1 phosphorylation, suggesting preferential activation of the mTOR-S6K1-S6 branch. Pharmacological studies showed that pan-PI3K inhibition abolished S6 phosphorylation, whereas MEK blockade did not affect it; pertussis toxin and PI3K{gamma}-selective inhibitors also did not affect S6, suggesting a Gi/o-independent PI3K/Akt signaling driving mTOR-S6K1-S6 activation. Collectively, these data identify a sarcomere-associated pool of PC1-CT that engages PI3K-Akt-mTOR-S6K1-S6 signaling to enhance transcriptional programs related to ribosome biogenesis and protein synthesis, without activating a canonical hypertrophic gene program. These findings reveal a mechanistic link between PC1-CT and cardiomyocyte growth.

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.

BackgroundWithin the kidney, (pro) renin receptor (PRR) is abundantly expressed in the collecting duct (CD) and modulate physiological and pathophysiological processes. We have recently reported that activation of CD PRR mediates obstructive renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO). The current study addresses the underlying mechanisms by examining the profibrotic pathway mediated by soluble PRR (sPRR)-dependent alternative macrophage activation. MethodsWe performed UUO or sham surgery on mice with CD-specific deletion of PRR (CD PRR KO) and floxed controls. After 7 days, we assessed fibrosis-related parameters, inflammatory cytokines, M1/M2 macrophage markers, other gene expression markers of kidney injury, and the concentration of plasma sPRR. We also administered vehicle or site-1 protease (S1P) inhibitor PF-429242 (PF) to C57BL/6 mice with UUO to determine the role of sPRR. Experiments were performed in vitro to examine the mechanism of sPRR-His-mediated macrophage M2 polarization and activation of potential target genes in bone-marrow-derived macrophages (BMDMs). ResultsCompared with the floxed control, CD PRR KO decreased macrophage accumulation, M2 polarization, and Yap/Taz expression while improving renal fibrosis and suppressing plasma sPRR levels following UUO. In BMDMs, sPRR-His treatment promoted macrophage M2 polarization, fibrosis, and Yap/Taz expression, which was dependent on angiotensin type 1 receptor (AT1R). ConclusionCD PRR-derived sPRR acts via ATR to promote macrophage M2 polarization and stimulates the AT1R/Yap/Taz axis, which leads to renal fibrosis during UUO.

Autophagy is a critical homeostatic mechanism in podocytes, maintaining cellular integrity under stress and proteostatic challenges. Dysregulation of autophagy has been implicated in different glomerular diseases such as diabetes and membranous glomerulonephropathy (MGN), yet the underlying molecular drivers remain incompletely understood. We identified microRNA-378a (miR-378a), previously found upregulated in MGN, as a functional enhancer of autophagic flux in human podocytes and tubular epithelial cells. While miR-378a did not directly alter transcription of canonical autophagy genes (ATG2A, ATG5, ATG7, ATG12), it increased autophagic flux through suppression of mTOR phosphorylation at Ser2448. Given that NPNT is a miR-378a target and a key glomerular basement membrane component, we investigated its role in autophagy regulation. NPNT knockdown reduced ATG2A, ATG7, and BCN1 expression, but paradoxically increased autophagic flux, independent of mTOR, accompanied by enhanced ERK1/2 phosphorylation. These findings reveal a dual-layered regulatory network in which miR-378a promotes autophagy via mTOR inhibition, whereas NPNT modulates autophagy probably through MAPK-dependent signaling. Our results highlight the complex interplay between miRs, extracellular matrix components, and intracellular signaling pathways in podocyte autophagy. Dysregulation of these pathways in kidney disease may reflect both adaptive and maladaptive responses, providing mechanistic insights and potential therapeutic targets to preserve glomerular filtration barrier integrity in immune-mediated kidney disease.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

The use of decellularized diseased livers in regenerative medicine is a promising approach for eliminating organ shortages. Bioengineering studies have shown that ECM can impact cell physiology, inducing cell activation, function, and ECM deposition, which suggests that the ECM has a "memory" that is involved in the outcome after recellularization. However, the effect of diseased ECM memory on new cells in vitro and in vivo has not been thoroughly investigated. Since it has been increasingly recognized that liver ECM changes due to different factors, it is comprehensively that diseased ECM obtained from discarded organs will ensure a distinct environment and impact cell survival and physiology. Thus, we aimed at investigating the impact of the memory of diseased ECM obtained from metabolic dysfunction-associated steatohepatitis (MASH)-derived organs on steatohepatitis establishment. To address this aim, we explored decellularized ECM obtained from rats and humans with MASH in different contexts. First, MASH ECM was characterized and then submitted to transplantation to investigate whether a MASH-derived ECM could be used as a scaffold for transplantation and to promote steatohepatitis features in control animals. Histological analysis revealed that the MASH-ECM was completely recellularized after transplantation in both control and MASH recipient rats. However, steatosis and fibrosis were observed in MASH ECM after transplantation in both groups. Molecular analysis showed that MASH ECM stimulates de novo lipogenesis and fibrosis 30 days after transplantation. Untargeted metabolomic analysis revealed that cells grown on MASH ECM had a similar metabolic profile, even when transplanted into healthy or MASH recipient rats. In addition, we observed that MASH ECM promoted impaired lipid oxidation and mitochondrial dysfunction when transplanted into healthy recipients. Altered lipid turnover and inflammatory signaling were observed in MASH ECM transplanted in MASH recipients. In vitro analysis revealed that MASH ECM induced lipid accumulation in HepG2 cells after 10 days of culture. Calcium signalling experiments obtained from HepG2 cells cultured in MASH ECM showed a lower response to ATP, a reduced calcium signalling amplitude, and a distinct response profile than that observed in healthy ECM. On the other hand, a diseased human-derived ECM could still provide an environment that allows cell development. Taken together, our data showed that MASH ECM impacts cell metabolism, promoting steatohepatitis maintenance. In conclusion, our data confirm that diseased ECM memory can impact cell physiology contributing to disease progression.

AimsThe activation of inflammatory cells, particularly macrophages, plays a pivotal role in the pathogenesis of cardiac remodelling and heart failure. Emerging evidence indicates that extracellular traps released from inflammatory immune cells contribute to the progression of various pathologies. However, the clinical relevance and mechanistic role of macrophage extracellular traps (METs) in heart failure remain to be elucidated. Methods and ResultsEndomyocardial biopsy specimens from 69 patients with heart failure were analysed by fluorescent immunostaining to identify and quantify METs. The numbers of METs per myocardial tissue area in patients with heart failure showed a negative correlation with left ventricular (LV) ejection fraction and a positive correlation with LV end-diastolic diameter. Patients with higher MET counts had significantly lower event-free survival from the composite cardiac events. In a murine model of pressure overload by transverse aortic constriction (TAC), METs were most abundantly observed at 3 days post-TAC and remained detectable throughout the 4-week observation period. In vitro, time-dependent MET formation was induced by an intrinsic trigger of mitochondrial DNA in bone marrow-derived macrophages from wild-type (WT) mice, but not in peptidyl arginine deiminase 4 (PAD4)-deficient macrophages, indicating that PAD4 activity is indispensable for MET formation. The recipient mice transplanted with bone marrow cells from PAD4 knockout mice showed more preserved cardiac function, reduced myocardial fibrosis, and improved survival in response to TAC, compared to those transplanted with WT mice. Ex vivo analyses demonstrated that conditioned medium containing METs from WT macrophages induced fibroblast-to-myofibroblast transition via Toll-like receptor 4 signalling. ConclusionsPAD4-dependent MET formation from bone marrow-derived macrophages represents a novel driver of cardiac remodelling. Targeting MET formation may offer a potential therapeutic strategy for heart failure. Translational PerspectiveMacrophage extracellular traps (METs) are abundant in myocardial tissue from patients with heart failure with reduced ejection fraction and are associated with adverse left ventricular remodelling and worse clinical outcomes. These findings support myocardial MET burden as a potential tissue biomarker to improve risk stratification in heart failure patients. In mice, pressure overload induces MET formation, and hematopoietic PAD4 deficiency suppresses myocardial METs, attenuates fibrosis, preserves cardiac function, and improves survival. Mechanistically, mitochondrial DNA-enriched cardiomyocyte-derived exophers trigger PAD4-dependent METs, which activate cardiac fibroblasts through TLR4 signalling. Suppressing METs represents a potential therapeutic strategy to attenuate the progression of heart failure. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711858v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1c6a637org.highwire.dtl.DTLVardef@caa356org.highwire.dtl.DTLVardef@1a994bforg.highwire.dtl.DTLVardef@6493bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

The left ventricle (LV) exhibits torsional deformation during systole, and mechanical relaxation begins during the isovolumic phase. Recent advances in imaging techniques, such as MRI, have revealed that myocardial tissue deformation and sarcomere length changes occur during the isovolumic relaxation phase, even when the chamber volume remains constant. Although such ventricular deformation during the isovolumic phase is considered important for blood ejection and filling efficiency, its mechanistic contribution to contraction and relaxation remains unresolved. In this study, we hypothesized that sarcomere length dynamics during the isovolumic phase affect the isovolumic contraction and relaxation time (IVCT and IVRT) by regulating the contraction force via the force-velocity relationship of ventricular myocytes. To investigate this hypothesis, we focused on experimentally reported differences in the relationship between sarcomere length and LV volume across the endocardial and epicardial layers, as described by Rodriguez et al. We constructed and compared two types of hemodynamic models within the same integrated framework consisting of a circulation model, a LV model, and a myocardial cell contraction model by Negroni-Lascano et al., which differ only in how sarcomere length is determined: a volume-based length model (VL model), in which sarcomere length is uniquely determined by LV volume, and a volume-force-coupled length model (VFL model), in which sarcomere length is determined by the balance between LV volume and contraction force. Simulation results showed that in the VFL model, compared to the VL model, sarcomere length changed during the isovolumic phase, leading to a decrease in contractile force and shortening of IVRT, which may contribute to improved hemodynamic efficiency. These results indicate that sarcomere length dynamics can mechanically regulate force decay during isovolumic relaxation, even under constant left ventricular volume. This study provides a theoretical framework for understanding the contributions of different layers within the LV wall to diastolic function during the isovolumic relaxation phase.

PurposeThis study examined the association between traditional physical practice participation and vision-related quality of life among junior secondary school students and tested the mediating roles of exercise self-efficacy and visual function anomalies within a serial mediation framework. MethodsA four-wave time-lagged survey was conducted among 1,579 students in Grades 7-9 from schools implementing traditional physical practice activities. Variables were assessed at two-week intervals. Mediation effects were tested using the bias-corrected percentile bootstrap method with 5,000 resamples. ResultsThe total effect of traditional physical practice participation on vision-related quality of life was significant ({beta} = 0.591, p < .001). After including the mediators, the direct effect remained significant ({beta} = 0.404, 95% CI [0.348, 0.457]), accounting for 68.36% of the total effect. The total indirect effect was significant ({beta} = 0.187, 95% CI [0.160, 0.218]), representing 31.64% of the total effect. The indirect effect via exercise self-efficacy was significant ({beta} = 0.088, 95% CI [0.068, 0.112], 14.89%), as was the indirect effect via visual function anomalies ({beta} = 0.065, 95% CI [0.048, 0.086], 11.00%). The serial mediation pathway through exercise self-efficacy and visual function anomalies was also significant ({beta} = 0.034, 95% CI [0.025, 0.045], 5.75%). All confidence intervals excluded zero, supporting partial mediation. ConclusionTraditional physical practice participation was associated with vision-related quality of life both directly and indirectly through exercise self-efficacy and visual function anomalies, including a significant serial mediation pathway. The findings highlight the combined psychological and functional mechanisms underlying adolescents vision-related quality of life.

Metabolic syndrome (MetS), characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension, affects a substantial proportion of the global population and increases the risk for cardiovascular disease, diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). Despite its prevalence, there are currently no effective pharmacological therapies targeting MetS, highlighting the need to identify novel etiological mechanisms, particularly within the gastrointestinal (GI) tract. Using a mouse model of MetS and healthy lean controls, we assessed the colonic microenvironment through metabolomic, transcriptomic, and microbiome analyses. Colonic organoids were cultured to further explore epithelial alterations. Additionally, human MetS fecal metabolomics data were cross-compared with the mouse model to validate translational relevance. MetS mice exhibited upregulation of colonic anabolic pathways, including glycolysis, the pentose phosphate pathway, and the tryptophan/kynurenine pathway, without evidence of intestinal inflammation. Microbiome analysis revealed an increased abundance of the genus Lactobacillus in MS NASH mice. Colonic organoids from MetS mice showed altered goblet cell differentiation. Comparative analysis with human MetS fecal metabolomics demonstrated similar dysregulated pathways, underscoring the translational relevance of these findings. Our study reveals significant metabolic and microbial alterations in the colon of MS NASH mice, implicating a dysfunctional GI tract as a potential etiological factor in MetS. These findings highlight specific metabolic pathways and microbial signatures that could serve as future therapeutic targets for MetS. NEW & NOTEWORTHYThis study identifies the colon as a metabolically active tissue affected in metabolic syndrome. Despite the absence of intestinal inflammation, MS NASH mice displayed altered colonic metabolism and microbiota composition, with conserved metabolite changes matching those seen in humans with metabolic syndrome. These findings highlight colonic metabolic dysfunction as a potential driver of gut dysbiosis and disease progression in metabolic syndrome and MASLD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/716131v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1b7c685org.highwire.dtl.DTLVardef@4a832aorg.highwire.dtl.DTLVardef@1e95c66org.highwire.dtl.DTLVardef@1b14209_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: The association between physical activity (PA) and chronic kidney disease (CKD) was initially explored; however, it remained unknown whether PA affected the incidence of CKD through the inflammation pathway. Moreover, objective PA measured by accelerometers was rarely considered for this association. Methods: This study was performed in a large-scale prospective cohort with two different sub-cohorts: the International Physical Activity Questionnaire (IPAQ)-measured cohort (N=314,694); and the device-measured cohort (N=79,454). Cox models were conducted to assess the association of PA with incident CKD. The mediating role of inflammation in such association was investigated by four inflammation metrics [C-reactive protein (CRP), white blood cell (WBC), a low-grade inflammation (INFLA) score, and monocyte to high-density lipoprotein cholesterol ratio (MHR)]. Results: In the questionnaire-measured cohort, compared to low PA, moderate and high PA reduced the risk of CKD by approximately 28.0% (95% CI 24.4[~]31.5%) or 37.6% (34.4[~]40.7%), respectively. Inflammation significantly mediated this association, with the mediation proportion was 4.1% (3.0[~]5.1%), 1.4% (1.1[~]1.7%), 9.8% (7.7[~]11.9%), and 1.4% (1.1[~]1.7%) for CRP, WBC, INFLA score, and MHR, respectively. Evidence from the device-measured cohort further strengthened the robustness of our findings, but the effects were somewhat attenuated, with the mediation proportion being 2.2% (1.2[~]3.2%), 0.8% (0.2[~]1.3%), 4.3% (2.5[~]6.0%), and 1.3% (0.6[~]2.1%) for CRP, WBC, INFLA score, and MHR, respectively. Conclusions: Our study reveals suggestive evidence for the association of active PA with reduced CKD risk and further demonstrates the mediating role of inflammation in such association, providing a novel perspective for the early prevention of CKD.

Exercise is a cornerstone therapy for diabetes because working skeletal muscles take up glucose at dramatically greater rates than postprandial insulin-stimulated glucose uptake and, notably, do so without a requirement for insulin. This remarkable ability of working muscles is preserved in diabetes, when muscles become resistant to insulin. However, the mechanism of insulin-independent glucose uptake by working muscles is not fully understood. Here we describe a previously unrecognized glucose uptake pathway in muscle, which we refer to as "mSGLT" based on shared properties with the Sodium Glucose Linked Transporter family. In contrast to the abundant GLUT4 transporter, mSGLT is not regulated by insulin, requires Na,K-ATPase-2 activity, and transports the hexose -methyl-D-glucoside (MDG), a glucose derivative that is handled by SGLTs but not GLUT4. The mSGLT pathway and GLUT transport pathways are independent and additive. In addition to exercise, mSGLT imports glucose under other conditions of adrenergic stimulation, which inhibits pancreatic insulin release and reduces the insulin sensitivity of muscle. SGLT2-specific antibodies recognize a protein in muscle of similar size to the kidney SGLT2; this protein localizes to the muscle t-tubules, together with Na,K-ATPase-2 and MAP17, the regulatory subunit of SGLT2. However, skeletal muscles do not express a full-length transcript of Slc5a2 (SGLT2), and SGLT2-specific inhibitors do not inhibit mSGLT with high affinity. The novel transporter may be a muscle variant of Slc5a2 that results from post-transcriptional or post-translational mechanisms. mSGLT and its regulation offer potential muscle-specific therapeutic targets for treating hyperglycemia and other conditions when insulin-stimulated glucose disposal into muscle is impaired.

Myocardial ischemia-reperfusion injury significantly exacerbates cardiac damage and worsens clinical outcomes, with oxidative stress in cardiomyocytes representing a central pathological mechanism. In this study, we reveal that APEX1, a key redox regulator, is markedly downregulated in cardiomyocytes under oxidative stress conditions. Functional analyses demonstrate that APEX1 knockdown intensifies oxidative stress-induced cardiomyocyte injury, whereas APEX1 overexpression confers robust protection against hypoxia reoxygenation mediated damage. Mechanistically, APEX1 exerts its cardioprotective effects by stabilizing the p53 protein and modulating its ubiquitination status. These findings establish APEX1 as a critical defender against oxidative injury in cardiomyocytes through direct regulation of p53 protein stability, highlighting its potential as a therapeutic target for ischemia-reperfusion related heart disease.

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

{beta}2-Adrenergic receptor (Adr{beta}2) is the most abundant form of adrenergic receptors in skeletal muscle. Our previous studies have shown that the ventromedial hypothalamic nucleus (VMH) regulates metabolic benefits of exercise, potentially by skeletal muscle Adr{beta}2. Although a large body of literature has shown the importance of Adr{beta}2 on skeletal muscle physiology, it remains unexplored whether skeletal muscle Adr{beta}2 contributes to metabolic benefits of exercise, such as prevention of diet-induced obesity (DIO). Here, we generated mice lacking Adr{beta}2 in skeletal muscle cells (SKMAdr{beta}2) and tested whether SKMAdr{beta}2 is required for metabolic benefits of exercise on DIO. Deletion of SKMAdr{beta}2 completely abolished the induction of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1) in skeletal muscle by {beta}2-agonist, which is a potent activator of Pgc-1. Exercise upregulates Pgc-1, which regulates a broad range of skeletal muscle physiology, including hypertrophy and mitochondrial function. Deletion of SKMAdr{beta}2 hampers augmented Pgc-1 in skeletal muscle by a single bout of exercise. Intriguingly, we found that deletion of SKMAdr{beta}2 increased endurance capacity. Further, our data showed that body weight in DIO mice lacking SKMAdr{beta}2 is comparable to that of control DIO mice during exercise training, suggesting that deletion of SKMAdr{beta}2 did not affect the metabolic benefits of exercise in DIO. Collectively, our data indicate that SKMAdr{beta}2 contributes to exercise-induced transcriptional changes and endurance capacity, however, it is not required for exercise benefits on bodyweight in DIO mice.

The ubiquitin conjugating enzyme UBE2J1/Ubc6e localizes to the endoplasmic reticulum where it mediates the ubiquitination and proteasomal degradation of terminally misfolded proteins. Although the protein is known to undergo phosphorylation at serine S184, we have considered modification at an additional site and used a bespoke anti-phospho antibody to confirm phosphorylation also at serine residue S266. Despite the well-described role of UBE2J1 in ER associated degradation (ERAD), we found no evidence for regulation at S266 during Unfolded Protein Response (UPR) induction by thapsigargin. Instead, our studies suggest that phosphorylation occurs independently at the S184 and S266 sites, with mutation at one site failing to disrupt basal phosphorylation at the second. We identified several contexts in which these two phosphorylations were differentially regulated. For example, ER localization, which is important for phosphorylation at S184, was not required for modification at S266, and sensitivity to proteasome inhibitors, which is regarded as a distinguishing feature of the S184 phospho-variant, was unaltered by the S266A mutation. Regarding regulation at S266 on the other hand, we found that pharmacological activation of protein kinase A resulted in rapid phosphorylation, with differential use of phospho-specific antibodies confirming that phosphorylation at S184 was unchanged by this treatment. Hormonal stimulation by glucagon resulted in a similar pattern of UBE2J1 phosphorylation, which occurred exclusively at S266 and could be inhibited by H89. The differential regulation demonstrated in these studies extends our understanding of the UBE2J1 enzyme, and may indicate a role in the integration of energy metabolism with environmental stress conditions.

Kidneys play an essential role in balancing fluid and electrolyte levels. Two mouse strains, C57Bl/6 and 129S2/SV, are routinely used to study renal physiology in laboratory settings, and prior observations suggest that significant differences in salt and water handling exist between them. This study aims to further establish the sources of these observed differences at both expressional and functional levels, in male and female mice. At baseline, male 129S2/SV mice displayed decreased Na+ and increased K+ plasma concentrations compared to C57Bl/6 males, while no statistical differences were observed between female mice. Interestingly, 129S2/SV male mice had lower glomerular density than C57Bl/6 males. Immunoblotting shows that 129S2/SV mice of both sexes had increased expression of NHE3 and NKCC2 compared to their C57Bl/6 counterparts. Both total and phosphorylated NCC were more abundant in female mice as compared to males, indicating sexual dimorphism. Furthermore, 129S2/SV females had higher expression of total and phosphorylated NCC compared to C57Bl/6 females. In contrast, the expression of SGLT2, ENaC subunits, and Na+/K+-ATPase were comparable between C57Bl/6 and 129S2/SV mice of both sexes. When challenged with diuretics intended to block NKCC2, NCC or ENaC, 129S2/SV male mice responded with a smaller diuresis and natriuresis than their C57Bl/6 counterparts. Taken together, our data suggest that differential expression of key Na+ transporters along the nephron contributes to differences in Na+/K+ homeostasis between these two mouse strains. NEW & NOTEWORTHYWe assessed the influence of genetic background on the expression of key Na+ transporters along the nephron in two commonly used inbred mouse strains, C57Bl/6 and 129S2/SV. We found that the kidney expression of NHE3, NKCC2, and NCC are strain dependent. Additionally, murine strain significantly contributes to the diuretic responses induced by hydrochlorothiazide, amiloride, and furosemide.