Introduction and Objective: Cardiac mitochondrial Akt1 activation modulates metabolic homeostasis by attenuating fatty liver and reducing whole body adiposity, though the underlying mechanism remains incompletely understood. Since gut microbiota plays a critical role in cardiovascular diseases pathogenesis, we investigated whether gut microbiota also mediates the whole-body metabolic benefits of cardiac mitochondrial Akt signaling. Methods: We used an inducible myocardium-specific transgenic mouse model harboring a mitochondria-targeting constitutively active Akt1 (CAMCAKT). To assess the role of gut microbiota, CAMCAKT mice were treated with broad-spectrum antibiotics (Ampicillin and Enrofloxacin) during high fat-high fructose diet (HFFD) feeding. Non-induced vehicle injected CAMCAKT mice were used as controls. Results: After 2 months on HFFD, antibiotic treated CAMCAKT mice had 7% higher body fat mass compared to controls. In contrast, on HFFD, body fat mass of untreated CAMCAKT was 8% lower compared to controls. Notably, antibiotics did not change the body composition of control mice on HFFD. Serum free fatty acid levels decreased in untreated HFFD-CAMCAKT mice but remained unchanged with antibiotic treatment. Furthermore, on HFFD, pronounced liver steatosis was comparable in control and antibiotic treated CAMCAKT mice, whereas untreated CAMCAKT mice exhibited significantly reduced hepatic steatosis by lipid area and droplet size. Conclusion: Microbiota depletion negates the metabolic and hepatoprotective benefits of cardiac mitochondrial Akt1 activation, reversing improvements in fatty liver and body composition. These results provide the first evidence that gut microbiota might be essential for mediating the metabolic effects of cardiac mitochondrial Akt1 activation, warranting further investigation into the specific microbial species and metabolites underlying this cardio-microbiome axis. Disclosure A. Pathak: None. A. Ta: None. R. Jenq: Other - patent royalties; Current; Seres Therapeutics. Consultant; Current; MaaT Pharma. Advisory Panel; Current; Postbiotics Plus. Consultant; Current; Nestlé. P.H. Wang: None.

BACKGROUND:Diabetic nephropathy (DN) is a leading cause of chronic kidney failure. We hypothesized that mitochondrial Akt1 dysfunction in renal proximal tubules plays a pathogenic role in DN development and that its activation may reverse DN progression. METHODS:To study this signaling pathway, we generated a transgenic mouse model harboring a renal tubule-specific, Tamoxifen-inducible, mitochondria-targeted constitutively active Akt1 (KMCAKT). Type 2 diabetes was induced by a high-fat, high fructose diet (HFFD) for 40 weeks. Renal histology and function were evaluated, and glucose metabolism was assessed using dynamic glucose testing. RESULTS:HFFD feeding resulted in development of DN in control non-induced KMCAKT mice, whereas KMCAKT mice with constitutively active mitochondrial Akt1, induced by Tamoxifen-injection (TAM), exhibited significant improvement of kidney dysfunction and histology. Urinary albumin, fasting plasma BUN levels, fibrosis and Jablonski scores were all markedly improved in HFFD-TAM-KMCAKT mice compared with controls, while levels of α-smooth muscle actin (αSMA) and transforming growth factor-β1 (TGFβ1) were significantly reduced. HFFD-TAM-KMCAKT mice exhibited lower fasting blood glucose and improved oral glucose tolerance, while basal and stimulated insulin levels were higher, along with increased beta cell mass and insulin secretion (HOMA-β) compared to controls. Hyperglycemic clamp studies confirmed increased insulin secretion in HFFD-TAM-KMCAKT mice. CONCLUSION:Tubular mitochondrial Akt1 plays a key role in DN progression. Restoring tubular mitochondrial Akt1 signaling may represent a novel approach to reverse development of chronic kidney disease. We also identified previously unrecognized metabolic crosstalk between renal tubular mitochondrial Akt1 and pancreatic insulin secretion that may modulate systemic glucose homeostasis.

Introduction and Objective: Diabetic nephropathy (DN) is a major cause of end-stage renal disease. We have recently shown that activation of mitochondrial Akt1 in proximal tubules played a renoprotective role in murine acute ischemia-reperfusion injury. However, whether activation of mitochondrial Akt1 in renal proximal tubules can protect against the development of DN remains to be elucidated. Methods: We have generated a transgenic mouse model harboring a renal proximal tubule-specific tamoxifen-inducible mitochondria-targeting constitutively active Akt1 (KMCAKT). Results: High fat+fructose diet (HFFD) induced DN, but KMCAKT mice attenuated renal dysfunction induced by HFFD. Urinary albumin/creatinine ratio, fasting plasma creatinine, glomerular mesangial expansion and fibrosis were all improved compared to controls. To dissect the underlying mechanism, we have determined the contents of renal αSMA and TGFβ1 and both were lower in KMCAKT mice than in controls (p<0.001). This indicated that activation of mitochondrial Akt1 in renal tubule protected against the development of DN induced by HFFD. Interestingly, HFFD-fed KMCAKT mice showed significant reduction in fasting plasma glucose and urinary glucose excretion with an improvement of oral glucose tolerance compared to controls. Fasting plasma insulin levels were higher in HFFD-fed KMCAKT mice as compared to controls, whereas insulin resistance (HOMA-IR) were unchanged from controls, suggesting that renal mitochondrial Akt1 positively modulated systemic glucose metabolism, at least partially, by enhancing beta cell function. Conclusion: These data demonstrated novel roles of renal tubular mitochondrial Akt1 in the regulation of whole-body glucose metabolism and insulin secretion beyond its local effect on kidney function in DN, which suggests renal function and glucose homeostasis could be mediated through a common pathway in renal tubular mitochondria. E. Salem: None. A. Ta: None. P.T. Fueger: None. P.H. Wang: None.

Disclosure: H. Lo: None. S. Bhattacharya: None. K. Shaw: None. Y. Chen: None. P. Fueger: None. A. Ta: None. J. Wang: None. P.H. Wang: None. Insulin-resistant females of reproductive age face increased risks of ovarian dysfunction. Insulin induces the translocation of activated AKT into mitochondria. This study aims to investigate the role of mitochondrial AKT in ovarian granulosa cells (GCs) during folliculogenesis. Two novel transgenic mouse models were developed with Cre-LoxP system to selectively inhibit mitochondrial AKT signaling in GCs (ovdnAKT) or augment mitochondrial AKT (constitutively active mitochondrial AKT) in GCs (ovcaAKT). Results: Single-cell RNA sequencing (scRNA-seq) identified seven distinct GC clusters representing sequential stages of folliculogenesis, from progenitor cells through the preantral and antral phases, and ending in luteinization or atresia. In ovdnAKT mice, compared to controls, the number of antral GCs was significantly reduced, while luteinizing GC numbers increased. Specific gene markers and pathways were identified, which explain the transcriptional and development changes observed in ovdnAKT GCs. Histological analysis revealed a significant increase in the total number of follicles and preantral follicles in ovdnAKT ovary, indicating impaired maturation of preantral to antral phase, thus aligns with scRNA-seq results. Histology also found preantral follicles consisted of various GC types and were surrounded by luteinizing GCs. Anti-Müllerian hormone (AMH), a biomarker for preantral follicles, was markedly elevated in ovdnAKT mice compared to controls. The estrous cycles of ovdnAKT mice were irregular and prolonged (11.9 ± 3.7 days vs. 4.5 ± 0.5 days in controls; p < 0.001). To further explore the effect of GC mitochondrial AKT, mice were fed a high-fat/high-fructose diet (HFFD) or a normal chow diet (NCD) to study metabolic syndrome-associated ovarian dysfunction. In control mice, HFFD significantly reduced insulin-induced AKT translocation to mitochondria in GCs and prolonged the estrous cycle (HFFD: 7.8 ± 2.1 days; NCD: 4.7 ± 0.5 days; p = 0.0022). Histological analysis showed an increased number of preantral follicles in the control mice after HFFD. In ovcaAKT mice, which express active mitochondrial AKT in GCs, the duration of the estrous cycle was normalized under HFFD, and follicle distribution in the ovaries restored. Summary: Mitochondrial AKT in GCs plays a critical role in ovarian follicle recruitment and maturation by modulating GC transcription of key reproduction genes. Disruption of GC mitochondrial AKT signaling impeded the maturation of preantral to antral follicles and accelerated follicle luteinization. Activating mitochondrial AKT in GCs restored normal folliculogenesis and estrous cycling in the HFFD-induced metabolic syndrome. Impact: Impaired mitochondrial AKT signaling in GCs is a novel mechanistic link between insulin resistance and ovarian dysfunction. Presentation: Sunday, July 13, 2025

Aims Following myocardial infarction (MI), the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix (ECM) by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure, and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors, and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human MI as well as non-ischaemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodelling is poorly understood. The aim of this study was to understand the role of Serpins in regulating scar formation after MI.Methods and results Using a SerpinA3n conditional knockout mice model, we observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homologue of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix, and significantly worse post-infarct cardiac function. Single-cell transcriptomics complemented with histology in SerpinA3n-deficient animals demonstrated increased inflammation, adverse myocyte hypertrophy, and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross-linking of ECM peptides consistent with increased proteolysis in SerpinA3n-deficient animals.Conclusion Our study demonstrates a hitherto unappreciated causal role of Serpins in regulating matrix function and post-infarct cardiac remodelling.

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA–promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial–nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

Activating PI3K/Akt pathway triggers translocation of activated AKT into mitochondria, and granulosa cells are enriched with mitochondria. The goals of this study were to determine the mechanistic roles of mitochondrial AKT in granulosa cells during folliculogenesis and progression of menstruation cycles, and to explore its role in the regulation of whole body metabolism. To simulate ovarian insulin resistance in mitochondria, we have generated a novel transgenic mice model with Cre-LoxP system to disrupt mitochondrial AKT signaling in granulosa cells. The transgenic mice express a dominant negative mitochondria-targeting AKT in the granulosa cells (ovdnAKT). We used histological analysis, biochemical assays, and scRNA-seq to investigate folliculogenesis. Body composition was determined by EchoMRI and glucose homeostasis by glucose tolerance test. Insulin stimulates AKT phosphorylation and translocation to the mitochondria in granulosa cells in vivo. When mitochondrial AKT was inhibited in granulosa cells in 8-week old ovdnAKT mice, menstrual cycle became irregular and prolonged (ovdnAKT, 11.9 ± 3.7 days; controls, 4.5 ± 0.5 days, p<0.001). Vaginal lavage showed predominant diestrus phase in ovdnAKT mice. The ovary weight was increased (ovdnAKT, 10.19 ± 0.89 mg; controls, 6.28 ± 2.40 mg, p=0.049). Histology analysis revealed increased total number of follicles (ovdnAKT, 533.00 ± 14.00; controls, 337.67 ± 53.38, p=0.001) and increased accumulation of preantral follicles in ovdnAKT mice (ovdnAKT, 438.67 ± 22.59; controls, 284.33 ± 41.50, p=0.002). But morphologically mature antral follicles were nearly absent in the ovdnAKT mice. Ovary scRNA-seq cluster annotation confirmed an increased pool of preantral granulosa cells in the ovdnAKT ovary, decreased number of mature granulosa cells and atretic granulosa cells, and increased number of luteinizing granulosa cells. The population of mesenchymal cells, immune cells, epithelial cells, theca cells, and endothelial cells were not changed. Transcriptional analysis in the granulosa cells at preantral phase showed altered folliculogenesis-promoting and steroidogenesis genes in the ovdnAKT mice. Mitochondrial AKT also modulated whole body metabolism, body weight increased by 20%, fat mass/weight ratio increased by 83%, and lean mass/weight ratio decreased by 11% in the ovdnAKT mice after 16 weeks, indicating development of obesity and metabolic syndrome. Insulin stimulated translocation of active AKT to mitochondria in granulosa cells. Mitochondrial AKT played a critical role in the recruitment and maturation of antral follicles through transcriptional modulation. Disrupting this pathway in granulosa cells led to obesity and metabolic syndrome, which suggests ovary-adipose metabolic crosstalk.

Distinct metabolic conditions rewire circadian-clock-controlled signaling pathways leading to the de novo construction of signal transduction networks. However, it remains unclear whether metabolic hallmarks unique to pluripotent stem cells (PSCs) are connected to clock functions. Reprogramming somatic cells to a pluripotent state, here we highlighted non-canonical functions of the circadian repressor CRY1 specific to PSCs. Metabolic reprogramming, including AMPK inactivation and SREBP1 activation, was coupled with the accumulation of CRY1 in PSCs. Functional assays verified that CRY1 is required for the maintenance of self-renewal capacity, colony organization, and metabolic signatures. Genome-wide occupancy of CRY1 identified CRY1-regulatory genes enriched in development and differentiation in PSCs, albeit not somatic cells. Last, cells lacking CRY1 exhibit differential gene expression profiles during induced PSC (iPSC) reprogramming, resulting in impaired iPSC reprogramming efficiency. Collectively, these results suggest the functional implication of CRY1 in pluripotent reprogramming and ontogenesis, thereby dictating PSC identity.

Insulin stimulates Akt1 translocation to cardiac mitochondria, and we have recently shown that inhibition of cardiac mitochondria Akt1 led to mitochondrial dysfunction and cardiomyopathy. However, whether activation of mitochondrial Akt1 in cardiac muscle can protect against the development of diabetic cardiomyopathy is not known. To this end, we have generated an inducible heart-specific transgenic mouse harboring a mitochondria-targeting constitutively active Akt1 (CAMCAKT). Long-term high fat-high fructose diet (HFFD) caused myocardial hypertrophy, fibrosis and ventricular dysfunction in control mice, but the impact of HFFD were attenuated in the CAMCAKT mice. After just two months on HFFD, expression of Col1a1 and Col3a1 in CAMCAKT mice were lower than controls (p<0.01). After five months on HFFD, ejection fraction in CA mice was 17 % higher than in controls while fractional shortening was 22% higher (p<0.05). This indicated that activation of mitochondrial Akt protected myocardium against the development of cardiomyopathy in diet-induced diabetes. Metabolically, cardiac mitochondrial Akt1 increased fatty acid uptake in the cardiac muscle, which resulted in redistribution of whole-body fatty acid metabolism. Dynamic PET scans revealed a 65% increase (p<0.001) in heart uptake of fatty acid reporter [18F]fluoro-4-thia-oleate in CAMCAKT mice. CAMCAKT mice had 15% less body fat, 30% lower total cholesterol and expended 13% more energy, compared to controls (p<0.05). Interestingly, fatty liver was reduced in the CAMCAKT mice (p<0.05), suggesting that cardiac mitochondrial Akt modulated fat deposition in the liver by higher myocardial fatty acid metabolism. In summary, activating cardiac mitochondrial Akt protected against diabetic cardiomyopathy, attenuated fatty liver, and induced whole body metabolism in this murine model of diet-induced diabetes. Disclosure A.Ta: None. Y.Chen: None. J.Li: None. E.Salem: None. P.H.Wang: Research Support; ViaCyte, Inc. Funding National Institutes of Health (R01HL096987); Ko Family Foundation

Diabetic nephropathy is a major cause of end-stage renal disease. We have recently shown translocation of AKT to mitochondria in proximal tubules played a renoprotective role in murine ischemia/reperfusion injury. In this study, we tested the hypothesis that activation of mitochondrial AKT (mito-AKT) signaling in renal proximal tubules improves kidney function in diabetic nephropathy and modulates overall glucose homeostasis. We used renal tubule-specific bigenic mice that harbor tamoxifen-inducible mitochondria-targeting constitutively active AKT1 (KMCAKT), which were fed with either normal chow diet or high fat and fructose diet (HFFD). Wild type mice fed with HFFD developed nephropathy after 24 wks. Activation of renal tubular mito-AKT significantly attenuated urinary albumin, creatinine, and glucose excretion in the KMCAKT mice on HFFD. Interestingly, activation of tubular mito-AKT also decreased fasting hyperglycemia, and improved glucose tolerance of HFFD-fed mice. Insulin resistance (HOMA-IR) was significantly reduced when tubular mito-AKT was activated. In addition, mito-AKT activation reduced plasma cholesterol. Furthermore, mito-AKT activation increased total body weight and fat mass of HFFD-fed mice, but had no effect on their water and food intake. To dissect the underlying mechanism, we used 18F-fluoro-4-thia-oleate PET scan to define organ specific fatty acid uptake, and revealed a significant increase of uptake in white and brown adipose tissues in KMCAKT mice on HFFD. In conclusion, this is the first report to demonstrate critical roles of mitochondrial AKT in renal tubules in the regulation of whole-body metabolism, insulin sensitivity, and body composition beyond its local effect on kidney function in diabetic nephropathy, which suggests kidney function and nephropathy associated metabolic dysfunction could be mediated through a common pathway in tubular mitochondria. Disclosure E.Salem: None. A.Ta: None. H.Y.H.Lin: None. J.Li: None. P.H.Wang: Research Support; ViaCyte, Inc. Funding Shannon & Amy Ko Foundation

BACKGROUND:The PI3K/AKT pathway transduces the majority of the metabolic actions of insulin. In addition to cytosolic targets, insulin-stimulated phospho-AKT also translocates to mitochondria in the myocardium. Mouse models of diabetes exhibit impaired mitochondrial AKT signaling but the implications of this on cardiac structure and function is unknown. We hypothesized that loss of mitochondrial AKT signaling is a critical step in cardiomyopathy and reduces cardiac oxidative phosphorylation.METHODS:To focus our investigation on the pathophysiological consequences of this mitochondrial signaling pathway, we generated transgenic mouse models of cardiac-specific, mitochondria-targeting, dominant negative AKT1 (CAMDAKT) and constitutively active AKT1 expression (CAMCAKT). Myocardial structure and function were examined using echocardiography, histology, and biochemical assays. We further investigated the underlying effects of mitochondrial AKT1 on mitochondrial structure and function, its interaction with ATP synthase, and explored in vivo metabolism beyond the heart.RESULTS:Upon induction of dominant negative mitochondrial AKT1, CAMDAKT mice developed cardiac fibrosis accompanied by left ventricular hypertrophy and dysfunction. Cardiac mitochondrial oxidative phosphorylation efficiency and ATP content were reduced, mitochondrial cristae structure was lost, and ATP synthase structure was compromised. Conversely, CAMCAKT mice were protected against development of diabetic cardiomyopathy when challenged with a high calorie diet. Activation of mitochondrial AKT1 protected cardiac function and increased fatty acid uptake in myocardium. In addition, total energy expenditure was increased in CAMCAKT mice, accompanied by reduced adiposity and reduced development of fatty liver.CONCLUSION:CAMDAKT mice modeled the effects of impaired mitochondrial signaling which occurs in the diabetic myocardium. Disruption of this pathway is a key step in the development of cardiomyopathy. Activation of mitochondrial AKT1 in CAMCAKT had a protective role against diabetic cardiomyopathy as well as improved metabolism beyond the heart.

After heart injury, dead heart muscle is replaced by scar tissue. Fibroblasts can electrically couple with myocytes, and changes in fibroblast membrane potential can lead to myocyte excitability, which suggests that fibroblast-myocyte coupling in scar tissue may be responsible for arrhythmogenesis. However, the physiologic relevance of electrical coupling of myocytes and fibroblasts and its impact on cardiac excitability in vivo have never been demonstrated. We genetically engineered a mouse that expresses the optogenetic cationic channel ChR2 (H134R) exclusively in cardiac fibroblasts. After myocardial infarction, optical stimulation of scar tissue elicited organ-wide cardiac excitation and induced arrhythmias in these animals. Complementing computational modeling with experimental approaches, we showed that gap junctional and ephaptic coupling, in a synergistic yet functionally redundant manner, excited myocytes coupled to fibroblasts.

Previous studies reported inconsistent findings on autophagy activation in skeletal muscles after acute exercise. In this study, we investigated the effect of a single bout of exhaustive treadmill exercise on AMPK and autophagy activations in mice gastrocnemius muscle in vivo. Male ICR/CD-1 mice were randomly divided into the control and exercise groups. The later was subjected to a single bout of exhaustive treadmill exercise. Changes of AMPK, phosphorylation of AMPKThr172 (pAMPKThr172 ), and autophagy markers including Beclin1, LC3II/LC3I and p62 mRNA and protein expressions in gastrocnemius muscle at different times (0, 6, 12, 24 h) after the exercise were analysed by quantitative real-time PCR and western blot. Our results demonstrated that a single bout of exhaustive treadmill exercise significantly induced AMPK content and AMPK activity at 0, 6 and 12 h after the exercise, and changed the expressions of autophagy markers at different time points in the recovery period, respectively. Moreover, we observed positive correlations between expressions of LC3II/LC3I ratio and pAMPKThr172 or AMPK, and a negative correlation between expressions of p62 and AMPK or pAMPKThr172 . In conclusion, a single bout of exhaustive treadmill exercise in mice caused a prolonged activation of AMPK and improved autophagy in the gastrocnemius muscle. The regulation of autophagic markers were related to enhanced AMPK activity. The findings indicate that acute exercise enhanced AMPK-related autophagy activation may be the underlying molecular mechanism that regulates cellular energy metabolism during exercise.

Kidney tubular dysfunction contributes to acute kidney injury and to the transition to chronic kidney disease. Although tubular mitochondria have been implicated in the pathophysiology of kidney failure, the mechanisms are not yet clear. Here, we demonstrated that ischemia-reperfusion injury induced acute translocation and activation of mitochondrial protein kinase B (also known as AKT1) in the kidney tubules. We hypothesized that mitochondrial AKT1 signaling protects against the development of acute kidney injury and subsequent chronic kidney disease. To test this prediction, we generated two novel kidney tubule-specific transgenic mouse strains with inducible expression of mitochondria-targeted dominant negative AKT1 or constitutively active AKT1, using a Cre-Lox strategy. Inhibition of mitochondrial AKT1 in mitochondria-targeted dominant negative AKT1 mice aggravated azotemia, tubular injuries, kidney fibrosis, glomerulosclerosis, and negatively impacted survival after ischemia-reperfusion injury. Conversely, enhancing tubular mitochondrial AKT1 signaling in mitochondria-targeted constitutively active AKT1 mice attenuated kidney injuries, protected kidney function, and significantly improved survival after ischemia-reperfusion injury (76.9% vs. 20.8%, respectively). Uncoupled mitochondrial respiration and increased oxidative stress was found in the kidney tubules when mitochondria AKT1 was inhibited, supporting the role of mitochondrial dysfunction in the pathophysiology of kidney failure. Thus, our studies suggest tubular mitochondrial AKT1 signaling could be a novel target to develop new strategies for better prevention and treatment of kidney injury.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.

This study aimed to elucidate the role of transcription factor EB (TFEB) in protecting C2C12 myotubes against palmitate (PA)‐induced insulin resistance (IR) and explored its mechanism associated with autophagy. PA treatment significantly decreased insulin sensitivity in myotubes and downregulated TFEB protein expression. TFEB overexpression significantly reversed the PA‐suppressed glucose transporter 4 ( GLUT4) protein expression and improved intracellular glucose uptake and consumption, and also alleviated the decrease of autophagy markers induced by PA. The effect of TFEB overexpression on GLUT4 was also abolished by the autophagy inhibitor 3‐MA. In addition, AMPKɑ2‐DN inhibited or abolished the effects of TFEB overexpression on upregulation of GLUT4 and PA‐induced decrease of autophagy marker expressions. Taken together, our data demonstrated that upregulation of TFEB improved PA‐induced IR in C2C12 myotubes by enhancing autophagy and upregulating AMPK activity. TFEB, as a critical regulator of glucose homeostasis in skeletal muscle cells, may be a potential therapeutic target for IR and Type 2 diabetes.

Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in the nucleus. Lsd1 KO reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.