Role of the mitochondrial protein cyclophilin D in skin wound healing and collagen secretion.

Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation has been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired reepithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, while its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif-KO mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.

Patient-specific iPSCs carrying an SFTPC mutation reveal the intrinsic alveolar epithelial dysfunction at the inception of interstitial lung disease.

Alveolar epithelial type 2 cell (AEC2) dysfunction is implicated in the pathogenesis of adult and pediatric interstitial lung disease (ILD), including idiopathic pulmonary fibrosis (IPF); however, identification of disease-initiating mechanisms has been impeded by inability to access primary AEC2s early on. Here, we present a human in vitro model permitting investigation of epithelial-intrinsic events culminating in AEC2 dysfunction, using patient-specific induced pluripotent stem cells (iPSCs) carrying an AEC2-exclusive disease-associated variant (SFTPCI73T). Comparing syngeneic mutant versus gene-corrected iPSCs after differentiation into AEC2s (iAEC2s), we find that mutant iAEC2s accumulate large amounts of misprocessed and mistrafficked pro-SFTPC protein, similar to in vivo changes, resulting in diminished AEC2 progenitor capacity, perturbed proteostasis, altered bioenergetic programs, time-dependent metabolic reprogramming, and nuclear factor κB (NF-κB) pathway activation. Treatment of SFTPCI73T-expressing iAEC2s with hydroxychloroquine, a medication used in pediatric ILD, aggravates the observed perturbations. Thus, iAEC2s provide a patient-specific preclinical platform for modeling the epithelial-intrinsic dysfunction at ILD inception.

Mitochondrial Proton Leak Regulated by Cyclophilin D Elevates Insulin Secretion in Islets at Nonstimulatory Glucose Levels.

Fasting hyperinsulinemia precedes the development of type 2 diabetes. However, it is unclear whether fasting insulin hypersecretion is a primary driver of insulin resistance or a consequence of the progressive increase in fasting glycemia induced by insulin resistance in the prediabetic state. Herein, we have discovered a mechanism that specifically regulates non-glucose-stimulated insulin secretion (NGSIS) in pancreatic islets that is activated by nonesterified free fatty acids, the major fuel used by ß-cells during fasting. We show that the mitochondrial permeability transition pore regulator cyclophilin D (CypD) promotes NGSIS, but not glucose-stimulated insulin secretion, by increasing mitochondrial proton leak. Islets from prediabetic obese mice show significantly higher CypD-dependent proton leak and NGSIS compared with lean mice. Proton leak-mediated NGSIS is conserved in human islets and is stimulated by exposure to nonesterified free fatty acids at concentrations observed in obese subjects. Mechanistically, proton leak activates islet NGSIS independently of mitochondrial ATP synthesis but ultimately requires closure of the KATP channel. In summary, we have described a novel nonesterified free fatty acid-stimulated pathway that selectively drives pancreatic islet NGSIS, which may be therapeutically exploited as an alternative way to halt fasting hyperinsulinemia and the progression of type 2 diabetes.

Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.

Chronic exposure of pancreatic ß cells to high concentrations of free fatty acids leads to lipotoxicity (LT)-mediated suppression of glucose-stimulated insulin secretion. This effect is in part caused by a decline in mitochondrial function as well as by a reduction in lysosomal acidification. Because both mitochondria and lysosomes can alter one another's function, it remains unclear which initiating dysfunction sets off the detrimental cascade of LT, ultimately leading to ß-cell failure. Here, we investigated the effects of restoring lysosomal acidity on mitochondrial function under LT. Our results show that LT induces a dose-dependent lysosomal alkalization accompanied by an increase in mitochondrial mass. This increase is due to a reduction in mitochondrial turnover as analyzed by MitoTimer, a fluorescent protein for which the emission is regulated by mitochondrial clearance rate. Mitochondrial oxygen consumption rate, citrate synthase activity, and ATP content are all reduced by LT. Restoration of lysosomal acidity using lysosome-targeted nanoparticles is accompanied by stimulation of mitochondrial turnover as revealed by mitophagy measurements and the recovery of mitochondrial mass. Remarkably, re-acidification restores citrate synthase activity and ATP content in an insulin secreting ß-cell line (INS-1). Furthermore, nanoparticle-mediated lysosomal reacidification rescues mitochondrial maximal respiratory capacity in both INS-1 cells and primary mouse islets. Therefore, our results indicate that mitochondrial dysfunction is downstream of lysosomal alkalization under lipotoxic conditions and that recovery of lysosomal acidity is sufficient to restore the bioenergetic defects.-Assali, E. A., Shlomo, D., Zeng, J., Taddeo, E. P., Trudeau, K. M., Erion, K. A., Colby, A. H., Grinstaff, M. W., Liesa, M., Las, G., Shirihai, O. S. Nanoparticle-mediated lysosomal reacidification restores mitochondrial turnover and function in ß cells under lipotoxicity.

Elamipretide Promotes Mitophagosome Formation and Prevents Its Reduction Induced by Nutrient Excess in INS1 ß-cells.

Elamipretide is a tetrapeptide that restores defects in mitochondrial function, binds to cardiolipin, and is being tested in clinical trials for mitochondria-related diseases. However, whether elamipretide modulates mitochondrial quality control and dynamics, processes essential to preserve mitochondrial function, is unclear. Thus, we tested the effects of elamipretide on mitochondrial morphology, mitophagosome formation, and their early disruption induced by excess nutrients in INS1 ß-cells. Elamipretide treatment was sufficient to increase engulfment of mitochondria into autophagosomes in control INS1 ß-cells, without inducing widespread changes in mitochondrial morphology or membrane potential. In an early pathogenic context mimicked by short-term exposure to nutrient excess, elamipretide treatment prevented both mitochondrial fragmentation and defects in the engulfment of mitochondria into autophagosomes. On the other hand, elamipretide did not prevent lysosomal defects induced by nutrient excess. Accordingly, elamipretide treatment did not entail benefits on pathogenic p62 and LC3II accumulation or on insulin secretory function. In conclusion, our data show that elamipretide selectively stimulates the engulfment of mitochondria into autophagosomes and prevents its defects induced by nutrient excess. Thus, we propose that improved selectivity of mitochondrial quality control processes might contribute to the benefits stemming from elamipretide treatments in other disease models.

Individual islet respirometry reveals functional diversity within the islet population of mice and human donors.

OBJECTIVE: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. METHODS: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). RESULTS: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 µm2 area (∼210 µm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. CONCLUSIONS: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.

Lysophosphatidic acid counteracts glucagon-induced hepatocyte glucose production via STAT3.

Hepatic glucose production (HGP) is required to maintain normoglycemia during fasting. Glucagon is the primary hormone responsible for increasing HGP; however, there are many additional hormone and metabolic factors that influence glucagon sensitivity. In this study we report that the bioactive lipid lysophosphatidic acid (LPA) regulates hepatocyte glucose production by antagonizing glucagon-induced expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK). Treatment of primary hepatocytes with exogenous LPA blunted glucagon-induced PEPCK expression and glucose production. Similarly, knockout mice lacking the LPA-degrading enzyme phospholipid phosphate phosphatase type 1 (PLPP1) had a 2-fold increase in endogenous LPA levels, reduced PEPCK levels during fasting, and decreased hepatic gluconeogenesis in response to a pyruvate challenge. Mechanistically, LPA antagonized glucagon-mediated inhibition of STAT3, a transcriptional repressor of PEPCK. Importantly, LPA did not blunt glucagon-stimulated glucose production or PEPCK expression in hepatocytes lacking STAT3. These data identify a novel role for PLPP1 activity and hepatocyte LPA levels in glucagon sensitivity via a mechanism involving STAT3.

The Mitochondrial Permeability Transition Pore Regulator Cyclophilin D Exhibits Tissue-Specific Control of Metabolic Homeostasis.

The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial function that has been implicated in the pathogenesis of metabolic disease. Cyclophilin D (CypD) is a critical regulator that directly binds to mPTP constituents to facilitate the pore opening. We previously found that global CypD knockout mice (KO) are protected from diet-induced glucose intolerance; however, the tissue-specific function of CypD and mPTP, particularly in the control of glucose homeostasis, has not been ascertained. To this end, we performed calcium retention capacity (CRC) assay to compare the importance of CypD in the liver versus skeletal muscle. We found that liver mitochondria are more dependent on CypD for mPTP opening than skeletal muscle mitochondria. To ascertain the tissue-specific role of CypD in metabolic homeostasis, we generated liver-specific and muscle-specific CypD knockout mice (LKO and MKO, respectively) and fed them either a chow diet or 45% high-fat diet (HFD) for 14 weeks. MKO mice displayed similar body weight gain and glucose intolerance compared with wild type littermates (WT), whereas LKO mice developed greater visceral obesity, glucose intolerance and pyruvate intolerance compared with WT mice. These findings demonstrate that loss of muscle CypD is not sufficient to alter whole body glucose metabolism, while the loss of liver CypD exacerbates obesity and whole-body metabolic dysfunction in mice fed HFD.

Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.

In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP3) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.

Dietary sugar intake increases liver tumor incidence in female mice.

Overnutrition can promote liver cancer in mice and humans that have liver damage caused by alcohol, viruses, or carcinogens. However, the mechanism linking diet to increased liver tumorigenesis remains unclear in the context of whether tumorigenesis is secondary to obesity, or whether nutrients like sugar or fat drive tumorigenesis independent of obesity. In male mice, liver tumor burden was recently found to correlate with sugar intake, independent of dietary fat intake and obesity. However, females are less susceptible to developing liver cancer than males, and it remains unclear how nutrition affects tumorigenesis in females. Herein, female mice were exposed to the liver carcinogen diethylnitrosamine (DEN) and fed diets with well-defined sugar and fat content. Mice fed diets with high sugar content had the greatest liver tumor incidence while dietary fat intake was not associated with tumorigenesis. Diet-induced postprandial hyperglycemia and fasting hyperinsulinemia significantly correlated with tumor incidence, while tumor incidence was not associated with obesity and obesity-related disorders including liver steatosis, glucose intolerance, or elevated serum levels of estrogen, ALT, and lipids. These results simplify the pathophysiology of diet-induced liver tumorigenesis by focusing attention on the role of sugar metabolism and reducing emphasis on the complex milieu associated with obesity.

Structure-activity relationships of furazano[3,4-b]pyrazines as mitochondrial uncouplers.

Chemical mitochondrial uncouplers are lipophilic weak acids that transport protons into the mitochondrial matrix via a pathway that is independent of ATP synthase, thereby uncoupling nutrient oxidation from ATP production. These uncouplers have potential for the treatment of diseases such as obesity, Parkinson's disease, and aging. We have previously identified a novel mitochondrial protonophore, named BAM15, which stimulates mitochondrial respiration across a broad dosing range compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Herein, we report our investigations on the structure-activity relationship profile of BAM15. Our studies demonstrate the importance of the furazan, pyrazine, and aniline rings as well as pKa in maintaining its effective protonophore activity.

Posttranslational modifications in connexins and pannexins.

Posttranslational modification is a common cellular process that is used by cells to ensure a particular protein function. This can happen in a variety of ways, e.g., from the addition of phosphates or sugar residues to a particular amino acid, ensuring proper protein life cycle and function. In this review, we assess the evidence for ubiquitination, glycosylation, phosphorylation, S-nitrosylation as well as other modifications in connexins and pannexin proteins. Based on the literature, we find that posttranslational modifications are an important component of connexin and pannexin regulation.