Testicular fat deposition attenuates reproductive performance via decreased follicle-stimulating hormone level and sperm meiosis and testosterone synthesis in mouse.

OBJECTIVE: Testicular fat deposition has been reported to affect animal reproduction. However, the underlying mechanism remains poorly understood. The present study explored whether sperm meiosis and testosterone synthesis contribute to mouse testicular fat depositioninduced reproductive performance. METHODS: High fat diet (HFD)-induced obesity CD1 mice (DIO) were used as a testicular fat deposition model. The serum hormone test was performed by agent kit. The quality of sperm was assessed using a Sperm Class Analyzer. Testicular tissue morphology was analyzed by histochemical methods. The expression of spermatocyte marker molecules was monitored by an immuno-fluorescence microscope during meiosis. Analysis of the synthesis of testosterone was performed by real-time polymerase chain reaction and reagent kit. RESULTS: It was found that there was a significant increase in body weight among DIO mice, however, the food intake showed no difference compared to control mice fed a normal diet (CTR). The number of offspring in DIO mice decreased, but there was no significant difference from the CTR group. The levels of follicle-stimulating hormone were lower in DIO mice and their luteinizing hormone levels were similar. The results showed a remarkable decrease in sperm density and motility among DIO mice. We also found that fat accumulation affected the meiosis process, mainly reflected in the cross-exchange of homologous chromosomes. In addition, overweight increased fat deposition in the testis and reduced the expression of testosterone synthesis-related enzymes, thereby affecting the synthesis and secretion of testosterone by testicular Leydig cells. CONCLUSION: Fat accumulation in the testes causes testicular cell dysfunction, which affects testosterone hormone synthesis and ultimately affects sperm formation.

Contribution of Hepatic Steatosis-Intensified Extracellular Vesicle Release to Aggravated Inflammatory Endothelial Injury in Liver-Specific Asah1 Gene Knockout Mice.

To study the mechanism by which nonalcoholic fatty liver disease (NAFLD) contributes to vascular endothelial Nod-like receptor pyrin domain 3 (NLRP3) inflammasome activation and neointima hyperplasia, NAFLD was established in high-fat diet (HFD)-treated Asah1fl/fl/Albcre (liver-specific deletion of the acid ceramidase gene Asah1) mice. Compared with Asah1 flox [Asah1fl/fl/wild type (WT)] and wild-type (WT/WT) mice, Asah1fl/fl/Albcre mice exhibited significantly enhanced ceramide levels and lipid deposition on HFD in the liver. Moreover, Asah1fl/fl/Albcre mice showed enhanced expression of extracellular vesicle (EV) markers, CD63 and annexin II, but attenuated lysosome-multivesicular body fusion. All these changes were accompanied by significantly increased EV counts in the plasma. In a mouse model of neointima hyperplasia, liver-specific deletion of the Asah1 gene enhanced HFD-induced neointima proliferation, which was associated with increased endothelial NLRP3 inflammasome formation and activation and more severe endothelial damage. The EVs isolated from plasma of Asah1fl/fl/Albcre mice on HFD were found to markedly enhance NLRP3 inflammasome formation and activation in primary cultures of WT/WT endothelial cells compared with those isolated from WT/WT mice or normal diet-treated Asah1fl/fl/Albcre mice. These results suggest that the acid ceramidase/ceramide signaling pathway controls EV release from the liver, and its deficiency aggravates NAFLD and intensifies hepatic EV release into circulation, which promotes endothelial NLRP3 inflammasome activation and consequent neointima hyperplasia in the mouse carotid arteries.

Regulation of exosome release by lysosomal acid ceramidase in coronary arterial endothelial cells: Role of TRPML1 channel.

Lysosomal acid ceramidase (AC) has been reported to determine multivesicular body (MVB) fate and exosome secretion in different mammalian cells including coronary arterial endothelial cells (CAECs). However, this AC-mediated regulation of exosome release from CAECs and associated underlying mechanism remain poorly understood. In the present study, we hypothesized that AC controls lysosomal Ca2+ release through TRPML1 channel to regulate exosome release in murine CAECs. To test this hypothesis, we isolated and cultured CAECs from WT/WT and endothelial cell-specific Asah1 gene (gene encoding AC) knockout mice. Using these CAECs, we first demonstrated a remarkable increase in exosome secretion and significant reduction of lysosome-MVB interaction in CAECs lacking Asah1 gene compared to those cells from WT/WT mice. ML-SA1, a TRPML1 channel agonist, was found to enhance lysosome trafficking and increase lysosome-MVB interaction in WT/WT CAECs, but not in CAECs lacking Asah1 gene. However, sphingosine, an AC-derived sphingolipid, was able to increase lysosome movement and lysosome-MVB interaction in CAECs lacking Asah1 gene, leading to reduced exosome release from these cells. Moreover, Asah1 gene deletion was shown to substantially inhibit lysosomal Ca2+ release through suppression of TRPML1 channel activity in CAECs. Sphingosine as an AC product rescued the function of TRPML1 channel in CAECs lacking Asah1 gene. These results suggest that Asah1 gene defect and associated deficiency of AC activity may inhibit TRPML1 channel activity, thereby reducing MVB degradation by lysosome and increasing exosome release from CAECs. This enhanced exosome release from CAECs may contribute to the development of coronary arterial disease under pathological conditions.

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia.

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1trg/ECcre) and their control littermates (Smpd1trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1trg/ECcre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1trg/ECcre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1trg/ECcre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1trg/ECcre mice than in their control littermates. Using ECs from Smpd1trg/ECcre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1ß levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.

Renomedullary exosomes produce antihypertensive effects in reversible two-kidney one-clip renovascular hypertensive mice.

The rapid fall in blood pressure following unclipping of the stenotic renal artery in the Goldblatt two-kidney one-clip (2K1C) model of renovascular hypertension is proposed to be due to release of renomedullary vasodepressor lipids, but the mechanism has remained unclear. In this study, we hypothesized that the hypotensive response to unclipping is mediated by exosomes released from the renal medulla. In male C57BL6/J mice made hypertensive by the 2K1C surgery, unclipping of the renal artery after 10 days decreased mean arterial pressure (MAP) by 23 mmHg one hr after unclipping. This effect was accompanied by a 556% increase in the concentration of exosomes in plasma as observed by nanoparticle tracking analysis. Immunohistochemical analysis of exosome markers, CD63 and AnnexinII, showed increased staining in interstitial cells of the inner medulla of stenotic but not contralateral control kidney of clipped 2K1C mice. Treatment with rapamycin, an inducer of exosome release, blunted the hypertensive response to clipping, whereas GW-4869, an exosome biosynthesis inhibitor, prevented both the clipping-induced increase in inner medullary exosome marker staining and the unclipping-induced fall in MAP. Plasma exosomes isolated from unclipped 2K1C mice showed elevated neutral lipid content compared to sham mouse exosomes by flow cytometric analysis after Nile red staining. Exosomes from 2K1C but not sham control mice exerted potent MAP-lowering and diuretic-natriuretic effects in both 2K1C and angiotensin II-infused hypertensive mice. These results are consistent with increased renomedullary synthesis and release of exosomes with elevated antihypertensive neutral lipids in response to increased renal perfusion pressure.

Release and Actions of Inflammatory Exosomes in Pulmonary Emphysema: Potential Therapeutic Target of Acupuncture.

BACKGROUND: Exosomes have been reported to mediate activation of the inflammatory response by secretion of inflammasome products such as IL-1ß or IL-18 and that changes in exosomes production or secretion may be a therapeutic target for treatment of a variety of different chronic diseases. The present study tested the hypothesis that exosome-mediated release of NLRP3 inflammasome products instigates the inflammatory response in the lung during emphysema, a type of chronic obstructive pulmonary disease (COPD) and that electroacupuncture (EA) may attenuate emphysema by inhibition of NLRP3 inflammasome activation and consequent inflammation. METHODS: The COPD mice model was developed by injecting porcine pancreatic elastase (PPE) via puncture tracheotomy and instillation. EA (4 Hz/20 Hz, 1 to 3 mA) was applied to the bilateral BL13 and ST36 for 30 min, once every other day for 2 weeks. Micro computed tomography (micro-CT) was performed to measure lung function. Histopathological changes in the lungs were displayed by HE staining. RESULTS: In a mouse model of porcine pancreatic elastase (PPE)-induced emphysema, the lung tissue was found to display several key features of emphysema, including alveolar septal thickening, enlarged alveoli, interstitial edema, and inflammatory cells infiltration. Lungs of mice receiving PPE exhibited substantially increased low attenuation area (LAA) in micro-CT images. The colocalization of NLRP3 vs ASC or caspase-1 detected by confocal microscopy was shown to increase in both bronchial and alveolar walls, indicating the increased formation of NLRP3 inflammasomes. IL-1ß, a prototype NLRP3 inflammasome activating product, was also found to have increased in the lung during emphysema, which was colocalized with CD63 (an exosome marker), an indicative of inflammatory exosome formation. By nanoparticle tracking analysis (NTA), IL-1ß-containing exosomes were shown to significantly increase in the bronchoalveolar lavage (BAL) from mice with emphysema. Therapeutically, IL-1ß production in the lung during emphysema was significantly reduced by EA at the acupoint Feishu (BL13) and Zusanli (ST36), accompanied by decreased colocalization of NLRP3 vs ASC or caspase-1. Increased exosome release into BAL during emphysema was shown to be significantly attenuated in EA-treated mice compared to their controls. However, EA of non-specific BL23 together with ST36 acupoint had no effects on NLRP3 inflammasome activation, exosome release and associated lung pathology during emphysema. CONCLUSION: NLRP3 inflammasome activation in concert with increased release of exosomes containing IL-1ß or other inflammasome products contributes to the development of lung inflammation and injury during PPE-induced emphysema and that EA of lung-specific acupoints attenuates inflammasome activation and exosome release, thereby reducing inflammatory response in the lung of mice with emphysema.

MSCFS: inferring circRNA functional similarity based on multiple data sources.

BACKGROUND: More and more evidence shows that circRNA plays an important role in various biological processes and human health. Therefore, inferring the circRNA's potential functions and obtaining circRNA functional similarity has become more and more significant. However, there is no effective approach to explore the functional similarity of circRNAs. METHODS: In this paper, we propose a new approach, called MSCFS, to calculate the functional similarity of circRNA by integrating multiple data sources. We combine circRNA-disease association, circRNA-gene-Gene Ontology association, and circRNA sequence information to explore the functional similarity of circRNA. Firstly, we employ different learning representation methods from three data sources to establish three circRNA functional similarity networks. Then we integrate the three networks to obtain the final circRNA functional similarity. RESULTS: We utilize circRNA-miRNA association similarity and circRNA co-expression similarity to evaluate the performance of MSCFS. The results show a positive correlation with miRNA association ([Formula: see text]) and circRNA co-expression similarity ([Formula: see text]). Finally, we construct a circRNA functional similarity network and perform case analysis. The result shows our method can be applied to infer new potential functions of circRNA and other associations. CONCLUSIONS: MSCFS combines multiple data sources related to circRNA functions. Correlation analysis and case analyses prove that MSCFS is a useful method to explore circRNA functional similarity.

Regulatory role of mammalian target of rapamycin signaling in exosome secretion and osteogenic changes in smooth muscle cells lacking acid ceramidase gene.

Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N-terminal nucleophile hydrolase family. This enzyme is located in the lysosome, which mediates conversion of ceramide (CER) into sphingosine and free fatty acids at acidic pH. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation. The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LEs)/lysosomes may control cargo selection, membrane biogenesis, and exosome secretion, which may be fine controlled by lysosomal sphingolipids such as CER. This lysosomal-CER-mTOR signaling may be a crucial molecular mechanism responsible for development of arterial medial calcification (AMC). Torin-1 (5 mg/kg/day), an mTOR inhibitor, significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin (OSP) and runt-related transcription factor 2 (RUNX2) and upregulation of smooth muscle 22α (SM22-α) in mice receiving high dose of Vitamin D (500 000 IU/kg/day). Asah1fl/fl /SMCre mice had markedly increased co-localization of mTORC1 with lysosome-associated membrane protein-1 (Lamp-1) (lysosome marker) and decreased co-localization of vacuolar protein sorting-associated protein 16 (VPS16) (a multivesicular bodies [MVBs] marker) with Lamp-1, suggesting mTOR activation caused reduced MVBs interaction with lysosomes. Torin-1 significantly reduced the co-localization of mTOR vs Lamp-1, increased lysosome-MVB interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using coronary artery smooth muscle cells (CASMCs), Pi -stimulation significantly increased p-mTOR expression in Asah1fl/fl /SMCre CASMCs as compared to WT/WT cells associated with increased calcium deposition and mineralization. Torin-1 blocked Pi -induced calcium deposition and mineralization. siRNA mTOR and Torin-1 significantly reduce co-localization of mTORC1 with Lamp-1, increased VPS16 vs Lamp-1 co-localization in Pi -stimulated CASMCs, associated with decreased exosome release. Functionally, Torin-1 significantly reduces arterial stiffening as shown by restoration from increased pulse wave velocity and decreased elastin breaks. These results suggest that lysosomal CER-mTOR signaling may play a critical role for the control of lysosome-MVB interaction, exosome secretion and arterial stiffening during AMC.

Downregulation of Lysosomal Acid Ceramidase Mediates HMGB1-Induced Migration and Proliferation of Mouse Coronary Arterial Myocytes.

High-mobility group box 1 protein (HMGB1) has been reported to trigger lysosome destabilization causing a wide of inflammatory diseases. The present study tested whether a lysosomal enzyme, acid ceramidase (AC), plays a critical role in HMGB1-induced alteration in ceramide metabolism and whether such HMGB1-AC interaction is associated with abnormal migration and proliferation of vascular smooth muscle cells (SMCs). We first observed that the expression of AC in the medial layer of mouse coronary arterial wall and colocalization of AC with a lysosome marker Lamp-1. In primary cultured coronary arterial myocytes (CAMs), AC expression and colocalization with Lamp-1 were significantly up-regulated by AC inducer, genistein, but down-regulated by AC inhibitor, N-oleoylethanolamine (NOE). HMGB1 dose-dependently decreased the colocalization of AC with Lamp-1 and reduced mRNA and protein expressions of AC in CAMs, but reversed by genistein. Consistently, HMGB1 significantly induced increases in the levels of long-chain ceramides in CAMs, which were not further enhanced by NOE but blocked by genistein. More importantly, HMGB1 promoted migration and proliferation of CAMs, which were not further increased by NOE but reduced by genistein. Lastly, CAMs isolated from smooth muscle-specific AC knockout mice (AC gene Asah1) exhibited increased ceramide levels and enhanced the migration and proliferation, which resembles the effects of HMGB1 on wild-type CAMs. Together, these results suggest that HMGB1 promotes SMC migration and proliferation via inhibition of AC expression and ceramide accumulation.

Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene.

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1-/- mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1-/- mice as compared to their wild-type littermates. Besides, Mcoln1-/- mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1-/- mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1-/- mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice.

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

Medial calcification in the arterial wall of smooth muscle cell-specific Smpd1 transgenic mice: A ceramide-mediated vasculopathy.

Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)-derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1trg /SMcre mice with SMC-specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1trg /SMwt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1trg /SMcre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome-MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1trg /SMcre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1trg /SMcre mice, increased Pi concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome-MVB interaction. However, amitriptyline prevented these changes in Pi -treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC.

Reversal of Endothelial Extracellular Vesicle-Induced Smooth Muscle Phenotype Transition by Hypercholesterolemia Stimulation: Role of NLRP3 Inflammasome Activation.

Recent studies reported that vascular endothelial cells (ECs) secrete NLR family pyrin domain-containing 3 (NLRP3) inflammasome products such as interleukin-1ß (IL-1ß) via extracellular vesicles (EVs) under various pathological conditions. EVs represent one of the critical mechanisms mediating the cell-to-cell communication between ECs and vascular smooth muscle cells (VSMCs). However, whether or not the inflammasome-dependent EVs directly participate in the regulation of VSMC function remains unknown. In the present study, we found that in cultured carotid ECs, atherogenic stimulation by oxysterol 7-ketocholesterol (7-Ket) induced NLRP3 inflammasome formation and activation, reduced lysosome-multivesicular bodies (MVBs) fusion, and increased secretion of EVs that contain inflammasome product IL-1ß. These EC-derived IL-1ß-containing EVs promoted synthetic phenotype transition of co-cultured VSMCs, whereas EVs from unstimulated ECs have the opposite effects. Moreover, acid ceramidase (AC) deficiency or lysosome inhibition further exaggerated the 7-Ket-induced release of IL-1ß-containing EVs in ECs. Using a Western diet (WD)-induced hypercholesterolemia mouse model, we found that endothelial-specific AC gene knockout mice (Asah1fl/fl/ECCre) exhibited augmented WD-induced EV secretion with IL-1ß and more significantly decreased the interaction of MVBs with lysosomes in the carotid arterial wall compared to their wild-type littermates (WT/WT). The endothelial AC deficiency in Asah1fl/fl/ECCre mice also resulted in enhanced VSMC phenotype transition and accelerated neointima formation. Together, these results suggest that NLRP3 inflammasome-dependent IL-1ß production during hypercholesterolemia promotes VSMC phenotype transition to synthetic status via EV machinery, which is controlled by lysosomal AC activity. Our findings provide novel mechanistic insights into understanding the pathogenic role of endothelial NLRP3 inflammasome in vascular injury through EV-mediated EC-to-VSMC regulation.

Suppression of Glucagon-Like Peptide-1 Release by Inhibition of Intestinal NLRP3 Inflammasome Activation in Asc-/- and Nlrp3-/- Mice.

The glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone secreted by intestinal enteroendocrine L-cells, which plays a crucial role in glucose control, regulation, and protection from different pathological conditions such as diabetes mellitus. The present study sought to test whether GLP-1 release increases gut injury with a high-fat diet (HFD) and whether this GLP-1 release is associated with NLRP3 inflammasome activation. Our results showed that the NLRP3 inflammasome is activated in the intestinal tissue of wild-type mice on a HFD, accompanied by GLP-1 overexpression. The number of intestinal L-cells and the GLP-1 level in serum are increased in WT mice with HFD. However, in the Asc-/- and Nlrp3-/- mice, these HFD-induced intestinal and serum GLP-1 changes were suppressed. Using confocal microscopy, the colocalization of GLP-1 and FLICA that labels activated caspase-1 in intestine was decreased in the Asc-/- and Nlrp3-/- mice compared to WT mice. Mechanistically, the inhibitor of caspase-1 or HMGB1 blocker is used to demonstrate the regulatory action of NRLP3 inflammasome in GLP-1 release. It was found that the level of GLP-1 and its colocalization with IL-1ß were reduced by inhibition of the caspase-1 activity, but not altered by blockade of HMGB1 action. Our results suggest that NLRP3 inflammasome activation triggers GLP-1 production from the intestine, which is associated with IL-1ß, but not with HMGB1. These findings for the first time provide evidence that the activation of NLRP3 inflammasome in the intestine increases GLP-1 release in mice, which may serve as an adaptive response to intestinal inflammation.

Endothelial acid ceramidase in exosome-mediated release of NLRP3 inflammasome products during hyperglycemia: Evidence from endothelium-specific deletion of Asah1 gene.

Exosomes have been demonstrated to be one of the mechanisms mediating the release of intracellular signaling molecules to conduct cell-to-cell communication. However, it remains unknown whether and how exosomes mediate the release of NOD-like receptor pyrin domain 3 (NLRP3) inflammasome products such as interleukin-1 beta (IL-1ß) from endothelial cells. The present study hypothesized that lysosomal acid ceramidase (AC) determines the fate of multivesicular bodies (MVBs) to control the exosome-mediated release of NLRP3 inflammasome products during hyperglycemia. Using a streptozotocin (STZ)-induced diabetes mouse model, we found that endothelium-specific AC gene knockout mice (Asah1fl/fl/ECcre) significantly enhanced the formation and activation of NLRP3 inflammasomes in coronary arterial ECs (CECs). These mice also had increased thickening of the coronary arterial wall and reduced expression of tight junction protein compared to wild-type (WT/WT) littermates. We also observed the expression of exosome markers such as CD63 and alkaline phosphatase (ALP) was augmented in STZ-treated Asah1fl/fl/ECcre mice compared to WT/WT mice, which was accompanied by an increased IL-1ß release of exosomes. In the primary cultures of CECs, we demonstrated that AC deficiency markedly enhanced the formation and activation of NLRP3 inflammasomes, but significantly down-regulated tight junction proteins when these cells were exposed to high levels of glucose. The CECs from Asah1fl/fl/ECcre mice had decreased MVB-lysosome interaction and increased IL-1ß-containing exosome release in response to high glucose stimulation. Together, these results suggest that AC importantly controls exosome-mediated release of NLRP3 inflammasome products in CECs, which is enhanced by AC deficiency leading to aggravated arterial inflammatory response during hyperglycemia.

Control of lysosomal TRPML1 channel activity and exosome release by acid ceramidase in mouse podocytes.

The transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca2+ release that is involved in Ca2+-dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested whether the TRPML1 channel in podocytes mediates lysosome trafficking, which is essential for multivesicular body (MVB) degradation by lysosomes. We first demonstrated the abundant expression of TRPML1 channel in podocytes. By GCaMP3 Ca2+ imaging, we characterized the lysosomal specificity of TRPML1 channel-mediated Ca2+ release in podocytes. Given the important role of acid ceramidase (AC) in lysosome function and podocyte injury, we tested whether AC regulates this TRPML1 channel-mediated Ca2+ release and consequent lysosome-dependent MVB degradation in podocytes. Pharmacologically, it was found that TRPML1 channel activity was remarkably attenuated by the AC inhibitor carmofur. Sphingosine, as an AC product, was demonstrated to induce TRPML1-mediated Ca2+ release, which was inhibited by a TRPML1 blocker, verapamil. Using a Port-a-Patch planar patch-clamp system, we found that AC-associated sphingolipids, sphingomyelin, ceramide, and sphingosine had different effects on TRPML1 channel activity in podocytes. Functionally, the inhibition of AC or blockade of TRPML1 channels was found to suppress the interaction of lysosomes and MVBs, leading to increased exosome release from podocytes. These results suggest that AC is critical for TRPML1 channel-mediated Ca2+ release, which controls lysosome-MVB interaction and exosome release in podocytes.

Rac1 GTPase Inhibition Blocked Podocyte Injury and Glomerular Sclerosis during Hyperhomocysteinemia via Suppression of Nucleotide-Binding Oligomerization Domain-Like Receptor Containing Pyrin Domain 3 Inflammasome Activation.

Elevated homocysteine (Hcy) levels have been shown to activate nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome leading to podocyte dysfunction and glomerular injury. However, it remains unclear how this inflammasome activation in podocytes is a therapeutic target for reversal of glomerular injury and ultimate sclerosis. The present study tested whether inhibition of Rac1 GTPase activity suppresses NLRP3 inflammation activation and thereby blocks podocyte injury induced by elevated Hcy. In cultured podocytes, we found that L-Hcy (the active Hcy form) stimulated the NLRP3 inflammasome formation, as shown by increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1, which was accompanied by increased interleukin-1ß production and caspase-1 activity, indicating NLRP3 inflammasome activation. Rac1 activator, uridine triphosphate (UTP), mimicked L-Hcy-induced NLRP3 inflammasome activation, while Rac1 inhibitor NSC23766 blocked it. This Rac1 inhibition also prevented L-Hcy-induced podocyte dysfunction. All these effects were shown to be mediated via lipid raft redox signaling platforms with nicotinamide adenine dinucleotide phosphate oxidase subunits and consequent O2- production. In animal studies, hyperhomocysteinemia (hHcy) induced by folate-free diet was shown to induce NLRP3 inflammasome formation and activation in glomeruli, which was also mimicked by UTP and inhibited by NSC23766 to a comparable level seen in Nlrp3 gene knockout mice. These results together suggest that Rac1 inhibition protects the kidney from hHcy-induced podocyte injury and glomerular sclerosis due to its action to suppress NLRP3 inflammasome activation in podocytes.

Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells.

Growth differentiation factor (GDF)11 has been reported to reverse age-related cardiac hypertrophy in mice and cause youthful regeneration of cardiomyocytes. The present study attempted to test a hypothesis that GDF11 counteracts the pathologic dedifferentiation of mouse carotid arterial smooth muscle cells (CASMCs) due to deficient autophagy. By real-time RT-PCR and Western blot analysis, exogenously administrated GDF11 was found to promote CASMC differentiation with increased expression of various differentiation markers (α-smooth muscle actin, myogenin, myogenic differentiation, and myosin heavy chain) as well as decreased expression of dedifferentiation markers (vimentin and proliferating cell nuclear antigen). Upregulation of the GDF11 gene by trichostatin A (TSA) or CRISPR-cas9 activating plasmids also stimulated the differentiation of CASMCs. Either GDF11 or TSA treatment blocked 7-ketocholesterol-induced CASMC dedifferentiation and autophagosome accumulation as well as lysosome inhibitor bafilomycin-induced dedifferentiation and autophagosome accumulation. Moreover, in CASMCs from mice lacking the CD38 gene, an autophagy deficiency model in CASMCs, GDF11 also inhibited its phenotypic transition to dedifferentiation status. Correspondingly, TSA treatment was shown to decrease GDF11 expression and reverse CASMC dedifferentiation in the partial ligated carotid artery of mice. The inhibitory effects of TSA on dedifferentiation of CASMCs were accompanied by reduced autophagosome accumulation in the arterial wall, which was accompanied by attenuated neointima formation in partial ligated carotid arteries. We concluded that GDF11 promotes CASMC differentiation and prevents the phenotypic transition of these cells induced by autophagosome accumulation during different pathological stimulations, such as Western diet, lysosome function deficiency, and inflammation. NEW & NOTEWORTHY The present study demonstrates that growth differentiation factor (GDF)11 promotes autophagy and subsequent differentiation in carotid arterial smooth muscle cells. Upregulation of GDF11 counteracts dedifferentiation under different pathological conditions. These findings provide novel insights into the regulatory role of GDF11 in the counteracting of sclerotic arterial diseases and also suggest that activation or induction of GDF11 may be a new therapeutic strategy for the treatment or prevention of these diseases.

Protective Role of Autophagy in Nlrp3 Inflammasome Activation and Medial Thickening of Mouse Coronary Arteries.

We hypothesized that autophagy and associated lysosome function serve as a critical modulator during Nod-like receptor family pyrin domain containing 3 (Nlrp3) inflammasome activation on proatherogenic stimuli. We first demonstrated that 7-ketocholesterol stimulated Nlrp3 inflammasome formation and activation as shown by increased colocalization of inflammasome components [Nlrp3 versus apoptosis associated speck-like protein (Asc) or caspase-1] and enhanced cleavage of caspase-1 into active caspase-1 to generate IL-1ß in coronary artery smooth muscle cells. Deletion of the CD38 gene (CD38-/-) that regulates lysosome function and autophagic flux also led to Nlrp3 inflammasome formation and activation. In the presence of rapamycin, the effects of either 7-ketocholesterol treatment or CD38 gene deletion were abolished. The autophagy inhibitor spautin-1 and the lysosome function blocker bafilomycin A1 also enhanced Nlrp3 inflammasome formation and activation. In animal experiments, we found that increased colocalization of Nlrp3 versus Asc or caspase-1 enhanced IL-1ß accumulation and caspase-1 activity in the coronary arterial wall of CD38-/- mice on the Western diet compared with CD38+/+ mice. This increased colocalization was blocked by treatment with rapamycin but enhanced by chloroquine, a water-soluble blocker of autophagic flux. Morphologic examinations confirmed that the media of coronary arteries was significantly thicker in CD38-/- mice on the Western diet than CD38+/+ mice. In conclusion, the deficiency of autophagic flux promotes Nlrp3 inflammasome formation and activation in coronary artery smooth muscle cells on proatherogenic stimulation, leading to medial thickening of the coronary arterial wall.

NLRP3 Inflammasome Formation and Activation in Nonalcoholic Steatohepatitis: Therapeutic Target for Antimetabolic Syndrome Remedy FTZ.

The Nod-like receptor protein 3 (NLRP3) inflammasome activation not only serves as an intracellular machinery triggering inflammation but also produces uncanonical effects beyond inflammation such as changing cell metabolism and increasing cell membrane permeability. The present study was designed to test whether this NLRP3 inflammasome activation contributes to the "two-hit" injury during nonalcoholic steatohepatitis (NASH) and whether it can be a therapeutic target for the action of Fufang Zhenzhu Tiaozhi (FTZ), a widely used herbal remedy for hyperlipidemia and metabolic syndrome in China. We first demonstrated that NLRP3 inflammasome formation and activation as well as lipid deposition occurred in the liver of mice on the high-fat diet (HFD), as shown by increased NLRP3 aggregation, enhanced production of IL-1ß and high mobility group box 1 (HMGB1), and remarkable lipid deposition in liver cells. FTZ extracts not only significantly reduced the NLRP3 inflammasome formation and activation but also attenuated the liver steatosis and fibrogenic phenotype changed. In in vitro studies, palmitic acid (PA) was found to increase colocalization of NLRP3 components and enhanced caspase-1 activity in hepatic stellate cells (HSCs), indicating enhanced formation and activation of NLRP3 inflammasomes by PA. PA also increased lipid deposition. Nlrp3 siRNA can reverse this effect by silencing the NLRP3 inflammasome and both with FTZ. In FTZ-treated cells, not only inflammasome formation and activation was substantially attenuated but also lipid deposition in HSCs was blocked. This inhibition of FTZ on lipid deposition was similar to the effects of glycyrrhizin, an HMGB1 inhibitor. Mechanistically, stimulated membrane raft redox signaling platform formation and increased O2•- production by PA to activate NLRP3 inflammasomes in HSCs was blocked by FTZ treatment. It is concluded that FTZ extracts inhibit NASH by its action on both inflammatory response and liver lipid metabolism associated with NLRP3 inflammasome formation and activation.