AAV induces hepatic necroptosis and carcinoma in diabetic and obese mice dependent on Pebp1 pathway.

Obesity and diabetes are risk factors for hepatocellular carcinoma (HCC); however, the underlying mechanisms are yet to be elucidated. Adeno-associated virus (AAV) frequently infects humans and has been widely used in gene therapy, but the risk of AAV infection such as HCC should be further evaluated. Here, we show that recombinant AAV injection caused liver injury, hepatic necroptosis, and HCC in db/db or high-fat diet-induced hyperglycemic and obese mice, but not in mice with only hyperglycemia or obesity. Prednisone administration or knockdown of Pebp1, highly expressed in db/db mice, alleviated hepatic injury and necroptosis induced by recombinant AAV in mice with diabetes and obesity. Inhibition of Pebp1 pathway also attenuated inflammation and necroptosis in vitro. Our findings show that AAV infection is a critical risk factor for HCC in patients with diabetes and obesity, and AAV gene therapy for these patients should be carefully evaluated. Both prednisone treatment and targeting Pebp1 pathway are promising strategies to alleviate inflammation and necroptosis that occurred in AAV gene therapy or related diseases.

Capsaicin receptor TRPV1 maintains quiescence of hepatic stellate cells in the liver via recruitment of SARM1.

BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.

High-protein diet prevents fat mass increase after dieting by counteracting Lactobacillus-enhanced lipid absorption.

Dietary restriction is widely used to reduce fat mass and lose weight in individuals with or without obesity; however, weight regain after dieting is still a big challenge, and the underlying mechanisms remain largely elusive. Here we show that refeeding after various types of dieting induces quick fat accumulation in mice and enhanced intestinal lipid absorption contributes to post-dieting fat mass increase. Moreover, refeeding after short-term dietary restriction is accompanied by an increase in intestinal Lactobacillus and its metabolites, which contributes to enhanced intestinal lipid absorption and post-dieting fat mass increase; however, refeeding a high-protein diet after short-term dietary restriction attenuates intestinal lipid absorption and represses fat accumulation by preventing Lactobacillus growth. Our results provide insight into the mechanisms underlying fat mass increase after dieting. We also propose that targeting intestinal Lactobacillus to inhibit intestinal lipid absorption via high-protein diet or antibiotics is likely an effective strategy to prevent obesity after dieting.

PARP1 Is Upregulated by Hyperglycemia Via N6-methyladenosine Modification and Promotes Diabetic Retinopathy.

Poly (ADP-ribose) polymerase 1 (PARP1) plays an irreplaceable role in the progression of diabetic retinopathy (DR). The m6A methylation in mRNA controls gene expression under various physiological and pathological conditions. However, effects of m6A methylation on PARP1 expression and DR progression at molecular level have not been documented. This study shows that the levels of PARP1, inflammatory factors, and fibrosis markers were significantly upregulated via evaluation by real-time PCR, western blotting, and immunofluorescence in both in vivo and in vitro experiments. EdU, CCK8, and apoptosis assays demonstrate that knockdown of PARP1 not only significantly improved the vitality of hRMECs (human retinal microvascular endothelial cells) even under high glucose conditions but also prevented glucose-induced inflammation, fibrosis, and angiogenesis in vivo. Mechanistically, dot blot, RNA pull-down, and immunoblots were implemented to explore the mechanism of m6A-mediated PARP1 stability and function. PARP1 is identified as a target of YTHDF2-mediated m6A modification. Overexpression of YTHDF2 substantially suppressed PARP1 mRNA m6A modification and inhibited its mRNA expression. Collectively, it has been demonstrated that PARP1 is frequently upregulated in human retinas and contributes to DR progression, and that YTHDF2-mediated m6A modification epigenetically regulates diabetes-induced PARP1 expression. Findings from this work may engender therapeutic targets for treating diabetic retinopathy.

Sustained Inflammation Induced by LPS Leads to Tolerable Anorexia and Fat Loss via Tlr4 in Mice.

Background: Sustained inflammation is implicated in a variety of pathological conditions like infection, obesity and type 2 diabetes. Lipid metabolism is crucial to support immune response during infection of bacteria. However, how sustained inflammation affects lipid metabolism, especially in white adipose tissue remains largely unknown. Methods: Sustained inflammation was induced by daily injection of Lipopolysaccharide (LPS). Tlr4 knockout mice were used to study the mechanism. Inflammation and lipid metabolism were evaluated by quantitative PCR, white blood cell counting, nuclear magnetic resonance, fat cell size quantification, lipolysis and fatty acid uptake assays, respiratory exchange ratio, and energy expenditure. Results: Here, we found that sustained inflammation leads to fat loss in mice with a quick loss and gradual increase manner. Moreover, LPS injection leads to inflammation, anorexia, decreased lipid anabolism, and increased lipid catabolism. Mechanically, we show that LPS induces fat loss, inflammation, anorexia, and alteration of lipid metabolism mainly dependent on Tlr4. Interestingly, sustained inflammation induces less fat loss, especially in epididymal white adipose tissue, than pair-feeding, and pair-feeding has no significant effect on inflammation and leads to less fatty acid uptake, more lipid catabolism and energy expenditure than LPS injection. In addition, we demonstrate that short-term sustained inflammation leads to relative long-term tolerance for LPS-induced anorexia, inflammation and altered lipid metabolism. Conclusion: These findings demonstrate that sustained inflammation induced by LPS leads to tolerable anorexia and fat loss via Tlr4 in mice, and provide new insights into the effect of sustained inflammation on lipid metabolism and subsequent tolerance.

KPNA4 is involved in cataract formation via the nuclear import of p53.

KPNA4 (also called importin-α3) belongs to the importin α adaptor proteins family, which orchestrates classical nuclear transport processes, importin-α/importin-ß1 pathway, and involves in cellular homeostasis. Disruption of balanced transport pathways may result in ectopic nuclear proteins and eventually cause diseases, mainly under the situation of cellular stress, such as oxidative stress. Little evidence is available on its cellular functions for high specific expression in lens. We firstly studied the role of KPNA4 in cataract formation. Lens defects were observed at an early age in kpna4 gene knockout zebrafish, generated by the CRISPR/Cas9 system. Those phenotype, including cloudy center part of the lens, via bright field microscopy, and the thinning of the LE layer, wider space between the adjacent LE and LF cells, irregular cells morphology and the increased number of holes inside the LE cells, which were detected by transmission electron microscopy, recapitulate the clinical features of cataract patients. As the p53-specific adaptor of the nuclear import, KPNA4 upregulated with the same pattern of p53 in hydrogen peroxide-induced apoptosis in human lens epithelia cells. Furthermore, the loss of Kpna4 resulted in the accumulation of p53 in the center of lens. Taken together, we showed that KPNA4 was involved in the formation of cataract, likely by mediating p53 nuclear transport.

The complete mitochondrial genome of Praia tianmunica (Hymenoptera: Cimbicidae) with related phylogenetic analysis.

The complete mitochondrial genome of Praia tianmunica is 15,556 bp long. It contains 37 genes and a control region of 505 bp in length. Gene rearrangement is present in the mitogenome of P. tianmunica. The maximum-likelihood and Bayesian phylogenetic analyses showed that the genus Praia was closely related to genus Labriocimbex.

Sarm1 Gene Deficiency Attenuates Diabetic Peripheral Neuropathy in Mice.

Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes, but any treatment toward the development of DPN is not yet available. Axon degeneration is an early feature of many peripheral neuropathies, including DPN. Delay of axon degeneration has beneficial effects on various neurodegenerative diseases, but its effect on DPN is yet to be elucidated. Deficiency of Sarm1 significantly attenuates axon degeneration in several models, but the effect of Sarm1 deficiency on DPN is still unclear. In this study, we show that Sarm1 knockout mice exhibit normal glucose metabolism and pain sensitivity, and deletion of the Sarm1 gene alleviates hypoalgesia in streptozotocin-induced diabetic mice. Moreover, Sarm1 gene deficiency attenuates intraepidermal nerve fiber loss in footpad skin; alleviates axon degeneration, the change of g-ratio in sciatic nerves, and NAD+ decrease; and relieves axonal outgrowth retardation of dorsal root ganglia from diabetic mice. In addition, Sarm1 gene deficiency markedly diminishes the changes of gene expression profile induced by streptozotocin in the sciatic nerve, especially some abundant genes involved in neurodegenerative diseases. These findings demonstrate that Sarm1 gene deficiency attenuates DPN in mice and suggest that slowing down axon degeneration is a potential promising strategy to combat DPN.

Short-term tamoxifen treatment has long-term effects on metabolism in high-fat diet-fed mice with involvement of Nmnat2 in POMC neurons.

Short-term tamoxifen treatment has effects on lipid and glucose metabolism in mice fed chow. However, its effects on metabolism in mice fed high-fat diet (HFD) and the underlying mechanisms are unclear. Here, we show that tamoxifen treatment for 5 days decreases fat mass for as long as 18 weeks in mice fed HFD. Tamoxifen alters mRNA levels of some genes involved in lipid metabolism in white adipose tissue and improves glucose and insulin tolerance as well as hepatic insulin signaling for 12-20 weeks. Proopiomelanocortin (POMC) neuron-specific deletion of nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) attenuates the effects of tamoxifen on glucose and insulin tolerance. These data demonstrate that short-term injection of tamoxifen has long-term effects on lipid and glucose metabolism in HFD mice with involvement of Nmnat2 in POMC neurons.

Hydrogen peroxide-induced apoptosis of human lens epithelial cells is inhibited by parthenolide.

AIM: To explore the effect of parthenolide on hydrogen peroxide (H2O2)-induced apoptosis in human lens epithelial (HLE) cells. METHODS: The morphology and number of apoptotic HLE cells were assessed using light microscopy and flow cytometry. Cell viability was tested by MTS assay. In addition, the expression of related proteins was measured by Western blot assay. RESULTS: Apoptosis of HLE cells was induced by 200 µmol/L H2O2, and the viability of these cells was similar to the half maximal inhibitory concentration (IC50), as examined by MTS assay. In addition, cells were treated with either different concentrations (6.25, 12.5, 25 and 50 µmol/L) of parthenolide along with 200 µmol/L H2O2 or only 50 µmol/L parthenolide or 200 µmol/L H2O2 for 24h. Following treatment with higher concentrations of parthenolide (50 µmol/L), fewer HLE cells underwent H2O2-induced apoptosis, and cell viability was increased. Further, Western blot assay showed that the parthenolide treatment reduced the expression of caspase-3 and caspase-9, which are considered core apoptotic proteins, and decreased the levels of phosphorylated nuclear factor-κB (NF-κB), ERK1/2 [a member of the mitogen-activated protein kinase (MAPK) family], and Akt proteins in HLE cells. CONCLUSION: Parthenolide may suppress H2O2-induced apoptosis in HLE cells by interfering with NF-κB, MAPKs, and Akt signaling.

Hemin Improves Insulin Sensitivity and Lipid Metabolism in Cultured Hepatocytes and Mice Fed a High-Fat Diet.

Hemin is a breakdown product of hemoglobin. It has been reported that the injection of hemin improves lipid metabolism and insulin sensitivity in various genetic models. However, the effect of hemin supplementation in food on lipid metabolism and insulin sensitivity is still unclear, and whether hemin directly affects cellular insulin sensitivity is yet to be elucidated. Here we show that hemin enhances insulin-induced phosphorylation of insulin receptors, Akt, Gsk3ß, FoxO1 and cytoplasmic translocation of FoxO1 in cultured primary hepatocytes under insulin-resistant conditions. Furthermore, hemin diminishes the accumulation of triglyceride and increases in free fatty acid content in primary hepatocytes induced by palmitate. Oral administration of hemin decreases body weight, energy intake, blood glucose and triglyceride levels, and improves insulin and glucose tolerance as well as hepatic insulin signaling and hepatic steatosis in male mice fed a high-fat diet. In addition, hemin treatment decreases the mRNA and protein levels of some hepatic genes involved in lipogenic regulation, fatty acid synthesis and storage, and increases the mRNA level and enzyme activity of CPT1 involved in fatty acid oxidation. These data demonstrate that hemin can improve lipid metabolism and insulin sensitivity in both cultured hepatocytes and mice fed a high-fat diet, and show the potential beneficial effects of hemin from food on lipid and glucose metabolism.

Experimental assessment of novel PAX6 splicing mutations in two Chinese families with aniridia.

Aniridia is a rare, congenital ocular disorder caused by the mutations of the paired box gene-6 (PAX6) (OMIM 607108), which encodes a highly conserved transcriptional regulator. In order to investigate the clinical characterizations and genetic defects of two Chinese families affected with aniridia, we recruited the family members and 200 ethnically matched controls. The entire exons and flanking intronic sequences of the PAX6 gene (NG_008679.1) were analyzed and effects of variants on splicing were assessed in silico and in vitro using exon trapping assay with pET01. The donor site (c.1183+1G>A) mutation identified in family 1 would result in a complete skipping of exon 12 and cause a frameshift and run-on translation past the normal termination codon, creating an enlarged PAX6 protein with extended COOH-terminal domain. Novel c.1033-1_1033delinsCT mutation was detected in family 2. This mutation provoked both complete exon 12 skipping and partial skipping of exon 12 deleting 7bp. This would lead to a frameshift translation and the introduction of pre-mature termination code, which resulted in severely truncated PAX6 protein likely to be degraded. Our study further expands the spectrum of genetic pathology underlying PAX6.

Metabolic syndrome risk factors and dry eye syndrome: a Meta-analysis.

AIM: To explore the relationship between metabolic risk factors and dry eye syndrome (DES). METHODS: Retrieved studies on the association of metabolic syndrome risk factors (hypertension, hyperglycemia, obesity, and hyperlipidemia) and DES were collected from PubMed, Web of Science, and the Cochrane Library in December 2015. Odds ratio (OR) with 95% confidence interval (CI) were pooled to evaluate the final relationship. Subgroup analyses were conducted according to diagnostic criteria of DES. RESULTS: Nine cross-sectional studies and three case-control studies were included in this Meta-analysis. The pooled results showed that people with hypertension, hyperglycemia, and hyperlipidemia had a higher risk of suffering from DES (P<0.05), especially the typical DES symptoms. On the other hand, obesity did not increase the risk of DES. CONCLUSION: The present Meta-analysis suggests that all metabolic risk factors except obesity were risk factors for DES.