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SUMMARYTo discover new drugs to combat coronavirus disease 2019 (COVID-19), an understanding of the molecular basis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated and was positively correlated with disease course and severity in COVID-19 patients. Correspondingly, SARS-CoV-2 pseudovirus infection increasedCTSLexpression in human cell lines and humanACE2transgenic mice, whileCTSLoverexpression, in turn, enhanced pseudovirus infection. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdownin vitroand application of CTSL inhibitor drugsin vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity and prevented SARS-CoV-2 pseudovirus infection. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

Identification of key regulators of energy homeostasis holds important therapeutic promise for metabolic disorders, such as obesity and diabetes. ACE2 cleaves angiotensin II (Ang II) to generate Ang-(1-7) which acts mainly through the Mas1 receptor. Here, we identify ACE2 pathway as a critical regulator in the maintenance of thermogenesis and energy expenditure. We found that ACE2 is highly expressed in brown adipose tissue (BAT) and that cold stimulation increases ACE2 and Ang-(1-7) levels in BAT and serum. Ace2 knockout mice (Ace2-/y) and Mas1 knockout mice (Mas1-/-) displayed impaired thermogenesis. Mice transplanted with brown adipose tissue from Mas1-/- display metabolic abnormalities consistent with those seen in the Ace2 and Mas1 knockout mice. In contrast, impaired thermogenesis of Leprdb/db obese diabetic mice and high-fat diet-induced obese mice were ameliorated by overexpression of Ace2 or continuous infusion of Ang-(1-7). Activation of ACE2 pathway was associated with improvement of metabolic parameters, including blood glucose, lipids, and energy expenditure in multiple animal models. Consistently, ACE2 pathway remarkably enhanced the browning of white adipose tissue. Mechanistically, we showed that ACE2 pathway activated Akt/FoxO1 and PKA pathway, leading to induction of UCP1 and activation of mitochondrial function. Our data propose that adaptive thermogenesis requires regulation of ACE2 pathway and highlight novel potential therapeutic targets for the treatment of metabolic disorders.

BackgroundPatients with severe acute pancreatitis (SAP) have a high mortality, thus early diagnosis and interventions are critical for improving survival. However, conventional tests are limited in acute pancreatitis (AP) stratification. We aimed to assess AP severity by integrating the informative clinical measurements with cell free DNA (cfDNA) methylation markers.MethodsOne hundred and seventy-five blood samples were collected from 61 AP patients at multiple time points, plus 24 samples from healthy individuals. Genome-wide cfDNA methylation profiles of all samples were characterized with reduced representative bisulfite sequencing. Clinical blood tests covering 93 biomarkers were performed on AP patients within 24 hours. SAP predication models were built based on cfDNA methylation and conventional blood biomarkers separately and in combination. ResultsWe identified 565 and 59 cfDNA methylation markers informative for acute pancreatitis and its severity. These markers were used to develop prediction models for AP and SAP with area under the receiver operating characteristic of 0.92 and 0.81, respectively. Twelve blood biomarkers were systematically screened for a predictor of SAP with a sensitivity of 87.5% for SAP, and a specificity of 100% in mild acute pancreatitis, significantly higher than existing blood tests. An expanded model integrating 12 conventional blood biomarkers with 59 cfDNA methylation markers further improved the SAP prediction sensitivity to 92.2%.ConclusionsWe have demonstrated accurate prediction of SAP by the integration of conventional and novel blood molecular markers, paving the way for early and effective SAP intervention through a non-invasive rapid diagnostic test.

Aims: Urinary haptoglobin (UHp) is a potential biomarker for predicting progress of diabetic kidney disease (DKD) to remedy the defects of currently used urinary albumin. The clinical application of UHp is however limited, owing to the extremely low level in urine. This study aims to establish an enzyme-linked immunosorbent assay (ELISA) kit specifically for detecting UHp in urine samples of patients with diabetes and DKD. Materials and Methods: Supersensitive human haptoglobin antibodies were generated for ELISA kit development, and the sensitivity, specificity and reproducibility of the kit was evaluated. This kit was used to detect UHp in 246 healthy individuals and 83 patients with type 2 diabetes (T2D). The interference of blood haptoglobin genotypes on UHp measurement was analysed. Results: The UHp ELISA kit had a standard curve ranging from 5 to 200 ng/ml. The low detection limit was 0.11 ng/ml. The coefficients of variation of intra- and interassay were 5.5% and 8.3%, respectively. The kit showed high accuracy with 100.9% mean recovery rate, and linearity R2 = 0.999. The reference range of UHp was 0–42.3 ng/g creatinine (0–Q95) in the healthy individuals. UHp level was significantly higher in T2D patients with microalbuminuria and macroalbuminuria than that in T2D without microalbuminuria (p

Aim PAX6 is a transcription factor involved in embryonic development of many organs, including the eyes and the pancreas. Mutations of PAX6 gene is the main cause of a rare disease, congenital aniridia (CA). This case-control study aims to investigate the effects of PAX6 mutations on glucose metabolism and insulin secretion in families with CA. Methods Twenty-one families with CA were screened by Sanger sequencing. Patients with PAX6 mutations and CA (cases) and age-matched healthy family members (controls) were enrolled. Oral glucose tolerance test (OGTT) were performed to detect diabetes or impaired glucose tolerance (IGT). Insulin and proinsulin secretion were evaluated. Results Among 21 CA families, heterozygous PAX6 mutations were detected in five families. Among cases (n=10) from the five families, two were diagnosed with newly identified diabetes and another two were diagnosed with IGT. Among controls (n=12), two had IGT. The level of HbA1c were 36 ± 4 mmol/mol (5.57 ± 0.46 %) and 32 ± 5 mmol/L (5.21 ± 0.54 %) in the cases and the controls, respectively (P = 0.049). More importantly, levels of proinsulin in the cases were significantly higher than that of the controls, despite similar levels of total insulin. The areas under the curve (AUCs) of proinsulin in the cases (6425 ± 4390) were significantly higher than that of the controls (3709 ± 1769) (P = 0.032). Conclusion PAX6 may participate in the production of proinsulin to insulin and heterozygous PAX6 mutations may be associated with glucose metabolism in CA patients.

Background. The pathogenesis Graves’ Orbitopathy (GO) is not yet fully understood. Here, we conducted a pathway analysis based on genome-wide DNA methylation data of Chinese GO patients to explore GO-related pathways and potential feature genes. Methods. Six GO patients and 6 age-matched control individuals were recruited, and a genome-scale screen of DNA methylation was measured using their peripheral blood sample. After extracting the differentially methylated regions (DMRs), we classified DMRs into three clusters with respect to median absolute deviation (MAD) for GO and control group, respectively. Then the extract tests were performed to identify significant pathways by comparing the counts of genes in each cluster between GO and control group in a pathway. For each significant pathway, we calculated the Methylation-based Inference of Regulatory Activity (MIRA) score to infer the regulatory activity of genes involved in the pathway. Furthermore, we took the significant pathways as the subsets and applied Random forests (RF) method to extract GO-related feature genes. Results. We identified four potential significant pathways associated with the occurrence and development of GO disease. There were Toxoplasmosis, Axon guidance, Focal adhesion, and Proteoglycans in cancer (p Conclusion. Our study suggested pathway analyses can help understand the potential relationships between the DNA methylation level of some certain genes and their regulation in Chinese GO patients.

Regulatory Factor X-box binding transcriptional factor 6 (Rfx6) plays an important role in the differentiation and development of pancreas in mammals. However, the direct target genes of Rfx6 to regulate this process were largely unknown. The present study aimed to investigate the function of Rfx6 on regulating pancreatic differentiation and development in a physiologically-relevant context. We performed the chromatin immunoprecipitation followed by the next generation sequencing analysis (ChIP-seq) using whole pancreatic tissue harvested from C57/BL6 adult mice to find target genes of Rfx6. We captured 4146 unique peaks in the genome region of the adult murine pancreas. Among all these binding peaks, a majority were located in intron or intergenic regions. We further annotated all peaks to their nearest gene, and over 1000 genes were captured as Rfx6-binding genes in the pancreas. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis found that Rfx6-binding genes to be associated with the pancreas developmental process. A portion of selected ChIP-seq targets related with pancreas differentiation including Pdx1, Neurod1, Hnf1a, Nkx6-1, St18 and Shox2 were selected and validated as true targets by independent qPCR experiments. In addition, Rfx6 can directly bind to upstream of MiR-145, MiR-195, and possibly other non-protein-coding functional RNAs to control adult mouse pancreatic differentiation. Interestingly, our study revealed that Rfx6 played an important role in insulin translation by binding to the Eif2ak1, Upf1, and Eif5. Our data provide direct target genes of Rfx6 during pancreas development and point to Rfx6 as a potential therapeutic target for improving insulin protein content.

Inflammation constitutes an important component of non-alcoholic fatty liver disease. STAT3 is a direct target of inflammatory cytokines, but also mediates glycolipid metabolism in the liver. As a potent inhibitor of STAT3, the effect of Nifuroxazide (Nifu) on glycolipid metabolism in liver has not been reported. In this study, we used palmitic acid (PA)-induced HepG2 cells to examine the expression of inflammatory factors and apoptosis-related proteins and the content of triglyceride (TG), total cholesterol (TC), and glycogen. The expression of hepatic lipogenic proteins (ACCα, SREBP-1c, FAS), gluconeogenesis enzymes (PEPCK, G6Pase, and IRS2), the IL-6/STAT3/SOCS3 inflammatory axis, and the insulin signaling pathway was determined. Our study shows that Nifu significantly improves lipid metabolism disorders in the PA-induced HepG2 cells, whereas, it remarkably reduced intracellular free fatty acid (FFA), TG, and TC content, suppressed lipid synthesis, and increased lipid decomposition. Our results also showed that Nifu significantly improved dysregulated glucose metabolism in the PA-treated HepG2 cells, increased glycogen content, and inhibited gluconeogenesis. Further research indicated that Nifu markedly inhibited activation of the IL-6/STAT3/SOCS3 signaling pathway. Finally, due to anti-inflammatory stress, Nifu enhanced insulin signaling in the PA-induced HepG2 cells. Therefore, Nifu can improve glucose and lipid metabolism in the PA-induced HepG2 cells, which provides new evidence that Nifu has a positive effect on PA-induced cellular hepatic steatosis and improves glucose metabolism in HepG2 cells, providing a new perspective for studying drug treatment of glucose and lipid metabolism disorders.

Mitochondrial metabolism plays an essential role in the regulation of insulin release and glucose homeostasis. Evidence demonstrated that the angiotensin-converting enzyme 2 (ACE2) participates in the regulation of glucose metabolism, however, its role in mitochondrial metabolism remains unclear. The purpose of our study was to determine if ACE2 can regulate mitochondrial function in pancreatic β-cells. We found that ACE2 over-expression restored glucose-stimulated insulin secretion (GSIS) and mitochondrial membrane potential (MMP) in the presence of H2O2 in INS-1 cells. PCR array demonstrated that ACE2 over-expression up-regulated 67 mitochondria-related genes in INS-1 cells. In pancreatic islets, ACE2 ablation attenuated intracellular calcium influx with a decrease in GSIS. Ace2−/y mice islets exhibited impaired mitochondrial respiration and lower production of ATP, along with decreased expression of genes involved in mitochondrial oxidation. In islets from db/db mice, ACE2 over-expression increased intracellular calcium influx and restored impaired mitochondrial oxidation, potentially causing an increase in GSIS. These results shed light on the potential roles of ACE2 in mitochondrial metabolism, moreover, may improve our understanding of diabetes.

To further investigate the application of positron emission tomography (PET) technology in acupuncture research field, with "PET" and "acupuncture" as keywords, the related literature published from 1997 to 2018 was searched in PubMed, CNKI and WANFANG database; then the literature was classified and analyzed. The results showed that in clinical and experimental studies, whether in physiological or pathological conditions, PET technology has verified the specificity of acupoints, bidirectional regulation of acupoints, and the clinical effect of

AimsBicarbonate is involved in many human essential metabolic processes, but little is known about the association between serum bicarbonate and glucose metabolism. This study aims to investigate the association between serum bicarbonate and the risk of progressing to impaired fasting glucose (IFG)/diabetes mellitus (DM).SettingThe data were obtained from a large-scale prospective cohort study in a single health centre in Beijing.ParticipantsA total of 5318 participants aged 18–70 years who underwent health examinations annually with baseline fasting plasma glucose (FPG) ranging from 3.9 to 5.5 mmol/L, without a history of either diabetes or concomitant chronic diseases, were enrolled in this 6-year observational study.Primary outcome measuresA logistic regression analysis was used to calculate ORs for progressing to IFG/DM by the category of baseline serum bicarbonate. In addition, an analysis of the receiver operating characteristic (ROC) curve for predicting IFG was performed.ResultsOf the 5318 participants, 210 developed IFG after a median 2.2 years of follow-up. After adjusting for sex, age, FPG, body mass index, systolic blood pressure, serum creatinine, serum alanine aminotransferase and low-density lipoprotein cholesterol at baseline, the participants in the first (OR 4.18, 95% CI 2.42 to 7.21; pConclusionsLower serum bicarbonate is associated with higher risk of the development of IFG/DM.

To observe the effect of twirling-reducing needling on changes of profile of differentially-expressed genes in the hypothalamus in rats with stress-induced prehypertension (SI-PHT), so as to explore its mechanism under-lying improvement of prehypertension.Wistar male rats were randomly divided into blank control group (After modeling, the systolic pressure levels were remarkably increased from the 3Twirling-reducing needling of acupuncture can lower blood pressure in prehypertension rats, which may be related to its effects in regulating expression of Acsm 3, Cgα, CryαB, Tshβ genes and in activating AITD pathway.

Summary: Glucose-stimulated insulin secretion from islet β cells is mediated by KATP channels. However, the role of non-KATP K+ channels in insulin secretion is largely unknown. Here, we show that a non-KATP K+ channel, KCNH6, plays a key role in insulin secretion and glucose hemostasis in humans and mice. KCNH6 p.P235L heterozygous mutation co-separated with diabetes in a four-generation pedigree. Kcnh6 knockout (KO) or Kcnh6 p.P235L knockin (KI) mice had a phenotype characterized by changing from hypoglycemia with hyperinsulinemia to hyperglycemia with insulin deficiency. Islets from the young KO mice had increased intracellular calcium concentration and increased insulin secretion. However, islets from the adult KO mice not only had increased intracellular calcium levels but also had remarkable ER stress and apoptosis, associated with loss of β cell mass and decreased insulin secretion. Therefore, dysfunction of KCNH6 causes overstimulation of insulin secretion in the short term and β cell failure in the long term. : Yang et al. show that KCNH6 plays a key role in insulin secretion and glucose hemostasis in humans and mice. Dysfunction of KCNH6 results in a hyperinsulinemia phenotype in the short term and hypoinsulinemia and diabetes in the long term. Keywords: maturity-onset diabetes of the young, MODY, monogenic diabetes, type 2 diabetes, islet, insulin secretion, KCNH6, KCNH2, KATP, Cas9

Blocking the renin-angiotensin system (RAS) can reduce the risk of diabetes. Meanwhile, the angiotensin (Ang)-converting enzyme-2 (ACE2)/Ang-(1-7)/Mas axis has recently been proposed to function as a negative regulator of the RAS. In previous studies, we first demonstrated that ACE2 knockout (ACE2(-/)(y)) mice exhibit impaired glucose tolerance or diabetes. However the precise roles of ACE2 on glucose metabolism are unknown. Here we show that the ACE2/Ang-(1-7)/Mas axis can ameliorate insulin resistance in the liver. Activation of the ACE2/Ang-(1-7)/Mas axis increases glucose uptake and decreases glycogen synthesis in the liver accompanied by increased expression of glucose transporters, insulin receptor substrates and decreased expression of enzymes for glycogen synthesis. ACE2 knockout mice displayed elevated levels of oxidative stress and exposure to Ang-(1-7) reduced the stress in hepatic cells. As a consequence of anti-oxidative stress, activation of the ACE2/Ang-(1-7)/Mas axis led to improved hepatic insulin resistance through the Akt/PI3K/IRS-1/JNK insulin signaling pathway. This is the first time documented that the ACE2/Ang-(1-7)/Mas axis can ameliorate insulin resistance in the liver. As insulin resistance in the liver is considered to be the primary cause of the development of type 2 diabetes, this axis may serve as a new diabetes target.

OBJECTIVE The predictive value of microalbuminuria (MAU) for kidney damage is limited in type 2 diabetes (T2D). We studied whether a urine proteome specific for sight-threatening proliferative diabetic retinopathy (PDR) is an indicator to predict chronic renal insufficiency (CRI) in patients with T2D. RESEARCH DESIGN AND METHODS A shotgun urine proteomic analysis was performed in patients with MAU and PDR (case subjects) and in patients with MAU and a duration of T2D for >10 years but without any degree of retinopathy (control subjects). In the cohort study, 210 patients with T2D with an estimated glomerular filtration rate (eGFR) ≥80 mL/min/1.73 m2 were followed for a median of 5.3 years. Urine proteins specific for PDR were used for predicting CRI (eGFR RESULTS The top two urine proteins with the highest difference in ratio of case subjects to control subjects were haptoglobin (8.7 times; P < 0.0001) and α-2-macroglobulin (5.7 times; P < 0.0001). In the cohort study, patients with baseline urinary haptoglobin ≥20 ng/min (haptoglobinuria) had a higher incidence of CRI than those without (hazard ratio [95% CI] 3.27 [1.41–7.58]; P = 0.006). The overall CRI rate was 3.2% for patients without haptoglobinuria or MAU, 9.5% for those with MAU, and 13.3% for those with haptoglobinuria. The highest rate for CRI (22.4%) was in patients with both MAU and haptoglobinuria (P < 0.001). CONCLUSIONS Urine haptoglobin, which is specific for PDR, is a novel biomarker and complement to urine albumin for predicting kidney damage in patients with T2D.

Background Previous works indicated that the stress on the endoplasmic reticulum (ER) affected nonalcoholic fatty liver disease (NAFLD). However, there is no clear evident on the effect of the regulation of ER stress by angiotensin-converting enzyme 2 (ACE2) on the prevention of NAFLD. Methods HepG2 cells were treated with thapsigargin (Tg) or palmitic acid (PA). We analysed ACE2 expression using Western-blotting analyses. ER stress-related proteins were detected in ACE2 knockout mice and Ad-ACE2-treated db/db mice by immunofluorescence or Western-blotting analyses. In ACE2-overexpression HepG2 cells, the triglyceride (TG), total cholesterol (TC), and glycogen content were detected by assay kits. Meanwhile, the expression of hepatic lipogenic proteins (ACCα, SREBP-1c, FAS, and LXRα), enzymes for gluconeogenesis (PEPCK, G6Pase, and IRS2), and IKKβ/NFκB/IRS1/Akt pathway were analysed by Western-blotting analyses. Results ACE2 was significantly increased in Tg/PA-induced cultured hepatocytes. Additionally, ACE2 knockout mice displayed elevated levels of ER stress, while Ad-ACE2-treated db/db mice showed reduced ER stress in liver. Furthermore, activation of ACE2 can ameliorate ER stress, accompanied by decreased TG content, increased intracellular glycogen, and downregulated expression of hepatic lipogenic proteins and enzymes for gluconeogenesis in Tg/PA-induced hepatocytes. As a consequence of anti-ER stress, the activation of ACE2 led to improved glucose and lipid metabolism through the IKKβ/NFκB/IRS1/Akt pathway. Conclusions This is the first time documented that ACE2 had a notable alleviating role in ER stress-induced hepatic steatosis and glucose metabolism via the IKKβ/NFκB/IRS1/Akt-mediated pathway. This study may further provide insight into a novel underlying mechanism and a strategy for treating NAFLD.

To study the characteristics of the chest X-ray images in children infected with enterovirus 71.A total of 120 children with enterovirus 71 infection between April, 2010 and July, 2011 were classified into three groups according to the disease condition: mild (31 cases), severe (43 cases) and life-threatening (46 cases). The period from the onset of clinical symptoms to the first chest X-ray imaging examination and the results of the first chest X-ray findings were compared among the three groups.The period from the onset of clinical symptoms to the first chest X-ray imaging examination in the mild, severe and life-threatening groups was 26-48 hrs (median 37 hrs), 10-36 h (median 23 hrs) and 2-36 hrs (median 19 hrs) respectively. Chest X-ray abnormalities were initially observed at 30 hrs after the onset of clinical symptoms in the mild group, at 23 hrs in the severe group and at 2 hrs in the life-threatening group (P0.01). The mild group presented an initial imaging abnormality rate of 5.8%, the severe group 81.3% and the life-threatening group 100%. The life-threatening group showed a significantly higher initial X-ray abnormality rate than the other two groups (P0.01). In terms of chest X-ray performance, the mild group usually presented lung marking thickening or vagueness. Most children in the severe group presented lung effusion and consolidation. Signs of pulmonary edema were found in the life-threatening group, and lesions in the life-threatening group were characterized by wide distribution and many lung lobe involvements.The interval between the onset of clinical symptoms and the initial chest X-ray examination, the period of time of, and the onset of clinical symptoms, at which chest X-ray abnormalities, the abnormality rate and the severity of chest X-ray findings may be paralleled to the clinical situation in children with enterovirus 71 infection.