CytoSIP: an annotated structural atlas for interactions involving cytokines or cytokine receptors.

Therapeutic agents targeting cytokine-cytokine receptor (CK-CKR) interactions lead to the disruption in cellular signaling and are effective in treating many diseases including tumors. However, a lack of universal and quick access to annotated structural surface regions on CK/CKR has limited the progress of a structure-driven approach in developing targeted macromolecular drugs and precision medicine therapeutics. Herein we develop CytoSIP (Single nucleotide polymorphisms (SNPs), Interface, and Phenotype), a rich internet application based on a database of atomic interactions around hotspots in experimentally determined CK/CKR structural complexes. CytoSIP contains: (1) SNPs on CK/CKR; (2) interactions involving CK/CKR domains, including CK/CKR interfaces, oligomeric interfaces, epitopes, or other drug targeting surfaces; and (3) diseases and phenotypes associated with CK/CKR or SNPs. The database framework introduces a unique tri-level SIP data model to bridge genetic variants (atomic level) to disease phenotypes (organism level) using protein structure (complexes) as an underlying framework (molecule level). Customized screening tools are implemented to retrieve relevant CK/CKR subset, which reduces the time and resources needed to interrogate large datasets involving CK/CKR surface hotspots and associated pathologies. CytoSIP portal is publicly accessible at https://CytoSIP.biocloud.top , facilitating the panoramic investigation of the context-dependent crosstalk between CK/CKR and the development of targeted therapeutic agents.

PinMyMetal: A hybrid learning system to accurately model metal binding sites in macromolecules.

Metal ions are vital components in many proteins for the inference and engineering of protein function, with coordination complexity linked to structural (4-residue predominate), catalytic (3-residue predominate), or regulatory (2-residue predominate) roles. Computational tools for modeling metal ions in protein structures, especially for transient, reversible, and concentration-dependent regulatory sites, remain immature. We present PinMyMetal (PMM), a sophisticated hybrid machine learning system for predicting zinc ion localization and environment in macromolecular structures. Compared to other predictors, PMM excels in predicting regulatory sites (median deviation of 0.34 Å), demonstrating superior accuracy in locating catalytic sites (median deviation of 0.27 Å) and structural sites (median deviation of 0.14 Å). PMM assigns a certainty score to each predicted site based on local structural and physicochemical features independent of homolog presence. Interactive validation through our server, CheckMyMetal, expands PMM's scope, enabling it to pinpoint and validates diverse functional zinc sites from different structure sources (predicted structures, cryo-EM and crystallography). This facilitates residue-wise assessment and robust metal binding site design. The lightweight PMM system demands minimal computing resources and is available at https://PMM.biocloud.top. While currently trained on zinc, the PMM workflow can easily adapt to other metals through expanded training data.

Obesity and Overweight Are Independently Associated with Greater Survival in Critically Ill Diabetic Patients: A Retrospective Cohort Study.

BACKGROUND: The relationship between obesity and the outcomes of critically ill diabetic patients is not completely clear. We aimed to assess the effects of obesity and overweight on the outcomes among diabetic patients in the intensive care unit (ICU). METHODS: Critically ill diabetic patients in the ICU were classified into three groups according to their body mass index. The primary outcomes were 30-day and 90-day mortality. ICU and hospital length of stay (LOS) and incidence and duration of mechanical ventilation were also assessed. Cox regression models were developed to evaluate the relationship between obesity and overweight and mortality. RESULTS: A total of 6108 eligible patients were included. The 30-day and 90-day mortality in the normal weight group were approximately 1.8 times and 1.5 times higher than in the obesity group and overweight group, respectively (P < 0.001, respectively). Meanwhile, the ICU (median (IQ): 2.9 (1.7, 5.3) vs. 2.7 (1.6, 4.8) vs. 2.8 (1.8, 5.0)) and hospital (median (IQ): 8.3 (5.4, 14.0) vs. 7.9 (5.1, 13.0) vs. 8.3 (5.3, 13.6)) LOS in the obesity group and overweight group were not longer than in the normal weight group. Compared with normal weight patients, obese patients had significantly higher incidence of mechanical ventilation (58.8% vs. 64.7%, P < 0.001) but no longer ventilation duration (median (IQ): 19.3 (7.0, 73.1) vs. 19.0 (6.0, 93.7), P = 1). Multivariate Cox regression showed that obese and overweight patients had lower 30-day (HR (95% CI): 0.62 (0.51, 0.75); 0.76 (0.62, 0.92), respectively) and 90-day (HR (95% CI): 0.60 (0.51, 0.70); 0.79 (0.67, 0.93), respectively) mortality risks than normal weight patients. CONCLUSIONS: Obesity and overweight were independently associated with greater survival in critically ill diabetic patients, without increasing the ICU and hospital LOS. Large multicenter prospective studies are needed to confirm our findings and the underlying mechanisms warrant further investigation.

Effectiveness and safety of human amnion/chorion membrane therapy for diabetic foot ulcers: An updated meta-analysis of randomized clinical trials.

Human amnion/chorion membrane therapy has shown advantages in the management of diabetic foot ulcers and its effectiveness has been evaluated in the systematic reviews and meta-analyses. However, the number of patients included in the previous literatures was small and the safety profile of human amnion/chorion membrane therapy was not concerned. Therefore, we conducted an updated meta-analysis to better understand the effectiveness and safety of human amnion/chorion membrane therapy for diabetic foot ulcers. The PubMed, Embase, Cochrane Library, and ClinicalTrial.gov databases were searched for any randomized clinical trials comparing human amnion/chorion membrane therapy and standard therapy in the treatment of diabetic foot ulcers. Ulcer healing rate was considered as the primary outcome and the secondary outcomes mainly included mean time to ulcer healing and adverse events. Nine RCTs with 541 patients were included. Compared with merely standard therapy, human amnion/chorion membrane therapy plus standard therapy improved the ulcer healing rates at 6 weeks (RR = 3.50, 95% CI: 2.35-5.21), 12 weeks (RR = 2.09, 95% CI: 1.53-2.85) and 16 weeks (RR = 1.70, 95% CI: 1.25-2.30), and also shortened the healing time (MD = -4.58, 95% CI: -5.70 to -3.46). Meanwhile, no significant difference was observed in the number of patients with adverse events (RR = 0.56, 95% CI: 0.31-1.03) between two groups. This meta-analysis suggests that human amnion/chorion membrane therapy as an adjuvant treatment could promote the healing of diabetic foot ulcers and has a safety profile. More evidence from large high-quality randomized clinical trials with long follow-up duration are in urgent need to further confirm our findings.

The Efficacy and Safety of Acellular Matrix Therapy for Diabetic Foot Ulcers: A Meta-Analysis of Randomized Clinical Trials.

BACKGROUND: Acellular matrix (AM) therapy has shown promise in the treatment of diabetic foot ulcers (DFUs) in several studies. The clinical effects of AM therapy were not well established. Therefore, we conducted a meta-analysis of randomized clinical trials (RCTs) to examine the efficacy and safety of AM therapy for patients with DFUs. METHODS: A literature search of 5 databases was performed to identify RCTs comparing AM therapy to standard therapy (ST) in patients with DFUs. The primary outcome was the complete healing rate and the secondary outcomes mainly included time to complete healing and adverse events. RESULTS: Nine RCTs involving 897 patients were included. Compared with ST group, patients allocated to AM group had a higher complete healing rate both at 12 weeks (risk ratio (RR) = 1.73, 95% confidence interval (CI): 1.31 to 2.30) and 16 weeks (RR = 1.56, 95% CI: 1.28 to 1.91), a shorter time to complete healing (mean difference (MD) = -2.41; 95% CI: -3.49 to -1.32), and fewer adverse events (RR = 0.64, 95% CI: 0.44 to 0.93). CONCLUSION: The present study suggests that AM therapy as an adjuvant treatment could further promote the healing of full-thickness, noninfected, and nonischemia DFUs. AM therapy also has a safety profile. More large well-designed randomized clinical trials with long follow-up duration are needed to further explore the efficacy and safety of AM therapy for DFUs.

Vasodilatory Actions of Glucagon-Like Peptide 1 Are Preserved in Skeletal and Cardiac Muscle Microvasculature but Not in Conduit Artery in Obese Humans With Vascular Insulin Resistance.

OBJECTIVE: Obesity is associated with microvascular insulin resistance, which is characterized by impaired insulin-mediated microvascular recruitment. Glucagon-like peptide 1 (GLP-1) recruits skeletal and cardiac muscle microvasculature, and this action is preserved in insulin-resistant rodents. We aimed to examine whether GLP-1 recruits microvasculature and improves the action of insulin in obese humans. RESEARCH DESIGN AND METHODS: Fifteen obese adults received intravenous infusion of either saline or GLP-1 (1.2 pmol/kg/min) for 150 min with or without a euglycemic insulin clamp (1 mU/kg/min) superimposed over the last 120 min. Skeletal and cardiac muscle microvascular blood volume (MBV), flow velocity and blood flow, brachial artery diameter and blood flow, and pulse wave velocity (PWV) were determined. RESULTS: Insulin failed to change MBV or flow in either skeletal or cardiac muscle, confirming the presence of microvascular insulin resistance. GLP-1 infusion alone increased MBV by ∼30% and ∼40% in skeletal and cardiac muscle, respectively, with no change in flow velocity, leading to a significant increase in microvascular blood flow in both skeletal and cardiac muscle. Superimposition of insulin to GLP-1 infusion did not further increase MBV or flow in either skeletal or cardiac muscle but raised the steady-state glucose infusion rate by ∼20%. Insulin, GLP-1, and GLP-1 + insulin infusion did not alter brachial artery diameter and blood flow or PWV. The vasodilatory actions of GLP-1 are preserved in both skeletal and cardiac muscle microvasculature, which may contribute to improving metabolic insulin responses and cardiovascular outcomes. CONCLUSIONS: In obese humans with microvascular insulin resistance, GLP-1's vasodilatory actions are preserved in both skeletal and cardiac muscle microvasculature, which may contribute to improving metabolic insulin responses and cardiovascular outcomes.

The Protective Effect of Fluorofenidone against Cyclosporine A-Induced Nephrotoxicity.

BACKGROUND/AIMS: Cyclosporine A (CsA) is an immunosuppressant drug that is used during organ transplants. However, its utility is limited by its nephrotoxic potential. This study aimed to investigate whether fluorofenidone (AKF-PD) could provide protection against CsA-induced nephrotoxicity. METHODS: Eighty-five male Sprague-Dawley rats were divided into 5 groups: drug solvent, CsA, CsA with AKF-PD (250, 500 mg/kg/day), and CsA with pirfenidone (PFD, 250 mg/kg/day). Tubulointerstitial injury index, extracellular matrix (ECM) deposition, expression of type I and IV collagen, transforming growth factor (TGF)-ß1, platelet-derived growth factor (PDGF), Fas ligand (FASL), cleaved-caspase-3, cleaved-poly(ADP-ribose) polymerase (PARP)-1, and the number of transferase-mediated nick end-labeling (TUNEL)-positive renal tubule cells were determined. In addition, levels of TGF-ß1, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of annexin V-positive cells were determined in rat proximal tubular epithelial cells (NRK-52E) treated with CsA (20 µmol/L), AKF-PD (400 µg/mL), PFD (400 µg/mL), and GW788388 (5 µmol/L). RESULTS: AKF-PD (250, 500 mg/kg/day) significantly reduced tubulointerstitial injury, ECM deposition, expression of type I and IV collagen, TGF-ß1, PDGF, FASL, cleaved-caspase-3, cleaved-PARP-1, and number of TUNEL-positive renal tubule cells in the CsA-treated kidneys. In addition, AKF-PD (400 µg/mL) significantly decreased TGF-ß1, FASL, cleaved-caspase-3, and PARP-1 expression in NRK-52E cells and further reduced the number of annexin V-positive cells. CONCLUSION: AKF-PD protect kidney from fibrosis and apoptosis in CsA-induced kidney injury.

Intravenous Injection of miR-34a Inhibitor Alleviates Diabetes Mellitus-Induced Vascular Endothelial Dysfunction by Targeting NOTCH1.

BACKGROUND: miR-34a is a multifunctional post-translational modulator, which is involved in several diabetes-related complications. However, miR-34a remains to be fully elucidated in the diabetic endothelium from rats. In this study, the role of miR-34a/NOTCH1 signaling in the progression of hyperglycemia-vascular endothelial dysfunction was investigated. METHODS: In intravenous injection of miR-34a mimics and inhibitors in streptozotocin (STZ)-induced diabetic rats, the biomarkers of endothelial dysfunction was measured. The targeted genes were predicted by a bioinformatics algorithm and confirmed by a dual luciferase reporter assay. The mRNA and protein levels were assayed by qRT-PCR and western blotting, respectively. Immunohistochemical staining was performed to measure NOTCH1 expression in the diabetic endothelium. RESULTS: miR-34a was significantly up-regulated, and NOTCH1 down-regulated, in the thoracic aorta from STZ-induced diabetic rats compared with control group. As compared to model group, the mRNA of NOTCH1 was significantly decreased or increased by miR-34a mimics or inhibitors ex vivo, respectively. Bioinformatics methods further demonstrated that NOTCH1 was a potential target of miR-34a, which was confirmed by dual-luciferase reporter assay. Moreover, both serum ET and NO were significantly increased in diabetic rats as compared to control group. miR-34a inhibitors ex vivo treatment resulted in significant down-regulation ofserum ET and NO levels in diabetic rats as compared to model group. CONCLUSION: These results provide evidence to support the use of miR-34a inhibitors as a therapeutic approach attenuating hyperglycemia-induced vascular endothelial dysfunction.

Direct Activation of Angiotensin II Type 2 Receptors Enhances Muscle Microvascular Perfusion, Oxygenation, and Insulin Delivery in Male Rats.

Angiotensin II receptors regulate muscle microvascular recruitment and the delivery of nutrients, oxygen, and insulin to muscle. Although angiotensin type 1 receptor antagonism increases muscle microvascular perfusion and insulin action, angiotensin type 2 receptor blockade markedly restricts muscle microvascular blood volume and decreases muscle delivery of insulin. To examine the effects of direct type 2 receptor stimulation using Compound 21 (C21) on microvascular perfusion, insulin delivery and action, and tissue oxygenation in muscle, overnight-fasted adult male rats were infused with C21 systemically. C21 potently increased microvascular blood volume without altering microvascular flow velocity or blood pressure, resulting in a net increase in microvascular blood flow in muscle. This was associated with a substantial increase in muscle interstitial oxygen saturation and insulin delivery into the skeletal and cardiac muscle. These effects were neutralized by coinfusion of the type 2 receptor antagonist or nitric oxide synthase inhibitor. Superimposing C21 infusion on insulin infusion increased insulin-mediated whole body glucose disposal by 50%. C21 significantly relaxed the preconstricted distal saphenous artery ex vivo. We have concluded that direct type 2 receptor stimulation markedly increases muscle microvascular perfusion through nitric oxide biosynthesis and enhances insulin delivery and action in muscle. These findings provide a physiologic mechanistic insight into type 2 receptor modulation of insulin action and suggest that type 2 receptor agonists might have therapeutic potential in the management of diabetes and its associated complications.

Efficacy and Safety of Hyperbaric Oxygen Therapy Used in Patients With Diabetic Foot: A Meta-analysis of Randomized Clinical Trials.

PURPOSE: The efficacy and safety profile of hyperbaric oxygen therapy (HBOT) in patients with diabetic foot ulcer have been controversial in recent years. Our meta-analysis was undertaken to evaluate the efficacy and safety profile of HBOT in patients with diabetic foot ulcer. METHODS: We searched the PubMed, Cochrane Library, EMBASE, and Clinical Trials.gov databases for controlled trials. The efficacy end points included the incidence of healed ulcers, major amputations, minor amputations, and reduction in the ulcer wound area. The tolerability end point was the incidence of adverse events. FINDINGS: Nine randomized clinical trials involving 526 patients met the inclusion criteria. No difference was found in the incidence of healed ulcers (risk ratio [RR] = 2.22; 95% CI, 0.87-5.62; P = 0.32; I2 = 81%), minor amputations (RR = 0.95; 95% CI, 0.39-2.29; P = 0.91; I2 = 74%), major amputations (RR = 0.47; 95% CI, 0.17-1.28; P = 0.14; I2 = 61%), and adverse events (RR = 1.00; 95% CI, 0.64-1.56; P = 0.99; I2 = 26%) between the HBOT and standard therapy (ST) groups. HBOT was associated with a greater reduction in the ulcer wound area versus ST (standard mean difference = 1.12; 95% CI, 0.20-2.04; P = 0.04; I2 = 70%). IMPLICATIONS: No differences existed between HBOT and ST with respect to the incidence of healed ulcers, risk of minor or major amputations, and adverse events. HBOT was associated with a greater reduction in the ulcer wound area than ST. HBOT is a clinically meaningful adjuvant therapy for patients with diabetic foot ulcer.

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice.

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150mg/L NaF in drinking water, 5.75g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration.

GPR109A Expression in the Murine Min6 Pancreatic Beta Cell Line, and Its Relation with Glucose Metabolism and Inflammation.

BACKGROUND: Nicotinic acid has been used clinically to manage dyslipidemia for many years, and its receptor, GPR109A, is expressed in various tissues or cells. It is not known if GPR109A is also expressed in pancreatic beta cells or if it takes part in maintaining homeostasis of glucose metabolism. AIMS: In this study, the expression of GPR109A was investigated in the murine Min6 pancreatic beta cell line. The anti-inflammatory role of GPR109A in MIN6 cells was also explored. METHODS: RT-PCR, western blotting, and immunocytochemical staining were used to detect the expression of GPR109A in MIN6 cells. Real-Time RT-PCR was used to investigate GPR109A mRNA levels influenced by IFN-γ and glucose. Cell viability and cytoplasmic nitrite levels were measured colorimetrically. RESULTS: We have identified that MIN6 cell, a mouse pancreatic beta cell line, expresses GPR109A transcripts and protein. GPR109A protein is mainly located in the cell membrane and cytoplasm. GPR109A mRNA increased more than 9-fold in MIN6 cells incubated with IFN-γ. High glucose inhibited GPR109A mRNA expression. Nitric oxide accumulation, induced by IFN-γ/TNF-α, was inhibited by nicotinic acid and 3-hydroxy-butyrate. CONCLUSIONS: Our results suggest that the expression of GPR109A in pancreatic beta cells is not only influenced by inflammation and glucose, but also plays a protective role under inflammatory conditions. Moreover, the MIN6 cell line may serve as a cellular model for further investigation of GPR109A-mediated signal transduction in modulating metabolism and diabetes.

The Protective Mechanism of Fluorofenidone in Renal Interstitial Inflammation and Fibrosis.

BACKGROUND: Deregulated inflammation has been implicated in the development of renal interstitial fibrosis and progressive renal failure. Previous work has established that fluorofenidone, a pyridone agent, attenuates renal fibrosis. However, the mechanism by which fluorofenidone prevents renal fibrosis remains unclear. The aim of this study was to investigate the in vivo effects of fluorofenidone on unilateral ureteral obstruction-induced fibrosis and the involved molecular mechanism in mouse peritoneal macrophages. METHODS: Renal fibrosis was induced in rat by unilateral ureteral obstruction for 3, 7 or 14 days. Ipsilateral kidneys were harvested for morphologic analysis. Leukocyte infiltration was assessed by immunohistochemistry staining. The expression of chemokines (MCP-1, RANTAS, IP-10, MIP-1α and MIP-1ß) and pro-inflammatory cytokines (TNF-α and IL-1ß) was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Mouse peritoneal macrophages and HK-2 cells were incubated with necrotic MES-13 cells or TNF-α in the presence or absence of fluorofenidone. The production of MCP-1 was measured by enzyme-linked immunosorbent assay, and phosphorylation of ERK1/2, p38 and JNK was quantified by Western blot. RESULTS: Fluorofenidone treatment hampered renal pathologic change and interstitial collagen deposition. Leukocyte infiltration and the expression of chemokines (MCP-1, RANTES, IP-10, MIP-1α and MIP-1ß) and pro-inflammatory cytokines (IL-1α) in kidney were significantly reduced by fluorofenidone treatment. Mechanistically, fluorofenidone significantly inhibited TNF-α or necrotic cell-induced activation of MAP kinase pathways in vitro. CONCLUSIONS: Fluorofenidone serves as a novel anti-inflammatory agent that attenuates ureteral obstruction-induced renal interstitial inflammation and fibrosis, possibly through the inhibition of the microtubule-associated protein kinase pathways.

Fluorofenidone attenuates hepatic fibrosis by suppressing the proliferation and activation of hepatic stellate cells.

Fluorofenidone (AKF-PD) is a novel pyridone agent. The purpose of this study is to investigate the inhibitory effects of AKF-PD on liver fibrosis in rats and the involved molecular mechanism related to hepatic stellate cells (HSCs). Rats treated with dimethylnitrosamine or CCl4 were randomly divided into normal, model, AKF-PD treatment, and pirfenidone (PFD) treatment groups. The isolated primary rat HSCs were treated with AKF-PD and PFD respectively. Cell proliferation and cell cycle distribution were analyzed by bromodeoxyuridine and flow cytometry, respectively. The expression of collagen I and α-smooth muscle actin (α-SMA) were determined by Western blot, immunohistochemical staining, and real-time RT-PCR. The expression of cyclin D1, cyclin E, and p27(kip1) and phosphorylation of MEK, ERK, Akt, and 70-kDa ribosomal S6 kinase (p70S6K) were detected by Western blot. AKF-PD significantly inhibited PDGF-BB-induced HSC proliferation and activation by attenuating the expression of collagen I and α-SMA, causing G0/G1 phase cell cycle arrest, reducing expression of cyclin D1 and cyclin E, and promoting expression of p27(kip1). AKF-PD also downregulated PDGF-BB-induced MEK, ERK, Akt, and p70S6K phosphorylation in HSCs. In rat liver fibrosis, AKF-PD alleviated hepatic fibrosis by decreasing necroinflammatory score and semiquantitative score, and reducing expression of collagen I and α-SMA. AKF-PD attenuated the progression of hepatic fibrosis by suppressing HSCs proliferation and activation via the ERK/MAPK and PI3K/Akt signaling pathways. AKF-PD may be used as a potential novel therapeutic agent against liver fibrosis.

Losartan increases muscle insulin delivery and rescues insulin's metabolic action during lipid infusion via microvascular recruitment.

Insulin delivery and transendothelial insulin transport are two discrete steps that limit muscle insulin action. Angiotensin II type 1 receptor (AT1R) blockade recruits microvasculature and increases glucose use in muscle. Increased muscle microvascular perfusion is associated with increased muscle delivery and action of insulin. To examine the effect of acute AT1R blockade on muscle insulin uptake and action, rats were studied after an overnight fast to examine the effects of losartan on muscle insulin uptake (protocol 1), microvascular perfusion (protocol 2), and insulin's microvascular and metabolic actions in the state of insulin resistance (protocol 3). Endothelial cell insulin uptake was assessed, using (125)I-insulin as tracer. Systemic lipid infusion was used to induce insulin resistance. Losartan significantly increased muscle insulin uptake (∼60%, P < 0.03), which was associated with a two- to threefold increase in muscle microvascular blood volume (MBV; P = 0.002) and flow (MBF; P = 0.002). Losartan ± angiotensin II had no effect on insulin internalization in cultured endothelial cells. Lipid infusion abolished insulin-mediated increases in muscle MBV and MBF and lowered insulin-stimulated whole body glucose disposal (P = 0.0001), which were reversed by losartan administration. Inhibition of nitric oxide synthase abolished losartan-induced muscle insulin uptake and reversal of lipid-induced metabolic insulin resistance. We conclude that AT1R blockade increases muscle insulin uptake mainly via microvascular recruitment and rescues insulin's metabolic action in the insulin-resistant state. This may contribute to the clinical findings of decreased cardiovascular events and new onset of diabetes in patients receiving AT1R blockers.

Glucagon-like peptide 1 recruits microvasculature and increases glucose use in muscle via a nitric oxide-dependent mechanism.

Glucagon-like peptide 1 (GLP-1) increases tissue glucose uptake and causes vasodilation independent of insulin. We examined the effect of GLP-1 on muscle microvasculature and glucose uptake. After confirming that GLP-1 potently stimulates nitric oxide (NO) synthase (NOS) phosphorylation in endothelial cells, overnight-fasted adult male rats received continuous GLP-1 infusion (30 pmol/kg/min) for 2 h plus or minus NOS inhibition. Muscle microvascular blood volume (MBV), microvascular blood flow velocity (MFV), and microvascular blood flow (MBF) were determined. Additional rats received GLP-1 or saline for 30 min and muscle insulin clearance/uptake was determined. GLP-1 infusion acutely increased muscle MBV (P < 0.04) within 30 min without altering MFV or femoral blood flow. This effect persisted throughout the 120-min infusion period, leading to a greater than twofold increase in muscle MBF (P < 0.02). These changes were paralleled with increases in plasma NO levels, muscle interstitial oxygen saturation, hind leg glucose extraction, and muscle insulin clearance/uptake. NOS inhibition blocked GLP-1-mediated increases in muscle MBV, glucose disposal, NO production, and muscle insulin clearance/uptake. In conclusion, GLP-1 acutely recruits microvasculature and increases basal glucose uptake in muscle via a NO-dependent mechanism. Thus, GLP-1 may afford potential to improve muscle insulin action by expanding microvascular endothelial surface area.

Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy.

Fluorofenidone (FD) is a novel pyridone agent with significant antifibrotic effects in vitro. The purpose of this study is to investigate the effects of FD on renal interstitial fibrosis in rats with obstructive nephropathy caused by unilateral ureteral obstruction (UUO). With pirfenidone (PD, 500 mg/kg/day) and enalapril (10 mg/kg/day) as the positive treatment controls, the rats in different experimental groups were administered with FD (500 mg/kg/day) from day 4 to day 14 after UUO. The tubulointerstitial injury, interstitial collagen deposition, and expression of type I and type III collagen, transforming growth factor-ß(1) (TGF-ß(1)), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), α-smooth muscle actin (α-SMA), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were assessed. FD treatment significantly attenuated the prominently increased scores of tubulointerstitial injury, interstitial collagen deposition, and protein expression of type I and type III collagen in ureter-obstructed kidneys, respectively. As compared with untreated rats, FD also significantly reduced the expression of α-SMA, TGF-ß(1), CTGF, PDGF, and inhibitor of TIMP-1 in the obstructed kidneys. Fluorofenidone attenuates renal interstitial fibrosis in the rat model of obstructive nephropathy through its regulation on fibrogenic growth factors, tubular cell transdifferentiation, and extracellular matrix.

Resveratrol recruits rat muscle microvasculature via a nitric oxide-dependent mechanism that is blocked by TNFα.

Resveratrol, a polyphenol found in many plants, has antioxidant and anti-inflammatory actions. It also improves endothelial function and may be cardioprotective. Tumor necrosis factor-α (TNFα) causes oxidative stress and microvascular endothelial dysfunction. Whether resveratrol affects microvascular function in vivo and, if so, whether inflammatory cytokines antagonize its microvascular action are not clear. In cultured bovine aortic endothelial cells (BAECs), resveratrol (100 nM) increased the phosphorylation of protein kinase B (Akt), endothelial nitric oxide (NO) synthase (eNOS), and ERK1/2 within 15 min by more than twofold, and this effect lasted for at least 2 h. Treatment of BAECs with TNFα (10 ng/ml) significantly increased the NADPH oxidase activity and the production of hydrogen peroxide and superoxide. Pretreatment of cells with resveratrol (100 nM) prevented each of these. Injection (ip) of resveratrol in rats potently increased muscle microvascular blood volume (MBV; P = 0.007) and flow (MBF; P < 0.02) within 30 min, and this was sustained for at least 2 h. The phosphorylation of Akt in liver or muscle was unchanged. Superimposed systemic infusion of L-NAME (NOS inhibitor) completely abolished resveratrol-induced increases in MBV and MBF. Similarly, systemic infusion of TNFα prevented resveratrol-induced muscle microvascular recruitment. In conclusion, resveratrol activates eNOS and increases muscle microvascular recruitment via an NO-dependent mechanism. Despite the potent antioxidant effect of resveratrol, TNFα at concentrations that block insulin-mediated muscle microvascular recruitment completely neutralized resveratrol's microvascular action. Thus, chronic inflammation, as seen in type 2 diabetes, may limit resveratrol's vasodilatory actions on muscle microvasculature.

Fluorofenidone protects mice from lethal endotoxemia through the inhibition of TNF-alpha and IL-1beta release.

The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release proinflammatory factors like TNF-alpha and IL-1beta. Fluorofenidone (AKF-PD) is a novel pyridone agent, which exerts a strong antifibrotic effect. In this work, we showed that AKF-PD also exert an inhibitory effect on acute systemic inflammatory response. AKF-PD treatment significantly increased survival in animals with established endotoxemia. In addition, AKF-PD treatment significantly reduced circulating levels of TNF-alpha and IL-1beta during endotoxemia. In macrophage cultures, AKF-PD inhibited the release of TNF-alpha and IL-1beta in a dose-dependent manner. In conclusion, these results indicate that AKF-PD inhibits the release of the proinflammatory cytokines (TNF-a and IL-1beta) and improves survival during lethal endotoxemia, which suggest this new pyridone agent can be a novel candidate for therapy of septic shock.

Fluorofenidone attenuates collagen I and transforming growth factor-beta1 expression through a nicotinamide adenine dinucleotide phosphate oxidase-dependent way in NRK-52E cells.

AIM: Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone) is a novel pyridone agent. The aim of the present study is to investigate the effects of fluorofenidone on angiotensin (Ang)II-induced fibrosis and the involved molecular mechanism in rat proximal tubular epithelial cells. METHODS: NRK-52E cells, a rat proximal tubular epithelial cell line, were incubated with medium containing AngII, with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), losartan, fluorofenidone (2, 4 and 8 mmol/L) and pirfenidone (8 mmol/L) for 24 h. Cells in the serum-free medium were controls. The expression of three subunits of NADPH oxidase, including p47phox, Nox-4 and p22phox, were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot. NADPH oxidase activity was measured directly by superoxide dismutase (SOD) inhibitable cytochrome C reduction assay. The generation of reactive oxygen species (ROS) was measured by dichlorofluorescein fluorescence analysis. The mRNA and protein expression of collagen I and transforming growth factor (TGF)-beta1 were determined by real-time RT-PCR and enzyme-linked immunosorbent assay. RESULTS: Fluorofenidone significantly inhibited TGF-beta1 and collagen I expression upregulation induced by AngII or TGF-beta1 respectively. Moreover, fluorofenidone greatly reduced the elevation of expression and activity of NADPH oxidase and inhibited ROS generation induced by AngII in rat proximal tubular epithelial cells. These responses were also attenuated by DPI, losartan, and pirfenidone. CONCLUSION: Fluorofenidone acted as an anti-oxidative and anti-fibrotic agent through the mechanisms of blocking NADPH oxidase-dependent oxidative stress and inhibiting TGF-beta1 expression in rat proximal tubular epithelial cells.