The antiangiogenic effect of total saponins of Panax japonicus C.A. Meyer in rheumatoid arthritis is mediated by targeting the HIF-1α/VEGF/ANG-1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese herbal medicine Panax japonicus C.A. Meyer has a long history in clinical treatment of rheumatoid arthritis (RA). Total saponins of Panax japonicus C.A. Meyer (TSPJs) were extracted from the root of Panax japonicus C.A. Meyer, and its anti-rheumatism mechanism is still unclear. AIM OF THE STUDY: To investigate whether TSPJs attenuated synovial angiogenesis in RA and explore the potential mechanisms. MATERIALS AND METHODS: Potential TSPJs targets involving gene function were predicted by network pharmacology related databases. Bioinformatics analysis and molecular docking technology were used to predict the mechanism of TSPJs in the treatment of RA. The predicted results were validated by cell experiments and a collagen-induced arthritis (CIA) mouse model. RESULTS: Bioinformatics analysis results showed that TSPJs may inhibit RA-related angiogenesis through the hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) pathways. In vitro, different doses of TSPJs showed a good inhibitory effect on the tube formation of EA.hy926 cells. The results of the cellular thermal shift assay indicated that TSPJs can bind to the HIF-1α, VEGFA, and angiopoietin-1 (ANG-1) proteins. In vivo, the administration of TSPJs alleviated the symptoms of CIA mice, including the arthritis index, hind paw thickness, and swollen joint count. The histological results demonstrated that TSPJs inhibited inflammation, angiogenesis, bone damage, and cartilage destruction. Furthermore, TSPJs decreased the number of vessels and the expression level of CD31. The mechanistic results revealed that TSPJs decreased the expression of HIF-1α, VEGFA, and ANG-1 in the serum or synovial tissues of CIA mice. CONCLUSION: These results suggest that TSPJs effectively inhibit angiogenesis in RA, and the mechanism may be related to inhibiting the HIF-1α/VEGF/ANG-1 axis.

A glucose-responsive insulin therapy protects animals against hypoglycemia.

Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.

Early echocardiographic predictors of outcomes in the mouse transverse aortic constriction heart failure model.

INTRODUCTION: Mouse transverse aortic constriction (TAC) is a widely-used model of pressure overload-induced heart failure. An intrinsic limitation of the model is variability in the response to pressure overload even when employing a standard severity of stenosis. Few literature studies have explicitly reported the use of entry criteria or early predictors to mitigate variability and enrich outcomes in this model. METHODS: Eleven-week-old male C57BL/6J mice underwent TAC or sham surgery. Left ventricular (LV) function and dimensions were assessed by M-mode echocardiography at baseline (pre) and 3, 9 and 12weeks post-procedure (end-study). At 24h post-procedure, transverse aortic flow velocities were obtained for estimating trans-TAC pressure gradients. Invasive LV hemodynamic assessments were performed and terminal heart and lung weights obtained at end-study. RESULTS: TAC mice displayed early development of LV hypertrophy and wall thickening followed by the later development of LV chamber dilation, and progressive development of LV systolic and diastolic dysfunction. The use of a pre-defined trans-TAC pressure gradient criterion of 45-60mmHg did not affect end-study organ weight, echocardiographic and invasive hemodynamic outcomes. A post-hoc receiver operator characteristic (ROC) analysis identified early 3week echocardiographic measures of LVmass(echo) and ejection fraction, with threshold changes of ~+30% and -10% normalized to baseline respectively, as good predictors for multiple end-study organ weight, echocardiographic and invasive hemodynamic outcomes. DISCUSSION: This ROC analysis has identified early predictive threshold changes which may serve, alone or in combination, as entry criteria to enrich outcome in this model.

Duodenal-jejunal bypass surgery induces hepatic lipidomic alterations associated with ameliorated hepatic steatosis in mice.

OBJECTIVE: Bariatric surgery induces weight loss and improvement of insulin resistance; one aspect of both is an amelioration of hepatic steatosis. This study was undertaken to assess the changes in the hepatic lipidome after duodenal-jejunal bypass (DJB) surgery. METHODS: A DJB surgical model was developed and characterized in diet-induced obese mice. In comparison with sham-operated mice, an unbiased lipidomic profiling of hepatic lipids was performed together with measurements of gene expression within key pathways of hepatic lipid metabolism. RESULTS: In the liver of DJB mice, a dramatic reduction (by 77%) in hepatic triacylglycerols was observed. Global lipidomic profiling identified marked decreases of triacylglycerols comprised of medium length fatty acids and with low double bond content. Specific diacylglycerol species were also among the most dramatic decreases in hepatic lipids, whereas lysophosphatidic acids and phosphatidic acids were increased. Expression of fatty acid transporter and lipogenic genes was down-regulated. CONCLUSIONS: From in-depth analysis of hepatic lipid composition, specific lipid intermediates were identified that are preferentially changed following DJB surgery. These changes were most likely due to DJB-induced weight loss, and only further studies will be able to distinguish weight loss-dependent from weight loss-independent changes.

Induction of miR-132 and miR-212 Expression by Glucagon-Like Peptide 1 (GLP-1) in Rodent and Human Pancreatic ß-Cells.

Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic ß-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2'-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic ß-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion.

Exploring the site of anorectic action of peripherally administered synthetic melanocortin peptide MT-II in rats.

Melanotan-II (MT-II), a cyclic heptapeptide, is a potent, non-selective melanocortinergic agonist. When administered centrally or systemically, MT-II elicited a profound inhibitory effect on food intake in rodents, presumably via activation of melanocortin-4-receptor (MC4R). In this study, we sought to investigate whether penetration of MT-II and iodo-MT-II into brain parenchyma is required for the anorectic effect following intravenous (IV) administration. Firstly, both MT-II and iodo-MT-II were effective at suppressing appetite in rats following their IV administration. We next surveyed by in vitro autoradiographic studies the distribution of selective (125)I-MT-II binding sites in multiple brain regions including areas important for feeding regulation such as the hypothalamus and caudal brainstem. Upon IV administration of (125)I-MT-II, significant radioactivity could not be detected in various brain regions by autoradiography except for a group of circumventricular organs (CVOs), which are anatomically situated outside the blood-brain barrier (BBB). The most intensely labeled CVOs include the subfornical organ, median eminence, area postrema and choroid plexus, and accumulation of radioactivity at these sites can be blocked by co-injection of excess unlabeled MT-II. Direct measurement of MT-II in the brain and plasma by LC-MS-MS following IV injection confirmed that the degree of MT-II penetration into the brain is negligible. Furthermore, when given peripherally under conditions that suppressed food intake, MT-II did not result in a detectable induction of c-Fos-like immunoreactivity in brain regions where a significantly elevated c-Fos expression was observed following intracerebroventricular injection of this peptide. Our results indicate that MT-II has a very limited brain penetration capability, and its effect on feeding behavior following systemic administration may be mediated by either the brain regions in close proximity to the CVOs or sites outside of the BBB, including CVOs or other peripheral systems.

The role of dipeptidyl peptidase IV in the cleavage of glucagon family peptides: in vivo metabolism of pituitary adenylate cyclase activating polypeptide-(1-38).

Dipeptidyl peptidase IV (DP-IV) is a cell surface serine dipeptidase that is involved in the regulation of the incretin hormones, glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). There is accumulating evidence that other members of the glucagon family of peptides are also endogenous substrates for this enzyme. To identify candidate substrates for DP-IV, a mass spectrometry-based protease assay was developed that measures cleavage efficiencies (kcat/Km) of polypeptides in a mixture, using only a few picomoles of each substrate and physiological amounts of enzyme in a single kinetic experiment. Oxyntomodulin and the growth hormone-(1-43) fragment were identified as new candidate in vivo substrates. Pituitary adenylate cyclase-activating polypeptide-(1-38) (PACAP38), a critical mediator of lipid and carbohydrate metabolism, was also determined to be efficiently processed by DP-IV in vitro. The catabolism of exogenously administered PACAP38 in wild type and DP-IV-deficient C57Bl/6 mice was monitored by tandem mass spectrometry. Animals lacking DP-IV exhibited a significantly slower clearance of the circulating peptide with virtually complete suppression of the inactive DP-IV metabolite, PACAP-(3-38). These in vivo results suggest that DP-IV plays a major role in the degradation of circulating PACAP38.

Potentiation of insulin signaling in tissues of Zucker obese rats after acute and long-term treatment with PPARgamma agonists.

Thiazolidinediones (TZDs), agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), improve insulin sensitivity in vivo, and the mechanism remains largely unknown. In this study, we showed that, in Zucker obese (fa/fa) rats, acute (1-day) treatment with both rosiglitazone (a TZD) and a non-TZD PPARgamma agonist (nTZD) reduced plasma free fatty acid and insulin levels and, concomitantly, potentiated insulin-stimulated Akt phosphorylation at threonine 308 (Akt-pT308) in adipose and muscle tissues. A similar effect on Akt was observed in liver after a 7-day treatment. The increase in Akt-pT308 was correlated with an increase in Akt phosphorylation at serine 473 (Akt-pS473), tyrosine phosphorylation of insulin receptor beta subunit and insulin receptor substrate-1, and serine phosphorylation of glycogen synthase kinase-3alpha/beta. The agonists appeared to potentiate Akt1 phosphorylation in muscle and liver and both Akt1 and Akt2 in adipose. Finally, potentiation of insulin signaling was also observed in isolated adipose tissue ex vivo and differentiated 3T3 L1 adipocytes in vitro, but not in rat primary hepatocytes in vitro. These results suggest that 1) PPARgamma agonists acutely potentiate insulin signaling in adipose and muscle tissues and such regulation may be physiologically relevant to insulin sensitization in vivo; 2) the agonists directly target adipose tissues; and 3) the metabolic and signaling effects of the agonists are mediated by structurally distinct PPARgamma agonists.

2-Arylindoles as gonadotropin releasing hormone (GnRH) antagonists: optimization of the tryptamine side chain.

A series of 2-arylindoles containing novel heteroaromatic substituents on the tryptamine tether, based on compound 1, was prepared and evaluated for their ability to act as gonadotropin releasing hormone (GnRH) antagonists. Successful modifications of 1 included chain length variation (reduction) and replacement of the pyridine with heteroaromatic groups. These alterations culminated in the discovery of compound 27kk which had excellent in vitro potency and oral efficacy in rodents.