Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium.

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa. The structure of 1 was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey's analysis) studies. Pure compound 1 was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small-cell lung cancer cells (IC50 = 1.1 µM). Molecular modeling studies suggested that one possible mechanism of action for 1 is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 µM and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity.

Peptidomic profiling of secreted products from pancreatic islet culture results in a higher yield of full-length peptide hormones than found using cell lysis procedures.

Peptide Hormone Acquisition through Smart Sampling Technique-Mass Spectrometry (PHASST-MS) is a peptidomics platform that employs high resolution liquid chromatography-mass spectrometry (LC-MS) techniques to identify peptide hormones secreted from in vitro or ex vivo cultures enriched in endocrine cells. Application of the methodology to the study of murine pancreatic islets has permitted evaluation of the strengths and weaknesses of the approach, as well as comparison of our results with published islet studies that employed traditional cellular lysis procedures. We found that, while our PHASST-MS approach identified fewer peptides in total, we had greater representation of intact peptide hormones. The technique was further refined to improve coverage of hydrophilic as well as hydrophobic peptides and subsequently applied to human pancreatic islet cultures derived from normal donors or donors with type 2 diabetes. Interestingly, in addition to the expected islet hormones, we identified alpha-cell-derived bioactive GLP-1, consistent with recent reports of paracrine effects of this hormone on beta-cell function. We also identified many novel peptides derived from neurohormonal precursors and proteins related to the cell secretory system. Taken together, these results suggest the PHASST-MS strategy of focusing on cellular secreted products rather than the total tissue peptidome may improve the probability of discovering novel bioactive peptides and also has the potential to offer important new insights into the secretion and function of known hormones.

A primary colonic crypt model enriched in enteroendocrine cells facilitates a peptidomic survey of regulated hormone secretion.

To enable the first physiologically relevant peptidomic survey of gastrointestinal tissue, we have developed a primary mouse colonic crypt model enriched for enteroendocrine L-cells. The cells in this model were phenotypically profiled using PCR-based techniques and showed peptide hormone and secretory and processing marker expression at mRNA levels that were increased relative to the parent tissue. Co-localization of glucagon-like peptide-1 and peptide YY, a characteristic feature of L-cells, was demonstrated by double label immunocytochemistry. The L-cells displayed regulated hormone secretion in response to physiological and pharmacological stimuli as measured by immunoassay. Using a high resolution mass spectrometry-based platform, more than 50 endogenous peptides (<16 kDa), including all known major hormones, were identified a priori. The influence of culture conditions on peptide relative abundance and post-translational modification was characterized. The relative abundance of secreted peptides in the presence/absence of the stimulant forskolin was measured by label-free quantification. All peptides exhibiting a statistically significant increase in relative concentration in the culture media were derived from prohormones, consistent with a cAMP-coupled response. The only peptides that exhibited a statistically significant decrease in secretion on forskolin stimulation were derived from annexin A1 and calcyclin. Biophysical interactions between annexin A1 and calcyclin have been reported very recently and may have functional consequences. This work represents the first step in characterizing physiologically relevant peptidomic secretion of gastrointestinally derived primary cells and will aid in elucidating new endocrine function.

Role of glycogen synthase kinase-3 alpha in insulin action in cultured human skeletal muscle cells.

An association between glycogen synthase kinase-3 (GSK3) in skeletal muscle and insulin resistance has been demonstrated in type 2 diabetic patients. In addition, inhibition of GSK3 improves insulin action. The aim of the present study was to elucidate the role of the alpha-isoform of GSK3 in insulin resistance in human skeletal muscle cells from nondiabetic subjects maintained in culture. Transfection of muscle cells with specific antisense oligonucleotides resulted in a 30-50% decrease of GSK3alpha protein expression (P < 0.05). Whereas neither the basal fractional velocity of glycogen synthase (GS FV) (an indicator of the activation state of the enzyme) nor glucose uptake (GU) were altered, reducing GSK3alpha expression resulted in increases in insulin stimulation of both GS FV and GU. GSK3alpha overexpression (60-100% increase over control) did not alter basal GS FV or GU but impaired insulin stimulation of both responses. Knockdown of GSK alpha also led to an increase in insulin receptor substrate-1 protein expression but did not alter insulin stimulation of pS473-Akt phosphorylation. However, GSK3alpha overexpression impaired insulin action on pS473-Akt. In summary, we concluded the following: 1) modulation of GSK3alpha expression has no effect on basal GU and glycogen synthase activities; 2) reduction of GSK3alpha expression results in improvements in insulin action; and 3) elevation of GSK3alpha in human skeletal muscle cells can induce insulin resistance for several responses. We conclude that GSK3alpha is an important regulator of muscle insulin action.

Exenatide (exendin-4) improves insulin sensitivity and {beta}-cell mass in insulin-resistant obese fa/fa Zucker rats independent of glycemia and body weight.

The effects of the incretin mimetic exenatide (exendin-4) on metabolic parameters, insulin sensitivity, and beta-cell mass were examined in nondiabetic, insulin-resistant obese fa/fa Zucker rats. After 6 wk of treatment, ad libitum-fed exenatide-treated (EX) and pair-fed vehicle control (PF) rats had comparable food intake, body weight, hemoglobin A(1c) (HbA(1c)), and fasting plasma concentrations of glucose, insulin, and lipids. Concurrent decreases in food intake and weight gain were observed in EX and PF rats, compared with ad libitum-fed vehicle control (CON) rats (P < 0.001). The increases in HbA(1c) and fasting plasma insulin concentrations that occur during the normal progression of this disease model were significantly reduced in EX and PF rats, compared with CON rats (P < 0.001). The insulin sensitivity index (ISI; glucose infusion rate to plasma insulin concentration) measured during a hyperinsulinemic euglycemic clamp was 224% higher in EX rats than CON rats (P < 0.001) and 61% higher in EX rats than PF rats (P < 0.004). The latter difference was despite comparable HbA(1c), fasting glucose, fasting insulin, total cholesterol, high-density lipoprotein, and daily food consumption between EX and PF animals. In the absence of exenatide, beta-cell mass was hyperbolically related to ISI (beta-cell mass * ISI was constant). Analogous to the disposition index, the beta-cell mass * ISI product was 63% greater in EX than PF rats (P < 0.05). Thus, exenatide increased beta-cell mass to a greater extent than would be expected in animals of comparable insulin resistance, suggesting a direct trophic effect on islet neogenesis in obese fa/fa rats independent of body weight and glycemia.

Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle.

Glycogen synthase kinase (GSK)-3 has been implicated in the regulation of multiple cellular physiological processes in skeletal muscle. Selective cell-permeable reversible inhibitors (INHs) of GSK-3 (CT98014 and CHIR98023 [Chiron, Emeryville, CA] and LiCl) were used to evaluate the role of GSK-3 in controlling glucose metabolism. Acute treatment (30 min) of cultured human skeletal muscle cells with either INH resulted in a dose-dependent activation of glycogen synthase (GS) with a maximally effective concentration of approximately 2 micromol/l. The maximal acute effect of either INH on GS (103 +/- 25% stimulation over basal) was greater than the maximal insulin response (48 +/- 9%, P < 0.05 vs. INH); LiCl was as effective as insulin. The GSK-3 inhibitor effect, like that of insulin, was on the activation state (fractional velocity [FV]) of GS. Cotreatment of muscle cells with submaximal doses of INH and insulin resulted in an additive effect on GS FV (103 +/- 10% stimulation, P < 0.05 vs. either agent alone). Glucose incorporation into glycogen was also acutely stimulated by INH. While prolonged (6-24 h) insulin exposure led to desensitization of GS, INH continued to activate GS FV for at least 24 h. Insulin and LiCl acutely activated glucose uptake, whereas INH stimulation of glucose uptake required more prolonged exposure, starting at 6 h and continuing to 24 h. Chronic (4-day) treatment with INH increased both basal (154 +/- 32% of control) and insulin-stimulated (219 +/- 74%) glucose uptake. Upregulation of uptake activity occurred without any change in total cellular GLUT1 or GLUT4 protein content. Yet the same chronic treatment resulted in a 65 +/- 6% decrease in GSK-3 protein and a parallel decrease (61 +/- 11%) in GSK-3 total activity. Together with the INH-induced increase in insulin-stimulated glucose uptake, there was an approximately 3.5-fold increase (P < 0.05) in insulin receptor substrate (IRS)-1 protein abundance. Despite upregulation of IRS-1, maximal insulin stimulation of Akt phosphorylation was unaltered by INH treatment. The results suggest that selective inhibition of GSK-3 has an impact on both GS and glucose uptake, including effects on insulin action, using mechanisms that differ from and are additive to those of insulin.

Effect of orally administered dipterinyl calcium pentahydrate on oral glucose tolerance in diet-induced obese mice.

Calcium pterins have been shown to be significant immunotherapeutic agents in models of breast cancer, hepatitis B, and tuberculosis (Bacillus Calmette-Guérin mycobacteria). These compunds modulate the immuno-enzyme indoleamine 2,3-dioxygenase (IDO) and the blood levels of several identified inflammatory cytokines. Recent research into the pathology of diabetes implicates inflammatory factors in the progression of the disease, leading the authors to study its possible control by one of the calcium pterins, dipterinyl calcium pentahydrate (DCP). The investigators tested DCP as a novel therapeutic for type 2 diabetes. Female C57BL/6 J mice with diet-induced obesity were fed a high-fat diet and were administered DCP in 0.4% carboxymethylcellulose for 21 days. Blood glucose was followed during the dosing period, and an oral glucose tolerance test (OGTT) was carried out on day 21. Measurements of plasma indoleamine 2,3-dioxygenase metabolites (tryptophan and kynurenine) and certain cytokines and chemokines were also taken. DCP 7 mg/kg/day reduced OGTT area under the curve (OGTT/ AUC) by 50% (P < 0.05). A significant multivariate regression (P = 0.013; R(2) = 0.571) of OGTT/ AUC was derived from DCP dosage and plasma Trp. Elevated plasma Trp concentration, likely from heterogeneity in diet and/or indoleamine 2,3-dioxygenase activity, was found to correlate with higher OGTT/AUC diabetic measures, possibly via inhibition of histamine degradation. In conclusion, an optimum dose of DCP 7 mg/kg/day significantly improved the OGTT diabetic state in these female diet-induced obese mice.

Tissue-specific expression and regulation of GSK-3 in human skeletal muscle and adipose tissue.

Glycogen synthase kinase-3 (GSK-3) is a ubiquitous kinase implicated in both insulin action and adipogenesis. To determine how these multiple roles may relate to insulin resistance, we studied the regulation of GSK-3 protein expression and phosphorylation in skeletal muscle and isolated adipocytes from nonobese healthy control (HC), obese control (OC), and obese type 2 diabetic (OT2D) subjects. At baseline there were no differences in the GSK-3 protein expression in adipocytes. OC subjects underwent a 6-mo caloric restriction resulting in a 7% decrease in body mass index (BMI) and a 21% improvement in insulin-stimulated whole body glucose disposal rate (GDR). GSK-3alpha and GSK-3beta expression decreased in adipocytes (P < 0.05), whereas GSK-3alpha protein expression increased in skeletal muscle (P < 0.05). OT2D subjects were treated with troglitazone or metformin for 3-4 mo. After troglitazone treatment GDR improved (P < 0.05) despite an increase in BMI (P < 0.05), whereas metformin had no significant effect on GDR. There was no significant change in GSK-3 expression in adipocytes following troglitazone, whereas both GSK-3alpha and -beta were decreased in skeletal muscle (P < 0.05). Metformin treatment had no significant impact on GSK-3 protein expression in either adipocytes or skeletal muscle. Neither treatment influenced GSK-3 serine phosphorylation in skeletal muscle or adipocytes. These results suggest that there is tissue specificity for the regulation of GSK-3 in humans. In skeletal muscle GSK-3 plays a role in control of metabolism and insulin action, whereas the function in adipose tissue is less clear.

PYY[3-36] administration decreases the respiratory quotient and reduces adiposity in diet-induced obese mice.

In rodents, weight reduction after peptide YY[3-36] (PYY[3-36]) administration may be due largely to decreased food consumption. Effects on other processes affecting energy balance (energy expenditure, fuel partitioning, gut nutrient uptake) remain poorly understood. We examined whether s.c. infusion of 1 mg/(kg x d) PYY[3-36] (for up to 7 d) increased metabolic rate, fat combustion, and/or fecal energy loss in obese mice fed a high-fat diet. PYY[3-36] transiently reduced food intake (e.g., 25-43% lower at d 2 relative to pretreatment baseline) and decreased body weight (e.g., 9-10% reduction at d 2 vs. baseline) in 3 separate studies. Mass-specific metabolic rate in kJ/(kg x h) in PYY[3-36]-treated mice did not differ from controls. The dark cycle respiratory quotient (RQ) was transiently decreased. On d 2, it was 0.747 +/- 0.008 compared with 0.786 +/- 0.004 for controls (P < 0.001); light cycle RQ was reduced throughout the study in PYY[3-36]-treated mice (0.730 +/- 0.006) compared with controls (0.750 +/- 0.009; P < 0.001). Epididymal fat pad weight in PYY[3-36]-treated mice was approximately 50% lower than in controls (P < 0.01). Fat pad lipolysis ex vivo was not stimulated by PYY[3-36]. PYY[3-36] decreased basal gallbladder emptying in nonobese mice. Fecal energy loss was negligible ( approximately 2% of ingested energy) and did not differ between PYY[3-36]-treated mice and controls. Thus, negative energy balance after PYY[3-36] administration in diet-induced obese mice results from reduced food intake with a relative maintenance of mass-specific energy expenditure. Fat loss and reduced RQ highlight the potential for PYY[3-36] to drive increased mobilization of fat stores to help meet energy requirements in this model.