TRPV1 Antagonists as Novel Anti-Diabetic Agents: Regulation of Oral Glucose Tolerance and Insulin Secretion Through Reduction of Low-Grade Inflammation?

With a global prevalence among adults over 18 years of age approaching 9%, Type 2 diabetes mellitus (T2DM) has reached pandemic proportions and represents a major unmet medical need. To date, no disease modifying treatment is available for T2DM patients. Accumulating evidence suggest that the sensory nervous system is involved in the progression of T2DM by maintaining low-grade inflammation via the vanilloid (capsaicin) receptor, Transient Receptor Potential Vanilloid-1 (TRPV1). In this study, we tested the hypothesis that TRPV1 is directly involved in glucose homeostasis in rodents. TRPV1 receptor knockout mice (Trpv1-/-) and their wild-type littermates were kept on high-fat diet for 15 weeks. Moreover, Zucker obese rats were given the small molecule TRPV1 antagonist, N-(4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), per os twice-a-day or vehicle for eight days. Oral glucose tolerance and glucose-stimulated insulin secretion was improved by both genetic inactivation (Trpv1-/- mice) and pharmacological blockade (BCTC) of TRPV1. In the obese rat, the improved glucose tolerance was accompanied by a reduction in inflammatory markers in the mesenteric fat, suggesting that blockade of low-grade inflammation contributes to the positive effect of TRPV1 antagonism on glucose metabolism. We propose that TRPV1 could be a promising therapeutic target in T2DM by improving glucose intolerance and correcting dysfunctional insulin secretion.

The sarcolipin-bound calcium pump stabilizes calcium sites exposed to the cytoplasm.

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic Ca(2+), initiated by the release of Ca(2+) from the sarcoplasmic reticulum and terminated by re-sequestration of Ca(2+) into the sarcoplasmic reticulum as the main mechanism of Ca(2+) removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic Ca(2+) enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the Ca(2+)-ATPase in a previously undescribed E1 state, with exposure of the Ca(2+) sites through an open cytoplasmic pathway stabilized by Mg(2+). The structure suggests a mechanism for selective Ca(2+) loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound Ca(2+). These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes.

Antimalarial screening via large-scale purification of Plasmodium falciparum Ca2+-ATPase 6 and in vitro studies.

The most severe form of human malaria is caused by the parasite Plasmodium falciparum. Despite the current need, there is no effective vaccine and parasites are becoming resistant to most of the antimalarials available. Therefore, there is an urgent need to discover new drugs from targets that have not yet suffered from drug pressure with the aim of overcoming the problem of new emerging resistance. Membrane transporters, such as P. falciparum Ca(2+)-ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA), have been proposed as potentially good antimalarial targets. The present investigation focuses on: (a) the large-scale purification of PfATP6 for maintenance of its enzymatic activity; (b) screening for PfATP6 inhibitors from a compound library; and (c) the selection of the best inhibitors for further tests on P. falciparum growth in vitro. We managed to heterologously express in yeast and purify an active form of PfATP6 as previously described, although in larger amounts. In addition to some classical SERCA inhibitors, a chemical library of 1680 molecules was screened. From these, we selected a pool of the 20 most potent inhibitors of PfATP6, presenting half maximal inhibitory concentration values in the range 1-9 µm. From these, eight were chosen for evaluation of their effect on P. falciparum growth in vitro, and the best compound presented a half maximal inhibitory concentration of ~ 2 µm. We verified the absence of an inhibitory effect of most of the compounds on mammalian SERCA1a, representing a potential advantage in terms of human toxicity. The present study describes a multidisciplinary approach allowing the selection of promising PfATP6-specific inhibitors with good antimalarial activity.

Beta-cell selective K(ATP)-channel activation protects beta-cells and human islets from human islet amyloid polypeptide induced toxicity.

BACKGROUND AND AIMS: In type 2 diabetes mellitus (T2DM) chronic beta-cell stimulation and oligomers of aggregating human islet amyloid polypeptide (h-IAPP) cause beta-cell dysfunction and induce beta-cell apoptosis. Therefore we asked whether beta-cell rest prevents h-IAPP induced beta-cell apoptosis. MATERIALS AND METHODS: We induced beta-cell rest with a beta-cell selective K(ATP)-channel opener (K(ATP)CO) in RIN cells and human islets exposed to h-IAPP versus r-IAPP. Apoptosis was quantified by time-lapse video microscopy (TLVM) in RIN cells and TUNEL staining in human islets. Whole islets were also studied with TLVM over 48h to examine islet architecture. RESULTS: In RIN cells and human islets h-IAPP induced apoptosis (p<0.001 h-IAPP versus r-IAPP). Concomitant incubation with K(ATP)CO inhibited apoptosis (p<0.001). K(ATP)CO also reduced h-IAPP induced expansion of whole islets (disintegration of islet architecture) by ~70% (p<0.05). Thioflavin-binding assays show that K(ATP)CO does not directly inhibit amyloid formation. CONCLUSIONS: Opening of K(ATP)-channels reduces beta-cell vulnerability to apoptosis induced by h-IAPP oligomers. This effect is not due to a direct interaction of K(ATP)CO with h-IAPP, but might be mediated through hyperpolarization of the beta-cell membrane induced by opening of K(ATP)-channels. Induction of beta-cell rest with beta-cell selective K(ATP)-channel openers may provide a strategy to protect beta-cells from h-IAPP induced apoptosis and to prevent beta-cell deficiency in T2DM.

The actions of a novel potent islet beta-cell specific ATP-sensitive K+ channel opener can be modulated by syntaxin-1A acting on sulfonylurea receptor 1.

Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K(ATP) channel openers.

Novel and established potassium channel openers stimulate hair growth in vitro: implications for their modes of action in hair follicles.

Although ATP-sensitive potassium (K(ATP)) channel openers, e.g., minoxidil and diazoxide, can induce hair growth, their mechanisms require clarification. Improved drugs are needed clinically. but the absence of a good bioassay hampers research. K(ATP) channels from various tissues contain subtypes of the regulatory sulfonylurea receptor, SUR, and pore-forming, K(+) inward rectifier subunits, Kir6.X, giving differing sensitivities to regulators. Therefore, the in vitro effects of established potassium channel openers and inhibitors (tolbutamide and glibenclamide), plus a novel, selective Kir6.2/SUR1 opener, NNC 55-0118, were assessed on deer hair follicle growth in serum-free median without streptomycin. Minoxidil (0.1-100 microM, p<0.001), NNC 55-0118 (1 mM, p<0.01; 0.1, 10, 100 microM, p<0.001), and diazoxide (10 microM, p<0.01) increased growth. Tolbutamide (1 mM) inhibited growth (p<0.001) and abolished the effect of 10 microM minoxidil, diazoxide and NNC 55-0118; glibenclamide (10 microM) had no effect, but prevented stimulation by 10 microM minoxidil. Phenol red stimulated growth (p<0.001), but channel modulator responses remained unaltered. Thus, deer follicles offer a practical, ethically advantageous in vitro bioassay that reflects clinical responses in vivo. The results indicate direct actions of K(ATP) channel modulators within hair follicles via two types of channels, with SUR 1 and SUR 2, probably SUR2B, sulfonylurea receptors.

H3 domain of syntaxin 1A inhibits KATP channels by its actions on the sulfonylurea receptor 1 nucleotide-binding folds-1 and -2.

The ATP-sensitive potassium (K(ATP)) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the K(ATP) channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K(+) and Ca(2+) channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K(ATP) channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell K(ATP) channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the K(ATP) channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the K(ATP) current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis.

Establishment and application of in vitro membrane potential assays in cell lines with endogenous or recombinant expression of ATP-sensitive potassium channels (Kir6.2/SUR1) using a fluorescent probe kit.

The flow of current through the adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) of the isoform Kir6.2/SUR1 regulates the resting membrane potential in the pancreatic beta-cell. In combination with the cellular glucose metabolism, it is an important minute-to-minute regulator of insulin secretion and whole-body glucose homeostasis. The same K(ATP) isoform is further reported to be present in glucagon-secreting alpha-cells, intestinal L-cells, and glucose-responsive neurons in the hypothalamus. All in all, this makes Kir6.2/SUR1 an interesting drug target. Using a commercially available fluorescent membrane potential probe kit and a conventional 96-well fluorescence plate reader, the authors have developed and established qualitative membrane potential assays used to screen for potassium channel closers (KCCs) and openers (KCOs) in insulin- and glucagon-secreting cell lines as well as in cells with recombinant expression of the human Kir6.2/SUR1 channel complex. Both glucose- and KCC-induced depolarization could be demonstrated. The magnitudes of these responses and KCO-induced repolarization at high glucose displayed some variation between the different cell lines but a similar rank order of test compounds. Some cell types required the presence of a KCC, such as tolbutamide, to display significant effects of KCOs. The authors find that robust and reliable functional in vitro assays compatible with medium-throughput screening and high-throughput screening can be developed as a base for finding new, more potent, and isoform-selective KCCs and KCOs.

Induction of beta-cell rest by a Kir6.2/SUR1-selective K(ATP)-channel opener preserves beta-cell insulin stores and insulin secretion in human islets cultured at high (11 mM) glucose.

In health, most insulin is secreted in pulses. Type 2 diabetes mellitus (TTDM) is characterized by impaired pulsatile insulin secretion with a defect in insulin pulse mass. It has been suggested that this defect is partly due to chronic overstimulation of beta-cells imposed by insulin resistance and hyperglycemia, which results in depletion of pancreatic insulin stores. It has been reported that in TTDM overnight inhibition of insulin secretion (induction of beta-cell rest) leads to quantitative normalization of pulsatile insulin secretion upon subsequent stimulation. Recently, decreased orderliness of insulin secretion has been recognized as another attribute of impaired insulin secretion in TTDM. In the current studies we sought to address at the level of the isolated islet whether chronic elevated glucose concentrations induce both defects involved in impaired insulin secretion in TTDM: deficiency and decreased orderliness of insulin secretion. We use the concept of beta-cell rest, induced by a novel beta-cell selective K(ATP)-channel opener (KCO), NN414 (6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide), to test whether preservation of insulin stores leads to normalization of both processes in response to glucose stimulation. Human islets were isolated from three cadaveric organ donors and studied in perifusion experiments and static incubation. Acute activation of K(ATP)-channels suppressed insulin secretion from perifused human islets by approximately 90% (P < 0.0001). This KCO also inhibited glucagon secretion in a dose-dependent manner (P = 0.01). Static incubation at 11 and 16 vs. 4 mM glucose for 96 h decreased islet insulin stores by approximately 80% and 85% (P < 0.0001, respectively). In subsequent perifusion experiments, total insulin secretion ( approximately 30%; P < 0.01) from these islets and insulin pulse mass ( approximately 40%; P < 0.05) were both decreased (11 vs. 4 mM). The inhibition of insulin secretion during static incubation with KCO reduced the loss of islet insulin stores in a dose-dependent manner (P < 0.0001) and resulted in increased total insulin secretion (2.6-fold; P < 0.01) and insulin pulse mass (2.5-fold; P < 0.05) during subsequent perifusion. The orderliness of insulin secretion was significantly reduced after chronic incubation of human islets at 11 mM glucose (P = 0.04), but induction of beta-cell rest at 11 mM failed to normalize the regularity of insulin secretion during subsequent perifusion. We conclude that physiological increased glucose concentrations (11 mM), which are frequently observed in diabetes, lead to a loss of islet insulin stores and defective pulsatile insulin secretion as well as reduced orderliness of insulin secretion. Induction of beta-cell rest by selective activation of beta-cell K(ATP)-channels preserves insulin stores and pulsatile insulin secretion without restoring the orderliness of insulin secretion. Therefore, the concept of beta-cell rest may provide a strategy to protect beta-cells from chronic overstimulation and to improve islet function. Impaired glucose-regulated insulin secretion in TTDM may, however, partially involve mechanisms that are distinct from insulin stores and insulin secretion rates.

NN414, a SUR1/Kir6.2-selective potassium channel opener, reduces blood glucose and improves glucose tolerance in the VDF Zucker rat.

A novel potassium channel opener compound, NN414, selective for the SUR1/Kir6.2 subtype of the ATP-sensitive potassium channel, was used to examine the effect of reducing beta-cell workload in the male Vancouver diabetic fatty (VDF) Zucker rat model of mild type 2 diabetes. Two chronic dosing protocols of NN414 of 3 weeks' duration were compared with appropriate vehicle-treated controls. In the first group, rats received NN414 (continued group; 1.5 mg/kg p.o. twice daily), during which an oral glucose tolerance test (OGTT) (on day 19 of dosing) was performed and insulin secretion from an in situ perfused pancreas preparation (on day 21) was measured. The second group received NN414 (discontinued group; same dose), but active treatment was replaced by vehicle treatment 2 days before the OGTT and for a further 2 days before the perfused pancreas study. Basal glucose was significantly reduced by NN414, with the fall averaging 0.64 mmol/l after 3 weeks of treatment (P < 0.0001). The glucose excursion and hyperinsulinemia during the OGTT were significantly different between the continued, discontinued, and vehicle groups (glucose area under the curve [AUC]: 640 +/- 29, 740 +/- 27, and 954 +/- 82 mmol. l(-1). min(-1), respectively, P < 0.0001; insulin AUC: 38.9 +/- 4.2, 44.2 +/- 4.2, and 55.1 +/- 2.6 nmol.l(-1).min(-1), respectively, P < 0.0001). Hyperinsulinemia during the pancreas perfusion with 4.4 mmol/l glucose was significantly reduced in both treatment groups versus vehicle (P < 0.0005). Insulin secretory responsiveness to a step increase in glucose from 4.4 to 16.6 mmol/l, calculated relative to basal, was significantly improved in the continued group versus vehicle (P < 0.01). In conclusion, administration of NN414 for 3 weeks in VDF rats reduces basal hyperglycemia, improves glucose tolerance, and reduces hyperinsulinemia during an OGTT and improves insulin secretory responsiveness ex vivo. NN414 may therefore represent a novel approach to the prevention and treatment of impaired glucose tolerance and type 2 diabetes.

Effect of factor X inhibition on coagulation activation and cytokine induction in human systemic inflammation.

Anticoagulants have gained increasing attention in the treatment of sepsis. This study used danaparoid to investigate the role of factor Xa in endotoxin-induced coagulation and inflammation and its effectiveness when coagulation activation has already occurred. Thirty healthy volunteers were enrolled in the randomized, placebo-controlled trial. Subjects received 2 ng/kg endotoxin and danaparoid 10 min or 3 h thereafter or placebo. Endotoxin increased prothrombin fragment 1+2 (F(1+2)) levels from 0.5 to 7.0 nmol/L at 5 h in the placebo group. Early danaparoid infusion inhibited endotoxin-induced thrombin formation: maximum F(1+2) levels reached only 1.8 nmol/L (P<.01, vs. baseline or placebo). Delayed danaparoid infusion effectively blocked further thrombin formation. However, danaparoid did not alter endotoxin-induced changes in the fibrinolytic system, cytokine levels, activation of leukocytes, or tissue factor expression on monocytes. Danaparoid therefore selectively attenuates endotoxin-induced coagulopathy, even with delayed administration when coagulation activation is well under way.

Beware the boogie board: blunt abdominal trauma from bodyboarding.

Riding waves on a bodyboard (boogie board) at the beach is popular with children. Three teenagers who sustained blunt abdominal trauma during bodyboarding are described. Two suffered lacerated livers, one a lacerated spleen. Serious blunt abdominal injuries from bodyboarding mishaps have not previously been reported. The usual method of riding a bodyboard may place the rider at risk of abdominal trauma.