Walter H. Hsu

Ion Channels and Insulin Secretion

We review the role of ion channels in regulating insulin secretion from pancreatic β-cells. By controlling ion permeability, ion channels at the membrane play a major role in regulating both electrical activity and signal transduction in the β-cell. A proximal step in the cascade of events required for stimulus-secretion coupling is the closure of ATP-sensitive K+ channels, resulting in cell depolarization. Of particular relevance is the finding that this channel is directly regulated by a metabolite of glucose, which is the primary insulin secretagogue. In addition, this channel, or a closely associated protein, contains the sulfonylurea-binding site. Another K+ channel, the Ca2+-activated K+ channel, may be involved in cell repolarization to create homeostasis. Voltage-dependent Ca2+ channels are activated by cell depolarization and regulate Ca2+ influx into the cell. By controlling cytosolic free-Ca2+ levels ([Ca2+]i), these channels play an important role in transducing the initial stimulus to the effector systems that modulate insulin secretion. The link between a rise in [Ca2+[(and the terminal event of exocytosis is the least-understood aspect of stimulus-secretion coupling. However, phosphorylation studies have identified substrate proteins that may correspond to those involved in smooth muscle contraction, suggesting an analogy in the processes of stimulus secretion and excitation contraction. The advent of new methodology, particulary the patch-clamp technique, has fostered a more detailed characterization of the β-cell ion channels. Furthermore, biochemical and molecular approaches developed for the structural analysis of ion channels in other tissues can now be applied to the isolation and characterization of the β-cell ion channels. This is of particular significance because there appear to be tissue-specific variations in the different types of ion channels. Given the importance of ion channels in cell physiology, a knowledge of the structure and properties of these channels in the β-cell is required for understanding the abnormalities of insulin secretion that occur in non-insulin-dependent diabetes mellitus. Ultimately, these studies should also provide new therapeutic approaches to the treatment of this disease.

Effect of amitraz and chlordimeform on heart rate and pupil diameter in rats: Mediated by α2-adrenoreceptors

Pupillary and cardiac responses to the insecticide/acaricide amitraz (0.03 to 1.0 mg/kg, iv) and chlordimeform (0.03 to 10.0 mg/kg, iv), as well as the alpha 2-adrenergic agonists clonidine (1 to 30 micrograms/kg, iv) and xylazine (10 to 300 micrograms/kg, iv), were investigated in rats anesthetized with an ether and pentobarbital combination. Amitraz, clonidine, and xylazine caused a dose-dependent mydriasis and bradycardia. The order of potency of the mydriatic and bradycardic effects was: clonidine greater than xylazine greater than amitraz. Chlordimeform did not cause mydriasis or bradycardia at the dosages studied. Amitraz-induced mydriasis and bradycardia were blocked by antagonists with alpha 2-adrenoreceptor blocking activity: yohimbine and phentolamine (2.5 mg/kg each, iv). In contrast, these effects of amitraz were not affected by prazosin (2.5 mg/kg, iv), an alpha 1-adrenoreceptor antagonist. In rats pretreated with reserpine (7.5 mg/kg, sc, 20 hr) and alpha-methyl-p-tyrosine (250 mg/kg, ip, 5 hr) to deplete catecholamine, amitraz (0.03-1.0 mg/kg, iv) produced mydriasis of similar magnitude as in the control animals. However, amitraz did not lower the heart rate in the pretreated animals as it did in the control animals. The results demonstrated that amitraz, a formamidine, induced mydriasis and bradycardia which were not observed with administration of another formamidine, chlordimeform. The data also suggest that amitraz-induced mydriasis is mediated by postsynaptic alpha 2-adrenoreceptors while amitraz-induced bradycardia is mediated by presynaptic alpha 2-adrenoreceptors.

The Impact of Food Viscosity on Eating Rate, Subjective Appetite, Glycemic Response and Gastric Emptying Rate

Understanding the impact of rheological properties of food on postprandial appetite and glycemic response helps to design novel functional products. It has been shown that solid foods have a stronger satiating effect than their liquid equivalent. However, whether a subtle change in viscosity of a semi-solid food would have a similar effect on appetite is unknown. Fifteen healthy males participated in the randomized cross-over study. Each participant consumed a 1690 kJ portion of a standard viscosity (SV) and a high viscosity (HV) semi-solid meal with 1000 mg acetaminophen in two separate sessions. At regular intervals during the three hours following the meal, subjective appetite ratings were measured and blood samples collected. The plasma samples were assayed for insulin, glucose-dependent insulinotropic peptide (GIP), glucose and acetaminophen. After three hours, the participants were provided with an ad libitum pasta meal. Compared with the SV meal, HV was consumed at a slower eating rate (P = 0.020), with postprandial hunger and desire to eat being lower (P = 0.019 and P<0.001 respectively) while fullness was higher (P<0.001). In addition, consuming the HV resulted in lower plasma concentration of GIP (P<0.001), higher plasma concentration of glucose (P<0.001) and delayed gastric emptying as revealed by the acetaminophen absorption test (P<0.001). However, there was no effect of food viscosity on insulin or food intake at the subsequent meal. In conclusion, increasing the viscosity of a semi-solid food modulates glycemic response and suppresses postprandial satiety, although the effect may be short-lived. A slower eating rate and a delayed gastric emptying rate can partly explain for the stronger satiating properties of high viscous semi-solid foods.

Increasing the number of masticatory cycles is associated with reduced appetite and altered postprandial plasma concentrations of gut hormones, insulin and glucose

To determine the influence of masticatory efficiency on postprandial satiety and glycaemic response, twenty-one healthy males were recruited for this randomised cross-over trial. The participants consumed a fixed amount of pizza provided in equal-sized portions by chewing each portion either fifteen or forty times before swallowing. Subjective appetite was measured by appetite questionnaires at regular intervals for 3 h after the meal and plasma samples were collected for the measurement of selected satiety-related hormones, glucose, insulin and glucose-dependent insulinotropic peptide (GIP) concentrations. An ad libitum meal was provided shortly after the last blood sample was drawn and the amount eaten recorded. Compared with fifteen chews, chewing forty times per portion resulted in lower hunger (P= 0·009), preoccupation with food (P= 0·005) and desire to eat (P= 0·002). Meanwhile, plasma concentrations of glucose (P= 0·024), insulin (P< 0·001) and GIP (P< 0·001) were higher following the forty-chews meal. Chewing forty times before swallowing also resulted in a higher plasma cholecystokinin concentration (P= 0·045) and a trend towards a lower ghrelin concentration (P= 0·051). However, food intake at the subsequent test meal did not differ (P= 0·851). The results suggest that a higher number of masticatory cycles before swallowing may provide beneficial effects on satiety and facilitate glucose absorption.

Xylazine-induced delay of small intestinal transit in mice

Subcutaneous injection of xylazine (0.1-3.0 mg/kg) produced a dose-dependent delay of small intestinal transit without affecting gastric emptying in the conscious mice. The xylazine-induced delay of small intestinal transit was antagonized by alpha 2-adrenoceptor antagonists, e.g., yohimbine, piperoxan and tolazoline. The antagonism of xylazine activity by yohimbine was dose-dependent, and the maximal antagonistic effect was seen at 1 mg/kg. Other adrenoceptor antagonists with only alpha 1-blocking activity, e.g., thymoxamine, prazosin and phenoxybenzamine at the doses studied did not reduce the depressant effect of xylazine on small intestinal transit. A beta-adrenergic antagonist, propranolol was not effective in reducing xylazine activity. The opioid antagonist, naloxone did not reduce the effective of xylazine, nor did yohimbine antagonize the morphine-induced delay of small intestinal transit. The xylazine-induced delay of small intestinal transit was not altered by atropine, hexamethonium, haloperidol, methysergide, chlorpheniramine or cimetidine. Pretreatment of mice with reserpine and alpha-methyl-p-tyrosine or 6-hydroxydopamine failed to reduce the intestinal effect of xylazine. These results suggest that xylazine-induced delay of small intestinal transit is mediated by postjunctional alpha 2-adrenoceptors and appears not to involve activation of opioid, cholinergic, dopaminergic, histaminergic, or serotonergic receptors.

TRPM4 impacts on Ca2+ signals during agonist-induced insulin secretion in pancreatic β-cells

TRPM4 is a Ca(2+)-activated non-selective cation (CAN) channel that functions in cell depolarization, which is important for Ca(2+) influx and insulin secretion in pancreatic beta-cells. We investigated TRPM4 expression and function in the beta-cell lines HIT-T15 (hamster), RINm5F (rat), beta-TC3 (mouse), MIN-6 (mouse) and the alpha-cell line INR1G9 (hamster). By RT-PCR, we identified TRPM4 transcripts in alpha- and beta-cells. Patch-clamp recordings with increasing Ca(2+) concentrations resulted in a dose-dependent activation of TRPM4 with the greatest depolarizing currents recorded from hamster-derived cells. Further, Ca(2+) imaging experiments revealed that inhibition of TRPM4 by a dominant-negative effect significantly decreased the magnitude of the Ca(2+) signals generated by agonist stimulation compared to control cells. The decrease in the [Ca(2+)](i) resulted in reduced insulin secretion. Our data suggest that depolarizing currents generated by TRPM4 are an important component in the control of intracellular Ca(2+) signals necessary for insulin secretion and perhaps glucagon from alpha-cells.

Effect of amitraz on heart rate and aortic blood pressure in conscious dogs: Influence of atropine, prazosin, tolazoline, and yohimbine

The effect of amitraz on heart rate (HR) and mean aortic blood pressure (MAP) were studied in five conscious male dogs. An iv injection of amitraz (1 mg/kg) caused a decrease in HR, which was accompanied by sinus arrhythmia for at least 60 min. Administration of amitraz also caused an increase in MAP for 20 min. Atropine sulfate (0.045 mg/kg, iv) increased HR and prevented amitraz-induced bradycardia. In addition, atropine potentiated amitraz-induced hypertension for 45 min. Yohimbine, an alpha 2-adrenoreceptor antagonist, given iv at 0.1 mg/kg, prevented hypertension, bradycardia, and sinus arrhythmia induced by amitraz. Tolazoline, a nonselective alpha-adrenoreceptor antagonist, given iv at 5 mg/kg, reduced the bradycardia and sinus arrhythmia caused by amitraz administration but did not change amitraz-induced hypertension. Tolazoline alone also increased both HR and MAP. Prazosin, an alpha 1-adrenoreceptor antagonist, given iv at 1 mg/kg, did not affect the cardiovascular actions of amitraz. The results suggest that (1) alpha 2-adrenoreceptors mediate amitraz-induced bradycardia and hypertension, and (2) yohimbine, but not atropine, can be used to control the untoward reactions of amitraz.

Characterization of receptors mediating AVP- and OT-induced glucagon release from the rat pancreas

We characterized the receptors that mediate arginine vasopressin (AVP)- and oxytocin (OT)-induced glucagon release by use of a number of antagonists in the perfused rat pancreas and the fluorescence imaging of the receptors. AVP and OT (3 pM-3 nM) increased glucagon release in a concentration-dependent manner. The antagonist with potent V(1b) receptor-blocking activity, CL-4-84 (10 nM), abolished AVP (30 pM)-induced glucagon release but did not alter OT (30 pM)-induced glucagon release. d(CH(2))(5)[Tyr(Me)(2)]AVP (10 nM), a V(1a) receptor antagonist, and L-366,948 (10 nM), a highly specific OT-receptor antagonist, failed to inhibit AVP-induced glucagon release. In contrast, L-366,948 (10 nM) abolished OT (30 pM)-induced glucagon release but did not change the effect of AVP. Fluorescent microscopy of rat pancreatic sections showed that fluorescence-labeled AVP and OT bound to their receptors in the islets of Langerhans and that the bindings were inhibited by 1 microM of Cl-4-84 and L-366,948, respectively. Because AVP and OT at physiological concentrations (3-30 pM) increased glucagon release, we conclude that AVP and OT increase glucagon release under the physiological condition through the activation of V(1b) and OT receptors, respectively.We characterized the receptors that mediate arginine vasopressin (AVP)- and oxytocin (OT)-induced glucagon release by use of a number of antagonists in the perfused rat pancreas and the fluorescence imaging of the receptors. AVP and OT (3 pM-3 nM) increased glucagon release in a concentration-dependent manner. The antagonist with potent V1b receptor-blocking activity, CL-4-84 (10 nM), abolished AVP (30 pM)-induced glucagon release but did not alter OT (30 pM)-induced glucagon release. d(CH2)5[Tyr(Me)2]AVP (10 nM), a V1a receptor antagonist, and L-366,948 (10 nM), a highly specific OT-receptor antagonist, failed to inhibit AVP-induced glucagon release. In contrast, L-366,948 (10 nM) abolished OT (30 pM)-induced glucagon release but did not change the effect of AVP. Fluorescent microscopy of rat pancreatic sections showed that fluorescence-labeled AVP and OT bound to their receptors in the islets of Langerhans and that the bindings were inhibited by 1 μM of Cl-4-84 and L-366,948, respectively. Because AVP and OT at physiological concentrations (3-30 pM) increased glucagon release, we conclude that AVP and OT increase glucagon release under the physiological condition through the activation of V1b and OT receptors, respectively.

Mycoplasma hyopneumoniae Increases Intracellular Calcium Release in Porcine Ciliated Tracheal Cells

ABSTRACT We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 ± 3 nM (n = 217 cells). The [Ca2+]i increased by 250 ± 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 μg/ml), and this increase lasted ∼60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 μg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 μM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 μM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins Gi and Go, increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to Gi or Go, which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.

A Short Antisense Oligonucleotide Ameliorates Symptoms of Severe Mouse Models of Spinal Muscular Atrophy

Recent reports underscore the unparalleled potential of antisense-oligonucleotide (ASO)-based approaches to ameliorate various pathological conditions. However, in vivo studies validating the effectiveness of a short ASO (<10-mer) in the context of a human disease have not been performed. One disease with proven amenability to ASO-based therapy is spinal muscular atrophy (SMA). SMA is a neuromuscular disease caused by loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Correction of aberrant splicing of the remaining paralog, SMN2, can rescue mouse models of SMA. Here, we report the therapeutic efficacy of an 8-mer ASO (3UP8i) in two severe models of SMA. While 3UP8i modestly improved survival and function in the more severe Taiwanese SMA model, it dramatically increased survival, improved neuromuscular junction pathology, and tempered cardiac deficits in a new, less severe model of SMA. Our results expand the repertoire of ASO-based compounds for SMA therapy, and for the first time, demonstrate the in vivo efficacy of a short ASO in the context of a human disease.