Search

Amyloid β 1-42 peptide (Aβ 1-42 ) accumulates in Alzheimer's disease (AD) that is toxic to the basal forebrain cholinergic (BFC) neurons in substantia innominata-nucleus basalis magnocellularis complex (SI-NBM). Transient Receptor Potential Ankyrin1 (TRPA1) receptor is present in murine brain, however its role in neurotoxic processes is unclear. We investigated the Aβ 1-42 -induced neurotoxicity in TRPA1 wild-type (TRPA1 +/+ ) and knockout (TRPA1 -/- ) mice. Expression and neuroanatomical localization of TRPA1 receptor were examined using RT qPCR. Cholinergic fibre loss was determined on acetylcholinesterase (AChE) stained brain slices, and choline acetyltransferase (ChAT) immunohistochemistry was used to assess the cholinergic cell loss. Novel object recognition (NOR), radial arm maze (RAM) and Y-maze tests were used to investigate memory loss. Aβ 1-42 -injected WT mice showed marked loss of cholinergic fibres and cell bodies, which was significantly attenuated in TRPA1 -/- animals. According to the NOR and RAM tests, pronounced memory loss was detected in Aβ 1-42 -injected TRPA1 +/+ mice, but not in TRPA1 -/- group. Our findings demonstrate that TRPA1 KO animals show substantially reduced morphological damage and memory loss after Aβ 1-42 injection in the SI-NBM. We conclude that TRPA1 receptors may play an important deteriorating role in the Aβ 1-42 -induced cholinergic neurotoxicity and the consequent memory loss in the murine brain., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Comparatively little attention has been paid to the role of free radical oxidation in acne vulgaris. Here, using the traditional abnormalities cited for acne, the authors address the role of free radical oxidation throughout the pathogenesis by detailing the chemistry that may contribute to clinical changes. To probe the effects of free radical oxidation and test an antioxidant, they conducted a preliminary study of topically applied vitamin E., Methods: Seventeen patients with mild-to-moderate acne vulgaris were evaluated over an eight-week period in two private dermatology practices in this open-label study. All patients enrolled were on the same baseline regimen of salicylic acid and benzoyl peroxide. This regimen was then supplemented with topical vitamin E in sunflower seed oil., Results: At the end of the eight-week period, all patients demonstrated clinical improvement, as indicated by a reduction in the number of lesions and global mean difference. A statistically significant reduction was noted as early as Week 2. Enrolled patients also expressed a positive experience due to good tolerability and easy application., Conclusion: Although the exact pathogenesis of acne vulgaris remains unknown, the presence of excessive reactive oxygen species can be implicated in each of the major abnormalities involved. This presence, along with the positive results of the authors' preliminary study, demonstrates the need for more exploration on the use of topical antioxidants in limiting free radical oxidation in the acne model. This paper is designed to stimulate academic discussion regarding a new way of thinking about the disease state of acne.

In situ sample preparation and preparative overpressured layer chromatography (OPLC) fractionation on a 0.5 mm thick adsorbent layer of chamomile flower methanol extract prepurified by conventional gravitation accelerated column chromatography were applied in searching for bioactive components. Sample cleanup in situ on the adsorbent layer subsequent to sample application was performed using mobile phase flow in the opposite direction (the input and output of the eluent was exchanged). The antibacterial effect of the fractions obtained from the stepwise gradient OPLC separation with the flow in the normal direction was evaluated by direct bioautography against two Gram-negative bacteria: the luminescence gene tagged plant pathogenic Pseudomonas syringae pv. maculicola, and the naturally luminescent marine bacterium Vibrio fischeri. The fractions having strong activity were analyzed by SPME-GC/MS and HPLC/MS/MS. Mainly essential oil components, coumarins, flavonoids, phenolic acids, and fatty acids were tentatively identified in the fractions.

Components of 50% aqueous ethanol chamomile (Matricaria recutica L.) flower extract, previously found antibacterial in a TLC-bioautographic study, were separated and isolated by the use of on-line overpressured layer chromatography (OPLC). This system consisted of an OPLC 50 BS system, an on-line coupled flow-through UV detector, and a manual fraction collector. The collected fractions were investigated by GC-MS analysis and by TLC re-chromatography with subsequent visualization, performed after use of the vanillin-sulphuric acid reagent, or under UV illumination, or applying bioautographic detection. The main compounds of the collected 11 fractions were identified by GC-MS. The results showed that the antibacterial effect of 50% aqueous ethanol extract of chamomile is ascribable to cis-, trans-spiroethers, and the coumarins like herniarin and umbelliferone.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC) are present in sensory neurons and vascular smooth muscle. PACAP infusion was found to trigger migraine-like headache in humans and we showed its central pro-nociceptive function in several mouse pain models. Nitroglycerol (NTG)-induced pathophysiological changes were investigated in this study in PACAP gene-deleted (PACAP(-/-)) and wildtype (PACAP(+/+)) mice. Chemical activation of the trigeminovascular system was induced by 10 mg/kg i.p. NTG. Light-aversive behavior was determined in a light-dark box, meningeal microcirculation by laser Doppler blood perfusion scanning and the early neuronal activation marker c-Fos with immunohistochemistry. NTG-induced photophobia both in the early (0-30 min) and late phases (90-120 min) due to direct vasodilation and trigeminal sensitization, respectively, was significantly reduced in PACAP(-/-) mice. Meningeal blood flow increased by 30-35% during 4 h in PACAP(+/+) mice, but only a 5-10% elevation occurred from the second hour in PACAP(-/-) ones. The number of c-Fos expressing cells referring to neuronal activation in the trigeminal ganglia and nucleus caudalis significantly increased 4h after NTG in PACAP(+/+), but not in PACAP(-/-) animals. Similar PAC1 receptor immunostaining was detected in both groups, which did not change 4 h after NTG treatment. PACAP-38 (300 μg/kg, i.p.) produced photophobia similarly to NTG and 30% meningeal vasodilatation for 30 min in PACAP(+/+), but not in PACAP(-/-) mice. It significantly increased neural activation 4h later in the trigeminal ganglia of both groups, but in the nucleus caudalis of only the PACAP(+/+) mice. We provide the first experimental results that PACAP is a pivotal mediator of trigeminovascular activation/sensitization and meningeal vasodilation related to migraine., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Transgenic mice with a small hairpin RNA construct interfering with the expression of transient receptor potential vanilloid 1 (TRPV1) were created by lentiviral transgenesis. TRPV1 expression level in transgenic mice was reduced to 8% while the expression of ankyrin repeat domain 1 (TRPA1) was unchanged. Ear oedema induced by topical application of TRPV1 agonist capsaicin was completely absent in TRPV1 knockdown mice. Thermoregulatory behaviour in relation to environmental thermopreference (30 vs. 35°C) was slightly impaired in male knockdown mice, but the reduction of TRPV1 function was not associated with enhanced hyperthermia. TRPV1 agonist resiniferatoxin induced hypothermia and tail vasodilatation was markedly inhibited in knockdown mice. In conclusion, shRNA-mediated knock down of the TRPV1 receptor in mice induced robust inhibition of the responses to TRPV1 agonists without altering the expression, gating function or neurogenic oedema provoked by TRPA1 activation. Thermoregulatory behaviour in response to heat was inhibited, but enhanced hyperthermia was not observed.

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in capsaicin-sensitive peptidergic sensory nerves, inflammatory and immune cells suggest its involvement in inflammation. However, data on its role in different inflammatory processes are contradictory and there is little known about its functions in the airways. Therefore, our aim was to examine intranasal endotoxin-induced subacute airway inflammation in PACAP gene-deficient (PACAP⁻/⁻) and wild-type (PACAP⁺/⁺) mice. Airway responsiveness to inhaled carbachol was determined in unrestrained mice with whole body plethysmography 6 h and 24 h after LPS. Myeloperoxidase (MPO) activity referring to the number of accumulated neutrophils and macrophages was measured with spectrophotometry and interleukin-1β (IL-1β) concentration with ELISA from the lung homogenates. Histological evaluation and semiquantitative scoring were also performed. Bronchial responsiveness, as well as IL-1β concentration and MPO activity markedly increased at both timepoints. Perivascular edema dominated the histological picture at 6 h, while remarkable peribronchial granulocyte accumulation, macrophage infiltration and goblet cell hyperplasia were seen at 24h. In PACAP⁻/⁻ mice, airway hyperreactivity was significantly higher 24 h after LPS and inflammatory histopathological changes were more severe at both timepoints. MPO increase was almost double in PACAP⁻/⁻ mice compared to the wild-types at 6 h. In contrast, there was no difference between the IL-1β concentrations of the PACAP⁺/⁺ and PACAP⁻/⁻ mice. These results provide evidence for a protective role for PACAP in endotoxin-induced airway inflammation and hyperreactivity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The transient receptor potential vanilloid 1 (TRPV1) is a noxious heat-sensitive, chemonociceptive cation channel which is expressed in primary sensory neurons of polymodal nociceptors. The present study is devoted to analyse the role of lipid raft constituents in calcium influx evoked by various TRPV1 agonists on sensory neurons and on rTRPV1-transfected CHO cell line. Depletion of cholesterol by methyl beta-cyclodextrin (MCD, 1-10mM) diminished the percent of the calcium uptake response of cultured trigeminal neurons to capsaicin (100nM) or resiniferatoxin (RTX, 3nM). In contrast, in TRPV1-transfected cells the inhibition was observed only when capsaicin or N-oleoyldopamine (OLDA, 10microM) was applied, but not when RTX, anandamide (AEA, 10microM) or pH 5.5 was used for gating. The magnitude of Ca(2+)-transients evoked by capsaicin (330nM) was also inhibited in both cell types. Treatment of rTRPV1-expressing cells with sphinomyelinase inhibited the capsaicin-evoked (45)Ca-uptake leaving the RTX-induced response unchanged. On the other hand, in trigeminal neurons the effect of both compounds was inhibited by sphingomyelinase treatment. Inhibition of ganglioside biosynthesis by d-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP, 10-20microM) or myriocyn (5-50nM) diminished similarly capsaicin- or RTX-evoked calcium uptake in both cultured trigeminal neurons and rTRPV1-expressing cells. The present study revealed that depletion of different constituents of lipid raft inhibited gating the TRPV1 cation channel by various vanilloid and non-vanilloid agents. Evidence for a supporting role of cholesterol, sphingomyelin and gangliosides were obtained both in native and TRPV1-transfected cells. Differential modulation of responses to capsaicin and RTX was often observed.

We have shown that somatostatin released from activated capsaicin-sensitive nociceptive nerve endings during inflammatory processes elicits systemic anti-inflammatory and analgesic effects. With the help of somatostatin receptor subtype 4 gene-deleted mice (sst(4)(-/-)), we provide here several lines of evidence that this receptor has a protective role in a variety of inflammatory disease models; several symptoms are more severe in the sst(4) knockout animals than in their wild-type counterparts. Acute carrageenan-induced paw edema and mechanical hyperalgesia, inflammatory pain in the early phase of adjuvant-evoked chronic arthritis, and oxazolone-induced delayed-type hypersensitivity reaction in the skin are much greater in mice lacking the sst(4) receptor. Airway inflammation and consequent bronchial hyperreactivity elicited by intranasal lipopolysaccharide administration are also markedly enhanced in sst(4) knockouts, including increased perivascular/peribronchial edema, neutrophil/macrophage infiltration, mucus-producing goblet cell hyperplasia, myeloperoxidase activity, and IL-1beta, TNF-alpha, and IFN-gamma expression in the inflamed lung. It is concluded that during these inflammatory conditions the released somatostatin has pronounced counterregulatory effects through sst(4) receptor activation. Thus, this receptor is a promising novel target for developing anti-inflammatory, analgesic, and anti-asthmatic drugs.

Three major flavonoid chamomile components (quercetin, apigenin-7-O-glucoside and rutin) were subjected to oxidative metabolism by cytochrome P-450 of rat liver microsomal preparations. Changes over time in their respective concentrations were followed using reversed-phase HPLC with UV detection. No clean-up had to be applied as only the specific flavonoid had to be separated from the background components originating from the rat liver microsome.Neither the concentration of apigenin-7-O-glucoside nor that of the diglycoside rutin decreased during one hour of exposure to rat microsomal treatment. In contrast, the concentration of quercetin, a lipophilic aglycon, decreased.Our analytical HPLC results complement the in silico calculated lipophilicity (logP) of these compounds; the relatively high lipophilicity of quercetin appears to predispose it to oxidative metabolism in order to decrease its fat solubility. In contrast the much less lipophilic compounds apigenin-7-O-glucoside and rutin were resistant in vitro to microsomal treatment.

The essential oil composition and genetic variability of common sage ( Salvia officinalis L.) and its three ornamental cultivars ('Purpurascens', 'Tricolor', and 'Kew Gold') as well as Judean sage ( Salvia judaica Boiss.) were analyzed by GC-FID, GC-MS, and random amplified polymorphic DNA (RAPD). Common sage and its cultivars contained the same volatile compounds; only the ratio of compounds differed. The main compounds were the sesquiterpene alpha-humulene and the monoterpenes beta-pinene, eucalyptol, and camphor. Judean sage contained mainly the sesquiterpenes beta-cubebene and ledol. All of the samples exhibited characteristic RAPD patterns that allowed their identification. Cluster analyses based on oil composition and RAPD markers corresponded very well to each other, suggesting that there is a strong relationship between the chemical profile and the genetic variability.

To assess whether an increased genetic predisposition for type 2 diabetes mellitus (T2DM) influences the contributions of insulin resistance and impaired insulin secretion to impaired glucose tolerance (IGT), 437 subjects not known to have T2DM underwent an oral glucose tolerance test and a 3-hour hyperglycemic clamp. Plasma insulin responses and insulin sensitivity were compared between all subjects (unselected for demographic or anthropometric characteristics) who had normal glucose homeostasis and no first-degree T2DM relative (n = 133), IGT with a first-degree T2DM relative (IGT/FH+, n = 74), or IGT without a first-degree T2DM relative (IGT/FH-, n = 50). Compared with those with normal glucose homeostasis, first- and second-phase plasma insulin responses were reduced approximately 45% and 30%, respectively (both P < .001), in IGT/FH+, whereas insulin sensitivity was only approximately 20% reduced (P = .011). In contrast, in IGT/FH-, first-phase plasma insulin responses were only approximately 20% reduced (P = .016), second-phase plasma insulin responses were not reduced, but insulin sensitivity was approximately 40% reduced (P < .001). The IGT/FH+ group differed significantly from the IGT/FH- group by having 25% to 30% lower first-phase plasma insulin responses (P = .026) and 25% to 30% greater insulin sensitivity (P = .027). Adjustment for obesity abolished the differences in insulin resistance but not plasma insulin responses. However, when the IGT groups were stratified into subgroups based on body mass index (BMI), first-phase plasma insulin responses were approximately 30% lower in IGT/FH+ with a BMI of at least 27 kg/m(2) (P = .018) but similar in IGT/FH+ with a BMI less than 27 kg/m(2) compared with the corresponding IGT/FH- subgroups. We conclude that, in IGT, an increased genetic predisposition for T2DM increases the contribution of impaired insulin secretion to its pathophysiology. This effect is enhanced by obesity.

Isoflavones are suitable guest molecules for inclusion complex formation with cyclodextrins (CDs). The molecular encapsulation with CDs results in a solid, molecularly dispersed form and in a significantly improved aqueous solubility of isoflavones. Genistein, a key isoflavone constituent of Ononidis spinosae radix was found to form a supramolecular, non-covalent inclusion complex with both beta-cyclodextrin (beta-CD) and gamma-cyclodextrin (gamma-CD), while it did not form a stable complex with alpha-CD. The guest genistein was found to spatially located in the less polar cavity of cyclodextrin. The isolated binary genistein/CD complexes appeared novel crystalline lattices. The in vitro dissolution of genistein entrapped into both beta- and gamma-CD, significantly surpassed that of the plain isoflavone.

N-oleoyldopamine (OLDA) has been identified as an agonist of the transient receptor potential vanilloid type 1 (TRPV1) receptor. A related fatty acid amide, N-oleoylethanolamide (OEA), was found to excite sensory neurons and produce visceral hyperalgesia via activation of the TRPV1 receptor, however, a recent study described this agent as an antinociceptive one. The aim of the present paper was to characterize two newly synthesized derivatives of N-oleoyldopamine, 3-methyl-N-oleoyldopamine (3-MOLDA) and 4-methyl-N-oleoyldopamine (4-MOLDA) as well as OEA with regard to their effects on the TRPV1 receptor. Radioactive 45Ca2+ uptake was measured in HT5-1 cells transfected with the rat TRPV1 receptor and intracellular Ca2+ concentration was monitored by fura-2 microfluorimetry in cultured trigeminal sensory neurons. Thermonociception was assessed by determining the behavioral noxious heat threshold in rats. 3-MOLDA induced 45Ca2+ uptake in a concentration-dependent manner, whereas 4-MOLDA and OEA were without effect. 4-MOLDA and OEA, however, concentration-dependently reduced the 45Ca2+ uptake-inducing effect of capsaicin. In trigeminal sensory neurons, 3-MOLDA caused an increase in intracellular Ca2+ concentration and this effect exhibited tachyphylaxis upon repeated application. Again, 4-MOLDA and OEA failed to alter intracellular Ca2+ levels. Upon intraplantar injection, 3-MOLDA caused an 8-10 degrees C drop of the noxious heat threshold in rats which was inhibited by the TRPV1 receptor antagonist iodo-resiniferatoxin. 4-MOLDA and OEA failed to alter the heat threshold but inhibited the threshold drop induced by the TRPV1 receptor agonist resiniferatoxin. These data show that 3-MOLDA behaves as an agonist, whereas 4-MOLDA and OEA appear to be antagonists, at the rat TRPV1 receptor.

Objective: To examine the effect of aging on insulin secretion (first- and second-phase insulin release) and insulin sensitivity in people with normal glucose tolerance (NGT) or impaired glucose tolerance (IGT)., Research Design and Methods: First- and second-phase insulin secretion and insulin sensitivity were assessed in hyperglycemic clamp experiments in 266 individuals with NGT and 130 individuals with IGT, ranging in age from approximately 20 to approximately 70 years. Changes in beta-cell function were compared using the disposition index to adjust for differences in insulin sensitivity., Results: As expected, both phases of insulin release and insulin sensitivity were reduced in individuals with IGT (all P < 0.01). Insulin sensitivity was not independently correlated with age in either group. In people with NGT, the disposition index for first- and second-phase insulin release decreased similarly at a rate of approximately 0.7% per year. In people with IGT, the disposition indexes for first- and second-phase insulin release decreased at greater rates ( approximately 2.2 and 1.4% per year, P = 0.002 and 0.009, respectively, vs. NGT), with the decrease in first phase being greater than that of second phase (P = 0.025)., Conclusions: Insulin secretion (both first and second phase) normally decreases at a rate of approximately 0.7% per year with aging; this decrease in beta-cell function is accelerated about two-fold in people with impaired glucose tolerance-first phase to a greater extent than second phase. Finally, aging per se has no effect on insulin sensitivity independent of changes in body composition.

In this work the essential oil composition of some less known Dracocephalum species was studied and compared the effectiveness, selectivity and influence of different extraction methods (hydrodistillation, Soxhlet extraction with organic solvents and supercritical fluid extraction) on essential oils. For investigations in Hungary and Transylvania cultivated plant material was used. The analysis of essential oils was carried out by GC and GC-MS methods. The components were identified by standard addition, retention factors and mass spectra. The percentile evaluation of each volatile constituents was made on basis of GC-FID chromatograms. The accuracy of measurements was characterized by relative standard deviation. In the essential oil of D. renati Emb. (studied firstly by us) 18.3% of limonene was measured and carvone, citrals and linalyl acetate monoterpenes, methyl chavicol and some sesquiterpene (e.g. bicyclovetivenol) determined in lower quantities. We established that more than 50% of essential oil of D. grandiflorum L. was formed by sesquiterpenes (beta-caryophyllene and- oxide, beta-bourbonene, beta-cubebene, aromadendrene) and the essential oil of D. ruyschiana L. contained pinocamphone isomers in more than 60%. The oxygenated acyclic monoterpenes, the characteristic constituents of Moldavian dragonhead were present in some tenth percent only in D. renati oil. We found significant differences in the composition of the SFE extract and traditional essential oil of D. moldavica L. The supercritical fractions collected at the beginning of the extraction process were richer in valuable ester component (geranyl acetate) than the essential oil obtained by hydrodistillation. The fractions collected at the end of supercritical were poor in oxygenated monoterpenes but rich in minor compounds of traditional oil, e.g. palmitic acid.

Objective: To compare the pathophysiology of impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) in a more comprehensive and standardized fashion than has hitherto been done., Research Design and Methods: We studied 21 individuals with isolated IFG (IFG/normal glucose tolerance [NGT]), 61 individuals with isolated IGT (normal fasting glucose [NFG]/IGT), and 240 healthy control subjects (NFG/NGT) by hyperglycemic clamps to determine first- and second-phase insulin release and insulin sensitivity. Homeostasis model assessment (HOMA) indexes of beta-cell function (HOMA-%B) and insulin resistance (HOMA-IR) were calculated from fasting plasma insulin and glucose concentrations., Results: Compared with NFG/NGT, IFG/NGT had similar fasting insulin concentrations despite hyperglycemia; therefore, HOMA-IR was increased approximately 30% (P < 0.05), but clamp-determined insulin sensitivity was normal (P > 0.8). HOMA-%B and first-phase insulin responses were reduced approximately 35% (P < 0.002) and approximately 30% (P < 0.02), respectively, but second-phase insulin responses were normal (P > 0.5). NFG/IGT had normal HOMA-IR but approximately 15% decreased clamp-determined insulin sensitivity (P < 0.03). Furthermore, HOMA-%B was normal but both first-phase (P < 0.0003) and second-phase (P < 0.0001) insulin responses were reduced approximately 30%. IFG/NGT differed from NFG/IGT by having approximately 40% lower HOMA-%B (P < 0.012) and approximately 50% greater second-phase insulin responses (P < 0.005)., Conclusions: Since first-phase insulin responses were similarly reduced in IFG/NGT and NFG/IGT, we conclude that IFG is due to impaired basal insulin secretion and preferential resistance of glucose production to suppression by insulin, as reflected by fasting hyperglycemia despite normal plasma insulin concentrations and increased HOMA-IR, whereas IGT mainly results from reduced second-phase insulin release and peripheral insulin resistance, as reflected by reduced clamp-determined insulin sensitivity.

In type 2 diabetes mellitus (T2DM), little is known about hormonal responses to hypoglycemia. In particular, beta-cell responses to hypoglycemia have not been carefully investigated and potentially because of confounding factors or insufficient power, conflicting data have been obtained regarding growth hormone responses. We therefore compared hormonal responses including rates of insulin secretion during a 2-hour hyperinsulinemic hypoglycemic clamp in a relatively large number of nondiabetic (n=21) and moderately insulin-deficient subjects with T2DM (homeostasis model assessment of beta-cell function [HOMA-%B], 751+/-160 vs 1144+/-83 [pmol/L]/[mmol/L], P<.04) (n=14) matched for age, sex, and body mass index. Subjects with T2DM were excluded for antecedent hypoglycemia, and baseline glycemia was controlled by a variable infusion of insulin overnight. Although both groups of subjects had indistinguishable plasma glucose levels at baseline and virtually identical levels of plasma insulin and glucose throughout the hypoglycemic clamp, insulin secretion decreased more slowly in the subjects with T2DM. The time required for insulin secretion to decline to half its baseline level was markedly increased (38.9+/-4.9 vs 22.3+/-1.3 minutes [SD], P<.01), and insulin secretion decreased to a lesser extent (-0.79+/-0.17 vs -1.51+/-0.09 [pmol/L]/kg per minute, P<.002). Moreover, responses of glucagon (28.3+/-7.3 vs 52.8+/-7.0 ng/L, P<.05) and growth hormone (2.9+/-0.8 vs 6.3+/-0.9 ng/mL, P<.04) were reduced in the subjects with T2DM, whereas responses of epinephrine, norepinephrine, and cortisol were similar to those in nondiabetic subjects (all P>0.6). We conclude that multiple defects exist in hormonal responses to hypoglycemia in T2DM with moderate beta-cell failure. These include delayed and reduced decreases in insulin secretion, and impaired increases of plasma glucagon and growth hormone.

To assess mechanisms for postprandial hyperglycemia, we used a triple-isotope technique ([\3-(3)H]glucose and [(14)C]bicarbonate and oral [6,6-dideutero]glucose iv) and indirect calorimetry to compare components of glucose release and pathways for glucose disposal in 26 subjects with type 2 diabetes and 15 age-, weight-, and sex-matched normal volunteers after a standard meal. The results were as follows: 1) diabetic subjects had greater postprandial glucose release (P<0.001) because of both increased endogenous and meal-glucose release; 2) the greater endogenous glucose release (P<0.001) was due to increased gluconeogenesis (P<0.001) and glycogenolysis (P=0.01); 3) overall tissue glucose uptake, glycolysis, and storage were comparable in both groups (P>0.3); 4) glucose clearance (P<0.001) and oxidation (P=0.004) were reduced, whereas nonoxidative glycolysis was increased (P=0.04); and 5) net splanchnic glucose storage was reduced by approximately 45% (P=0.008) because of increased glycogen cycling (P=0.03). Thus in type 2 diabetes, postprandial hyperglycemia is primarily due to increased glucose release; hyperglycemia overcomes the effects of impaired insulin secretion and sensitivity on glucose transport, but intracellular defects persist so that pathways of glucose metabolism are abnormal and glucose is shunted away from normal sites of storage (e.g., liver and muscle) into other tissues.

Severe hypoglycemia, the most serious side effect of sulfonylurea therapy, has been reported to occur more frequently with glyburide than glimepiride. The present studies were undertaken to test the hypothesis that a differential effect on glucagon secretion may be involved. We performed hyperinsulinemic hypoglycemic (approximately 2.5 mmol/L) clamps in 16 healthy volunteers who received in randomized order placebo, glyburide (10 mg), and glimepiride (4 mg) just before beginning the insulin infusion and measured plasma glucagon, insulin, C-peptide, glucagon, epinephrine, cortisol, and growth hormone levels during the clamp and during a 3-hour recovery period after discontinuation of the insulin infusion. Neither sulfonylurea altered glucagon responses or those of other counterregulatory hormones (except cortisol) during the clamp. However, glyburide delayed plasma glucose recovery from hypoglycemia (plasma glucose at end of recovery period: control, 4.9 +/- 0.2 mmol/L; glyburide, 3.7 +/- 0.2 mmol/L; P = .0001; glimepiride, 4.5 +/- 0.2 mmol/L; P = .08). Despite lower plasma glucose levels, glyburide stimulated insulin secretion during this period (0.89 +/- 0.13 vs 1.47 +/- 0.15 pmol x kg(-1) x min(-1), control vs glyburide; P = .001), whereas glimepiride did not (P = .08). Short-term administration of glyburide or glimepiride did not alter glucagon responses during hypoglycemia. In contrast, during recovery from hypoglycemia, glyburide but not glimepiride inappropriately stimulates insulin secretion at low plasma glucose levels. This differential effect on insulin secretion may be an important factor in explaining why glyburide causes severe hypoglycemia more frequently than glimepiride.

A new high-performance liquid chromatographic method is described for tropane alkaloid analysis in genetically transformed root cultures of Datura innoxia Mill. and Atropa belladonna L. Sample preparation, tropane alkaloid extraction with chloroform-methanol-concentrated ammonia 15:5:1 (v/v/v), was followed by solid-phase extraction on Supelclean LC-18 cartridges. Optimized conditions and careful pH control resulted in high recovery and reproducibility. Simultaneous determination of apoatropine, 6beta-hydroxyhyoscyamine, hyoscyamine and scopolamine was performed by HPLC on C18 (2) reversed-phase column. The application of Luna new-generation silica-based stationary phase resulted in excellent peak shapes using an ion-pair reagent and triethanolamine free mobile phase and allowed to exploit the full power of pH-dependent selectivity. Simplicity and improved selectivity make this method a preferred alternative of published ion-pair chromatographic methods. Validation studies proved that the global method has good repeatability and satisfactory recovery. Absolute limits of detection were 0.6, 0.6, and 0.8 ng for hyoscyamine, 6beta-hydroxyhyoscyamine, and scopolamine respectively.

Type 2 diabetes mellitus (T2DM) is a heterogeneous disorder caused by a combination of genetic and acquired abnormalities that affect insulin sensitivity and insulin secretion. Currently available data suggest that insulin resistance is the acquired defect largely secondary to unhealthy lifestyles and that the major genetic factor is impaired insulin secretion. The latter is the result of both reduced beta-cell mass and functional abnormalities makes the beta-cell unable to compensate for increased insulin requirements caused by insulin resistance. Targeting both insulin resistance and impaired insulin secretion is therefore appropriate to prevent T2DM and to improve glycemic control in those with the disorder.

Objective: Animal and in vitro studies indicate that a decrease in beta-cell insulin secretion, and thus a decrease in tonic alpha-cell inhibition by intraislet insulin, may be an important factor for the increase in glucagon secretion during hypoglycemia. However, in humans this role of decreased intraislet insulin is still unclear., Research Design and Methods: We studied glucagon responses to hypoglycemia in 14 nondiabetic subjects on two separate occasions. On both occasions, insulin was infused from 0 to 120 min to induce hypoglycemia. On one occasion, somatostatin was infused from -60 to 60 min to suppress insulin secretion, so that the decrement in intraislet insulin during the final 60 min of hypoglycemia would be reduced. On the other occasion, subjects received an infusion of normal saline instead of the somatostatin., Results: During the 2nd h of the insulin infusion, when somatostatin or saline was no longer being infused, plasma glucose ( approximately 2.6 mmol/l) and insulin levels ( approximately 570 pmol/l) were comparable in both sets of experiments (both P > 0.4). In the saline experiments, insulin secretion remained unchanged from baseline (-90 to -60 min) before insulin infusion and decreased from 1.20 +/- 0.12 to 0.16 +/- 0.04 pmol . kg(-1) . min(-1) during insulin infusion (P < 0.001). However, in the somatostatin experiments, insulin secretion decreased from 1.18 +/- 0.12 pmol . kg(-1) . min(-1) at baseline to 0.25 +/- 0.09 pmol . kg(-1) . min(-1) before insulin infusion so that it did not decrease further during insulin infusion (-0.12 +/- 0.10 pmol . kg(-1) . min(-1), P = 0.26) indicating the complete lack of a decrement in intraislet insulin during hypoglycemia. This was associated with approximately 30% lower plasma glucagon concentrations (109 +/- 7 vs. 136 +/- 9 pg/ml, P < 0.006) and increments in plasma glucagon above baseline (41 +/- 8 vs. 67 +/- 11 pg/ml, P < 0.008) during the last 15 min of the hypoglycemic clamp. In contrast, increases in plasma growth hormone were approximately 70% greater during hypoglycemia after somatostatin infusion (P < 0.007), suggesting that to some extent the increases in plasma glucagon might have reflected a rebound in glucagon secretion., Conclusions: These results provide direct support for the intraislet insulin hypothesis in humans. However, the exact extent to which a decrement in intraislet insulin accounts for the glucagon responses to hypoglycemia remains to be established.