Maximal Secretion Rate Research Articles

Brij-58 supplementation enhances menaquinone-7 biosynthesis and secretion in Bacillus natto.

Menaquinone-7 is a form of vitamin K2 that has been shown to have numerous healthy benefits. In this study, several surfactants were investigated to enhance the production of menaquinone-7 in Bacillus natto. Results showed that Brij-58 supplementation influenced the cell membrane via adsorption, and changed the interfacial tension of fermentation broth, while the changes in the state and the composition of the cell membrane enhanced the secretion and biosynthesis of menaquinone-7. The total production and secretion rate of menaquinone-7 increased by 48.0% and 56.2% respectively. During fermentation, the integrity of the cell membrane decreased by 82.9% while the permeability increased by 158% when the maximum secretory rate was reached. Furthermore, Brij-58 supplementation induced the stress response in bacteria, resulting in hyperpolarization of the membrane, and increased membrane ATPase activity. Finally, changes in fatty acid composition increased membrane fluidity by 30.1%. This study provided an effective strategy to enhance menaquinone-7 yield in Bacillus natto and revealed the mechanism of Brij-58 supplementation in menaquinone-7 production. KEY POINTS: • MK-7 yield in Bacillus natto was significantly increased by Brij-58 supplementation. • Brij-58 could be adsorbed on cell surface and change fermentation environment. • Brij-58 supplementation could affect the state and composition of the cell membrane.

Abstract 447: Cholesterol Excretion In Whole-body And Liver-specific ABCG5 ABCG8-deficient Mice

Background: The ABCG5 ABCG8 sterol transporter is the body’s primary defense against the accumulation of dietary sterols. Loss of function mutations in ABCG5 or ABCG8 result in Sitosterolemia, a rare disorder that presents as familial hypercholesterolemia, but is distinguished by recessive genetics and the accumulation of phytosterols in plasma and tissues. Biliary cholesterol is reduced by greater than 80% in ABCG5 ABCG8-deficient mice, but fecal neutral sterols are unaffected, suggesting a G5G8-independent pathway for cholesterol excretion. The objectives of the present study were to determine the ability of whole-body and liver-specific G5G8-deficient mice to maintain fecal neutral sterol output when challenged with cholesterol -enriched diet and determine maximal rates of biliary cholesterol secretion. Methods: Wild-type, whole-body and liver-specific ABCG5 ABCG8-deficient mice were maintained on cereal-based mixed diet, a phytosterol-free purified diet, or a purified diet supplemented with 0.2% cholesterol. Following acclimatization to each diet, fecal neutral sterol output was determined over a three-day period by gas chromatography/mass spectrometry. Maximal biliary cholesterol secretion rates were determined by biliary diversion with bile acid infusion over a range of 0 to 1000 nmol/minute. Biliary lipid secretion was determined by enzymatic colorimetric assays and molecular coupling of cholesterol to bile acid and phospholipid secretion calculated by linear regression. Results & Conclusions: Whole-body G5G8-deficient mice exhibited a 40% reduction in fecal neutral sterol output compared to wild-type mice when maintained on cereal-based or purified, low cholesterol containing diets. Cholesterol feeding resulted in a 10-fold increase in fecal neutral sterols in wild-type mice, an effect not observed in G5G8-deficient mice. These data indicate that ABCG5 ABCG8 is indispensable in opposing the accumulation of dietary cholesterol and that alternative pathways cannot compensate for G5G8 deficiency.

Decreased maximal cortisol secretion rate in patients with cirrhosis: Relation to disease severity

Background & AimsHepatic enzymes play a major role in the metabolic elimination of cortisol, and reduced rates of cortisol clearance have been consistently observed in patients with chronic liver disease. It is less clear whether there are concomitant abnormalities of adrenocortical function in patients with cirrhosis. In the present study, we sought to assess adrenocortical function in patients with cirrhosis using measures of free cortisol appearance and elimination rates that are independent of serum concentrations of cortisol binding proteins.MethodsPost hoc analysis used computer-assisted numerical and modelling methods with serial total and free cortisol concentration data to obtain rates of free cortisol appearance and elimination. Rate parameters were obtained in 114 patients with chronic liver disease, including Child-Pugh (CP) ≤8 (n = 53) and CP >8 (n = 61).ResultsMaximal cortisol secretion rate (CSRmax) was significantly decreased (p = 0.01) in patients with cirrhosis with CP >8 (0.28 nM/s; 95% CI 0.24–0.34) compared with those with CP ≤8 (0.39 nM/s; 95% CI 0.33–0.46), and CSRmax was negatively correlated with CP score (r = −0.19, p = 0.01). Free cortisol elimination rate was significantly (p = 0.04) decreased in the CP >8 group (0.16 ± 0.20 min−1) compared with that in the CP ≤8 group (0.21 ± 0.21 min−1), and free cortisol elimination rates were negatively correlated with CP score (r = −0.23, p = 0.01). A significant correlation between CSRmax and free cortisol elimination rate (r = 0.88, p <0.001) was observed.ConclusionsCSRmax and free cortisol elimination rates were significantly reduced according to severity of cirrhosis. In contrast to stimulated total cortisol concentrations, CSRmax estimates were independent of cortisol-binding protein concentrations. Results provide additional evidence of subnormal adrenocortical function in patients with cirrhosis.Lay summaryWe applied numerical analytic methods to characterise adrenocortical function in patients with varying stages of chronic liver disease. We found that patients with more severe cirrhosis have decreased rate of free cortisol elimination and decreased maximal cortisol secretion rate, which is a measure of adrenocortical function. In contrast to conventional measures of adrenocortical function, those obtained using numerical methods were not affected by variation in corticosteroid binding globulin and albumin concentrations. We conclude that patients with cirrhosis demonstrate measurable abnormalities of adrenocortical function, evidence of which supports aspects of the hepatoadrenal syndrome hypothesis.

Clinical observation on prevention and treatment of radiation-induced parotid-gland injury in patients with nasopharyngeal carcinoma by Compound Sarcandra Glabra

ObjectiveTo investigate the protective effect of the Compound Sarcandra Glabra on radiation injury of parotid glands in patients with nasopharyngeal carcinoma. MethodsSixty patients with primary nasopharyngeal carcinoma in the First Affiliated Hospital of Guangxi Medical University from June 2018 to January 2019 were collected, which were divided into treatment group and control group using a random number table. All patients were treated by a combination of 9-field intense-modulated radiotherapy and chemotherapy. The treatment group was given the Compound Sarcandra Glabra orally every day from 3 days before radiotherapy to the end. The degree of xerostomia (dry mouth) of patients was compared between the two groups at the end of radiotherapy and 3 months after radiotherapy by the chi-square test. The maximal adsorption and secretion rates of the parotid glands were compared between groups before radiotherapy, at the end of radiotherapy and 3 months after radiotherapy by the independent sample t-test. ResultsThe symptoms of dry mouth were alleviated in the treatment group, and the degree of dry mouth significantly differed between groups at the end of radiotherapy and 3 months after radiotherapy (χ2 ​= ​6.696 and 10.977, P ​< ​0.05). There were no significant differences in the maximal adsorption and secretion rates of the parotid glands between groups before radiotherapy (t ​= ​0.974 and −4.646, P ​> ​0.05); however, both variables were significantly higher in the treatment group at the end of radiotherapy and 3 months after radiotherapy (t ​= ​2.920 and 5.037; t ​= ​2.820 and 2.213, P ​< 0.05). Patients presented no liver and kidney injury after radiotherapy. ConclusionsOral administration of the Compound Sarcandra Glabra can effectively prevent radiation injury of the parotid glands in patients with nasopharyngeal carcinoma, thereby alleviating the degree of dry mouth after radiotherapy.

Time Course of Normalization of Functional β-Cell Capacity in the Diabetes Remission Clinical Trial After Weight Loss in Type 2 Diabetes.

To assess functional β-cell capacity in type 2 diabetes during 2 years of remission induced by dietary weight loss. A Stepped Insulin Secretion Test with Arginine was used to quantify functional β-cell capacity by hyperglycemia and arginine stimulation. Thirty-nine of 57 participants initially achieved remission (HbA1c <6.5% [<48 mmol/mol] and fasting plasma glucose <7 mmol/L on no antidiabetic drug therapy) with a 16.4 ± 7.7 kg weight loss and were followed up with supportive advice on avoidance of weight regain. At 2 years, 20 participants remained in remission in the study. A nondiabetic control (NDC) group, matched for age, sex, and weight after weight loss with the intervention group, was studied once. During remission, median (interquartile range) maximal rate of insulin secretion increased from 581 (480-811) pmol/min/m2 at baseline to 736 (542-998) pmol/min/m2 at 5 months, 942 (565-1,240) pmol/min/m2 at 12 months (P = 0.028 from baseline), and 936 (635-1,435) pmol/min/m2 at 24 months (P = 0.023 from baseline; n = 20 of 39 of those initially in remission). This was comparable to the NDC group (1,016 [857-1,507] pmol/min/m2) by 12 (P = 0.064) and 24 (P = 0.244) months. Median first-phase insulin response increased from baseline to 5 months (42 [4-67] to 107 [59-163] pmol/min/m2; P < 0.0001) and then remained stable at 12 and 24 months (110 [59-201] and 125 [65-166] pmol/min/m2, respectively; P < 0.0001 vs. baseline) but lower than that of the NDC group (250 [226-429] pmol/min/m2; P < 0.0001). A gradual increase in assessed functional β-cell capacity occurred after weight loss, becoming similar to that of NDC group participants by 12 months. This result was unchanged at 2 years with continuing remission of type 2 diabetes.

Concentration-Dependency and Time Profile of Insulin Secretion: Dynamic Perifusion Studies With Human and Murine Islets.

The detailed characterization and quantification of the kinetics of glucose-stimulated insulin secretion (GSIS) by normal pancreatic islets is of considerable interest for characterizing β-cell dysfunction, assessing the quality of isolated islets, and improving the design of artificial pancreas devices. Here, we performed dynamic evaluation of GSIS by human and mouse islets at high temporal resolution (every minute) in response to different glucose steps using an automated multichannel perifusion instrument. In both species, insulin responses were biphasic (a transient first-phase peak followed by a sustained second-phase), and the amount of insulin released showed a sigmoid-type dependence on glucose concentration. However, compared to murine islets, human islets have (1) a less pronounced first-phase response, (2) a flat secretion rate during second-phase response, (3) a left-shifted concentration response (reaching half-maximal response at 7.9 ± 0.4 vs. 13.7 ± 0.6 mM), and (4) an ~3-fold lower maximal secretion rate (8.3 ± 2.3 vs. 23.9 ± 5.1 pg/min/islet at 30 mM glucose). These results can be used to establish a more informative protocol for the calculation of the stimulation index, which is widely used for islet assessment in both research and clinical applications, but without an accepted standard or clear evidence as to what low- to high-glucose steps can provide better characterization of islet function. Data obtained here suggest that human islet functionality might be best characterized with a dynamic stimulation index obtained with a glucose step from a low of 4–5 to a high of 14–17 mM (e.g., G4 → G16).

Glucose but not KCl diminishes submembrane granule turnover in mouse beta-cells.

KCl depolarization is widely used to mimic the depolarization during glucose-stimulated insulin secretion. Consequently, the insulin secretion elicited by KCl is often regarded as the equivalent of the first phase of glucose-induced insulin secretion. Here, the effects of both stimuli were compared by measuring the secretion of perifused mouse islets, the cytosolic Ca2+ concentration of single beta-cells and the mobility of submembrane insulin granules by TIRF microscopy of primary mouse beta-cells. Two cargo-directed granule labels were used namely insulin-EGFP and C-peptide-emGFP. The granule behaviour common to both was used to compare the effect of sequential stimulation with 40 mM KCl and 30 mM glucose and sequential stimulation with the same stimuli in reversed order. At the level of the cell secretory response, the sequential pulse protocol showed marked differences depending on the order of the two stimuli. KCl produced higher maximal secretion rates and diminished the response to the subsequent glucose stimulus, whereas glucose enhanced the response to the subsequent KCl stimulus. At the level of granule behaviour, a difference developed during the first stimulation phase in that the total number of granules, the short-term resident granules and the arriving granules, which are all parameters of granule turnover, were significantly smaller for glucose than for KCl. These differences at both the level of the cell secretory response and granule behaviour in the submembrane space are incompatible with identical initial response mechanisms to KCl and glucose stimulation.

Characterization of Cortisol Secretion Rate in Secondary Adrenal Insufficiency.

Context:In secondary adrenal insufficiency (SAI), chronic deficiency of adrenocorticotropin (ACTH) is believed to result in secondary changes in adrenocortical function, causing an altered dose-response relationship between ACTH concentration and cortisol secretion rate (CSR).Objective:We sought to characterize maximal cortisol secretion rate (CSRmax) and free cortisol half-life in patients with SAI, compare results with those of age-matched healthy controls, and examine the influence of predictor variables on ACTH-stimulated cortisol concentrations.Design:CSRmax was estimated from ACTH1-24 (250 μg)–stimulated cortisol time-concentration data. Estimates for CSRmax and free cortisol half-life were obtained for both dexamethasone (DEX) and placebo pretreatment conditions for all subjects.Setting:Single academic medical center.Patients:Patients with SAI (n = 10) compared with age-matched healthy controls (n = 21).Interventions:The order of DEX vs placebo pretreatment was randomized and double-blind. Cortisol concentrations were obtained at baseline and at intervals for 120 minutes after ACTH1-24.Main Outcome Measures:CSRmax and free cortisol half-life were obtained by numerical modeling analysis. Predictors of stimulated cortisol concentrations were evaluated using a multivariate model.Results:CSRmax was significantly (P < 0.001) reduced in patients with SAI compared with controls for both placebo (0.17 ± 0.09 vs 0.46 ± 0.14 nM/s) and DEX (0.18 ± 0.13 vs 0.43 ± 0.13 nM/s) conditions. Significant predictors of ACTH1-24–stimulated total cortisol concentrations included CSRmax, free cortisol half-life, and baseline total cortisol, corticosteroid-binding globulin, and albumin concentrations (all P < 0.05).Conclusions:Our finding of significantly decreased CSRmax confirms that SAI is associated with alterations in the CSR-ACTH dose-response curve. Decreased CSRmax contributes importantly to the laboratory diagnosis of SAI.

Systematic evaluation of salivary gland damage following I-131 therapy in differentiated thyroid cancer patients by quantitative scintigraphy and clinical follow-up.

Radioiodine therapy is a common adjunct to thyroidectomy in papillary thyroid cancer treatment. However, a variety of associated adverse effects have been reported. In this study, we assessed radioiodine-induced salivary gland dysfunction using quantitative scintigraphy, and evaluated the associated complications. Patients were divided into five groups on the basis of the cumulative I-131 dosage received. Scintigraphic dynamic images of the salivary glands were obtained and converted into clinically relevant parameters: uptake index (UI), maximum secretion rate (%SR), and combined gland function scores. Patients were followed up for 3-66 months and interviewed for side effects including xerostomia, taste alteration, bitter taste, dental caries, xerophthalmia, and pain/swelling. An increase in I-131 doses resulted in a reduction in the UI and %SR and an increase in the combined scintigraphy score. Parotid glands were more affected than submandibular glands. A cumulative dosage of greater than 600 mCi resulted in complete loss of %SR in the parotid glands. No significant difference in either the UI or the %SR was observed between nontreated patients and patients receiving an I-131 dosage of up to 150 mCi. The occurrence of xerostomia was significantly correlated with the gland scintigraphic score, the number of treatment cycles, and I-131 dosage. The occurrence of pain and swelling was extremely low and only lasted for a short time. Although the side effects associated with radioiodine treatment were apparent, they were usually small and temporary. Nevertheless, more consideration should be placed on careful dosing of I-131.

Reversible increase in maximal cortisol secretion rate in septic shock.

Cortisol clearance is reduced in sepsis and may contribute to the development of impaired adrenocortical function that is thought to contribute to the pathophysiology of critical illness-related corticosteroid insufficiency. We sought to assess adrenocortical function using computer-assisted numerical modeling methodology to characterize and compare maximal cortisol secretion rate and free cortisol half-life in septic shock, sepsis, and healthy control subjects. Post hoc analysis of previously published total cortisol, free cortisol, corticosteroid-binding globulin, and albumin concentration data. Single academic medical center. Subjects included septic shock (n = 45), sepsis (n = 25), and healthy controls (n = 10). I.v. cosyntropin (250 μg). Solutions for maximal cortisol secretion rate and free cortisol half-life were obtained by least squares solution of simultaneous, nonlinear differential equations that account for free cortisol appearance and elimination as well as reversible binding to corticosteroid-binding globulin and albumin. Maximal cortisol secretion rate was significantly greater in septic shock (0.83 nM/s [0.44, 1.58 nM/s] reported as median [lower quartile, upper quartile]) compared with sepsis (0.51 nM/s [0.36, 0.62 nM/s]; p = 0.007) and controls (0.49 nM/s [0.42, 0.62 nM/s]; p = 0.04). The variance of maximal cortisol secretion rate in septic shock was also greater than that of sepsis or control groups (F test, p < 0.001). Free cortisol half-life was significantly increased in septic shock (4.6 min [2.2, 6.3 min]) and sepsis (3.0 min [2.3, 4.8 min] when compared with controls (2.0 min [1.2, 2.6 min]) (both p < 0.004). Results obtained by numerical modeling are consistent with comparable measures obtained by the gold standard stable isotope dilution method. Septic shock is associated with generally not only higher levels but also greater variance of maximal cortisol secretion rate when compared with control and sepsis groups. Additional studies would be needed to determine whether assessment of cortisol kinetic parameters such as maximal cortisol secretion rate and free cortisol half-life is useful in the diagnosis or management of critical illness-related corticosteroid insufficiency.

Type 2 Diabetes

Reversal of type 2 diabetes to normal metabolic control by either bariatric surgery or hypocaloric diet allows for the time sequence of underlying pathophysiologic mechanisms to be observed. In reverse order, the same mechanisms are likely to determine the events leading to the onset of hyperglycemia and permit insight into the etiology of type 2 diabetes. Within 7 days of instituting a substantial negative calorie balance by either dietary intervention or bariatric surgery, fasting plasma glucose levels can normalize. This rapid change relates to a substantial fall in liver fat content and return of normal hepatic insulin sensitivity. Over 8 weeks, first phase and maximal rates of insulin secretion steadily return to normal, and this change is in step with steadily decreasing pancreatic fat content. The difference in time course of these two processes is striking. Recent information on the intracellular effects of excess lipid intermediaries explains the likely biochemical basis, which simplifies both the basic understanding of the condition and the concepts used to determine appropriate management. Recent large, long-duration population studies on time course of plasma glucose and insulin secretion before the diagnosis of diabetes are consistent with this new understanding. Type 2 diabetes has long been regarded as inevitably progressive, requiring increasing numbers of oral hypoglycemic agents and eventually insulin, but it is now certain that the disease process can be halted with restoration of normal carbohydrate and fat metabolism. Type 2 diabetes can be understood as a potentially reversible metabolic state precipitated by the single cause of chronic excess intraorgan fat. Type 2 diabetes has long been known to progress despite glucose-lowering treatment, with 50% of individuals requiring insulin therapy within 10 years (1). This seemingly inexorable deterioration in control has been interpreted to mean that the condition is treatable but not curable. Clinical guidelines recognize …

β- and α-Cell Dysfunctions in Africans With Ketosis-Prone Atypical Diabetes During Near-Normoglycemic Remission

OBJECTIVEKetosis-prone atypical diabetes (KPD) is a subtype of diabetes in which the pathophysiology is yet to be unraveled. The aim of this study was to characterize β- and α-cell functions in Africans with KPD during remission.RESEARCH DESIGN AND METHODSWe characterized β- and α-cell functions in Africans with KPD during remission. The cohort comprised 15 sub-Saharan Africans who had been insulin-free for a median of 6 months. Patients in remission were in good glycemic control (near-normoglycemic) and compared with 15 nondiabetic control subjects matched for age, sex, ethnicity, and BMI. Plasma insulin, C-peptide, and glucagon concentrations were measured in response to oral and intravenous glucose and to combined intravenous arginine and glucose. Early insulin secretion was measured during a 75-g oral glucose tolerance test. Insulin secretion rate and glucagon were assessed in response to intravenous glucose ramping.RESULTSEarly insulin secretion and maximal insulin secretion rate were lower in patients compared with control participants. In response to combined arginine and glucose stimulation, maximal insulin response was reduced. Glucagon suppression was also decreased in response to oral and intravenous glucose but not in response to arginine and insulin.CONCLUSIONSPatients with KPD in protracted near-normoglycemic remission have impaired insulin response to oral and intravenous glucose and to arginine, as well as impaired glucagon suppression. Our results suggest that β- and α-cell dysfunctions both contribute to the pathophysiology of KPD.

The cytokines interleukin-1β and tumor necrosis factor-α stimulate CFTR-mediated fluid secretion by swine airway submucosal glands

The airway is kept sterile by an efficient innate defense mechanism. The cornerstone of airway defense is mucus containing diverse antimicrobial factors that kill or inactivate pathogens. Most of the mucus in the upper airways is secreted by airway submucosal glands. In patients with cystic fibrosis (CF), airway defense fails and the lungs are colonized by bacteria, usually Pseudomonas aeruginosa. Accumulating evidence suggests that airway submucosal glands contribute to CF pathogenesis by failing to respond appropriately to inhalation of bacteria. However, the regulation of submucosal glands by the innate immune system remains poorly understood. We studied the response of submucosal glands to the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α. These are released into the airway submucosa in response to infection with the bacterium P. aeruginosa and are elevated in CF airways. Stimulation with IL-1β and TNF-α increased submucosal gland secretion in a concentration-dependent manner with a maximal secretion rate of 240 ± 20 and 190 ± 40 pl/min, respectively. The half maximal effective concentrations were 11 and 20 ng/ml, respectively. The cytokine effect was dependent on cAMP but was independent of cGMP, nitric oxide, Ca(2+), or p38 MAP kinase. Most importantly, IL-1β- and TNF-α-stimulated secretion was blocked by the CF transmembrane conductance regulator (CFTR) blocker, CFTRinh172 (100 μmol/l) but was not affected by the Ca(2+)-activated Cl(-) channel blocker, niflumic acid (1 μmol/l). The data suggest, that during bacterial infections and resulting release of proinflammatory cytokines, the glands are stimulated to secrete fluid, and this response is mediated by cAMP-activated CFTR, a process that would fail in patients with CF.

Estimation of Maximal Cortisol Secretion Rate in Healthy Humans

Cortisol secretion is related to ACTH concentration by a sigmoidal dose-response curve, in which high ACTH concentrations drive maximal cortisol secretion rates (CSR(max)). We sought to estimate CSR(max) and free cortisol half-life in healthy humans (n = 21) using numerical methods applied to data acquired during cosyntropin (250 μg) stimulation. We also evaluated the effect of overnight dexamethasone (DEX; 1 mg) vs. placebo on estimates of CSR(max) and free cortisol half-life. This study was a double-blind, placebo-controlled, randomized order of overnight DEX vs. placebo, cosyntropin (250 μg) stimulation with frequent serum cortisol sampling and computer-assisted numerical analysis. The study was conducted at a single academic medical center. Twenty-one healthy adult subjects (15 females and six males), mean aged 46 yr, participated in the study. Intervention in the study included DEX vs. placebo pretreatment, cosyntropin (250 μg) iv with frequent cortisol sampling. CSR(max) and free cortisol half-life estimates, R² for goodness of fit, were measured. Mean ± sd CSR(max) was 0.44 ± 0.13 nm/second, with free cortisol half-life of 2.2 ± 1.1 min. DEX did not significantly affect estimates of CSR(max) or free cortisol half-life. Our model accounts for most of the variability of measured cortisol concentrations (overall R² = 90.9 ±11.0%) and was more accurate (P = 0.004) during DEX suppression (R² = 94.6 ± 4.6%) compared with placebo (R² = 87.2 ± 8.7%). Application of a mass-action model under conditions of cosyntropin stimulation provides a relatively simple method for estimation CSR(max) that accurately predicts measured cortisol concentrations. DEX administration did not significantly affect estimates of CSR(max) or free cortisol half-life.

Structure, function and secretory products of the peltate glands of Centrolobium tomentosum (Fabaceae, Faboideae)

The glandular structures of Centrolobium tomentosum Guill. ex Benth. have been little studied despite the economic importance of this species. We describe here the distribution, development, structure and ultrastructure of the secretory cells of the peltate glands found on the vegetative organs of this species. Stem apices and leaves in various stages of development were collected and prepared for examination by light, scanning and transmission electron microscopy. Chemical analyses and conventional histochemical tests to determine the chemical nature of the secretory products were also carried out. Peltate glands occur on aerial vegetative organs during their primary growth stage. These trichomes are structurally stable, persisting throughout the development of the organ. During the initial stages of the gland development, cell separation creates a central space that expands as secretions accumulate. Maximum secretion rates occur during this phase and the secreting cells characteristically have well developed smooth and rough endoplasmic reticulum, and high numbers of plastids and mitochondria. During the later stages of the secretory phase, the central cells show symptoms of cell death and are incorporated in to the secretions. At trichome maturity, the central space is delimited by a uniseriate epithelium. In addition to the resin, which is the main secretory product, an extensive three-dimensional carbohydrate matrix was observed that extended throughout the central space, apparently giving support to the resin droplets. The terpenic nature of the secretion was confirmed by thin-layer chromatography. Given the terpenic nature of the secretion and the permanence of trichomes throughout all phases of leaf development, it is postulated that the resin-secreting trichomes act to protect the plant against herbivores.

A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype

The development of in vitro models able to support the long-term viability and function of acinar cells is critical for exploring pancreatic pathophysiology. Despite considerable efforts, no long-term culture models for non-transformed pancreatic acini exist. Our aim was to develop and validate culture conditions for this purpose. An explant outgrowth culture design was established in which mouse pancreatic explants were cultured at the gas–liquid interphase. An enriched culture medium, pH 7.8, was employed to promote the selective outgrowth of acinar cells and to support their differentiated phenotype. After 7 days, the outgrown primary acinar cells were subcultured and maintained up to an additional 7 days as secondary monolayers on tissue culture plastic. Measurements of basal and caerulein-induced amylase secretion, phase-contrast microscopy and immunohistochemical analyses were used to characterize the cultures. Explants retained their pancreatic cytoarchitecture for 2 days in vitro. A triphasic dose response to caerulein was detected in 7-day primary cultures. The maximal rate of secretion was 1.2-fold versus basal ( p = 0.009) and 1.7-fold versus 1 pM caerulein ( p = 0.014). In secondary cultures the response was biphasic with maximal rates of secretion being 1.9-fold in 3- to 4-day cultures at 0.01 nM ( p = 0.049) and 2-fold in 6- to 7-day cultures at 0.1 nM ( p = 0.003). The present culture model provides a means to obtain functionally competent normal mouse acinar cells for long-term in vitro experimentation.

CAMP-secretion coupling is impaired in diabetic GK/Par rat β-cells: a defect counteracted by GLP-1

cAMP-raising agents with glucagon-like peptide-1 (GLP-1) as the first in class, exhibit multiple actions that are beneficial for the treatment of type 2 diabetic (T2D) patients, including improvement of glucose-induced insulin secretion (GIIS). To gain additional insight into the role of cAMP in the disturbed stimulus-secretion coupling within the diabetic β-cell, we examined more thoroughly the relationship between changes in islet cAMP concentration and insulin release in the GK/Par rat model of T2D. Basal cAMP content in GK/Par islets was significantly higher, whereas their basal insulin release was not significantly different from that of Wistar (W) islets. Even in the presence of IBMX or GLP-1, their insulin release did not significantly change despite further enhanced cAMP accumulation in both cases. The high basal cAMP level most likely reflects an increased cAMP generation in GK/Par compared with W islets since 1) forskolin dose-dependently induced an exaggerated cAMP accumulation; 2) adenylyl cyclase (AC)2, AC3, and G(s)α proteins were overexpressed; 3) IBMX-activated cAMP accumulation was less efficient and PDE-3B and PDE-1C mRNA were decreased. Moreover, the GK/Par insulin release apparatus appears less sensitive to cAMP, since GK/Par islets released less insulin at submaximal cAMP levels and required five times more cAMP to reach a maximal secretion rate no longer different from W. GLP-1 was able to reactivate GK/Par insulin secretion so that GIIS became indistinguishable from that of W. The exaggerated cAMP production is instrumental, since GLP-1-induced GIIS reactivation was lost in the presence the AC blocker 2',5'-dideoxyadenosine. This GLP-1 effect takes place in the absence of any improvement of the [Ca(2+)](i) response and correlates with activation of the cAMP-dependent PKA-dependent pathway.

Obesity due to Melanocortin 4 Receptor (MC4R) Deficiency Is Associated with Increased Linear Growth and Final Height, Fasting Hyperinsulinemia, and Incompletely Suppressed Growth Hormone Secretion

Melanocortin receptor 4 (MC4R) deficiency is characterized by increased linear growth greater than expected for the degree of obesity. The objective of the investigation was to study the somatotroph axis in obese MC4R-deficient patients and equally obese controls. We obtained anthropometric measurements and insulin concentrations in 153 MC4R-deficient subjects and 1392 controls matched for age and severity of obesity. We measured fasting IGF-I, IGF-II, IGF binding protein (IGFBP)-1, IGFBP-3, and acid-labile subunit levels in a subset of 33 MC4R-deficient patients and 36 control subjects. We examined pulsatile GH secretion in six adult MC4R-deficient subjects and six obese controls. Height sd score was significantly greater in MC4R-deficient children under 5 yr of age compared with controls (mean ± SEM: 2.3 ± 0.06 vs. 1.8 ± 0.04, P < 0.001), an effect that persisted throughout childhood. Final height (cm) was greater in MC4R-deficient men (mean ± SEM 173 ± 2.5 vs. 168 ± 2.1, P < 0.001) and women (mean 165 ± 2.1 vs. 158 ± 1.9, P < 0.001). Fasting IGF-I, IGF-II, acid-labile subunit, and IGFBP-3 concentrations were similar in the two groups. GH levels were markedly suppressed in obese controls, but pulsatile GH secretion was retained in MC4R deficiency. The mean maximal GH secretion rate per burst (P < 0.05) and mass per burst (P < 0.05) were increased in MC4R deficiency, consistent with increased pulsatile and total GH secretion. Fasting insulin levels were markedly elevated in MC4R-deficient children. In MC4R deficiency, increased linear growth in childhood leads to increased adult final height, greater than predicted by obesity alone. GH pulsatility is maintained in MC4R deficiency, a finding consistent with animal studies, suggesting a role for MC4R in controlling hypothalamic somatostatinergic tone. Fasting insulin levels are significantly higher in children carrying MC4R mutations. Both of these factors may contribute to the accelerated growth phenotype characteristic of MC4R deficiency.

Hormone-dependent dissociation of blood flow and secretion rate in the lingual salt glands of the estuarine crocodile, Crocodylus porosus

Salt and water balance in the estuarine crocodile, Crocodylus porosus, involves the coordinated action of both renal and extra-renal tissues. The highly vascularised, lingual salt glands of C. porosus excrete a concentrated sodium chloride solution. In the present study, we examined the in vivo actions of vasoactive intestinal peptide (VIP), B-type natriuretic peptide (BNP) and angiotensin II (ANG II) on the secretion rate and blood perfusion of the lingual salt glands. These peptides were selected for their vasoactive properties in addition to their reported actions on salt gland activity in birds and turtles and rectal gland activity in elasmobranchs. The femoral artery was cannulated in seven juvenile crocodiles for delivery of peptides and measurement of mean blood pressure and heart rate. In addition, secretion rate of, and blood flow to, the salt glands were recorded simultaneously using laser Doppler flowmetry. VIP stimulated salt secretion was coupled to an increase in blood flow and vascular conductance of the lingual salt glands. BNP was a potent stimulant of salt gland secretion, resulting in a maximal secretion rate of more than 15-fold higher than baseline; however, this was not coupled to an increase in perfusion rate, which remained unchanged. ANG II failed to stimulate salt gland secretion and there was a transient decrease in salt gland blood flow and vascular conductance. It is evident from this study that blood flow to, and secretion rate from, the lingual salt glands of C. porosus are regulated independently; indeed, it is apparent that maximal secretion from the salt glands may not require maximal blood flow.

788 Effect of Genetic Ablation of the Acid/Base Transporters PNBC1, NHE1, NHE2, and Slc26a7, On Gastric Epithelial Functions

Background: Recent investigations have revealed the expression of a multitude of basolateral acid/base transporters in gastric parietal cells, but the physiological significance of many of them is not established. Aim and methods: The study was designed to elucidate the physiological role of the Na/HCO3 cotransporter pNBC1, the Na+/H+ exchangers NHE1 and NHE2, all abundantly expressed in parietal cells. Isolated gastric mucosae from ten days old mice (as both NBC1and NHE1-deficient mice have a short life expectancy) were placed in classical Ussing chambers and basal and forskolin-stimulated acid secretory rate determined by back titration. Identical experiments were performed with selective pharmacological inhibitors of NBC and NHEs. Parietal cell ultrastructure was examined by electron microscopy, expression of H+/K+ATPase was assessed by quantitative RT-PCR and immunohistochemistry, epitheial cell apoptosis assessed by the TUNEL assay, and proliferation by immunohistochemistry with anti-Ki67, anti-histone. Results: Forskolin-stimulated acid secretion was significantly reduced (by >50%) in NBC1-deficient gastric mucosa, or after pharmacological inhibition of NBC1, but remained unaltered in NHE1or Slc26a7 deficient gastric mucosa in ten days old mice, or after pharmacological inhibition of NHE1 and NHE2. Parietal cell number, ultrastructure, H+/K+-ATPase expression and epithelial cell proliferation were normal in NBC1as well as NHE1 deficient mice. This indicates that pNBC1 likely serves as an additional base extrusion pathway in addition to the Cl/HCO3 exchanger AE2. In contrast, NHE2-deficient mucosae secreted acid only as a short burst, and displayed a reduction of H+/K+-ATPase but not AE2 expression. At postnatal day 10, no difference in apoptotic rate, proliferative indices, histology, and parietal cell numbers were detected in NHE2 +/+ and -/mice, whereas a complete change of the gastric proliferative zone and marked loss of parietal cell number was seen in adult stomach. Summary and conclusions: The electrogenic Na/HCO3 cotransporter NBC1 is an important base extruder during stimulation of acid secretion, and its absence or inhibition leads to a significant reduction in maximal acid secretory rates. In contrast, NHE1 and SLC26a7 anion transporter are not essential components of the ion transport machinery for gastric acid secretion in young mice. NHE2 ablation leaves parietal cell structure intact in young mice, but renders them non-functional.