Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder.

ANK3, encoding the adaptor protein Ankyrin-G (AnkG), has been implicated in bipolar disorder by genome-wide association studies. ANK3 has multiple alternative first exons, and a bipolar disorder-associated ANK3 variant has been shown to reduce the expression of exon 1b. Here we identify mechanisms through which reduced ANK3 exon 1b isoform expression disrupts neuronal excitation-inhibition balance. We find that parvalbumin (PV) interneurons and principal cells differentially express ANK3 first exon subtypes. PV interneurons express only isoforms containing exon 1b, whereas excitatory principal cells express exon 1e alone or both 1e and 1b. In transgenic mice deficient for exon 1b, PV interneurons lack voltage-gated sodium channels at their axonal initial segments and have increased firing thresholds and diminished action potential dynamic range. These mice exhibit an Ank3 gene dosage-dependent phenotype including behavior changes modeling bipolar disorder, epilepsy and sudden death. Thus ANK3's important association with human bipolar susceptibility may arise from imbalance between AnkG function in interneurons and principal cells and resultant excessive circuit sensitivity and output. AnkG isoform imbalance is a novel molecular endophenotype and potential therapeutic target.

Pre-vaccination screening strategies for the use of the CYD-TDV dengue vaccine: A meeting report.

The first licensed dengue vaccine, CYD-TDV (Dengvaxia) is efficacious in seropositive individuals, but increases the risk for severe dengue in seronegative persons about two years after administration of the first dose. For countries considering the introduction of Dengvaxia, WHO recommends a pre-vaccination screening strategy whereby only persons with evidence of a past dengue infection would be vaccinated. Policy-makers need to consider the risk-benefit of vaccination strategies based on such screening tests, the optimal age to introduce the vaccine, communication and implementation strategies. To address these questions, the Global Dengue and Aedes-transmitted diseases Consortium (GDAC) organized a 3-day workshop in January 2019 with country representatives from Asia and Latin America. The meeting discussions highlighted many challenges in introducing Dengvaxia, in terms of screening test characteristics, costs of such tests combined with a 3-dose schedule, logistics, achieving high coverage rates, vaccine confidence and communication; more challenges than for any other vaccine introduction programme. A screening test would require a high specificity to minimize individual risk, and at the same time high sensitivity to maximize individual and population benefit. The underlying seroprevalence dependent positive predictive value is the best indicator for an acceptable safety profile of a pre-vaccination screening strategy. The working groups discussed many possible implementation strategies. Addressing the bottlenecks in school-based vaccine introduction for Dengvaxia will also benefit other vaccines such as HPV and booster doses for tetanus and pertussis. Levels of public trust are highly variable and context specific, and understanding of population perceptions and concerns is essential to tailor interventions, monitor and mitigate risks.

Effect of age and environmental factors on insulin release from the perfused pancreas of the rat.

In this study we examined the effect of age and various age-related environmental factors on maximal glucose-stimulated insulin release by the intact perfused pancreas. Male Sprague-Dawley rats were maintained from 40 d to 12 mo of age on standard chow, or on a sucrose-rich or calorie-restricted diet. At 12 mo, studies were carried out on the isolated pancreas of each animal to determine maximal (300 mg/ml) glucose-stimulated insulin secretion. After these studies were completed, each pancreas was perfused with formalin fixative and processed for morphometric estimation of the mass of the endocrine pancreas. Data from these older animals were compared with data from 2-mo-old control rats. The results indicate that maximal glucose-stimulated insulin secretion per unit endocrine pancreas was markedly reduced in all three groups of 12-mo-old rats, and was only 25-33% of that of 2-mo-old rats. Thus, aging led to a decline in insulin secretion per beta cell that was not modifiable by environmental manipulation. On the other hand, environmental factors can influence the development of endocrine tissue within the pancreas, and in so doing, modify total pancreatic insulin secretion. The mass of the endocrine pancreas of 12-mo-old rats fed either sucrose or chow was between three and four times that of 2-mo-old control rats, and these older rats were able to maximally secrete as much insulin per total pancreas as the young rats. In contrast, the endocrine cell mass of the calorie-restricted rats had not enlarged to this extent, and the maximally stimulated perfused pancreas from these rats secreted less insulin. These data suggest that the aging animal, challenged in vivo to secrete insulin, can overcome the loss of the beta cell response by expanding its pancreatic pool of beta cells. Although this compensation is successful in the 12-mo-old, obese, middle-aged rat, it is not yet clear what effect further aging would have on these events.

Oxytocin-dependent consolation behavior in rodents.

Consolation behavior toward distressed others is common in humans and great apes, yet our ability to explore the biological mechanisms underlying this behavior is limited by its apparent absence in laboratory animals. Here, we provide empirical evidence that a rodent species, the highly social and monogamous prairie vole (Microtus ochrogaster), greatly increases partner-directed grooming toward familiar conspecifics (but not strangers) that have experienced an unobserved stressor, providing social buffering. Prairie voles also match the fear response, anxiety-related behaviors, and corticosterone increase of the stressed cagemate, suggesting an empathy mechanism. Exposure to the stressed cagemate increases activity in the anterior cingulate cortex, and oxytocin receptor antagonist infused into this region abolishes the partner-directed response, showing conserved neural mechanisms between prairie vole and human.

Data errors in the National Hip Fracture Database: a local validation study.

AIMS: We present an audit comparing our level I major trauma centre's data for a cohort of patients with hip fractures in the National Hip Fracture Database (NHFD) with locally held data on these patients. PATIENTS AND METHODS: A total of 2036 records for episodes between July 2009 and June 2014 were reviewed. RESULTS: The demographics of nine patients were recorded incorrectly. The rate of incorrect data in operation codes was most significant with overall accuracy of 0.637 (95% CI 0.615 to 0.658). The sensitivity of NHFD coding ranged from 0.250 to 1.000 and the specificity 0.879 to 0.999. The recording of cementation had a sensitivity of 0.932 and specificity of 0.713. The recording of total hip arthroplasty had a sensitivity of 0.739 and specificity of 0.983. The overall accuracy of mortality data was 0.942 (95% CI 0.931 to 0.952), with sensitivity of 0.967 and specificity of 0.419. CONCLUSION: This paper highlights the need for local audit of the integrity of data uploaded to the NHFD. Cite this article: Bone Joint J 2016;98-B:1406-9.

Effect of age and sex on rat endocrine pancreas.

Maximal glucose-stimulated insulin secretion was quantified in perfused pancreases of 11-wk-old and 12-mo-old female and male rats. In addition, measurements were made of body weight, total pancreatic weight, and percentage of the pancreas occupied by islet tissue. Body weight (mean +/- SE) of male rats was greater than that of female rats at both 11 wk (319 +/- 3 vs. 237 +/- 13 g) and 12 mo (684 +/- 17 vs. 376 +/- 13 g) of age. Pancreatic weight and percentage of the pancreas occupied by islet tissue were also greater in male rats and increased in approximate proportion to the gain in weight. The first phase and the second phase of maximal glucose-stimulated insulin secretion were both qualitatively and quantitatively similar in all four groups of rats. However, because islet cell mass increased with age, maximal glucose-stimulated insulin secretion declined with age in rats of both sexes when expressed per unit islet tissue. Although the fall in insulin secretion (per islet cell mass) with age was observed in perfused pancreases from both male and female rats, the pancreases of female rats contained relatively less islet tissue and secreted more insulin per unit islet cell mass than pancreases of male rats at either age. Thus, there are sex differences in both islet cell structure and function, but the effect of age on endocrine pancreatic function seems to be independent of sex.

Synthesis-secretion coupling of insulin. Effect of cyclosporin.

This study investigated the effects of cyclosporin (Cs) on insulin secretion and synthesis from the endocrine pancreas. With in vitro perfused pancreases from control and Cs-treated rats (1, 5, 10, or 25 mg.kg-1.day-1 for 2 wk), a dose-response relationship between Cs dose and inhibition of insulin secretion was demonstrated. Examination of the dynamic secretory response to a glucose stimulus (200 mg/dl) over a 3-h perfusion revealed an inhibition of all three secretory phases. Similarly, the ability of the pancreases to synthesize insulin decreased as a function of Cs dose. Reversibility of Cs toxicity on the pancreas was established by 2 wk after cessation of treatment. To evaluate the effect of Cs treatment in vivo, intravenous glucose tolerance tests were performed. Rats treated with 25 mg.kg-1.day-1 Cs for 2 wk had significantly lower k values (slope of log glucose concentration over time) than controls. At 10 mg.kg-1.day-1, although curves that appeared abnormal were observed, k values were not significantly different from those of controls. In summary, this study demonstrates the profound inhibitory effect of Cs on the endocrine pancreas.

Intravesical chondroitin sulphate for interstitial cystitis/painful bladder syndrome.

INTRODUCTION: Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic inflammatory condition of the bladder. Bladder instillation is one avenue of treatment but evidence for its effectiveness is limited. Chondroitin sulphate solution 2.0% (Urocyst) is a glycosaminoglycan (GAG) replenishment therapy instilled for patients with IC/PBS. We assessed its effectiveness for treating IC/PBS in Northern Ireland. METHODS: Patients with IC/PBS were assessed with the O'Leary-Sant interstitial cystitis index score and global response assessment questionnaire prior to commencing treatment. Assessment with these questionnaires was performed after 6 treatments (10 weeks) and again after 10 treatments (24 weeks). Assessment end points were pain, urgency, symptom score and problem score. RESULTS: Data was collected on 10 patients, 9 female and 1 male. 6 patients had failed RIMSO-50 dimethyl sulphoxide (DMSO) 50% treatment prior. At baseline the mean pain score was 6.6, urgency score 7.00, symptom score 13.5 and problem score 12.5. After 24 weeks the mean pain score fell to 2.0, urgency score to 1.80, symptom score to 6.89 and problem score to 5.67. At 10 weeks the global response to treatment was 100%. Nocturia was the first symptom to improve with urgency and pain following. No side effects were noted during instillation and all patients tolerated the treatments. CONCLUSION: IC/PBS is a difficult disease to treat. It requires a multimodal approach. We found that intravesical chondroitin sulphate reduced pain, urgency and O'Leary-Sant symptom and problem scores in patients with IC/PBS. All patients tolerated the treatment and no side effects were reported.

Effects of endurance exercise on bone mass and mechanical properties in intact and ovariectomized rats.

Exercise may play a role in the prevention of bone fractures in postmenopausal osteoporosis. The effects of endurance exercise on bone properties were assessed in 9-month-old sham-operated (SH) and ovariectomized (OVX) rats. The rats were either kept sedentary (SED) or were exercised (EX) on a rodent treadmill at 21 m/minute, 7% grade, 40 minutes/day, 4 days/week for 3 months. Bone mineral (by ash weight), morphometry, and biomechanical properties (by three-point bending) were evaluated after excision of bones at sacrifice. Ovariectomy resulted in a loss of bone mineral in femur, tibia, and fourth lumbar vertebra (L4), but biomechanical (force, deformation, stress, strain, and modulus of elasticity) and morphometric (length, cortical and medullary area, and moment of inertia) properties of femur were maintained. The ash weight of femur and tibia, but not L4, as well as femur yield and maximum force and moment of inertia, were improved in OVX-EX rats compared to OVX-SED animals. In SH rats exercise had no influence on ash weight of any of the three bones or femur morphometric properties, yet femur maximum force and plastic deformation were significantly enhanced compared to SH-SED rats. The results of the present study suggest that endurance exercise has beneficial effects on the bone mineral as well as biomechanical properties (femur yield and maximum force) during early stages after ovariectomy and improves the bending strength of the intact femur without an effect on bone mineral in sham-operated rats.

1,25(OH)2D3 regulates protein kinase C activity through two phospholipid-dependent pathways involving phospholipase A2 and phospholipase C in growth zone chondrocytes.

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.

Insulin content and insulinogenesis by the perfused rat pancreas: effects of long term glucose stimulation.

The dynamic response of the perfused pancreas differed between pancreases from fed and fasted rats. Insulin secretion was significantly lower in pancreases from fasted rats during the first 40 min of perfusion at glucose levels of 200 and 300 mg/dl. Thereafter, from 40-90 min, insulin secretion was similar by pancreases from both fed and fasted rats. The typical biphasic insulin secretory profile, consisting of a transient spike of insulin secretion followed by a slowly rising secretory phase, was observed in pancreases from fasted rats. In contrast, the transition from first to second phase secretion was accelerated in pancreases from fed rats. This suggests that transport of intracellular insulin stores may be accentuated due to the fact that insulinogenic sites (e.g. Golgi) in pancreases from fed rats may be fully primed for optimal secretion. Total pancreatic insulin measurements support this concept. Total pancreatic insulin content was determined under fed and 24-h fasted conditions after various times of perfusion (0, 60, and 90 min and 6 h) and in response to various glucose levels (0, 200, and 300 mg/dl). Fasting resulted in a significant decrease in insulin content at zero time compared with pancreases from fed rats (39.2 +/- 2.4 vs. 61.6 +/- 9.8 micrograms). In the fed rat pancreases, total insulin content decreased slightly after a 60-min glucose stimulus of 300 mg/dl, but returned to the basal level after 90 min and remained at that level during a 6-h period of perfusion. In the fasted state, insulin content remained constant as a function of time until 60 min, but increased by 90 min to a level comparable to that in pancreases from fed rats. The response to lower levels of glucose stimulation (200 mg/dl) was qualitatively similar by pancreases from fed and fasted rats compared to the response to a higher glucose dose (300 mg/dl), except that secretion was less. Insulin content remained relatively constant for periods of perfusion up to 60 min. Insulinogenesis (defined as de novo synthesis and conversion of existing preproinsulin and proinsulin to insulin, less intracellular degradation of insulin) was increased as a function of glucose concentration and differed temporally as a function of the food intake of the animal. At no time of perfusion with any level of glucose stimulation did the insulin content exceed the zero time value in pancreases from fed rats. This suggests that insulin secretion is the rate-limiting step for insulinogenesis.

Synthesis-secretion coupling of insulin: effect of aging.

Synthesis-secretion coupling of insulin was measured in four age groups of perfused pancreases taken from Sprague-Dawley rats ranging in age from 2-12 months. The effect of long term (6 h) near-maximal glucose stimulation (300 mg/dl) on both insulin secretion and net insulinogenesis demonstrated an age-related increase in both parameters. Net insulinogenesis as well as total insulin secretion increased linearly as a function of aging. Compared to that in 2-month-old rats, total net insulin synthesis was more than 3-fold greater in 12-month-old rats, slightly less than 3-fold greater in 8-month-old rats, and twice as much in 4-month-old rats. Compared to that in 2-month-old rats, total glucose-stimulated insulin secretion was 3-fold greater in 12-month-old rats, approximately 2.2-fold greater in 8-month-old rats, and about 1.7-fold greater in 4-month-old rats. A shorter term (90 min) glucose stimulation at 150 mg/dl produced an age-related increase in insulin secretion which was relatively comparable to the higher glucose stimulus. Of equal importance is that fact that pancreases from the older rats exhibited the same degree of secretory responsiveness to changing glucose levels as did pancreases from the younger rats. Regardless of age, first phase insulin secretion was approximately twice as much in response to the higher glucose level as to the lower. Similarly, second phase insulin secretion was almost 3 times greater regardless of age. When normalized and reported in terms of insulin content, total insulin secretion was no different as a function of aging during the first 1 h of glucose stimulation (i.e. the first two phases of secretion), but it was significantly elevated in the third secretory phase (2-6 h) by the older rat groups. Total 6-h net insulinogenesis was also greater in the older rat groups. When normalized and reported in terms of total body weight, both insulin synthesis and total insulin secretion became comparable and showed no specific age-related difference. Thus, there is no indication that aging results in an uncoupling of relatively long term (6-h) insulin synthesis-secretion, since both glucose-induced responses parallel one another as a function of aging. Furthermore, reporting insulin secretion and synthesis on the basis of body weight, rather than age, totally normalizes synthesis-secretion coupling of insulin.

Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes.

Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.

Dynamics of insulin and glucagon release in rats: influence of dietary manganese.

The effect of manganese on endocrine pancreatic function was examined in manganese-sufficient (control) and manganese-deficient (Mn-) Sprague-Dawley rats. Pancreatic insulin release was lower (P less than 0.05) in Mn- rats than in controls in response to both a 300 mg/dl and a 100 mg/dl glucose stimulus. The 300 mg/dl glucose stimulus induced the synthesis of 19.4 micrograms insulin/g pancreas in control rats. Additionally, no appreciable intracellular degradation of insulin occurred over an 80-min perfusion period. By contrast, in Mn- rats, there occurred an intracellular insulin degradation amounting to 7.8 micrograms/g pancreas. This enhanced degradation was partially compensated by a net insulin synthesis of only 3.4 micrograms insulin/g pancreas. Initial (min 1-3) insulin release by Mn- rats in response to 10 mM arginine was lower (P less than 0.05) than that observed in controls. Pancreatic glucagon release in response to 10 mM arginine was not affected by manganese deficiency. These findings demonstrate that manganese deficiency results in depressed pancreatic insulin synthesis and enhanced degradation. These factors may be responsible for the abnormal carbohydrate metabolism observed in Mn- animals.

Magnesium modulation of glucose-induced insulin secretion by the perfused rat pancreas.

Increasing levels of magnesium were found to cause a marked depression of glucosestimulated insulin secretion at fixed calcium levels, particularly at levels which bracketed the concentration of ultrafiltrable magnesium found in normal rat plasma (1.3 meq/l), i.e., increasing magnesium from 0.6 to 1.2 meq/l depressed secretion, and increasing magnesium from 1.2 to 2.4 meq/l resulted in a further depression. Paradoxically, when magnesium was omitted from the perfusing medium, insulin secretion was also depressed. The data strongly suggest that the calcium/magnesium ratio is a primary regulator of the insulin secretory process, since a relatively slight alteration of the physiologic ratio of calcium to magnesium (approximately 2.5) results in a marked alteration of total insulin secretion. In addition, small amounts of magnesium are necessary for optimum secretion, possibly reflecting the requirement for magnesium in several enzymatic processes. Thus, magnesium may play an important role in the regulation of insulin secretion by altering the sensitivity of the beta cells of the Islets of Langerhans to glucose.

Autonomic nervous system mediation of the pancreatic polypeptide response to insulin-induced hypoglycemia in conscious rats.

To investigate the neural regulation of pancreatic polypeptide (PP) secretion during hypoglycemia in the rat, insulin was administered to chronically cannulated rats, and plasma PP responses were compared between saline-treated animals and animals pretreated with a ganglionic blocking agent (hexamethonium), a muscarinic antagonist (atropine), combined alpha- and beta-adrenergic receptor blockade (propranolol + tolazoline), or combined adrenergic blockade + atropine. PP was measured using a new RIA which selectively detects PP in rat plasma. In control rats (n = 10), plasma PP increased from a baseline level of 30 +/- 3 pg/ml to 271 +/- 41 pg/ml during hypoglycemia (plasma glucose = 29 +/- 2 mg/dl) (delta PP = +241 +/- 42 pg/ml, P less than 0.0005), demonstrating that in rats, as in other species, insulin-induced hypoglycemia is a potent stimulus for PP release. PP only increased by 31 +/- 10 pg/ml during similar hypoglycemia in 7 hexamethonium-treated rats (P less than 0.01 vs. control animals). Thus, at least 90% of the PP response to hypoglycemia is neurally mediated. The plasma PP response to hypoglycemia was +85 +/- 24 pg/ml in atropine-treated rats (P 0.01 vs. control rats), suggesting that approximately 65% of the PP response is mediated via muscarinic acetylcholine receptors on the islet F cell. The PP response to hypoglycemia in rats with combined adrenergic blockade (delta = +168 +/- 32 pg/ml) was slightly, but not significantly smaller than that in control rats. The combination of combined blockade + atropine resulted in a PP response (delta = +26 +/- 7 pg/ml) to hypoglycemia that was similar to that in hexamethonium-treated rats (P less than 0.01 vs. control rats). These results suggest: 1) The PP response to hypoglycemia is predominantly the result of muscarinic, cholinergic activation. 2) There is a minor adrenergic contribution to the response. 3) The plasma PP response may be useful as an index of autonomic neural input to the islet during hypoglycemia.

Stimulus-secretion coupling and insulin secretion in aging: evaluation of glucose oxidation and ATP-sensitive potassium channel responsiveness.

The purpose of this study was to evaluate possible age-related alterations in the glucose-stimulus/insulin-secretion coupling mechanism of islets of Langerhans. To this end, the interaction among insulin secretion, glucose oxidation, and ATP-sensitive potassium (K ATP) channel responsiveness was determined in islets of Langerhans isolated from 6-, 12-, and 26-month-old male Fischer 344 (F344) rats. Groups of 20 islets were incubated for 40 min at 37 degrees C in a buffer containing: (1) either 1.7 or 11.1 mM glucose; (2) 1.2 microM glyburide and either 1.7 or 11.1 mM glucose; (3) 5 microM epinephrine and 11.1 mM glucose; or (4) 1.2 microM glyburide, 5 microM epinephrine, and either 1.7 or 11.1 mM glucose. Insulin release of islets incubated in the presence of glyburide and 1.7 or 11.1 mM glucose was greater than that of islets incubated in glucose alone, while glucose oxidation did not increase. Epinephrine inhibited insulin release and glucose oxidation at 11.1 mM glucose and abolished glyburide-enhanced insulin release at 11.1 mM glucose. No effect of age was observed in any of the treatment categories. These results indicate that if age-related alterations are occurring in glucose-stimulus/insulin-secretion coupling, then such alterations are not associated with changes in K ATP channel-mediated responsiveness.

Inhibition by cyclosporine of insulin secretion--a beta cell-specific alteration of islet tissue function.

We have reported the potent inhibitory effect of cyclosporine on glucose-induced insulin release in in vitro perfused pancreases. Suppression of both phases of release indicates inhibition of secretion and synthesis. Further studies were performed to examine the effect of high extracellular Ca2+ (4.875 mM). High Ca2+ failed to potentiate release in CsA-treated pancreases, thus we are focusing on the integrity of Ca(2+)-dependent signals in the beta cell. In this study, four groups of pancreases were perfused at 16.7 mM glucose: Control +/- somatostatin (SRIF) and CsA-treated +/- SRIF (60 nM). In control rats, the total 2-hr release decreased 40% with SRIF, from 42.7 +/- 5.5 to 25.5 +/- 3.9 micrograms/300 g body weight (P less than .05). In CsA-treated rats, release decreased 55% with SRIF, from 8.9 +/- 1.1 to 4.0 +/- 0.6 micrograms/300 g body weight. Further, at every time point of these CsA-treated rats, there was approximately 15% greater inhibition by SRIF than in controls. Pancreatic insulin contents were determined, indicating marked depletion of insulin stores in CsA-treated rats (190 +/- 9 vs. 76 +/- 5 micrograms/300 g body weight, P less than .01). Arginine-stimulated secretion of insulin and glucagon was also examined in control and CsA-treated pancreases. CsA exerted no effect on arginine-stimulated glucagon release, yet inhibited insulin approximately 50%. From these studies, we conclude that normal SRIF inhibitory mechanisms must be at least partially intact in CsA-treated pancreases during glucose-induced insulin release, and that CsA inhibition is specific for insulin release, as glucagon stores and arginine-stimulated glucagon release are unaffected by CsA.

Dynamics of insulin release by perfused hamster )Mesocricetus auratus) pancreases: effects of hypophysectomy, bovine and human growth hormone, and prolactin.

The hamster exhibits a biphasic pattern of insulin secretion; however, the dynamic response differs qualitatively from that of the rat in that there is a steady-state second release phase. A marked attenuation of insulin secretion as a result of hypophysectomy was observed after 3 weeks, but not after 2 weeks. This depression of insulin secretion was restored to near or above normal levels by bovine growth hormone, human growth hormone, and prolactin.

Altered cellular heterogeneity as a possible mechanism for the maintenance of organ function in senescent animals.

We tested the hypothesis that an alteration in the functional heterogeneity of cell populations (i.e., changes occurring in sensitivity and responsiveness to external stimuli among individual cells) may be a mechanism by which some organs are able to resist age-related decrements in function. To this end, changes in cytoplasmic free calcium concentration ([Ca2+]i) following glucose stimulation of individual pancreatic beta cells isolated from male F344 rats of ages 6, 12, and 26 mo were used as a model for evaluating responsiveness and sensitivity. Changes in [Ca2+]i of individual beta cells were monitored using fura-2 microspectrofluorimetry. No differences were observed in [Ca2+]i or in insulin secretion per beta cell among the age groups at any of the glucose concentrations. However, the percentage of beta cells that were responsive to a stimulatory glucose concentration (> 5.5 mM) was significantly greater in islets from the 26-mo-old rats (76%) as compared to the 6- and 12-mo-old animals (63% and 65%, respectively). Of the responsive beta cells, a significantly greater percentage of those from the 26-mo-old rats (72%) responded at the lowest stimulatory glucose concentration (7.5 mM) as compared to the 6- and 12-mo-old animals (58% and 60%, respectively). These data suggest that the maintenance of organ function in older rats at a level comparable to that of younger animals may be accomplished, in part, by an increase in the percentage of cells that are responsive to stimuli and/or by an increase in the sensitivity of the responsive cells.