Nesfatin-1 ameliorates type-2 diabetes-associated reproductive dysfunction in male mice.

PURPOSE: The present study was aimed to demonstrate the recuperative effect of nesfatin-1 on testicular dysfunction in the high-fat diet (HFD)/streptozotocin (STZ)-induced type-2 diabetes mellitus (T2DM) mice. METHOD AND RESULTS: Three experimental groups were formed: (1) vehicle control (VC), (2) T2DM mice, (3) T2DM + nesf-1. The mice with blood glucose level higher than 300 mg/dL following HFD and a single dose of STZ were used for the experiment. The T2DM mice showed increases in body mass, blood glucose and insulin levels, reductions in spermatogenesis and steroidogenesis, production of antioxidative enzymes, and disturbed lipid profile. These alterations were all ameliorated by administration of nesfatin-1 at 20 µg/Kg BW for 15 days. Nesfatin-1 treatment also increased the production of testosterone (T), improved insulin sensitivity, and effectively ameliorated the testicular aberrations, and increased spermatogenesis and steroidogenesis. In addition, nesfatin-1 treatment upregulated the PCNA and Bcl2 expression and inhibited the caspase-3 and prohibitin expression in T2DM mice. Nesfatin-1 increased insulin receptor (IR) and GLUT8 expressions, and lactate production, the changes that further substantiate the increase of energy influx to the testis. CONCLUSION: Altogether, the results suggest the ameliorative effect of nesfatin-1 against T2DM-associated testicular dysfunctions and improved insulin sensitivity along with promoting T production and fertility in T2DM mice.

Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1.

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP-CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

IL-10 gene transfer upregulates arcuate POMC and ameliorates hyperphagia, obesity and diabetes by substituting for leptin.

BACKGROUND: Obesity and metabolic syndrome are the major risk factors for cardiovascular disease. Obesity is caused by increased food intake and/or decreased energy expenditure. Leptin potently inhibits food intake and promotes energy expenditure. These effects of leptin involve the activation of proopiomelanocortin (POMC) neurons in the hypothalamus arcuate nucleus (ARC). Disruption of leptin signaling in POMC neuron is considered one of the major causes for obesity. AIMS: The present study aimed to examine whether overexpression of interleukin-10 (IL-10) could substitute for the leptin action and ameliorate obesity in leptin-deficient Lep(ob/ob) mice. DESIGN: Adeno-associated virus (AAV) expressing murine IL-10 (AAV-mIL-10) was injected into the skeletal muscle to overexpress IL-10 in mice. These mice were subsequently subjected to analysis of body weight, food intake, glucose metabolism and underlying mechanisms. RESULTS: In Lep(ob/ob) mice, AAV-IL-10 ameliorated hyperphagia, obesity, glucose intolerance and insulin resistance, as well as attenuated tumor necrosis factor-α expression. The IL-10 treatment also improved glucose-induced insulin release. Furthermore, IL-10 treatment increased POMC mRNA expression in ARC and phosphorylation of signal transducer and activator of transcription-3 (STAT3) in ARC and white adipose tissue (WAT). In neuron-specific STAT3-null mice that exhibited obesity and hyperphagia, AAV-mIL-10 administration failed to affect food intake, body weight and phosphorylation of STAT3 in WAT. CONCLUSIONS: These results demonstrate that peripheral overexpression of IL-10 induces STAT3 phosphorylation in ARC POMC neurons, and thereby ameliorates hyperphagia and obesity caused by leptin deficiency. IL-10 gene transfer may provide an effective approach for preventing progression of metabolic syndrome due to leptin resistance.

Ghrelin signalling in ß-cells regulates insulin secretion and blood glucose.

Insulin secretion from pancreatic islet ß-cells is stimulated by glucose. Glucose-induced insulin release is potentiated or suppressed by hormones and neural substances. Ghrelin, an acylated 28-amino acid peptide, was isolated from the stomach in 1999 as the endogenous ligand for the growth hormone (GH) secretagogue-receptor (GHS-R). Circulating ghrelin is produced predominantly in the stomach and to a lesser extent in the intestine, pancreas and brain. Ghrelin, initially identified as a potent stimulator of GH release and feeding, has been shown to suppress glucose-induced insulin release. This insulinostatic action is mediated by Gα(i2) subtype of GTP-binding proteins and delayed outward K⁺ (Kv) channels. Interestingly, ghrelin is produced in pancreatic islets. The ghrelin originating from islets restricts insulin release and thereby upwardly regulates the systemic glucose level. Furthermore, blockade or elimination of ghrelin enhances insulin release, which can ameliorate glucose intolerance in high-fat diet fed mice and ob/ob mice. This review focuses on the insulinostatic action of ghrelin, its signal transduction mechanisms in islet ß-cells, ghrelin's status as an islet hormone, physiological roles of ghrelin in regulating systemic insulin levels and glycaemia, and therapeutic potential of the ghrelin-GHS-R system as the target to treat type 2 diabetes.

Seasonal changes in gene expression of corticoid receptors in anadromous and non-anadromous strains of rainbow trout Oncorhynchus mykiss.

To clarify the regulation of expression of corticoid receptor (CR) genes during period of parr-smolt transformation of salmonids, seasonal changes in mRNA levels of glucocorticoid receptor (GR)-1, GR-2 and mineralocorticoid receptor (MR) were examined in gill, leucocytes, spleen and brain of anadromous and non-anadromous forms of Oncorhynchus mykiss. Increases in gill Na(+) , K(+) ATPase activity, plasma thyroxine levels and hypo-osmoregulatory ability assessed by 24 h seawater challenge test represented characteristics of smoltification in anadromous O. mykiss from May to June, whereas there was no apparent increase in the values of non-anadromous O. mykiss. Plasma cortisol levels of anadromous O. mykiss were higher than levels of non-anadromous O. mykiss from April to June. In gill of non-anadromous O. mykiss, there were significant increases in mRNA levels of three types of CR in spring. Although there were significant seasonal variations of CR mRNA levels in gill of anadromous O. mykiss, they appear to be less clear than those variations in non-anadromous O. mykiss. In anadromous O. mykiss, significant elevations in mRNA levels of the three types of CR were observed especially in the spleen. In both preoptic area and basal hypothalamus of the brain, there were tendencies to increase in CR mRNA levels from spring to summer in both anadromous and non-anadromous O. mykiss. These results showed difference in regulation of CR gene expression between the two forms of O. mykiss for osmoregulatory, immune and central nervous systems.

Predictive value of endoscopic findings in the diagnosis of active intestinal amebiasis.

Endoscopic diagnosis of amebic colitis can be difficult because its appearance may mimic other forms of colonic disease. The aim of this study was to identify predictive endoscopic findings for amebic colitis. Patients with suspected amebic colitis based on distinctive endoscopic findings such as aphthae or erosions, ulcers, exudates, or a bump, were included in the study. A total of 157 patients were selected, 50 of whom had amebic colitis. The sensitivity and specificity of endoscopic findings that were significantly associated with amebic colitis were: cecal lesions (80% and 54%), multiple number of lesions (96% and 29%), presence of aphthae or erosions (84% and 37%), and presence of exudate (88% and 74%). Multivariate analysis revealed that the best combination of findings to predict amebic colitis was the presence of cecal lesions, multiple lesions, and exudates, which corresponded to an area under the receiver operating characteristic curve of 0.89 (95% confidence interval 0.82-0.95).

Pebbles and sand on asteroid (162173) Ryugu: In situ observation and particles returned to Earth.

The Hayabusa2 spacecraft investigated the C-type (carbonaceous) asteroid (162173) Ryugu. The mission performed two landing operations to collect samples of surface and subsurface material, the latter exposed by an artificial impact. We present images of the second touchdown site, finding that ejecta from the impact crater was present at the sample location. Surface pebbles at both landing sites show morphological variations ranging from rugged to smooth, similar to Ryugu's boulders, and shapes from quasi-spherical to flattened. The samples were returned to Earth on 6 December 2020. We describe the morphology of >5 grams of returned pebbles and sand. Their diverse color, shape, and structure are consistent with the observed materials of Ryugu; we conclude that they are a representative sample of the asteroid.

Positional effects of polymorphisms in probe-target sequences on genoplot images of oligonucleotide microarrays.

Single nucleotide polymorphisms (SNPs) present in probe-target sequences (SPTS) have been shown to be associated with abnormal genoplot images. We explored the effects of SPTS positions on genoplot images using a data set from a genome-wide association study typed on an Illumina Human Hap300 platform. We screened the physical genomic positions of 308,330 autosomal probes to identify SPTS candidates deposited in dbSNP. The genoplot images across 293 individuals were inspected further in SNPs bearing an SPTS candidate. We identified 35,185 SNPs bearing a single SPTS candidate, including 264 SNPs showing abnormal genoplot images. The frequencies of SPTS at distances within 10 bases from the target SNP were significantly higher in the 264 SNPs showing abnormal genoplot images, than in the remaining 34,921 SNPs (49.62 vs 12.87%; Fisher exact test; P = 2.2 x 10(-16)). Of these 264 SNPs, we randomly selected 20 SNPs and resequenced them in 97 individuals. An SPTS within 10 bases of the target SNP was confirmed in all 20 SNPs, except for one SNP with a small deletion (7 bases) in the probe-target sequence. Taken together, these results suggest an association of a proximal SPTS with an abnormal genoplot image, which could result in spurious genotype detections, highlighting the importance of minimizing systematic errors in microarray experiments.

Effect of pituitary adenylate cyclase-activating polypeptide in islet transplantation.

INTRODUCTION: Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion similar to exendin-4. It has been reported that systemic administration of PACAP maintained beta-cell mass, delayed the onset of hyperglycemia, and protected beta cells from glucose toxicity. Moreover, PACAP increases glucose-stimulated insulin release in vitro and in vivo. In this study, we investigated the possibility of PACAP use in human islet transplantation. METHODS: Human islets were cultured in the presence or absence of PACAP (10(-12) mol/L) for 48 hours. We assessed beta-cell viability using FACS, cellular composition analysis by iCys/LSC, and glucose-stimulated insulin secretion. In vivo, islets were transplanted beneath the kidney capsule of Streptozotocin-induced diabetic immunodeficient mice. An intravenous glucose tolerance test (IVGTT) was also performed in the presence or absence of PACAP (Peptide International, Louisville, Ky, United States; 1.3 nmol/kg). RESULTS: There were significant improvements in terms of beta-cell viability and cellular composition between islets cultured with or without PACAP, respectively (P < .05). Moreover, glucose-stimulated insulin secretion significantly improved in islets cultured with PACAP compared with controls, respectively (P < .05). Treatment of recipient mice with PACAP resulted in beneficial effects on insulin secretion (PACAP vs control, 13.2 vs 1.9 mU/L), in IVGTT. However, no significant difference was observed in glucose levels between the 2 groups. CONCLUSIONS: Our study indicated that PACAP significantly improved beta-cell viability and survival during culture, and increased insulin secretion in vitro and in vivo. However, blood glucose levels in vivo after an IVGTT did not significantly improve, probably due to increased glucagon secretion from alpha cells. PACAP supplementation to culture medium could be of assistance to improve clinical islet transplantation outcomes.

Reconstruction of transcription-translation dynamics with a model of gene networks.

A transcription-translation model of gene networks and a method to reconstruct it from gene expression data are proposed. The model is a hybrid system based on the Glass network with continuous-time dynamics and logical interactions. Transcription-translation dynamics is introduced into the Glass network. The reconstruction of gene networks is reduced to the problem of estimating logical functions from binary representations of quantities of mRNAs and proteins. The reconstruction method is applied to the gene expression data of circadian rhythms. The response characteristics of the reconstructed gene network to periodic stimuli are analyzed. The results suggest the existence of a receiver gene that responds to an external signal, consistently with biological knowledge.

Weaning stage hyperglycemia induces glucose-insensitivity in arcuate POMC neurons and hyperphagia in type 2 diabetic GK rats.

Hyperphagia triggers and accelerates diabetes, and prevents proper dietary control of glycemia. Inversely, the impact of hyperglycemia on hyperphagia and possible mechanistic cause common for these two metabolic disorders in type 2 diabetes are less defined. The present study examined the precise developmental process of hyperglycemia and hyperphagia and explored the alterations in the hypothalamic arcuate nucleus (ARC), the primary feeding and metabolic center, in Goto-Kakizaki (GK) rats with type 2 diabetes and nearly normal body weight. At mid 3 to 4 weeks of age, GK rats first exhibited hyperglycemia, and then hyperphagia and reduced mRNA expressions for anorexigenic pro-opiomelanocortin (POMC) and glucokinase in ARC. Furthermore, [Ca2+]i responses to high glucose in ARC POMC neurons were impaired in GK rats at 4 weeks. Treating GK rats from early 3 to mid 6 weeks of age with an anti-diabetic medicine miglitol not only suppressed hyperglycemia but ameliorated hyperphagia and restored POMC mRNA expression in ARC. These results suggest that the early hyperglycemia occurring in weaning period may lead to impaired glucose sensing and neuronal activity of POMC neurons, and thereby induce hyperphagia in GK rats. Correction of hyperglycemia in the early period may prevent and/or ameliorate the progression of hyperphagia in type 2 diabetes.

Thermal and impact histories of 25143 Itokawa recorded in Hayabusa particles.

Understanding the origin and evolution of near-Earth asteroids (NEAs) is an issue of scientific interest and practical importance because NEAs are potentially hazardous to the Earth. However, when and how NEAs formed and their evolutionary history remain enigmas. Here, we report the U-Pb systematics of Itokawa particles for the first time. Ion microprobe analyses of seven phosphate grains from a single particle provide an isochron age of 4.64 ± 0.18 billion years (1σ). This ancient phosphate age is thought to represent the thermal metamorphism of Itokawa's parent body, which is identical to that of typical LL chondrites. In addition, the incorporation of other particles suggests that a significant shock event might have occurred 1.51 ± 0.85 billion years ago (1σ), which is significantly different from the shock ages of 4.2 billion years of the majority of shocked LL chondrites and similar to that of the Chelyabinsk meteorite. Combining these data with recent Ar-Ar studies on particles from a different landing site, we conclude that a globally intense impact, possibly a catastrophic event, occurred ca. 1.4 Ga ago. This conclusion enables us to establish constraints on the timescale of asteroid disruption frequency, the validity of the crater chronology and the mean lifetime of small NEAs.

Ghrelin stimulates phagocytosis and superoxide production in fish leukocytes.

To clarify the role of ghrelin in the fish immune system, the in vitro effect of ghrelin was examined in phagocytic leukocytes of rainbow trout (Oncorhynchus mykiss). Administration of trout ghrelin and des-VRQ-trout ghrelin, in which three amino acids are deleted from trout ghrelin, increased superoxide production in zymosan-stimulated phagocytic leukocytes from the head kidney. Gene expression of growth hormone (GH) secretagogue-receptor (GHS-R) was detected by RT-PCR in leukocytes. Pretreatment of phagocytic leukocytes with a GHS-R antagonist, [D-Lys3]-GHRP-6, abolished the stimulatory effects of trout ghrelin and des-VRQ-trout ghrelin on superoxide production. Ghrelin increased mRNA levels of superoxide dismutase and GH expressed in trout phagocytic leukocytes. Immunoneutralization of GH by addition of anti-salmon GH serum to the medium blocked the stimulatory effect of ghrelin on superoxide production. These results suggest that ghrelin stimulates phagocytosis in fish leukocytes through a GHS-R-dependent pathway, and also that the effect of ghrelin is mediated, at least in part, by GH secreted by leukocytes.

Young adult-specific hyperphagia in diabetic Goto-kakizaki rats is associated with leptin resistance and elevation of neuropeptide Y mRNA in the arcuate nucleus.

The present study aimed to examine whether hyperphagia, which is frequently observed in type 1 diabetic patients and model animals, also occurs in type 2 diabetic Goto-Kakizaki (GK) rats and, if so, to explore underlying abnormalities in the hypothalamus. GK rats at postnatal weeks 6-12, compared to control Wistar rats, exhibited hyperphagia, hyperglycaemia, hyperleptinemia and increased visceral fat accumulation, whereas body weight was unaltered. The ability of leptin to suppress feeding was reduced in GK rats compared to Wistar rats of these ages. In GK rats, leptin-induced phosphorylation of signal transducer and activator of transcription 3 was significantly reduced in the cells of the hypothalamic arcuate nucleus (ARC), but not of the ventromedial hypothalamus, whereas the mRNA level of functional leptin receptor was unaltered. By real-time polymerase chain reaction and in situ hybridisation, mRNA levels of neuropeptide Y, but not pro-opiomelanocortin and galanin-like peptide, were significantly increased in the ARC of GK rats at 11 weeks, but not 26 weeks. Following i.c.v. injection of a NPY Y1 antagonist, 1229U91, the amount of food intake in GK rats was indistinguishable from that in Wistar rats, thus eliminating the hyperphagia of GK rats. These results demonstrate that young adult GK rats display hyperphagia in association with leptin resistance and increased NPY mRNA level in the ARC.

Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats.

Leptin, an adipocytokine encoded by an obesity gene and expressed in adipose tissue, affects feeding behavior, thermogenesis, and neuroendocrine status via leptin receptors distributed in the brain, especially in the hypothalamus. Leptin may also modulate the synaptic plasticity and behavioral performance related to learning and memory since: leptin receptors are found in the hippocampus, and both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines that modulate long-term potentiation (LTP) in the hippocampus. We therefore examined the effect of leptin on (1) behavioral performance in emotional and spatial learning tasks, (2) LTP at Schaffer collateral-CA1 synapses, (3) presynaptic and postsynaptic activities in hippocampal CA1 neurons, (4) the intracellular Ca(2+) concentration ([Ca(2+)](i)) in CA1 neurons, and (5) the activity of Ca(2+)/calmodulin protein kinase II (CaMK II) in the hippocampal CA1 tissue that exhibits LTP. Intravenous injection of 5 and/or 50mug/kg, but not of 500mug/kg leptin, facilitated behavioral performance in passive avoidance and Morris water-maze tasks. Bath application of 10(-12)M leptin in slice experiments enhanced LTP and increased the presynaptic transmitter release, whereas 10(-10)M leptin suppressed LTP and reduced the postsynaptic receptor sensitivity to N-methyl-d-aspartic acid. The increase in the [Ca(2+)](i) induced by 10(-10)M leptin was two times greater than that induced by 10(-12)M leptin. In addition, the facilitation (10(-12)M) and suppression (10(-10)M) of LTP by leptin was closely associated with an increase and decrease in Ca(2+)-independent activity of CaMK II. Our results show that leptin not only affects hypothalamic functions (such as feeding, thermogenesis, and neuroendocrine status), but also modulates higher nervous functions, such as the behavioral performance related to learning and memory and hippocampal synaptic plasticity.

HeLa cells have histamine H1-receptors which mediate activation of the K+ conductance.

HeLa cells responded to exogenous histamine with a transient hyperpolarization due to increased membrane conductance to K+. After successive applications of histamine, the cell membrane became virtually unresponsive (desensitized). The responses were blocked by pyrilamine but not by cimetidine. Thus, it appears that HeLa cells have H1-receptors which mediate an increase in the K+ conductance.

Synchronous oscillation of the cytoplasmic Ca2+ concentration and membrane potential in cultured epithelial cells (Intestine 407).

Cultured epithelial Intestine 407 cells exhibit regular oscillations of the membrane potential with repeated hyperpolarizations. These hyperpolarizations were inhibited not only by K+ channel blockers (tetraethylammonium and nonyltriethylammonium) but also by inhibitors of the Ca2+-activated K+ channel (quinine and quinidine). Using Ca2+-selective microelectrodes, cyclic increases in the cytosolic free Ca2+ concentration of more than 1 X 10(-6) M were found to coincide with the cyclic membrane hyperpolarizations. Thus, it appears that the potential oscillation is brought about by the oscillation of the intracellular free Ca2+ level which induces periodic activation of the Ca2+-dependent K+ channels. Neither the deprivation of extracellular Ca2+ nor the application of Ca2+ channel blockers (Co2+ and Ni2+) abolished the potential oscillation. Mitochondrial inhibitors (KCN, NaN3, antimycin A, FCCP and dinitrophenol) inhibited the potential oscillation, whereas glycolytic inhibitors (iodoacetic acid and NaF) had no effects. Caffeine and oxalate, which affect the microsomal Ca2+ transport, failed to exert any effect upon the potential oscillation. It is concluded that the cytosolic Ca2+ oscillation results from cyclic releases of Ca2+ from the intracellular storage site, which depends upon mitochondrial activities.

Isolation and characterization of type IX collagen-proteoglycan from the Swarm rat chondrosarcoma.

Type IX collagen was partially purified from the Swarm rat chondrosarcoma by a series of a conventional salting-out procedures. The preparation was further separated by anion exchange chromatography into an unbound and a bound fraction in an A230 ratio of about 5:1. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the bound fraction appeared as a broad band, whose molecular mass ranged from 250 to 270 kDa. Digestion with chondroitinase ABC reduced the apparent molecular mass of the bound fraction to about 250 kDa, a value comparable to the molecular mass of the unbound fraction. Tryptic peptide maps of the protein moieties of unbound and bound forms showed that their molecular structures were basically identical. A monoclonal antibody specific for LMW, one of the pepsin-resistant fragments of the rat sarcoma type IX, reacted with both the unbound and bound fractions. Together the results indicate that the unbound and bound fractions represent a type IX collagen devoid of the chondroitin sulfate chain and its proteoglycan form with covalently bound chondroitin sulfate, respectively. The extent of glycosaminoglycan attachment to type IX collagen molecules in rat chondrosarcoma (about 16%) is quite different from the extents described in chick embryo cartilage (about 80%), chick vitreous humour (100%) and bovine cartilage (less than 5%). Further studies on the neoplastic tissue will offer additional information regarding the biological basis and biological consequences of the glycosaminoglycan attachment to type IX collagen molecules.

Induction and repair of strand breaks and 3'-hydroxy terminals in the DNA of mouse brain following gamma irradiation.

DNA was isolated from mouse brain after in vivo gamma-ray irradiation, treated with endonuclease S1 from Aspergillus oryzae if necessary, and analysed further by alkaline and neutral sucrose gradient centrifugation. In parallel, its template activity was determined by DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay as described previously. Similar experiments were performed with cultured mouse leukaemia cells (L5178Y) irradiated in vitro at 0 degrees C. Irradiation induced single- and double-strand breaks in the DNA of the brain with a yield of 1.0 and 0.1 break per 10(12) dalton per rad (100 eV/break and 770 eV/break), respectively. The yield of single-strand breaks in the brain was lower than that found in the cultured cells, whereas the yield of double-strand breaks was found to be almost the same in both cases. Treatment of irradiated DNA with single-strand-specific S1 endonuclease gave rise to further breaks detected on neutral sucrose gradient analysis. The yield of these breaks was also higher in the brain compared to the cultured cells. The increase per unit dose in the template activity of the DNA from the brain was found to be five times as much as that found in the cultured cells. Then, the average number of deoxyribonucleotides incorporated per break was determined on DNA which had experienced different treatments. The value for the brain DNA irradiated in vivo was found to be five times as much as that found for DNA treated with pancreatic deoxyribonuclease and 10 times as much as those found for DNA from the cultured cells and isolated brain nuclei irradiated in vitro at 0 degrees C. Thus, in vivo irradiation seemed to induce gaps with 3'-OH terminals in addition to simple breaks with or without 3'-OH terminals found in the cultured cells. Radiation-induced single-strand breaks and 3'-OH terminals in the DNA of the brain were repaired following irradiation. Approx. 20--40% of the terminals or breaks induced were, however, remaining at 3 h or more after irradiation, depending on the dose administered.

cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta-cells.

cAMP and the insulinotropic peptides that raise cAMP glucose-dependently increase the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells, which is tightly linked to the potentiation of glucose-induced insulin release. We examined whether cAMP increases [Ca2+]i in specific cooperation only with glucose or also with other insulin secretagogues that act through different mechanisms. [Ca2+]i in single rat pancreatic beta-cells was measured by dual-wavelength fura-2 microfluorometry. In the presence of a stimulatory concentration of glucose (8.3 mmol/l) and the moderate elevation in [Ca2+]i induced by it, forskolin, an activator of adenylyl cyclase, or dibutyryl cAMP produced a marked additional increase in [Ca2+]i but was ineffective at the basal 2.8 mmol/l glucose. These cAMP-elevating agents also potentiated the effect of tolbutamide on [Ca2+]i. The cAMP-induced increase in [Ca2+]i was completely and selectively inhibited by a blocker of cAMP-dependent protein kinase A (PKA), and by nitrendipine, a blocker of the L-type Ca2+ channel. However, in the presence of high KCl and the [Ca2+]i elevation induced by it, a rise in cAMP failed to further increase [Ca2+]i, whereas BAY K8644, an agonist of L-type Ca2+ channels, evoked an additional increase in [Ca2+]i. Under low Na+ conditions, the [Ca2+]i response to cAMP was observed in the majority of the cells. In the cells in which glucose at 4.5-5 mmol/l was inadequate to increase [Ca2+]i, the glucose together with a rise in cAMP often increased [Ca2+]i. Likewise, tolbutamide and a rise in cAMP acted in concert to increase [Ca2+]i. Thus, cAMP left-shifted the concentration-[Ca2+]i response relationship for glucose and tolbutamide. In conclusion, the cAMP-PKA pathway acts in selective synergism with glucose and tolbutamide to initiate [Ca2+]i signals in pancreatic beta- cells. cAMP appears to regulate beta-cell sensitivity to glucose and tolbutamide. In contrast, cAMP fails to cooperate with high KCl to increase [Ca2+]i. It is suggested that cAMP acts mainly on a site that is more proximal but functionally linked to the L-type Ca2+ channel, thereby finally increasing Ca2+ influx through this channel.