Ringer Bicarbonate Buffer Research Articles

WATER EXTRACT OF CAESALPINIA PULCHERRIMA FLOWERS INCREASES GLUCOSE UPTAKE IN INSULIN RESISTANT ADIPOCYTES IN A DOSEDEPENDENT MANNER

Caesalpinia pulcherrima (CP) flower extract has been shown to have antidiabetic effects on pancreatic beta cell regeneration and improved skeletal muscle glucose utilisation. However, little is known about its effect on adipocytes, particularly insulin resistant adipocytes. This study investigated the possibility of improving glucose uptake in adipocytes obtained from chickens which are naturally insulin resistant via direct effects of water extract of Caesalpinia pulcherrima flowers. The extract was prepared by boiling air dried CP flowers in a water based modified Krebs Ringer Bicarbonate buffer for 5mins and sterilizing the filtrate via vacuum filtration. A glucose load of 18mM and 10% ITS was added to the extract. In the absence of adipocytes, the glucose concentration of the CP extract remained unchanged (p=0.5087). Insulin resistance in the chicken adipocytes was confirmed by delayed and limited glucose uptake (maximum 2.9% + 1.62% in 60mins). This was reversed via the addition of the water extract of the CP flowers as concentrations as low as 2.8mg/ml doubled glucose uptake and concentrations as low as 5.6mg/ml increased the rate of glucose uptake to that of insulin sensitive cells. These results suggest that water extract of CP flowers directly modulate glucose uptake into insulin resistant cells at concentrations as low as 2.8mg/ml following a 5mins decoction preparation.

HYPOGLYCEMIC ACTIVITY OF THE AQUEOUS EXTRACT OF CAESALPINIA PULCHERRIMA FLOWERS IS INDEPENDENT OF INSULIN STIMULATION AND SUPPRESSED IN THE PRESENCE OF METFORMIN

Considering the high prevalence of insulin resistance, antidiabetic strategies that enhance insulin action or act independent of insulin are desirable. Caesalpinia pulcherrima (CP) flowers are known to have antidiabetic properties, but more work is required with respect to this action in insulin resistant adipocytes, particularly, its dependence on insulin and its therapeutic equivalence and/or interactions with other antidiabetic drugs. The purpose of this study was therefore to investigate the insulindependency of the water extract of CP flowers (CP extract) hypoglycemic effects, compare its antidiabetic action in diabetic and non-diabetic glucose loads, and explore its therapeutic equivalence and interactions with metformin. CP extract was prepared by boiling the air-dried flowers in cell culture media prepared in Krebs Ringer Bicarbonate buffer for 5mins. Metformin solution was prepared from a Metformin hydrochloride extended-release tablet to obtain low and therapeutic levels of metformin (0.8-2.4mg/L and). Insulin resistant (IR) adipocytes were exposed to CP extract in cell culture containing either 8mM or 18mM glucose and one of three insulin concentrations. CPextract allowed an efficient glucose disposal in the IR adipocytes in an insulin independent manner (p<0.0001). The percentage of glucose uptake did not significantly differ by models of diabetic and non-diabetic conditions (p=0.4727) although the significantly higher glucose concentration taken up by the IR adipocytes in the presence of IR adipocytes suggest an enhancement of antidiabetic action in hyperglycemic conditions. Expectedly metformin had a higher potency than the CP extract with its therapeutic dose of 1.8-2.4mg/L corresponding to 280mg/l of CP extract (p=0.9996). Additionally, metformin and CP extract appear to compete for similar sites which suppressed the hypoglycemic activity of CPextract.

68: Rapid Viability and Potency Assessment of Encapsulated Islets and Insulin-producing Cells

Introduction: Diabetes and its insidious complications sicken and kill millions of people worldwide. Our group has developed macroencapsulation devices capable of providing supplemental oxygen to macroencapsulated transplanted tissue. These devices house and protect islets and insulin-producing cells (IPCs) from the immune system, eliminating the need for immunosuppression of the transplant recipient. We predict these devices will facilitate transplant of small footprint devices containing high cell densities of islets or IPCs that can reverse diabetes in humans by fostering increased viability and functionality of transplanted islets and IPCs. However, in order to a) determine the number of cells encapsulation devices can support, b) assess the effects of encapsulation on islets and IPCs, c) meet FDA standards, and d) ensure the quality of a cellular transplant product, the viability and potency of islets and IPCs within encapsulation devices must be determined before clinical application. Materials and Methods: We have developed 2 bioreactor systems integrated with BioRep perifusion machines capable of simultaneous measurement of cellular oxygen consumption rate (OCR) and hormone secretion. One bioreactor can be used to assay free cells and the other, macroencapsulated cells. Each bioreactor contains two integrated FOSPOR oxygen sensors (Ocean Optics) at the inlet and outlet of the bioreactor. Oxygen concentration is derived from the fluorescence lifetime of the sensor measured by fiber optic cables linked to a phase fluorometer (Ocean Optics). Media flow, temperature and collection are controlled by the BioRep perifusion machine. Figure 1 depicts the general design of the integrated bioreactor systems. Results and Discussion: We have determined that cellular density and encapsulation can affect the viability and function of islets. Islet density greater than 500 islet equivalents/cm2 (without supplemental oxygen) was detrimental to islet viability during a 7-day transplant period. In vitro, densely encapsulated islets displayed delayed and diminished glucose-stimulated insulin secretion (GSIS) relative to free islets (Figure 2A), an important consideration for physiological control of glycemia with engrafted tissue. Furthermore, as expected in Krebs Ringer Bicarbonate Buffer, glucose-stimulated insulin secretion (GSIS) was correlated with an increased OCR (Figure 2B) highlighting the link between OCR and GSIS in this medium and the utility of OCR, an inexpensive real time assay as an islet potency indicator. Conclusions: In concert, oxygen consumption and hormone secretion are excellent indicators of islet and IPC health and function. Our bioreactor will allow us to optimize encapsulation densities, cellular function, and supplemental oxygen delivery strategies ensuring a clinical high-grade cellular product and, together with our encapsulation devices, improve the treatment of diabetes. BioRep, Inc. Juvenile Diabetes Research Foundation Award # #2-SRA-2018-685-S-B. NIH Diabetes Impact Award #1DP3DK106933-01.

Real-Time Monitoring of Insulin Using a Graphene Field-Effect Transistor Aptameric Nanosensor.

This paper presents an approach to the real-time, label-free, specific, and sensitive monitoring of insulin using a graphene aptameric nanosensor. The nanosensor is configured as a field-effect transistor, whose graphene-based conducting channel is functionalized with a guanine-rich IGA3 aptamer. The negatively charged aptamer folds into a compact and stable antiparallel or parallel G-quadruplex conformation upon binding with insulin, resulting in a change in the carrier density, and hence the electrical conductance, of the graphene. The change in the electrical conductance is then measured to enable the real-time monitoring of insulin levels. Testing has shown that the nanosensor offers an estimated limit of detection down to 35 pM and is functional in Krebs-Ringer bicarbonate buffer, a standard pancreatic islet perfusion medium. These results demonstrate the potential utility of this approach in label-free monitoring of insulin and in timely prediction of accurate insulin dosage in clinical diagnostics.

Cross Regulation Between cGMP-dependent Protein Kinase and Akt in Vasodilatation of Porcine Pulmonary Artery

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

Testicular Perfusion Apparatus

A reproductive biology laboratory requires to provide reliable instruments for regular use of research workers. To understand an organ and perform scientific study and reach on a conclusion good instruments and techniques are essential. Such a suitable one is not available in case of testicular study. The present study is aimed to clear this lacuna. A new testicular perfusion apparatus for rabbit and rat is designed and fabricated with glass. The glass blowing instrument available is used. The perfusion fluid preferred is Krebs Ringer bicarbonate buffer (pH 7.4). Quartz wool provided in stem tube filters emboli present in it. Perfusate temperature is maintained to that of testis. A 26-gauge needle is introduced into the spermatic artery to allow entry of perfusate into testis. Method for the collection of perfusate leaving testis is available. This permits to measure the level of an injected substance before its entry through perfusion fluid into the testis, which shall be compared in the fluid after completion of perfusion. Initially few animal testes were used and found the instrument to be functioning well. A new simple perfusion apparatus for testes of rabbit and rat is designed. The apparatus made out of glass could be prepared with the help of glass blowing facility and used regularly in any laboratory.

The perfused swine uterus model: long-term perfusion

BackgroundIt has previously been shown that the viability of swine uteri can be maintained within the physiological range in an open perfusion model for up to 8 hours. The aim of this study was to assess medium- to long-term perfusion of swine uteri using a modified Krebs–Ringer bicarbonate buffer solution (KRBB) in the established open perfusion model.MethodsIn an experimental study at an infertility institute, 30 swine uteri were perfused: group 1: n = 11, KRBB; group 2: n = 8, modified KRBB with drainage of perfusate supernatant; group 3: n = 11, modified KRBB with drainage of perfusate every 2 h and substitution with fresh medium. Modified and conventional KRBB were compared with regard to survival and contraction parameters: intrauterine pressure (IUP), area under the curve (AUC), and frequency of contractions (F).ResultsModified KRBB showed significantly higher IUP, AUC, and F values than perfusion with conventional KRBB. In group 3, the organ survival time of up to 17 h, with a 98% rate of effective contraction time, differed significantly from group 1 (P < 0.001).ConclusionsUsing modified KRBB in combination with perfusate substitution improves the open model for perfusion of swine uteri with regard to survival time and quality of contraction parameters. This model can be used for medium- to long-term perfusion of swine uteri, allowing further metabolic ex vivo studies in a cost-effective way and with little logistic effort.

Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs–Ringer bicarbonate buffer only, 0–1 mM [3,4- 13C 2]-4-hydroxynonanoate, and 0–2 mM [5,6,7- 13C 3]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7- 13C 3]heptanoate perfusates.

Metformin and the intestine

To the Editor: The antihyperglycaemic effect of metformin is generally attributed to a decrease in hepatic glucose output, with some additional effects that increase peripheral glucose uptake and utilisation [1]. The intestine also makes an important contribution to the glucose-lowering effect of metformin, but this is often overlooked because of a paucity of clinical data [2]. Animal studies indicate that metformin can cause intestinal glucose absorption to be delayed and occur more distally along the tract [3, 4]. However, animal studies have also shown that metformin increases glucose utilisation by the intestine, particularly anaerobic glucose metabolism [5–7], and this contributes to an apparent shortfall in the passage of glucose from the luminal to the serosal side of the intestine [3]. Extra lactate delivered into the portal vein is at least partly converted into glucose, increasing glucose turnover after administration of metformin [5,7,8]. We demonstrate here that metformin increases lactate production in human intestinal mucosa, similar to reports in animal studies. Animal studies have long established that very high concentrations of metformin accumulate in the wall of the intestine [9], which may at least partly explain the increase in anaerobic metabolism. We provide confirmatory evidence herein that similarly high concentrations of metformin accumulate in the human intestinal mucosa. In the present study, eight recently diagnosed, drug-naive, obese type 2 diabetic patients [five men, three women; age 55±3 years (mean±SEM); BMI 33±1 kg/m] attended on three occasions after an overnight fast. Patients gave informed consent, and ethical approval was granted by the research ethics committee. Avenous blood sample was taken for plasma glucose assay (automated glucose oxidase method), and a mucosal biopsy sample (approximately 25 mg) was taken on each occasion from the proximal jejunum, under light midazolam sedation, using an adapted paediatric gastroscope. On the first occasion, tissue was washed, divided into two portions, weighed and then incubated for 2 h at 37°C in 1 ml of Krebs Ringer bicarbonate buffer supplemented with 20 mg/ml serum albumin, 10 mmol/l glucose, 10 mol/l insulin with or without 10 mol/l metformin hydrochloride, and maintained in an atmosphere of 95% O2, 5% CO2 [4, 5]. Lactate production was measured as the concentration of lactate in the medium (manual spectrophotometric method). Metformin therapy was initiated at a dosage of 850 mg once daily and titrated up to twice daily after 2–3 weeks. Two repeat proximal jejunal mucosal biopsies were obtained after 6–8 weeks of therapy. A pre-dose sample was obtained in the morning, 12–16 h after the last metformin tablet (taken the evening before), and a post-dose sample was taken 3 h after the morning tablet. Tissue samples were washed in buffered saline, blotted and weighed, and then metformin was extracted with acetonitrile and assayed by gas chromatography [10]. The results showed that incubation of the jejunal biopsy tissue with 10 mol/l metformin increased lactate producDiabetologia (2008) 51:1552–1553 DOI 10.1007/s00125-008-1053-5

Glial Cell Line-Derived Neurotrophic Factor Increases β-Cell Mass and Improves Glucose Tolerance

Pancreatic beta-cell mass increases in response to increased demand for insulin, but the factors involved are largely unknown. Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that plays a role in the development and survival of the enteric nervous system. We investigated the role of GDNF in regulating beta-cell survival. Studies were performed using the beta-TC-6 pancreatic beta-cell line, isolated mouse pancreatic beta cells, and in vivo in transgenic mice that overexpress GDNF in pancreatic glia. GDNF receptor family alpha1 and c-Ret receptor expression were assessed by reverse-transcription polymerase chain reaction and immunofluorescence microscopy. Apoptosis was evaluated by assessing caspase-3 cleavage. Phosphoinositol-3-kinase signaling pathway was analyzed by Akt phosphorylation. Glucose homeostasis was assessed by performing intraperitoneal glucose tolerance tests. Insulin sensitivity was assessed using intraperitoneal injection of insulin. We demonstrate the presence of receptors for GDNF, GFRalpha1, and c-Ret on beta cells. GDNF promoted beta-cell survival and proliferation and protected them from thapsigargin-induced apoptosis (P<.0001) in vitro. Exposure of beta-cells to GDNF also resulted in phosphorylation of Akt and GSK3beta. Transgenic mice that overexpress GDNF in glia exhibit increased beta-cell mass, proliferation, and insulin content. No differences in insulin sensitivity and c-peptide levels were noted. Compared with wild-type mice, GDNF-transgenic mice have significantly lower blood glucose levels and improved glucose tolerance (P<.01). GDNF-transgenic mice are resistant to streptozotocin-induced beta-cell loss (P<.001) and subsequent hyperglycemia. We demonstrate that over expression of GDNF in pancreatic glia improves glucose tolerance and that GDNF may be a therapeutic target for improving beta-cell mass.

Kinetic properties of the redox switch/redox coupling mechanism as determined in primary cultures of cortical neurons and astrocytes from rat brain

We investigate the mechanisms underlying the redox switch/redox coupling hypothesis by characterizing the competitive consumption of glucose or lactate and the kinetics of pyruvate production in primary cultures of cortical neurons and astrocytes from rat brain. Glucose consumption was determined in neuronal cultures incubated in Krebs ringer bicarbonate buffer (KRB) containing 0.25-5 mM glucose, in the presence and absence of 5 mM lactate as an alternative substrate. Lactate consumption was measured in neuronal cultures incubated with 1-15 mM lactate, in the presence and absence of 1 mM glucose. In both cases, the alternative substrate increased the K(m) (mM) values for glucose consumption (from 2.2 +/- 0.2 to 3.6 +/- 0.1) or lactate consumption (from 7.8 +/- 0.1 to 8.5 +/- 0.1) without significant changes on the corresponding V(max). This is consistent with a competitive inhibition between the simultaneous consumption of glucose and lactate. When cultures of neurons or astrocytes were incubated with increasing lactate concentrations 1-20 mM, pyruvate production was observed with K(m) (mM) and V(max) (nmol/mg/h) values of 1.0 +/- 0.1 and 109 +/- 4 in neurons, or 0.28 +/- 0.1 and 342 +/- 54 in astrocytes. Thus, astrocytes or neurons are able to return to the incubation medium as pyruvate, a significant part of the lactate consumed. Present results support the reversible exchange of reducing equivalents between neurons and astrocytes in the form of lactate or pyruvate. Monocarboxylate exchange is envisioned to operate under near equilibrium, with the transcellular flux directed thermodynamically toward the more oxidized intracellular redox environment.

Rapid responses to thyroxine in the testis: Active protein synthesis-independent pathway

We investigated the involvement of protein synthesis in the stimulatory action of thyroid hormones on amino acid accumulation and characterized K + currents involved in the hyperpolarizing effect of thyroxine ( T 4) on Sertoli cells. Immature rat testes were incubated in Krebs Ringer-bicarbonate buffer (KRb) in the presence of [ 14C]methylaminoisobutyric acid with and without T 4, 3,5,3′- l-triiodothyronine (T 3) and/or cycloheximide. Sertoli cells were monitored by intracellular recording in a chamber perfused with KRb with and without T 4, T 3 and/or blockers, and the membrane potential was monitored. T 4 and T 3 stimulated amino acid accumulation and protein synthesis. Treatment with cycloheximide diminished T 3 stimulatory actions on amino acid accumulation but had no effect on T 4 action. Both hormones elicited a hyperpolarization of the Sertoli cell membrane potential which involved K + channels, since TEA and apamin abolished this effect. These findings on rapid membrane actions of thyroid hormone in the testis suggest that some effects of T 4 are modulated by non-genomic mechanisms.

Ganglionic Adrenergic Action Modulates Ovarian Steroids and Nitric Oxide in Prepubertal Rat

Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. The purpose of this work was to analyse the ganglionic adrenergic influence on the ovarian release of steroids and NO and the possible steroids/NO relationship. The experiments were carried out in the ex vivo coeliac ganglion-superior ovarian nerve (SON)-ovary system of prepubertal rats. The coeliac ganglion-SON-ovary system was incubated in Krebs Ringer-bicarbonate buffer in presence of adrenergic agents in the ganglionic compartment. The accumulation of progesterone, androstenedione, oestradiol and NO in the ovarian incubation liquid was measured. Norepinephrine in coeliac ganglion inhibited the liberation of progesterone and increased androstenedione, oestradiol and NO in ovary. The addition of alpha and beta adrenergic antagonists also showed different responses in the liberation of the substances mentioned before, which, from a physiological point of view, reveals the presence of adrenergic receptors in coeliac ganglion. In relation to propranolol, it does not revert the effect of noradrenaline on the liberation of progesterone, which leads us to think that it might also have a "per se" effect on the ganglion, responsible for the ovarian response observed for progesterone. Finally, we can conclude that the ganglionic adrenergic action via SON participates on the regulation of the prepubertal ovary in one of two ways: either increasing the NO, a gaseous neurotransmitter with cytostatic characteristics, to favour the immature follicles to remain dormant or increasing the liberation of androstenedione and oestradiol, the steroids necessary for the beginning of the near first estral cycle.

Anti-lipolytic Action of AMP-activated Protein Kinase in Rodent Adipocytes

Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the alpha(1) isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase alpha(1) knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect.

17beta-estradiol stimulates ascorbic acid and LHRH release from the medial basal hypothalamus in adult male rats.

In the present investigation, 17beta-estradiol (E(2)) and tamoxifen, an antiestrogen, were evaluated for their effects on the release of ascorbic acid (AA) and luteinizing hormone-releasing hormone (LHRH). Medial basal hypothalami (MBH) from adult male rats were incubated with graded concentrations of E(2) (10 (-9) to 10(-6) M) or a combination of E(2) (10(-7) M) and tamoxifen (10(-7) and 10(-6) M ) in 0.5 ml of Krebs Ringer bicarbonate buffer for 1 hr. AA and LHRH in the incubation medium were measured by high-performance liquid chromatography and radioimmunoassay, respectively. E(2) significantly elevated both AA and LHRH release and the minimal effective dose was 10(-7) M. A combination of E(2) (10(-7) M) and tamoxifen (10(-6) M) totally blocked E(2)-induced AA and LHRH release. The stimulatory effect of E(2) was also suppressed in the presence of N(G)-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase (NOS), illustrating that the release is mediated by nitric oxide (NO). To further characterize the role of NO, the tissues were incubated with E(2) or a combination of E(2) + (6 anilino-5, 8-quinolinedione) LY 83583 (10(-6) and 10(-5) M), an inhibitor of NOS. LY 83583 was effective in suppressing E(2)-induced AA and LHRH release, demonstrating that the effect was mediated by cyclic GMP. Incubation of the tissues with E(2) or a combination of E(2) + 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (O.D.Q.) (10(-5) and 10(-4) M), a specific inhibitor of soluble guanylyl cyclase failed to alter AA release but significantly suppressed LHRH release. The role of a prostaglandin synthesis blocker in E(2)-induced AA and LHRH release was tested by incubating the tissues with E(2) or a combination of E(2) + indomethacin (1.8 x 10 (-7) or 1.8 x 10(-6) M). Indomethacin produced a significant decrease in E(2)-induced AA and LHRH release, suggesting that the release process required prostaglandins as an intracellular mediator. In conclusion, E(2) stimulated both AA and LHRH release and the effect was mediated by NO and prostaglandins.

Effects of leptin on gonadotropin-releasing hormone release from hypothalamic–infundibular explants and gonadotropin release from adenohypophyseal primary cell cultures: further evidence that fully nourished cattle are resistant to leptin

In rodents and pigs, leptin stimulates the release of gonadotropin-releasing hormone (GnRH) from hypothalamus, gonadotropins from adenohypophyseal (AP) explants and cells, and luteinizing hormone (LH) from full-fed animals. In the current studies, we investigated whether leptin could stimulate the release of GnRH from bovine hypothalamic–infundibular (HYP) explants and gonadotropins from bovine adenohypophyseal cells. In Experiment 1A, HYP explants collected from 17 bulls and seven steers were incubated with Krebs–Ringer bicarbonate buffer (KRB) containing 0, 10, 100, or 1000 ng/ml recombinant ovine leptin (oleptin) for 30 min after a 3-h period of equilibration. None of the doses of leptin affected ( P>0.05) GnRH release into the media. In Experiment 1B, HYP explants collected from six steers were incubated with KRB containing 0 or 1000 ng/ml oleptin for two consecutive 30-min periods and challenged with 60 mM K + afterwards. Leptin did not affect ( P>0.05) basal or K +-stimulated release of GnRH. In Experiment 2, adenohypophyses from steers were collected at slaughter and cells dispersed and cultured for 4 days. On day 5, cells were treated with media alone (control) or media containing 10 −11, 10 −10, 10 −9, and 10 −8 M oleptin. Three independent replications were performed. None of the doses of leptin stimulated ( P>0.05) the release of LH. Although leptin at 10 −11, 10 −10, and 10 −9 M increased ( P<0.03) slightly the release of FSH compared to control-treated cells in one replicate, this effect was not confirmed in the other two replicates. Results support the hypothesis that leptin has limited effects on the release of GnRH and gonadotropins in full-fed cattle and reiterate important species differences in responsiveness to leptin.

Brevinin-1 and multiple insulin-releasing peptides in the skin of the frog Rana palustris.

Few studies have comprehensively examined amphibian granular gland secretions for novel insulinotropic peptides. This study involved isolation and characterisation of biologically active peptides from the skin secretions of Rana palustris frogs. Crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC on a semipreparative Vydac C18 column, yielding 80 fractions. These fractions were assayed for insulin-releasing activity using glucose-responsive BRIN-BD11 cells. Acute 20 min incubations were performed in Krebs Ringer bicarbonate buffer supplemented with 5.6 mmol/l glucose in the absence (control) and presence of various fractions. Fractions 29-54 and fractions 68-75 showed significant 2.0-6.5-fold increases in insulin-releasing activity (P<0.001). The fractions showing most prominent insulinotropic activity were further purified to single homogeneous peaks, which, on testing, evoked 1.5-2.8-fold increases in insulin release (P<0.001). The structures of the purified peptides were determined by mass spectrometry and N-terminal amino acid sequencing. Electrospray ionisation ion-trap mass spectrometry analysis revealed molecular masses of 2873.5-8560.4 Da. Sufficient material was isolated to determine the primary amino acid sequence of the 2873.5 Da peptide, revealing a 27 amino acid sequence, ALSILRGLEKLAKMGIALTNCKATKKC, repressing palustrin-1c. The database search for this peptide showed a 48% homology with brevinin-1, an antimicrobial peptide isolated from various Rana species, which itself stimulated insulin release from BRIN-BD11 cells in a concentration-dependent manner. In conclusion, the skin secretions of R. palustris frogs contain a novel class of peptides with insulin-releasing activity that merit further investigation.

Effects of leptin on gonadotropin-releasing hormone release from hypothalamic?infundibular explants and gonadotropin release from adenohypophyseal primary cell cultures: further evidence that fully nourished cattle are resistant to leptin

In rodents and pigs, leptin stimulates the release of gonadotropin-releasing hormone (GnRH) from hypothalamus, gonadotropins from adenohypophyseal (AP) explants and cells, and luteinizing hormone (LH) from full-fed animals. In the current studies, we investigated whether leptin could stimulate the release of GnRH from bovine hypothalamic–infundibular (HYP) explants and gonadotropins from bovine adenohypophyseal cells. In Experiment 1A, HYP explants collected from 17 bulls and seven steers were incubated with Krebs–Ringer bicarbonate buffer (KRB) containing 0, 10, 100, or 1000 ng/ml recombinant ovine leptin (oleptin) for 30 min after a 3-h period of equilibration. None of the doses of leptin affected (P>0.05) GnRH release into the media. In Experiment 1B, HYP explants collected from six steers were incubated with KRB containing 0 or 1000 ng/ml oleptin for two consecutive 30-min periods and challenged with 60 mM K+ afterwards. Leptin did not affect (P>0.05) basal or K+-stimulated release of GnRH. In Experiment 2, adenohypophyses from steers were collected at slaughter and cells dispersed and cultured for 4 days. On day 5, cells were treated with media alone (control) or media containing 10−11, 10−10, 10−9, and 10−8 M oleptin. Three independent replications were performed. None of the doses of leptin stimulated (P>0.05) the release of LH. Although leptin at 10−11, 10−10, and 10−9 M increased (P<0.03) slightly the release of FSH compared to control-treated cells in one replicate, this effect was not confirmed in the other two replicates. Results support the hypothesis that leptin has limited effects on the release of GnRH and gonadotropins in full-fed cattle and reiterate important species differences in responsiveness to leptin.

Influence of substrate and/or neurohormonal mimic on in vitro pancreatic enzyme release from calves postruminally infused with partially hydrolyzed starch and/or casein.

Our objectives were to determine the effects of neuroendocrine challenge and substrates on in vitro alpha-amylase and trypsin release in pancreatic tissue collected from Holstein calves (n = 24; 88 +/- 3 kg) abomasally infused for 10 d with tap water (control), partially hydrolyzed starch (SH; 4 g/[kg of BW x d]) and/ or casein (0.6 g/[kg of BW x d]). The caudal portion of the pancreas was removed, rinsed with ice-cold saline, cut into approximately 2 x 2-mm segments, and incubated in oxygenated Krebs Ringer bicarbonate buffer containing no substrate (control), glucose, amino acids, or VFA at 39 degrees C. After 60 min of incubation, neurohormonal mimics (none; control), carbachol (acetylcholine analog; 10 microM final), or caerulein (cholecystokinin mimic; 100 nM final) were added to the flasks and tissue was incubated for 60 min. Pancreatic tissue concentrations and in vitro release of alpha-amylase and trypsin decreased (P < 0.001) in calves abomasally infused with SH. Carbachol increased (P < 0.10) alpha-amylase and trypsin release in tissue collected from all calves. An effect of caerulein to increase alpha-amylase release (P < 0.10) was only observed with prior exposure to abomasal casein infusion in vivo or with simultaneous incubation with amino acids in vitro. Caerulein increased (P < 0.10) trypsin release in tissue collected from all calves except for those receiving SH + casein. Glucose decreased (P < 0.10) alpha-amylase release from pancreatic tissue collected from calves receiving abomasal control and casein treatments. Amino acids decreased (P < 0.10) alpha-amylase and trypsin release from pancreatic tissue collected from calves receiving the abomasal control treatment. Glucose, amino acids, and VFA decreased (P < 0.10) trypsin release from tissue collected from calves receiving abomasal SH. These data indicate that carbachol can stimulate pancreatic enzyme release in vitro. Caerulein, however, is only effective in stimulating in vitro pancreatic enzyme release in tissue from calves with an increased postruminal protein supply or in tissue incubated with amino acids. The results indicate that postruminal and local nutrients might be important in altering the responsiveness to a neuroendocrine challenge and could be an important regulatory event involved with dietary adaptation in ruminants.

Brain energy metabolism is compromised by the metabolites accumulating in homocystinuria

Homocystinuria is an inborn error of metabolism caused by severe deficiency of cystathionine β-synthase activity. It is biochemically characterized by tissue accumulation of homocysteine (Hcy) and methionine (Met). Homocystinuric patients present a variable degree of neurological dysfunction whose pathophysiology is poorly understood. In the present study, we investigated the in vitro effect of Hcy and Met on some parameters of energy metabolism in hippocampus of rats. CO 2 production from [U- 14 C ] acetate, glucose uptake and lactate release were assessed by incubating hippocampus prisms from 28-day-old rats in Krebs–Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of Hcy (10–500 μM) or Met (0.2–2.0 mM). Hcy and Met decreased CO 2 production in a dose-dependent manner and increased lactate release. In contrast, glucose uptake was not altered by the metabolites. The effect of Hcy and Met on cytochrome c oxidase activity was also studied. It was observed that Met did not alter this enzyme activity, in contrast with Hcy, which significantly inhibited cytochrome c oxidase activity. It is suggested that impairment of brain energy metabolism caused by the metabolites accumulating in homocystinuria may be related to the neurological symptoms present in homocystinuric patients.