Leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis.

To test the hypothesis that leptin secreted from adipose tissue is a mediator linking obesity and pancreatic islet hypertrophy, we examined the effects of leptin on proliferative and apoptotic responses in rat islet cells. Rat pancreatic islets were isolated and incubated with 0, 1, 5, or 75 nM leptin for 24 h under serum-deprived conditions. Cell viability was assessed with 2,5-diphenyltetrazolium bromide and trypan blue dye exclusion tests. Cell proliferation and apoptosis were evaluated with 5-bromo-2'-deoxyuridine incorporation into DNA and DNA ladder formation, respectively. Incubation for 24 h with 1 and 5 nM leptin, the concentrations observed in obese subjects, increased the viability of isolated pancreatic islet cells. Five nanomolar concentrations of leptin did not stimulate 5-bromo-2'-deoxyuridine incorporation into incubated islet cells, indicating no influence on cell proliferation, but did inhibit DNA ladder formation, a hallmark of cell apoptosis. Moreover, 5 nM leptin reduced the triglyceride content and suppressed inducible nitric oxide synthase mRNA expression in incubated islets. These results suggest that leptin increased viable cell numbers via suppression of apoptosis in isolated pancreatic islet cells under these experimental conditions. This mechanism might account at least in part for an obesity-induced increase in pancreatic beta-cell mass.

Troglitazone treatment increases plasma vascular endothelial growth factor in diabetic patients and its mRNA in 3T3-L1 adipocytes.

Troglitazone is one of the thiazolidinediones, a new class of oral antidiabetic compounds that are ligands of peroxisome proliferator-activated receptor-gamma. This study on vascular endothelial growth factor (VEGF), also known as vascular permeability factor, was prompted by our clinical observation that the characteristics of troglitazone-induced edema were very similar to those caused by vascular hyperpermeability. When Japanese diabetic patients were screened for plasma VEGF, we found levels to be significantly (P < 0.001) increased in troglitazone-treated subjects (120.1 +/- 135.0 pg/ml, n = 30) compared with those treated with diet alone (29.2 +/- 36.1 pg/ml, n = 10), sulfonylurea (25.8 +/- 22.2 pg/ml, n = 10), or insulin (24.6 +/- 19.0 pg/ml, n = 10). Involvement of troglitazone in increased VEGF levels was further supported by the plasma VEGF levels in five patients before treatment (20.2 +/- 7.0 pg/ml), after 3 months of troglitazone treatment (83.6 +/- 65.9 pg/ml), and 3 months after discontinuation (28.0 +/- 11.6 pg/ml). We further demonstrated that troglitazone, as well as rosiglitazone, at the plasma concentrations observed in patients, increased VEGF mRNA levels in 3T3-L1 adipocytes. VEGF is an angiogenic and mitogenic factor and is currently considered the most likely cause of neovascularization and hyperpermeability in diabetic proliferative retinopathy. Although increased VEGF may be beneficial for subjects with macroangiopathy and troglitazone is currently not available for clinical use, vascular complications, especially diabetic retinopathy, must be followed with great caution in subjects treated with thiazolidinediones.

Non-competitive inhibition of kainate-induced currents by diethylstilbestrol in acutely isolated mouse CA1 hippocampal neurons.

The effect of a synthetic estrogen, diethylstilbestrol (DES), on kainate-induced currents was investigated in the hippocampal CA1 pyramidal neurons acutely dissociated from the mice using the nystatin-perforated patch-clamp recording configuration under voltage-clamp conditions. DES inhibited the current evoked by 100 microM kainate in a concentration-dependent manner with a half-maximum inhibitory concentration of 8.8 microM. The action of DES was voltage-independent. Since DES produced a suppression of the maximum response of the kainate concentration-response curve, the inhibition by DES of the kainate-induced current appears to be non-competitive.

[Rosiglitazone (BRL-49653)].

Rosiglitazone, a thiazolidinedion antidiabetic agent, improves insulin resistance in patients with type 2 diabetes mellitus. Rosiglitazone binds to PPAR-gamma with high affinity and the in vivo antidiabetic potency of rosiglitazone is correlated with its high biding affinity. In animal models of insulin resistence, rosiglitazone decreased plasma glucose, triglyceride and insulin levels and also prevented diabetic nephropathy and pancreatic islet cell degeneration. In clinical trials in patients with type 2 diabetes mellitus, rosiglitazone, 2 to 12 mg/day (as a single daily dose or 2 divided daily doses), improved glycemic control as demonstrated, by decreases in fasting plasma glucose and HbA1C levels. Rosiglitazone did not appear to increase the risk of hypoglycemia and there was no evidence of hepatotoxicity in pre-clinical trials.

Evidence for stimulation of adenylyl cyclase by an activated G(s) heterotrimer in cell membranes: an experimental method for controlling the G(s) subunit composition of cell membranes.

Heterotrimeric (alphabetagamma) G(s) mediates agonist-induced stimulation of adenylyl cyclase (AC). Cholera toxin (CTx) will ADP-ribosylate the alpha-subunit of G(s) (G(s)alpha). G(s)alpha-deficient cyc(-) membranes were "stripped" of Gbeta. When the stripped cyc(-) were incubated with G(s)alpha and/or Gbetagamma, each was incorporated into the membranes independently of the other. Both G(s)alpha and Gbetagamma had to be present in the membranes, and they had to be able to form a heterotrimer in order for CTx to ADP-ribosylate G(s)alpha, indicating that the membrane bound G(s) heterotrimer is a substrate for CTx, but the G(s)alpha subunit by itself is not. When G(s)alpha was completely and irreversibly activated with GTPgammaS and incorporated into stripped cyc(-), it was a poor substrate for CTx and a weak stimulator of AC unless Gbetagamma was also incorporated. Furthermore, the level of AC stimulation corresponded to the amount of G(s) heterotrimer that was formed in the membranes from GTPgammaS-activated G(s)alpha and Gbetagamma. These data suggest that AC is stimulated by an activated G(s) heterotrimer in cell membranes.

Overexpression of dominant negative mutant hepatocyte nuclear factor (HNF)-1alpha inhibits arginine-induced insulin secretion in MIN6 cells.

AIMS/HYPOTHESIS: To explain the mechanisms whereby mutations in the HNF-1alpha gene cause insulin secretory defects. METHODS: A truncated mutant HNF-1alpha (HNF-1alpha288t) was overexpressed in hepatoma cells (HepG2) and murine insulinoma cells (MIN6) using a recombinant adenovirus system and expression of the HNF-1alpha target genes and insulin secretion were examined. RESULTS: Expression of phenylalanine hydroxylase and alpha1-antitrypsin genes, the target genes of HNF-1alpha, was suppressed in HepG2 cells by overexpression of HNF-1alpha288t. In MIN6 cells, overexpression of HNF-1alpha288t did not change insulin secretion stimulated by glucose (5 mmol/l and 25 mmol/l) or leucine (20 mmol/l). Potentiation of insulin secretion by arginine (20 mmol/l, in the presence of 5 mmol/l or 25mmol/l glucose) was, however, reduced (p < 0.0001 and p = 0.027, respectively). Similarly reduced responses were observed when stimulated with homoarginine. Expression of the cationic amino acid transporter-2 was not reduced and insulin secretory response to membrane depolarization by 50 mmol/l KCl was intact. CONCLUSION/INTERPRETATION: The HNF-1alpha288 t, which is structurally similar to the mutant HNF-1alpha expressed from the common MODY3 allele, P291fsinsC, exerts a dominant negative effect. Suppression of HNF-1alpha in MIN6 cells severely impaired potentiation of insulin secretion by arginine, whereas glucose-stimulated and leucine-stimulated insulin secretion was intact. Our findings delineate the complex nature of beta-cell failure in patients with MODY3. This cell model will be useful for further investigation of the mechanism of insulin secretory defects in these patients.

Direct stimulation of basal insulin secretion by physiological concentrations of leptin in pancreatic beta cells.

We examined a possible mechanism underlying the link between obesity and hyperinsulinemia, focusing on leptin, a peptide released from adipocytes which affects the satiety center in the brain. The leptin receptor isoforms, Ob-Ra and Ob-Rb, are present in the pancreatic beta cell line MIN6 and in rat pancreatic islets, based on RT-PCR. A 2 hr, but not a 30 min, incubation with 1 nM recombinant mouse leptin, the concentration observed in obese subjects, stimulated basal (at 5 mM glucose) insulin secretion by approximately 40% in both MIN6 and rat islets. Stimulatory effects were not observed without glucose or when the incubation medium containing 1 nM leptin had been preincubated with the immobilized leptin antibody. In contrast to the stimulatory effects on basal insulin secretion at 1 nM, the maximally stimulated insulin secretion at 25 mM glucose was not significantly changed by 1 nM leptin in isolated rat islets. In addition, 10 and 100 nM leptin exerted small but significant inhibitory effects on 16.7 mM glucose-stimulated insulin secretion. Thus, leptin acts directly on pancreatic beta cells, and stimulation of basal insulin secretion by physiological concentrations of leptin may account in part for the fasting hyperinsulinemia observed in obese subjects.

Leptin induces proliferation of pancreatic beta cell line MIN6 through activation of mitogen-activated protein kinase.

Leptin at 1-5 nM, the concentrations observed in obese subjects, caused an increase in the active form of mitogen-activated protein kinase (MAPK) that was accompanied by increased tyrosine phosphorylation of STAT-1 and STAT-3 in a mouse pancreatic beta cell line, MIN6. Leptin also increased DNA synthesis and cell viability in MIN6 cells based on the results of [3H]-thymidine incorporation and colorimetric MTT assay, respectively. The specific MAPK-inhibitor PD98059 blocked not only the MAPK activation but also the increment in DNA synthesis and cell viability caused by leptin. Thus, leptin stimulates both the MAPK and the Janus kinase (JAK)-STAT cascade as well as inducing proliferation through the MAPK cascade in MIN6 cells. This mechanism might account, at least in part, for obesity-induced pancreatic islet hypertrophy.

Donor leukocyte transfusions and discontinuation of immunosuppressants to achieve an initial remission after allogeneic bone marrow transplantation in a patient with primary refractory acute leukemia.

We present a female patient who received an allogeneic bone marrow transplantation for primary refractory Philadelphia-positive acute biphenotypic leukemia. Since leukemic blasts were persistently present in peripheral blood and bone marrow, in spite of the evidence for engraftment of male donor hematopoiesis, we performed donor leukocyte transfusions and discontinued immunosuppression. An initial complete remission was obtained 15 weeks after allogeneic bone marrow transplantation, and lasted for 24 weeks. We concluded that the prominent mechanism for the eradication of the refractory leukemic clone in the patient was the graft-versus-leukemia effect.

GTP binding to Gs does not promote subunit dissociation.

The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.

Altered Gs alpha N-terminus affects Gs activity and interaction with the G beta gamma subunit complex in cell membranes but not in solution.

The stimulatory G protein (Gs) mediates activation of adenylylcyclase by a ligand-receptor complex. Gs is heterotrimeric (alpha beta gamma) and activation can be accomplished by dissociation of the alpha-subunit (Gs alpha) from the beta gamma-subunit complex (G beta gamma). Gs alpha is also a substrate for choleragen catalyzed ADP-ribosylation when it is associated with G beta gamma but not as free Gs alpha. Using recombinant DNA techniques we modified the cDNA for the 52,000 M(r) form of Gs alpha (Gs alpha 52) to produce a protein with a 2,400 M(r) N-terminal extension (Gs alpha 54.4). This N-terminal extension could be removed with the protease Factor Xa. In vitro transcription and translation of the recombinant plasmid containing the cDNA's for Gs alpha 52 and Gs alpha 54.4 produced a 52,000 M(r) and a 54,000 M(r) protein, respectively. In solution the properties of Gs alpha 52 and Gs alpha 54.4 were indistinguishable. Both proteins: (a) formed a heterotrimer with G beta gamma and their affinities for the subunit complex were the same; (b) could be ADP-ribosylated by choleragen in the presence but not in the absence of G beta gamma; (c) bound the non-hydrolyzable GTP analogue, GTP gamma S, and were protected from chymotryptic proteolysis by the guanine nucleotide; and (d) could activate in vitro translated type IV adenylylcyclase. Gs alpha 54.4 and Gs alpha 52 were incorporated into S49 cyc-membranes, which lack Gs alpha. After incorporation, both Gs alpha 52 and Gs alpha 54.4 were protected from chymotryptic proteolysis when GTP gamma S was present, revealing that both proteins were able to bind the nucleotide and undergo a conformational change characteristic of Gs alpha activation. When Gs alpha 52 was incorporated into cyc-membranes it could mediate both hormone and GTP gamma S stimulation of adenylylcyclase and could be ADP-ribosylated by choleragen, but Gs alpha 54.4 could do neither of these things, indicating that the properties of Gs alpha 54.4 were altered by the membrane. Deletion of the N-terminal extension by treatment with Factor Xa in solution converted Gs alpha 54.4 to Gs alpha 52, and upon incorporation into cyc-membranes it behaved like Gs alpha 52 in every regard, showing that the effect of the N-terminal extension was reversible. A lack of other differences in the functional properties of Gs alpha 52 and Gs alpha 54.4 suggests a correlation between the interaction of Gs alpha with G beta gamma and its ability to activate adenylylcyclase.

Choleragen catalyzes ADP-ribosylation of the stimulatory G protein heterotrimer but not its free alpha-subunit.

The heterotrimeric (alpha beta gamma) stimulatory G protein (Gs) mediates activation of adenylylcyclase. Gs is inactive when GDP is bound to the guanine nucleotide binding site of the alpha-subunit (Gs alpha). Gs can be activated by fluoroaluminate or by binding GTP or GTP analogues, (e.g., GTP gamma S) in place of GDP. Magnesium ion is also required for the activation of Gs, and Gs alpha is a substrate for ADP-ribosylation catalyzed by choleragen (CT). Gs activation can also be accompanied by dissociation of Gs alpha from the G beta gamma-subunit complex. When dissociated Gs subunits were separated by chromatography, isolated Gs alpha could not be ADP-ribosylated by CT unless G beta was added back. RM/1 antiserum against Gs alpha was used to immunoprecipitate Gs, and the subunit composition of the immunoprecipitate was determined. When Gs was incubated with 2 mM MgCl2, the Gs heterotrimer was immunoprecipitated, and Gs alpha could be ADP-ribosylated by CT. Activation of Gs with GTP gamma S or fluoroaluminate in the presence of 2 mM MgCl2 did not cause Gs subunit dissociation nor did it affect the ability of Gs alpha to be ADP-ribosylated by CT. MgCl2 caused a dose-dependent decrease in the amount of G beta that coprecipitated with Gs alpha in the absence as well as the presence of GTP gamma S or fluoroaluminate. Gs subunit dissociation was accompanied by a corresponding decrease in CT-catalyzed ADP-ribosylation of Gs alpha regardless of whether or not GTP gamma S or fluoroaluminate was bound to Gs alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelofibrosis and systemic lupus erythematosus: reversal of fibrosis with high-dose corticosteroid therapy.

A case of myelofibrosis in association with systemic lupus erythematosus (SLE) is reported. Acute thrombocytopenia and a bleeding tendency developed in a 24-year-old woman with SLE. Bone marrow aspiration was unsuccessful due to myelofibrosis. Pulse therapy with methylprednisolone reversed both thrombocytopenia and myelofibrosis. A review of the literature revealed that the coexistence of SLE and myelofibrosis is a rare occurrence. Only 7 cases, to our knowledge, have ever been reported in detail. The present case is the 3rd in which myelofibrosis was reversed by corticosteroids.

Effect of brain-gut peptides upon neurons in centrally regulating sites for drinking.

The effects of angiotensin II (A II) and ANP on spontaneously active neurons in the subfornical organ (SFO), anteroventral third ventricle (AV3V) and supraoptic (SON) and paraventricular nucleus (PVN) were investigated using slice preparations and extracellular recordings. Application of A II (10(-7)M) excited the neural activity of 66% of the SFO neurons, 28% of the AV3V neurons and 44% of the SON neurons. The threshold concentration to produce responses in SFO and AV3V neuron was less than 10(-10)M, while that in SON neurons was 10(-9)M. The excitatory effects of A II were reversibly antagonized by saralasin and persisted after synaptic blockade in a low Ca2+ and high Mg2+ medium. Application of ANP (10(-7)M) inhibited the neural activity of 41% of the AV3V neurons, 22% of the PVN neurons and only 14% of the SFO neurons but had no effect on SON neurons. The threshold concentration for ANP in the AV3V was 10(-11)M. Interestingly, ANP inhibited A II induced excitation in most of the SFO neurons (87%), while ANP had little effects on their spontaneous firing rates. These results show that both peptides of A II and ANP have direct central actions on hypothalamic neurons although ANP can not directly influence magnocellular neurons, suggesting that these blood borne peptides are detected in the SFO and AV3V and that they are acting as a neurotransmitter or a neuromodulator in the central nervous system to regulate water homeostasis.

Fructose-6-phosphate,2-kinase activity in human erythrocytes.

The hemolysate partially purified from human red cells was demonstrated to be capable of synthesizing fructose-2,6-bisphosphate (F-2,6-P2) from fructose-6-phosphate in the presence of adenosine triphosphate (ATP) indicating that human red cells contain fructose-6-phosphate,2-kinase. The effect of F-2,6-P2 on the rate-limiting enzymes of glycolysis, ie, hexokinase, phosphofructokinase (PFK), and pyruvate kinase, has also been examined. PFK was activated by this metabolite and the half-maximum activation was obtained at a concentration of 10(-7) mol/L. Neither hexokinase nor pyruvate kinase was affected by F-2,6-P2. These results suggest that human erythrocytes may contain this metabolite as one of the positive effectors for PFK.