Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice.

AIMS/HYPOTHESIS: Metformin, the major target of which is liver, is commonly used to treat type 2 diabetes. Although metformin activates AMP-activated protein kinase (AMPK) in hepatocytes, the mechanism of activation is still not well known. To investigate AMPK activation by metformin in liver, we examined the role of reactive nitrogen species (RNS) in suppression of hepatic gluconeogenesis. METHODS: To determine RNS, we performed fluorescence examination and immunocytochemical staining in mouse hepatocytes. Since metformin is a mild mitochondrial complex I inhibitor, we compared its effects on suppression of gluconeogenesis, AMPK activation and generation of the RNS peroxynitrite (ONOO(-)) with those of rotenone, a representative complex I inhibitor. To determine whether endogenous nitric oxide production is required for ONOO(-) generation and metformin action, we used mice lacking endothelial nitric oxide synthase (eNOS). RESULTS: Metformin and rotenone significantly decreased gluconeogenesis and increased phosphorylation of AMPK in wild-type mouse hepatocytes. However, unlike rotenone, metformin did not increase the AMP/ATP ratio. It did, however, increase ONOO(-) generation, whereas rotenone did not. Exposure of eNOS-deficient hepatocytes to metformin did not suppress gluconeogenesis, activate AMPK or increase ONOO(-) generation. Furthermore, metformin lowered fasting blood glucose levels in wild-type diabetic mice, but not in eNOS-deficient diabetic mice. CONCLUSIONS/INTERPRETATION: Activation of AMPK by metformin is dependent on ONOO(-). For metformin action in liver, intra-hepatocellular eNOS is required.

Exendin-4 protects pancreatic beta cells from the cytotoxic effect of rapamycin by inhibiting JNK and p38 phosphorylation.

It has been reported that the immunosuppressant rapamycin decreases the viability of pancreatic beta cells. In contrast, exendin-4, an analogue of glucagon-like peptide-1, has been found to inhibit beta cell death and to increase beta cell mass. We investigated the effects of exendin-4 on the cytotoxic effect of rapamycin in beta cells. Incubation with 10 nM rapamycin induced cell death in 12 h in murine beta cell line MIN6 cells and Wistar rat islets, but not when coincubated with 10 nM exendin-4. Rapamycin was found to increase phosphorylation of c-Jun amino-terminal kinase (JNK) and p38 in 30 minutes in MIN6 cells and Wistar rat islets while exendin-4 decreased their phosphorylation. Akt and extracellular signal-regulated kinase (ERK) were not involved in the cytoprotective effect of exendin-4. These results indicate that exendin-4 may exert its protective effect against rapamycin-induced cell death in pancreatic beta cells by inhibiting JNK and p38 signaling.

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells.

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.

An insulinotropic effect of vitamin D analog with increasing intracellular Ca2+ concentration in pancreatic beta-cells through nongenomic signal transduction.

The effect of 1alpha,25-dihydroxylumisterol3 (1alpha,25(OH)2lumisterol3) on insulin release from rat pancreatic beta-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1Alpha,25(OH)2lumisterol3, a specific agonist of mVDR, dose-dependently augmented 16.7 mM glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca2+ concentration ([Ca2+]i), though not increasing Ca2+ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1beta,25-dihydroxyvitamin D3 (1beta,25(OH)2D3), or by a blocker of voltage-dependent Ca2+ channels, nitrendipine. Moreover, both [Ca2+]i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1Alpha,25(OH)2lumisterol3, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca2+ influx through voltage-dependent Ca2+ channels on the plasma membrane, being also linked to metabolic signals derived from glucose in pancreatic beta-cells. However, further investigations will be needed to discuss physiologically the meaning of insulinotropic effects of vitamin D through mVDR.

Spinal fluid cells and protein in amyotrophic lateral sclerosis.

The cerebrospinal fluid total protein in 385 cases of sporadic amyotrophic lateral sclerosis showed no relationship to survival, but it was related to survival time in 34 cases of familial amyotrophic lateral sclerosis. Infrequent and mild pleocytosis, and oligoclonal bands seemed to have no clinical significance in well established cases of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis. Lack of central chromatolytic response of motor neurocytons corresponding to active axonal degeneration.

Ventral spinal roots and anterior horn cells in the lateral nuclear group of the fourth lumbar segment from 21 patients with amyotrophic lateral sclerosis (ALS) and 23 control patients were morphometrically analyzed. The number of large myelinated fibers was remarkably decreased, while small myelinated fibers were well preserved. The population of large myelinated fibers significantly correlated with the population of anterior horn cells. Numerous axonal degenerations were observed in the ventral spinal roots of patients with ALS, even in patients with severe loss of neurons and axons. In spite of this high frequency of active axonal degeneration, the incidence of central chromatolysis of anterior horn cells remained at the control level.

A pedigree with protanopia and bulbospinal muscular atrophy.

We have studied one family of a pedigree in which bulbospinal muscular atrophy and protanopia were segregated. Genotypes for these disorders were obtained on three generations, and the maximum likelihood estimate of recombination fraction was 0.4 with the lod score method. The results indicated that the two loci are not close; the locus for bulbospinal muscular atrophy is not located near the end of the long arm of the X chromosome.

The insulinotropic mechanism of the novel hypoglycaemic agent JTT-608: direct enhancement of Ca(2+) efficacy and increase of Ca(2+) influx by phosphodiesterase inhibition.

We examined the effects of the novel hypoglycaemic agent JTT-608 [trans-4-(4-methylcyclohexyl)-4-oxobutyric acid] on insulin secretion using rat pancreatic islets, and analysed the mechanism of its effect. JTT-608 augmented 8.3 mM glucose-induced insulin secretion dose-dependently, and there was a stimulatory effect of 100 microM JTT-608 at both moderate and high concentrations (8.3, 11. 1 and 16.7 mM) of glucose, but not at low concentrations (3.3 and 5. 5 mM). In perifusion experiments, both phases of insulin release were enhanced, and the effect was eliminated 10 min after withdrawal of the agent. In the presence of 200 microM diazoxide and a depolarizing concentration (30 mM) of K(+), there was an augmentation of insulin secretion by 100 microM JTT-608, not only under high levels of glucose but also under low levels, and the effects were abolished by 10 microM nitrendipine. JTT-608 augmented insulin secretion from electrically permeabilized islets in the presence of stimulatory concentrations (0.3 and 1.0 microM) of Ca(2+), and the intracellular Ca(2+) concentration ([Ca(2+)](i)) response under 16.7 mM glucose, 200 microM diazoxide, and 30 mM K(+) was also increased. The cyclic AMP content in the islets was increased by 100 microM JTT-608, and an additive effect to 1 microM forskolin was observed, but not to 50 microM 3-isobutyl-1-methylxanthine (IBMX). JTT-608 inhibited phosphodiesterase (PDE) activity dose-dependently. We conclude that JTT-608 augments insulin secretion by enhancing Ca(2+) efficacy and by increasing Ca(2+) influx. This appears to be a result of the increased intracellular cyclic AMP concentration due to PDE inhibition.

Peripheral nerve involvement in familial chorea-acanthocytosis.

Myelinated fibers and neuronal cell bodies of the ventral spinal outflow and primary sensory neurons were histopathologically examined in a patient with familial chorea-acanthocytosis and age-matched controls. The patient exhibited a marked loss of large myelinated axons and their neuronal cell bodies in the ventral spinal outflow, while there was frequent occurrence of axonal sprouts. Large myelinated fibers in sensory afferents were also decreased in number. Segmental de- and remyelination was markedly increased in teased fiber preparations of both motor and sensory peripheral nerves.

Pathology of myelinated fibers in cervical and lumbar ventral spinal roots in amyotrophic lateral sclerosis.

Pathological alterations were evaluated by morphometry and by a teased-fiber study on the 6th cervical (C6) and the 4th lumbar (L4) ventral spinal roots of cases of amyotrophic lateral sclerosis (ALS). The large-diameter fibers were severely affected in both spinal segments. However, small-diameter myelinated fibers were numerically well preserved. The number of large fibers in C6 and L4 ventral roots was strongly correlated to the strength of muscles innervated by C6 or L4 segments. There was no correlation of the number of small fibers with muscle strength. Teased fiber studies revealed a marked increase in the incidence of fibers showing axonal degeneration. Fibers considered to be regenerative were rarely observed. These observations suggest that large myelinated fibers, which correspond to alpha-motoneuron fibers, are selectively affected, and that small myelinated fibers, which are considered to correspond to gamma-motoneuron fibers, are preserved to some extent in the C6 and L4 ventral spinal roots in ALS.

Subclinical phenotypic expressions in heterozygous females of X-linked recessive bulbospinal neuronopathy.

Four of 8 definite heterozygous female carriers determined by PCR amplification of tandem CAG repeat of the AR gene, from 4 families of X-linked recessive bulbospinal neuronopathy (X-BSNP) showed extensive high amplitude motor unit potentials in examined muscles although all subjects were neurologically normal. Plasma creatine kinase, myoglobin, myosin light chain, lactate and pyruvate were all normal even in the carriers who showed EMG abnormalities. Muscle biopsy showed a type 2 fiber preponderance and possible very mild type 2 fiber grouping in a carrier with an EMG abnormality. These results suggest that a mutant AR gene may express subclinical phenotypic manifestations in a subpopulation of the heterozygous females of X-BSNP.

Onset, natural history and outcome in idiopathic adult motor neuron disease.

Cases of adult-onset idiopathic motor neuron disease (MND) identified from January 1970 through December 1986 were studied in a defined area of California. The patients were followed prospectively throughout the illness in 99% of cases. Among 708 cases aged 25-74 years at onset, the most common type (86%) was typical, sporadic amyotrophic lateral sclerosis (SporALS). The risk of bulbar onset and shorter survival times increased with age in both men and women. About 4%, mainly younger men, experienced unusually long courses with milder paralysis, but could not be identified early in the illness. They probably represent one extreme of the ALS spectrum rather than a distinct subtype. Familial ALS (FamALS) was diagnosed in 7%. It developed earlier in life but ran a slightly longer course, which suggests a different disease process. Overall there was a statistically significant predominance of males, especially in 17 cases (2%) of progressive muscular atrophy (PMA). There were 26 cases (4%) classified as primary lateral sclerosis (PLS). Progressive bulbar palsy was not found; that diagnosis usually denotes merely the bulbar onset of ALS.

Aberrant androgen action and increased size of tandem CAG repeat in androgen receptor gene in X-linked recessive bulbospinal neuronopathy.

Plasma levels of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) after 3 or 6 days of administration of the synthetic androgenic hormone fluoxymesterone (10 mg/day) were measured in 26 patients with X-linked recessive bulbospinal neuronopathy (X-BSNP) and 22 age-matched male controls. The testosterone, LH and FSH levels in the controls were markedly suppressed after administration, but in the patients with X-BSNP, they were suppressed significantly less. The level of suppression varied considerably with the patients, and those of plasma testosterone and FSH were significantly correlated with the number of CAG repeats in the androgen receptor gene. These findings suggest that the androgen action was aberrantly transduced in the target organs in the patients with X-BSNP and which is related to the elongated CAG repeat in the androgen receptor gene.

CAG repeat expansion of Machado-Joseph disease in the Japanese: analysis of the repeat instability for parental transmission, and correlation with disease phenotype.

Machado-Joseph disease (MJD) is caused by abnormal expansion of an unstable CAG repeat in a novel gene locating on chromosome 14q32.1. We analysed this CAG repeat polymorphism with 66 Japanese MJD patients. All the patients were selectively associated with abnormal expansion of the CAG repeat. Repeat length of the mutant allele did not overlap that of normal allele and closely correlated with not only age at onset but also with clinical phenotypes. CAG repeat size is apparently related to a wide variety of phenotypic presentations in MJD.

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets.

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K(+) channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 microM) and higher (250 microM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca(2+) concentration ([Ca(2+)](i)) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 microM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca(2+)](i), PKC activation elicited glucose-induced insulin secretion at 100 and 250 microM diazoxide, while PKA activation did so only at 100 microM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 microM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.

Comparative study of symptoms and neuroendocrine-immune network mediator levels between rheumatoid arthritis patients and healthy subjects.

OBJECTIVE: In order to understand the disturbances in the neurophysiological, endocrine (including the hypothalamic-pituitary-adrenal axis), and immune systems objectively and in detail, we measured and compared various test items in the peripheral blood which were considered to reflect the state of these systems, in patients with rheumatoid arthritis and in control subjects. METHODS: The levels of beta-endorphin, methionine-enkephalin, epinephrin, norepinephrin (NE), dopamine, corticotropin releasing factor (CRF), adrenocoricotropic hormone (ACTH), cortisol, CD4/CD8 ratio, CD57, NK cell activity and IL-6 in the peripheral blood, which are considered to reflect the activity of this neuroendocrine-immune network, were measured and compared between 49 patients with rheumatoid arthritis (RA) and 54 healthy control subjects. The face scale (to measure mood) and the Cornell medical index (CMI) health questionnaire were administered to both groups, and pain scores were measured using a visual analog scale in the RA group. RESULTS: The serum levels of NE, dopamine, IL-6 and CD4/CD8 ratio were higher, whereas the levels of beta-endorphine, ACTH and NK cell activity were lower in the RA subjects than in the control subjects. On the other hand, the serum levels of Met-enk, epinephrin, CRF, cortisol and CD57 were not significantly different between the two groups. In RA patients a positive correlation was observed between the face scale score and the serum cortisol level and between the pain score and the serum IL-6 level. The more severe the pain, the higher the NK cell activity and IL-6 concentrations in the peripheral blood. On the other hand, in healthy females none of the measured items in the peripheral blood were significantly correlated with the face scale results or the responses to the CMI health questionnaire. CONCLUSION: In RA patients the hypothalamic-pituitary-adrenal (HPA) axis is altered and this condition is correlated to a deterioration in symptoms.

Enhanced delivery of mitomycin C derivatives through hairless mouse and rat skins.

The percutaneous permeation characteristics of two lipophilic mitomycin C derivatives with aromatic moieties were determined using excised hairless mouse and rat skins and compared with those of mitomycin C (MMC). 1a-N-Benzylmitomycin C penetrated more readily through both kinds of skin than MMC without metabolic conversion. 1a-N-Benzyloxycarbonylmitomycin C effected a 4-fold increase in the delivery of MMC through the rat skin and was completely converted to MMC by an enzyme in rat skin. However, saturation of the metabolic conversion activity was observed in the hairless mouse skin.

[Development and evaluation of a local area network system to make drug information paper for inpatient--adaptation to characteristics of diseases, and expansibility to other hospitals and the Internet].

We developed and evaluated a local area network system to make drug information papers. Because pharmacists should explain drug information both orally and using papers to patients in response to their understanding and characteristics of their diseases. The merit of this system is as follows: pharmacists can use it without any education for its operation; most data can be inputted by selection from the lists and automatic reference; the data are adjustable for individuals; different contents are available for different wards; multiple users can access the same data file. Input data in this system are available for making explanation papers on the medical examination of outpatients, and making a plan for pharmaceutical care and guidance services. This system is also available in other hospitals, and on the internet. First, we surveyed the time required for preparing drug information papers. Making a hand written paper for typical patient takes 395 sec. for manuscript, 160 sec. with the system using text data only, and 178 sec. with the system using text and picture data. Next, we surveyed 27 patients' views on the addition of drug pictures to drug information papers, and with the result that 19 patients thought it very good. Last, we surveyed 13 ward pharmacists about their usage of this system. We found that 8 pharmacists used frequently this system, and 3 pharmacists used the same file in the same hours at the maximum.

[Development and assessment of the pharmaceutical management and guidance services for inpatients considering clinical value and economy].

We developed pharmaceutical management and guidance services for inpatients in a ward of circulatory medicine, considering clinical and economical standpoints. In these services, pharmacists deliver drugs prescribed for inpatients with individual drug information papers, explain to them about their drugs using information papers and give counsel. Since most of the patients were aged people, developing many kinds of diseases and taking many kinds of drugs, they had many problems such as lack of knowledge of the effects of drugs. First, we surveyed views of patients, physicians and nurses on these services. Consequently, all of them advised us that "pharmacists should explain to patients about the prescribed drugs using information papers." The patients preferred pharmacists as expositors of drugs to physicians or nurses. The physicians considered that "pharmacists have to attach importance to clinical information and package-inserts of drugs and explain to patients about drug information using pamphlet in response to the understanding of patients." The nurses wanted to cooperate with pharmacists in "improving medication compliance." On the basis of these views, we improved our services. Next, we made a survey of patients' knowledge about their drugs. We found that in the patients the level of knowledge concerning "ways," "effects" and "reasons" of taking drugs and that of "compliance" and "satisfaction in taking drugs" were improved through these services. The patients reentered in the hospital kept a high level. The ratio of patients taking drugs by themselves increased. Last, we also applied this method to wards of "blood and collagen diseases" and "pediatrics." The demand for these services increased smoothly. We compared these services based on our method with all other services by hospital pharmacists from the viewpoint of economy. We found that only our service method was beneficial.

[Improvement of method to estimate guidance by pharmacists and trial to obtain standard pharmaceutical management and guidance services program].

The level of understanding of taking medicine was examined at the start and the end of medicine-taking guidance, based on the estimation by pharmacists and inpatients. Six items for the estimation, such as "the way of taking medicines" were evaluated using a five-grade system. The significant improvement was observed in all items, suggesting that inpatients' understanding is improved by the guidance. A markedly positive correlation was found between the estimation by inpatients and that by pharmacists (p < 0.001). This confirms the appropriateness of estimation by pharmacists. Inpatients whose estimation was significantly different from that by pharmacists (difference of two grades or more) were examined to identify clear and potential problems with taking medicine. The problems were classified into such seven categories as "types of medicine". For each item with differences in estimation (two grades or more), problems were biased in some specific categories. On the basis of such a bias, a flow chart was prepared to clarify problems. In addition, a standard pharmaceutical management and guidance services program was drafted, in which measures in observation, treatment and education against problems by pharmacists were described according to the frequency of occurrence. Information about the program available on the Internet enables its use by other hospitals.