Global gene expression analysis in liver of obese diabetic db/db mice treated with metformin.

AIMS/HYPOTHESIS: Metformin is widely used as a hypoglycaemic reagent for type 2 diabetes. While the reduction of hepatic gluconeogenesis is thought to be a key effect, the detailed molecular mechanism of action of metformin remains to be elucidated. To gain insight into this, we performed a global gene expression profiling study. MATERIALS AND METHODS: We performed DNA microarray analysis to study global gene expression in the livers of obese diabetic db/db mice 2 h after a single administration of metformin (400 mg/kg). RESULTS: This analysis identified 14 genes that showed at least a 1.5-fold difference in expression following metformin treatment, including a reduction of glucose-6-phosphatase gene expression. The mRNA levels of glucose-6-phosphatase showed one of the best correlations with blood glucose levels among 12,000 genes. Enzymatic activity of glucose-6-phosphatase was also reduced in metformin-treated liver. Moreover, intensive analysis of the expression profile revealed that metformin effected significant alterations in gene expression across at least ten metabolic pathways, including those involved in glycolysis-gluconeogenesis, fatty acid metabolism and amino acid metabolism. CONCLUSIONS/INTERPRETATION: These results suggest that reduction of glucose-6-phosphatase activity, as well as suppression of mRNA expression levels of this gene, in liver is of prime importance for controlling blood glucose levels in vivo, at least at early time points after metformin treatment. Our results also suggest that metformin not only affects expression of specific genes, but also alters the expression level of multiple genes linked to the metabolic pathways involved in glucose and lipid metabolism in the liver.

The effects of brain-derived neurotrophic factor on insulin signal transduction in the liver of diabetic mice.

AIM/HYPOTHESIS: We previously reported that repeated subcutaneous or intracerebroventricular injection of brain-derived neurotrophic factor (BDNF) reduces blood glucose concentrations in obese diabetic C57BL/KsJ-db/db mice. In this study, we assessed the effects of BDNF on insulin action in peripheral tissues of diabetic mice. METHODS: First, brain-derived neurotrophic factor (20 mg/kg) was subcutaneously given to male db/db mice for 14 days and then the insulin-stimulated tyrosine phosphorylation of insulin receptors and insulin-stimulated phosphatidylinositol (PI) 3-kinase activity in peripheral tissues was assessed. Second, we examined the effects of a single subcutaneous or intracerebroventricular brain-derived neurotrophic factor injection on insulin responsiveness in liver and skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Third, the effects of brain-derived neurothrophic factor on insulin action were also examined in cultured cells. RESULTS: Repeated injection of BDNF to db/db mice for 14 days enhanced insulin-stimulated tyrosine phosphorylation of insulin receptors in liver and insulin-stimulated PI 3-kinase activity in liver, skeletal muscle and interscapular brown adipose tissue. We then examined the rapid effect of BDNF on insulin signalling in vivo. A single subcutaneous or intracerebroventricular injection of BDNF rapidly increased insulin-stimulated tyrosine phosphorylation of insulin receptors and PI 3-kinase activity in liver of STZ-mice. No direct effect of brain-derived neurothrophic factor was observed on insulin signalling in primary cultured hepatocytes, L6 muscle cells or 3T3-L1 adipocytes. Brain-derived neurothrophic factor did not affect either glucose uptake or gluconeogenesis in these cells. CONCLUSION/INTERPRETATION: These data indicate that brain-derived neurothrophic factor rapidly enhances insulin signal transduction in liver and shows hypoglycaemic action in diabetic mice.

Brain-derived neurotrophic factor reduces blood glucose level in obese diabetic mice but not in normal mice.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. However, it is not yet known if BDNF works on the endocrine system itself. Here we report that BDNF improves hyperglycemia in obese diabetic animals. BDNF reduced the blood glucose level in obese db/db diabetic mice in which the effect of BDNF was age-dependent and high under the condition of hyperinsulinemia, while BDNF showed no effect on non-diabetic db/m mice. These results suggest that BDNF ameliorates insulin resistance by enhancing insulin action in peripheral tissues. Furthermore, BDNF was found to reduce the plasma insulin level in db/db mice. Among the neurotrophin family, NT-3 also reduced the blood glucose level in db/db mice. These results provide a novel insight that neurotrophin functions on the endocrine system as well as the nervous system.

Source of ATP for hexokinase-catalyzed glucose phosphorylation in tumor cells: dependence on the rate of oxidative phosphorylation relative to that of extramitochondrial ATP generation.

We isolated highly intact and tightly coupled mitochondria from the rat ascites hepatoma cell line AH130 by disruption of the cell membrane by nitrogen cavitation. These isolated mitochondria were found to have essentially the same functional properties as rat liver mitochondria, but unlike the latter, hexokinase (HK) was bound to their membrane. Using the tumor mitochondrial preparation, we examined the source of ATP for phosphorylation of glucose by HK under conditions in which intra- and extramitochondrial ATP-generation systems operated separately or together. Results showed that the membrane-bound HK utilized ATP derived from the most efficiently operating ATP generation system, i.e., oxidative phosphorylation. However, when the rate of extramitochondrial ATP generation was much greater than that of oxidative phosphorylation, HK used ATP from the extramitochondrial ATP-generation system.

Nucleotide sequence of the 5'-flanking region of the rat type II hexokinase gene.

In a previous study, we found that the steady state transcript level of type II hexokinase was specifically and remarkably elevated in rat hepatoma AH130 cells. To determine the molecular mechanism of transcriptional control of the type II hexokinase gene, we examined the nucleotide sequence of its 5'-flanking region and analyzed putative transcription factor binding sites. We also examined the type II hexokinase promoter activity by the chloramphenicol acetyltransferase (CAT) assay.

Steady state transcript levels of the type II hexokinase and type 1 glucose transporter in human tumor cell lines.

The steady state transcript levels of two hexokinase isozymes and type 1 glucose transporter in human tumor cell lines were analyzed. In HepG2 cells, both type II hexokinase and type 1 glucose transporter were highly expressed. However, in cell lines A431 and HeLa, in which the expression level of type 1 glucose transporter was lower than that in HepG2 cells, the amount of type II hexokinase transcript was almost negligible.

Remarkably enhanced expression of the type II hexokinase in rat hepatoma cell line AH130.

Expression of mRNAs encoding hexokinase isozymes was studied in various cells such as rat brain, liver, skeletal muscle, kidney and heart, and the rat hepatoma cell line AH130 by Northern blotting. High specific expression of type II hexokinase was observed only with AH130 cells. In contrast, specific expression of type I hexokinase was detected in energy-requiring normal tissue cells such as brain and heart. These results suggest that the expression of hexokinase isozyme in the tumor cells is different from that in normal cells.

Effect of vasoconstrictor and vasodilator on isolated human umbilical vein.

The mechanical properties of the longitudinal and circular muscle tissues of the human umbilical vein and the effects of nicardipine were investigated. Spontaneous contractions were observed in the isometric condition. When high concentrations of potassium (118mM-K+), oxytocin and serotonin were applied, these substances evoked contractions. Oxytocin (10(-4)-10(-2) U/ml) enhanced the spontaneous contractions. Serotonin produced contractions at 10(-9)-10(-4) M in both the longitudinal and circular muscle strips. When nicardipine (10(-5) M) was applied, contractions induced by 118mM-K+ were completely inhibited, but contractions induced by serotonin could not be completely abolished, i.e., tonic contractions ceased but small phasic contractions continued. Nicardipine (10(-8) M) did not inhibit the amplitude and frequency of spontaneous contractions, but decreased the basal tonus. These results suggested that nicardipine might improve feto-placental blood flow while decreasing the spontaneous basal tonus.

Modification by magnesium of the excitatory effect of oxytocin in electrical and mechanical activities of pregnant human myometrium.

The modification by magnesium of the excitatory effect of oxytocin (10(-5)-10(-2) U/mL) on electrical and mechanical activities of pregnant human myometrium was examined. The excitatory effect of oxytocin was enhanced by external magnesium, and the dose-response curve between oxytocin and relative tension in the presence of 118 mM potassium in tiny muscle strips shifted to the left with increases in magnesium from 0 to 2.4 mM. Oxytocin potentiates spontaneous contractions by enhancing the plateau part of action potentials, and the plateau potential induced in 2.4-mM magnesium was larger than that in magnesium-free solution. In potassium contracture experiments, the muscle contraction was potentiated in accordance with the concentration of preloaded magnesium when 10(-3) U/mL oxytocin was added at the tonic phase. These results suggest that magnesium might primarily potentiate the excitatory effect of oxytocin in electrical and mechanical activities of pregnant human myometrium at superficial sites of the plasma membrane, allowing the possibility of its intracellular action.

Gestational changes in mechanical properties of skinned muscle tissues of human myometrium.

We developed a technique to obtain very thin myometrial muscle strips that allowed comparison of characteristic features of contraction from the same strip under intact and membrane-permeable ("skinned") conditions. Absolute tension development per unit of cross-sectional area induced by high potassium and oxytocin concentrations in the intact condition, or by Ca2+ in the skinned condition, markedly increased in human myometrium at term compared with the nonpregnant state. The maximum tension in skinned strips was 8.3 mN/mm2 in nonpregnant strips and 47.5 mN/mm2 at term (both obtained at 10 mumol/L Ca2+). The Ca2+ sensitivity of the contractile proteins in skinned strips also increased at term compared with the nonpregnant state; the half-maximal response of Ca2+ sensitivity was 0.7 mumol/L in the nonpregnant state and 0.3 mumol/L at term. These results suggest that in human myometrium both the amount of contractile proteins and their sensitivity to Ca2+ may increase at term compared with the nonpregnant state.

[Fundamental studies on transcutaneous bilirubinometry in newborn infants using an organ scanning spectrophotometer].

In vivo reflectance spectra in the visible region were recorded on the blanched skin on the foreheads, chests, arms and legs of 140 newborn infants using an organ scanning spectrophotometer, and corresponding total serum bilirubin levels were determined. An elevation in the absolute spectra was observed in the 460 nm region for the higher bilirubin level, and the isosbestic point was located in the 510 nm region. A linear relationship was observed between the reflectance spectra difference and the serum bilirubin level. Although the best analysis results were obtained from the measurements of the reflectance spectra difference between 460 nm and 510 nm on the chests (r = 0.954, Y = 40.62X + 1.36), or on sums of 4 measuring sites (r = 0.973, Y = 46.59X + 0.61), there was no statistical difference from the values obtained between 460 nm and 550 nm on the foreheads (r = 0.913, Y = 33.55X + 5.30). The slopes and the Y-intercepts of the linear regressions differed considerably at various measuring sites. Marked decreases in the reflectance spectra differences were observed during the phototherapies and prominent rebounds followed for 24 hours after the cessation of phototherapies. The noninvasive and simple reflectance method was sensitive to the bilirubin level in the tissue and might be a better indicator of neonatal risk of kernicterus. However, the optical method has a certain limitations in representing the bilirubin concentration in the blood stream, especially during, and for 24 hours, after phototherapies.