Hiroshi Tsuneki

Effect of green tea on blood glucose levels and serum proteomic patterns in diabetic (db/db) mice and on glucose metabolism in healthy humans

BackgroundGreen tea is widely consumed in Asian countries and is becoming increasingly popular in Western countries. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The present study was aimed at providing evidence of improvement in glucose metabolism in diabetic mice and healthy humans upon green tea consumption.ResultsGreen tea promoted glucose metabolism in healthy human volunteers at 1.5 g/body in oral glucose tolerance tests. Green tea also lowered blood glucose levels in diabetic db+/db+ mice and streptozotocin-diabetic mice 2–6 h after administration at 300 mg/kg without affecting serum insulin level, whereas no effect was observed in control mice (+m/+m and normal ddY mice). The serum protein profiles of db+/db+ and +m/+m mice were analyzed for the first time by SELDI (surface-enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), and then compared to investigate any effects of oral green tea administration on serum proteins. The protein profiles in db+/db+ mice showed that the spectral peak intensities at the mass/charge ratios (m/z) of 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those of 13075, 17406, 17407, 17418, 17622, 18431 and 26100 were >3 times higher than respective peak intensities in +m/+m mice. When green tea was administered to db+/db+ mice, the peak intensities were markedly decreased at m/z 11651 and 11863, and slightly decreased at m/z 4212. The peak intensities at 7495, 7595, 7808, 14983, 15614, 31204 were markedly increased after the administration.ConclusionThe present study provides evidence that green tea has an antidiabetic effect. Although we could not find simple reversed effect of green tea on the diabetes-induced modifications of the levels of several serum proteins, we found that the 4211 (4212) Da protein level that was decreased in the diabetic state was further decreased after green tea administration. This is the first report demonstrating that a certain serum protein may be involved in the antihyperglycemic effect of green tea. The contribution of this protein should be further studied.

Spironolactone improves glucose and lipid metabolism by ameliorating hepatic steatosis and inflammation and suppressing enhanced gluconeogenesis induced by high-fat and high-fructose diet.

Recent evidence suggests that treatment with mineralocorticoid receptor antagonist suppressed local inflammation in vascular tissues or cardiomyocytes; therefore, we examined the effect of spironolactone on glucose and lipid metabolism in a mouse model with diet-induced diabetes and nonalcoholic fatty liver disease. C57BL/6 mice were fed either the control diet, 60% fat diet with 30% fructose water (HFFD), or HFFD with spironolactone for 8 wk. HFFD mice demonstrated apparent phenotypes of metabolic syndrome, including insulin resistance, hypertension, dyslipidemia, and fatty liver. Although treatment with spironolactone did not affect the increased calorie intake and body weight by HFFD, the increments of epididymal fat weight, blood pressure, serum triglyceride, free fatty acids, leptin, and total cholesterol levels were significantly suppressed. Elevation of blood glucose during glucose and insulin tolerance tests in HFFD mice was significantly lowered by spironolactone. Notably, increased glucose levels during pyruvate tolerance test in HFFD mice were almost completely ameliorated to control levels by the treatment. Staining with hematoxylin-eosin (HE) and Oil-red-O demonstrated marked accumulation of triglycerides in the centrilobular part of the hepatic lobule in HFFD mice, and these accumulations were effectively improved by spironolactone. Concomitantly HFFD feeding markedly up-regulated hepatic mRNA expression of proinflammatory cytokines (TNFalpha, IL-6, and monocyte chemoattractant protein-1), gluconeogenic gene phosphoenolpyruvate carboxykinase, transcription factor carbohydrate response element binding protein, and its downstream lipogenic enzymes, all of which were significantly suppressed by spironolactone. These results indicate that inhibition of mineralocorticoid receptor might be a beneficial therapeutic approach for diet-induced phenotypes of metabolic syndrome and fatty liver.

Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice.

Aims/hypothesisOrexin/hypocretin is a hypothalamic neuropeptide that regulates motivated behaviours, such as feeding and arousal, and, importantly, is also involved in energy homeostasis. The aim of this study was to reveal the role of orexin in the regulation of insulin sensitivity for glucose metabolism.MethodsOrexin knockout mice fasted overnight underwent oral glucose tolerance testing and insulin tolerance testing. The impact of orexin deficiency on insulin signalling was studied by Western blotting to measure levels of Akt phosphorylation and its upstream and downstream molecules in the hypothalamus, muscle and liver in orexin knockout mice.ResultsWe found that orexin deficiency caused the age-related development of impaired glucose tolerance and insulin resistance in both male mice without obesity and female mice with mild obesity, fed a normal chow diet. When maintained on a high-fat diet, these abnormalities became more pronounced exclusively in female orexin knockout mice that developed severe obesity. Insulin signalling through Akt was disrupted in peripheral tissues of middle-aged (9-month-old) but not young adult (2-to-3-month-old) orexin knockout mice fed a normal chow diet. Moreover, basal levels of hypothalamic Akt phosphorylation were abnormally elevated in orexin knockout mice at every age studied, and insulin stimulation failed to increase the level of phosphorylation. Similar abnormalities were observed with respect to GSK3β phosphorylation in the hypothalamus and peripheral tissues of middle-aged orexin knockout mice.Conclusions/interpretationOur results demonstrate a novel role for orexin in hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with age.

Aldosterone Inhibits Insulin-Induced Glucose Uptake by Degradation of Insulin Receptor Substrate (IRS) 1 and IRS2 via a Reactive Oxygen Species-Mediated Pathway in 3T3-L1 Adipocytes

Serum aldosterone level is clinically known to correlate with body weight and insulin resistance. Because the underlying molecular mechanism is largely unknown, we examined the effect of aldosterone on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. Aldosterone reduced the amounts of insulin receptor substrate (IRS) 1 and IRS2 in a time- and dose-dependent manner. As a result, insulin-induced phosphorylation of Akt-1 and -2, and subsequent uptake of 2-deoxyglucose were decreased. Degradation of IRSs was effectively prevented by a glucocorticoid receptor antagonist and antioxidant N-acetylcysteine, but not by a mineralocorticoid receptor antagonist. Because aldosterone induced phosphorylation of IRS1 at Ser(307), responsible kinases were investigated, and we revealed that rapamycin and BMS345541, but neither SP600125 nor calphostin C, conferred for degradation of IRSs. Although lactacystin prevented the degradation of IRSs, glucose uptake was not preserved. Importantly, sucrose-gradient-sediment intracellular fraction analysis revealed that lactacystin did not effectively restore the reduction of IRS1 in the low-density microsome fraction, important for the transduction of insulins metabolic signaling. These results indicate that aldosterone deteriorates metabolic action of insulin by facilitating the degradation of IRS1 and IRS2 via glucocorticoid receptor-mediated production of reactive oxygen species, and activation of IkappaB Kinase beta and target of rapamycin complex 1. Thus, aldosterone appears to be a novel key factor in the development of insulin resistance in visceral obesity.

Central versus peripheral impact of estradiol on the impaired glucose metabolism in ovariectomized mice on a high-fat diet

Age-related loss of ovarian function promotes adiposity and insulin resistance in women. Estrogen (E(2)) directly enhances insulin sensitivity and suppresses lipogenesis in peripheral tissues. Recently, the central actions of E(2) in the regulation of energy homeostasis are becoming clearer; however, the functional relevance and degree of contribution of the central vs. peripheral actions of E(2) are currently unknown. Therefore, we prepared and analyzed four groups of mice. 1) CONTROL: sham-operated mice fed a regular diet, 2) OVX-HF: ovariectomized (OVX) mice fed a 60% high-fat diet (HF), 3) E2-SC: OVX-HF mice subcutaneously treated with E(2), and 4) E2-ICV: OVX-HF mice treated with E(2) intracerebroventricularly. OVX-HF mice showed increased body weight with both visceral and subcutaneous fat volume enlargement, glucose intolerance, and insulin resistance. Both E2-SC and E2-ICV equally ameliorated these abnormalities. Although the size of adipocytes and number of CD11c-positive macrophages in perigonadal fat in OVX-HF were reduced by both E(2) treatments, peripherally administered E(2) decreased the expression of TNFα, lipoprotein lipase, and fatty acid synthase in the white adipose tissue (WAT) of OVX-HF. In contrast, centrally administered E(2) increased hormone-sensitive lipase in WAT, decreased the hepatic expression of gluconeogenic enzymes, and elevated core body temperature and energy expenditure with marked upregulation of uncoupling proteins in the brown adipose tissue. These results suggest that central and peripheral actions of E(2) regulate insulin sensitivity and glucose metabolism via different mechanisms, and their coordinated effects may be important to prevent the development of obesity and insulin resistance in postmenopausal women.

Role of orexin in the regulation of glucose homeostasis.

Orexin‐A (hypocretin‐1) and orexin‐B (hypocretin‐2) are hypothalamic neuropeptides that play key roles in the regulation of wakefulness, feeding, reward, autonomic functions and energy homeostasis. To control these functions indispensable for survival, orexin‐expressing neurones integrate peripheral metabolic signals, interact with many types of neurones in the brain and modulate their activities via the activation of orexin‐1 receptor or orexin‐2 receptor. In addition, a new functional role of orexin is emerging in the regulation of insulin and leptin sensitivities responsible for whole‐body glucose metabolism. Recent evidence indicates that orexin efficiently protects against the development of peripheral insulin resistance induced by ageing or high‐fat feeding in mice. In particular, the orexin receptor‐2 signalling appears to confer resistance to diet‐induced obesity and insulin insensitivity by improving leptin sensitivity. In fact, the expression of orexin gene is known to be down‐regulated by hyperglycaemia in the rodent model of diabetes, such as ob/ob and db/db mice. Moreover, the levels of orexin receptor‐2 mRNA have been shown to decline in the brain of mice along with ageing. These suggest that hyperglycaemia due to insulin insensitivity during ageing or by habitual consumption of a high‐fat diet leads to the reduction in orexin expression in the hypothalamus, thereby further exacerbating peripheral insulin resistance. Therefore, orexin receptor controlling hypothalamic insulin/leptin actions may be a new target for possible future treatment of hyperglycaemia in patients with type 2 diabetes.

The Disrupted-in-Schizophrenia-1 Ser704Cys polymorphism and brain morphology in schizophrenia

The Disrupted-in-Schizophrenia-1 (DISC1) polymorphism is a strong candidate for a schizophrenia-susceptibility gene as it is widely expressed in cortical and limbic regions, but the effect of its genotype variation on brain morphology in schizophrenia is not well known. This study examined the association between the DISC1 Ser704Cys polymorphism and volumetric measurements for a broad range of fronto-parietal, temporal, and limbic-paralimbic regions using magnetic resonance imaging in a Japanese sample of 33 schizophrenia patients and 29 healthy comparison subjects. The Cys carriers had significantly larger volumes of the medial superior frontal gyrus and short insular cortex than the Ser homozygotes only for healthy comparison subjects. The Cys carriers tended to have a smaller supramarginal gyrus than the Ser homozygotes in schizophrenia patients, but not in healthy comparison subjects. The right medial superior frontal gyrus volume was significantly correlated with daily dosage of antipsychotic medication in Ser homozygote schizophrenia patients. These different genotype effects of the DISC1 Ser704Cys polymorphism on the brain morphology in schizophrenia patients and healthy comparison subjects suggest that variation in the DISC1 gene might be, at least partly, involved in the neurobiology of schizophrenia. Our findings also suggest that the DISC1 genotype variation might have some relevance to the medication effect on brain morphology in schizophrenia.

Robust stimulation of TrkB induces delayed increases in BDNF and Arc mRNA expressions in cultured rat cortical neurons via distinct mechanisms.

In cultures of rat cortical neurons, we found that stimulation of tyrosine receptor kinase B (TrkB) with brain‐derived neurotrophic factor (BDNF) induced a biphasic expression of BDNF exon IV–IX mRNA, which became obvious 1–3 h (primary induction) and 24–72 h (delayed induction) after the stimulation, and characterized the delayed induction in relation to the mRNA expression of activity‐regulated cytoskeleton‐associated protein (Arc). Withdrawal of BDNF from the medium after stimulation for 3 h allowed the delayed induction, which was caused at the transcriptional level and dependent upon the initial contact between exogenously added BDNF and TrkB, the effect of which was time‐ and dose‐dependent. The primary induction was controlled by the extracellular signal‐regulated kinase/mitogen‐activated protein kinase (ERK/MAPK) whereas the secondary induction by the calcium (Ca2+) signaling pathway. The enhanced Arc or Zif268 mRNA expression was controlled by activation of the ERK/MAPK pathway, both of which were repressed by blocking the binding of endogenously synthesized BDNF to TrkB. Thus, robust stimulation of TrkB autonomously induces delayed BDNF mRNA expression in an activity‐dependent manner in rat cortical neurons, resulting in the stimulation of Arc mRNA expression through endogenously synthesized BDNF, the process being orchestrated by the Ca2+ and ERK/MAPK signaling pathways.

Immunohistochemical localization of neuronal nicotinic receptor subtypes at the pre-and postjunctional sites in mouse diaphragm muscle

The existence of neuronal nicotinic acetylcholine receptor (nAChR) subunits was investigated in the cryostat sections of mouse diaphragm muscles using the indirect immunofluorescence technique. The specific immunolabelings with monoclonal antibodies (mAbs) to beta 2 and to alpha 8 subunits of neuronal nAChR were observed at the endplate determined by labeling with a fluorescent dye (BODIPY)-conjugated alpha-bungarotoxin. The immunoreactivity of mAb to the alpha 3 subunit of neuronal nAChR was detected on the motor nerve fibers including the nerve terminals. These results provide evidence that the subtypes of postsynaptic nAChR, recognized by the anti-beta 2 and/or anti-alpha 8 mAbs, and the presynaptic nAChR recognized by the anti-alpha 3 mAb, are present at the neuromuscular junction, in addition to the classical muscle nAChR.

Association between the brain-derived neurotrophic factor Val66Met polymorphism and brain morphology in a Japanese sample of schizophrenia and healthy comparisons

Magnetic resonance imaging was used to investigate the relation between the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and volumetric measurements for the medial temporal lobe structures (amygdala, hippocampus, and parahippocampal gyrus) and prefrontal sub-regions (the superior frontal gyrus, middle frontal gyrus, inferior frontal gyrus, ventral medial prefrontal cortex, orbitofrontal cortex, and straight gyrus) in a Japanese sample of 33 schizophrenia patients and 29 healthy subjects. For the controls, the Met carriers had significantly smaller parahippocampal and left superior frontal gyri than the Val homozygotes. The schizophrenia patients carrying the Met allele had a significantly smaller right parahippocampal gyrus than those with the Val/Val genotype, but the genotype did not affect the prefrontal regions in schizophrenia patients. These findings might reflect different genotypic effects of BDNF on brain morphology in schizophrenia patients and healthy controls, implicating the possible role of the brain morphology as an endophenotype for future genetic studies in schizophrenia.