Experimental Biology and Medicine

Implications of mastication in energy intake and expenditure regulated by histamine (HA) neurons were Investigated in rats. Depletion of neuronal HA from the mesencephalic trigeminal sensory nucleus (Me5) reduced eating speed, but that from a satiety center of the ventromedial hypothalamus (VMH) increased both meal size and its duration leaving eating speed unaffected. Turnover of neuronal HA in the Me5 was elevated at the early phase of feeding and that in the VMH was at the later phase. This elevated turnover was abolished by gastric intubations of an isocaloric liquid diet or an equivolume of water. Mastication-induced activation of HA neurons suppressed physiological food intake through H₁-receptor in the hypothalamic paraventricular nucleus (PVN) and the VMH. On the other hand, the HA neurons activation accelerated lipolysis particularly in the visceral adipose tissues and up-regulated mRNA expression of uncoupling protein family through sympathetic efferent nerve. Mastication thus plays an important role as a potent input signal to activate HA neurons. Our recent findings have evidently shown how tightly and elegantly HA neurons are concordant with leptin signaling system through a negative feedback loop.

Obesity and insulin resistance have been recognized as leading causes of major health issues. We have endeavored to depict the molecular mechanism of insulin resistance, focusing on the function of adipocyte.

We have investigated a role of PPARγ on the pathogenesis of Type II diabetes. Heterozygous PPARγ-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Moreover, a Pro12Ala polymorphism in the human PPARγ2 gene was associated with decreased risk of Type II diabetes in Japanese. Taken together with these results, PPARγ is proved to be a thrifty gene mediating Type II diabetes. Pharmacological inhibitors of PPARγ/RXR ameliorate high-fat diet-induced insulin resistance in animal models of Type II diabetes.

We have performed a genome-wide scan of Japanese Type 2 diabetic families using affected sib pair analysis. Our genome scan reveals at least 9 chromosomal regions potentially harbor susceptibility genes of Type II diabetes in Japanese. Among these regions, 3q26-q28 appeared to be very attractive one, because of the gene encoding adiponectin, the expression of which we had found enhanced in insulin-sensitive PPARγ-deficient mice. Indeed, the subjects with the G/G genotype of SNP276 in the adiponectin gene were at increased risk for Type II diabetes compared with those having the T/T genotype. The plasma adiponectin levels were lower in the subjects with the G allele, suggesting that genetically inherited decrease in adiponectin levels predispose subjects to insulin resistance and Type II diabetes. Our work also confirmed that replenishment of adiponectin represents a novel treatment strategy for insulin resistance and Type II diabetes using animal models. Further investigation will be needed to clarify how adiponectin exerts its effect and to discover the molecular target of therapies.

Visceral fat accumulation is accompanied by several metabolic disorders. Here, we investigate the improvement of visceral fat accumulation in the early phase of diet. Hyperlipidemic obese patients received a low-calorie diet (1000 kcal/day) for 14 days. Visceral and subcutaneous fat accumulation was analyzed using ultrasonography. After 14 days of the diet, the average visceral fat of obese patients obviously decreased (P < 0.05), as well as the visceral fat-related secreted proteins, whereas subcutaneous fat did not decrease in these patients. These results show that visceral fat is reduced significantly in the early phase of diet therapy in humans. Therefore, to clarify its mechanism, we analyzed the expression of lipid metabolism-related genes in visceral and subcutaneous fat using obese rats. The Long-Evans Tokushima Otsuka (LETO) rats, as an obese model, were divided into two groups: fasting and non-fasting. The gene expressions in visceral and subcutaneous fat were measured by reverse transcriptase–polymerase chain reaction (RT-PCR). The expression of β₃-adrenergic receptor (AR), hormone sensitive lipase (HSL), peroxisome proliferator–activated receptor (PPAR)-γ, and uncoupling protein (UCP)-2 genes increased by 3.2-, 2.3-, 2.2-, and 2-fold in visceral fat (P < 0.01), but remained almost unchanged in subcutaneous fat. Taken together, the responsiveness of lipid metabolism-related genes to fasting is more sensitive in visceral fat than in subcutaneous fat in rats, suggesting that the different responsiveness to calorie restriction in fat tissues is due to the different induction of metabolism-related gene expression.

Ghrelin is a new orexigenic peptide primarily produced by the stomach but also present in the hypothalamus. It has adipogenic effects when it is chronically injected in rodents but in obese humans, its plasma concentration is decreased. It can reverse the anorectic effects of leptin when it is co-injected with this peptide in the brain ventricles. The Zucker fa/fa rat is a genetic model of obesity related to a default in the leptin receptor. It is characterized by a large dysregulation of numerous hypothalamic peptides but the ghrelin status of this rat has not yet been determined. Through several experiments, we determine in lean and obese Zucker rats its circulating form in the plasma, its tissue levels and/or expression, and studied the influence of different feeding conditions and its light/dark variations. Ghrelin expression was higher in the obese stomach and hypothalamus (P < 0.05 and P < 0.02, respectively). The ratio of [Octanoyl-Ser³]-ghrelin (active form) to [Des-Octanoyl-Ser³]-ghrelin (inactive form) was approximately 1:1 in the stomach and 2:1 in the plasma in lean and obese rats (no differences). After fasting, plasma ghrelin concentrations increased significantly in lean (+ 64%; P < 0.001) and obese (+ 60%; P < 0.02) rats. After 24 hours of refeeding, they returned to their initial ad lib levels. Ghrelin concentrations were higher in obese rats by 69% (P < 0.005), 65% (P < 0.02), and 73% (P < 0.005) in the ad libitum, fast, and refed states respectively. These results indicate that the obese Zucker rat is characterized by increases in the stomach mRNA expression and in peptide release in the circulation. They clearly support a role for ghrelin in the development of obesity in the absence of leptin signaling.

Leptin regulates feeding behavior and energy metabolism by affecting hypothalamic neuromodulators. The present study was designed to examine hypothalamic neuronal histamine, a recently identified mediator of leptin signaling in the brain, in genetic obese animals. Concentrations of hypothalamic histamine and tele-methylhistamine (t-MH), a major histamine metabolite, were significantly lower in obese (ob/ob) and diabetic (db/db) mice, and Zucker fatty (fa/fa) rats, leptln-deficient and leptin-receptor defective animals, respectively, relative to lean littermates (P < 0.05 for each). A bolus infusion of leptin (1.0 μg) into the lateral ventricle (ilvt) significantly elevated the turnover rate of hypothalamic neuronal histamine, as assessed by pargyline-induced accumulation of t-MH, in ob/ob mice compared with phosphate-buffered saline (PBS) infusions (P < 0.05). However, this same treatment did not affect hypothalamic histamine turnover in db/db mice. In agouti yellow (A^y/a) mice, animals defective in pro-opiomelanocortin (POMC) signaling, normal levels of histamine, and t-MH were seen in the hypothalamus at 4 weeks of age when obesity had not yet developed. These amine levels in A^y/a mice showed no change until 16 weeks of age, although the mice were remarkably obese by this time. Infusions of corticotropin releasing hormone (CRH), one of neuropeptide related to leptin signaling, into the third ventricle (i3vt) increased histamine turnover in the hypothalamus of Wistar King A rats (P < 0.05 versus PBS infusion). Infusion of neuropeptide Y (NPY) or α-melanocyte stimulating hormone (MSH), a POMC-derived peptide failed to increase histamine turnover. These results indicate that lowered activity of hypothalamic neuronal histamine in ob/ob and db/db mice, and fa/fa rats may be due to insufficiency of leptin action in the brains of these animals. These results also suggest that disruption of POMC signaling in A^y/a mice may not impact on neuronal histamine. Moreover, CRH but neither POMC-derived peptide nor NPY may act as a signal to neuronal histamine downstream of the leptin signaling pathway.

Mammalian muscles synthesize L-carnosine, but its roles were unknown. Previously, we found in rats that the administration of a certain amount of L-carnosine elicited an inhibition of the hyperglycemia induced by the Injection of 2-deoxy-D-glucose (2DG) into the lateral cerebral ventricle (LCV), and that intravenous injection of L-carnosine inhibited sympathetic nerves and facilitated the parasympathetic nerve. Moreover, the suppressive effect of L-carnosine on the hyperglycemia induced by 2DG was eliminated by thioperamide, a histaminergic H3 receptor. These findings suggested that L-carnosine might control the blood glucose level through regulating autonomic nerves via H3 receptor. To further clarify the function of L-carnosine, we examined its role in the control of the blood glucose. In this experiment, the following results were observed in rats: (i) A certain amount (0.01% or 0.001%) but not a larger amount (0.1%) of L-carnosine given as a diet suppressed the hyperglycemia induced by LCV-injection of 2DG (2DG-hyperglycemia); (ii) LCV-injection but not the injection into the intraperitoneal space (IP) of a certain amount of L-histidlne suppressed the 2DG-hyperglycemia; (iii) treatments of diphenhydramine, an H1 antagonist, and α-fluoromethylhistidine, an inhibitor of histamine-synthesizing enzyme, reduced the 2DG-hyperglycemia; (iv) the plasma L-carnosine concentration and carnosinase activity showed daily changes; (v) the plasma L-carnosine concentration was significantly lower in the streptozotocin-dlabetic rats; (vi) exercise by a running wheel tended to increase carnosine synthase activity in the gastrocnemius muscle and elevated the plasma L-carnosine concentration in the dark (active) period, and enbanced the plasma carnosinase activity in the light period; (vii) IP-injection of certain amount of L-carnosine stimulated the feeding response to IP-injection of 2DG. These findings suggest a possibility that L-carnosine released from muscles due to exercise functions to reduce the blood glucose level through the regulation of the autonomic nerves.

A sugar acid, 2-B40, has been found to increase from 3.5 to 13 μM in rat serum at 36 h after food deprivation. Injections of 2-B4O (2.5 μM) into the rat III cerebral ventricle (III ICV) suppress food intake and single neuronal activity in the lateral hypothalamic area (LHA). 2-B4O is effective even in 72 h food-deprived rats. 2-B40 hyperpolarlzes glucose-sensitive neurons in the LHA via Na⁺-K⁺ pump activation, but depolarizes glucoreceptor neurons in the ventromedial nucleus (VMH) via closure of ATP-sensitive K channels. The plasma levels of glucose, corticosterone, and catecholamines, and the firing rate in both parvocellular neurons in the paraventricular nucleus (PVN) and sympathetic efferent nerves, all increase 2-B40 intravenous (iv) injection, indicating activation of the hypothalamo-pituitaryadrenal axis. A 2-B40 iv injection facilitates emotional and spatial learning and memory, and pretreatment with anti-acidic fibroblast growth factor (aFGF) antibody ICV eliminates these effects. aFGF is released from ependymal cells in the III cerebral ventricle in response to the glucose increase in CSF induced by 2-B40 iv injection. 2-B40 also suppresses the clinical symptoms of experimental allergic encephalomyelitis (EAE) in Lewis rats [induced by immunization with a myelin basic protein (MBP)], a model for human multiple sclerosis. After immunization with MBP, the delayed-type hypersensitivity response to MBP is also reduced in 2-B40-treated rats. 2-B40 thus suppresses autoimmune responses. These results indicate that 2-B40 is not only a powerful satiety substance, but also effective as an activator of the hypothalamo-pituitary-adrenal axis and sympathetic efferent outflow, and as a memory facilitation and a modulator of immune functions.

Obese individuals with glucose intolerance present with high serum levels of glucose, insulin, and leptin. These substances are potent inhibitors of feeding in the brain. Obese subjects still present with over-feeding despite elevation of the above factors. To elucidate the mechanism of this paradox, the effects of insulin and glucose on the anorectic action of leptin in the hypothalamus were examined. Adult male Sprague-Dawley rats (weighing 2850–320 g) were pretreated with intracerebroventricular injection of insulin, glucose, or saline, followed by leptin (7.5 μg) or phosphate-buffered saline (PBS) injection into the third cerebral ventricle (icv). The cumulative food intakes were measured 24 hr after leptin icv. The tyrosine phosphorylation of signal transducer and activator transcription factor 3 (STAT3) in the hypothalamus was determined by Western blotting. In rats pretreated with saline and stimulated with leptin (saline/LEPTIN group), food intake diminished to about 50% of that of the saline/PBS group (P < 0.005). Food intake in the insulin/LEPTIN group was significantly higher compared with the saline/LEPTIN group (P < 0.005) and reached the level seen in the saline/PBS group. Similar data were obtained in glucose pretreatment experiments. Insulin and glucose icv resulted in reduction of leptin-induced STAT3 tyrosine phosphorylation compared with saline. Infusion of insulin and glucose icv did not alter peripheral blood glucose levels in all groups. High insulin or glucose levels in the brain could result in leptin resistance as manifested by food intake, which is probably due to the attenuation of STAT3 phosphorylation downstream the leptin receptor.

Leptin, a product of the ob gene, decreases food intake and body weight in both Wistar and Zucker obese rats when administered centrally or peripherally. To examine whether these leptin effects might be mediated through a neuropeptide Y (NPY) signaling pathway in the medial part of the arcuate nucleus of the hypothalamus (vmARC), the effects of leptin on vmARC neurons in Wistar and Zucker obese rats were examined electrophysiologically using brain slice preparations. Bath application of leptin inhibited about 60% of the vmARC neurons recorded in slices from Wistar rats. Similar inhibitory effects of leptin on vmARC neurons were also observed under low-Ca²⁺, high-Mg²⁺ Ringer's solution. However, inhibitory effects were almost absent under Ringer's solution containing a protein kinase C inhibitor, chelerythrine chloride. In slices from Zucker obese rats, leptin inhibited only about 25% of the vmARC neurons recorded, and the proportion of neurons inhibited was significantly smaller for these rats than for Wistar rats. These results suggest that reductions in food intake and body weight induced by leptin in both Wistar and Zucker obese rats are partly mediated via inhibition of an NPY signaling pathway in the vmARC.

Various stressors are known to cause eating disorders. However, it is not known in detail about the neural network and molecular mechanism that are involved in the stress-induced changes of feeding behavior in the central nervous system. Many novel feeding-regulated peptides such as orexins/hypocretins and ghrelin have been discovered since the discovery of leptin derived from adipocytes as a product of the ob gene. These novel peptides were identified as endogenous ligands of orphan G protein–coupled receptors. The accumulating evidence reveals that these peptides may be involved in stress responses via the central nervous system, as well as feeding behavior. The possible lnvolvement of novel feeding-related peptides in neuroendocrine responses to stress is reviewed here.

The world is experiencing an epidemic of obesity and its concomitant health problems. One implication is that the normally robust negative feedback system that controls energy homeostasis must be responding to different inputs than in the past. In this review we discuss the influence of gender on the efficacy of adiposity hormones as they interact with food intake control systems in the brain. Specifically, the levels of insulin and leptin in the blood are correlated with body fat, insulin being related mainly to visceral fat and ieptin to subcutaneous fat. Since females carry more fat subcutaneously and males carry more fat viscerally, leptin correlates better with total body fat in females and insulin correlates better in males. High visceral fat and plasma insulin are also risk factors for the complications of obesity, including type-2 diabetes, cardiovascular problems, and certain cancers, and these are more prevalent in males. Consistent with these systemic differences, the brains of females are more sensitive to the catabolic actions of low doses of leptin whereas the brains of males are more sensitive to the catabolic action of low doses of insulin. The implications of this are discussed.

This review discusses the regulation of the intestinal and hypothalamic apolipoprotein A-IV (apo A-IV) gene and protein expression. Apo A-IV is a glycoprotein secreted together with triglyceride-rich lipoproteins by the small intestine. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. This stimulation of intestinal apo A-IV synthesis is attenuated by intravenous leptin infusion. Chronic ingestion of a high-fat diet blunts the intestinal apo A-IV in response to dietary lipid. Intestinal apo A-IV synthesis is also stimulated by members of the pancreatic polypeptide family, including peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). Recently, apo A-IV was demonstrated to be present in the hypothalamus as well. Hypothalamic apo A-IV level was reduced by food deprivation and restored by lipid feeding. Intracerebroventricular administration of apo A-IV antiserum stimulated feeding and decreased the hypothalamic apo A-IV mRNA level, implying that feeding is intimately regulated by endogenous hypothalamic apo A-IV. Central administration of NPY significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner.

It has been mentioned in the field of aromatherapy that the fragrance of grapefruit has a refreshing and exciting effect, which suggests an activation of sympathetic nerve activity. It also can be assumed that the activation of sympathetic nerve innervating the white adipose tissue (WAT) facilitates lipolysis, then results in a suppression of body weight gain. This study addressed the effect of olfactory stimulation with the scent of grapefruit oil and lemon oil on the efferent nerve activity in the sympathetic branch of the WAT of the epididymis in the anesthetized rat. The results of the experiments demonstrated that the flavor of the citron group increased sympathetic nerve activity to WAT in anaesthetized rat, which suggests an increase in lipolysis and a suppression in body weight gain.

Cloning technology has potential to be a valuable tool in basic research, clinical medicine, and agriculture. However, it is critical to determine the consequences of this technique in resulting offspring before widespread use of the technology. Mammalian cloning using somatic cells was first demonstrated in sheep in 1997 and since then has been extended to a number of other species. We examined development, behavior, physiology, and longevity in B6C3F1 female mice cloned from adult cumulus cells. Control mice were naturally fertilized embryos subjected to the same in vitro manipulation and culture conditions as clone embryos. Clones attained developmental milestones similar to controls. Activity level, motor ability and coordination, and learning and memory skills of cloned mice were comparable with controls. Interestingly, clones gained more body weight than controls during adulthood. Increased body weight was attributable to higher body fat and was associated with hyperleptinemia and hyperinsulinemia indicating that cloned mice are obese. Cloned mice were not hyperphagic as adults and had hypersensitive leptin and melanocortin signaling systems. Longevity of cloned mice was comparable with that reported by the National Institute on Aging and the causes of death were typical for this strain of mouse. These studies represent the first comprehensive set of data to characterize cloned mice and provide critical information about the long-term effects of somatic cell cloning.

We investigated the effects of auricular acupuncture stimulation on non-obese healthy volunteers and mildly obese patients. Subjects (n = 55 and 5, respectively) averaged 34.5 years old, and BMI was 24.3 and less than 27.5 kg/m², respectively. We also studied the effects of single-blind sham treatment in approximately 500 age-, sex-, and BMI-matched subjects. Small (0.15 × 2.0 mm) auricular needles were placed intracutaneously into the bilateral cavum conchae identified by having a resistance of less than 100 kΩ/cm². In the 2-week pretreatment the period, in which body weight was measured without auricular acupuncture stimulation, 57.1% of the subjects showed a reduction in body weight. This indicates that charting one's own body weight might itself be a useful method of weight control. In the auricular acupuncture treatment period, 35 healthy subjects of 55 (63.6%) showed a decreased body weight, 11 (20%) showed an increased body weight, and 9 (16.4%) showed no change in body weight. The obese patients showed individual variation, but all achieved weight reduction, with a highly significant correlation between body weight and fat volume. The CT/MRI cross-sectional pictures supported these findings. Sham treatment had no statistically significant effect on body weight. These results suggest that success in achieving weight reduction can be partly attributed to the act of charting of one's own weight pattern. Bilateral auricular acupuncture stimulation can help reduce body weight both in mildly obese patients and in healthy non-obese subjects. In conclusion, this is in accord with the bilateral auricular acupuncture stimulation that it may be useful in the treatment of the obesity. We propose a possible mechanism for the weight-reducing effects of bilateral auricular acupuncture stimulation.

The beneficial effects of physical exercise on the decreased insulin sensitivity caused by detrimental lifestyle were reviewed based on experimental evidences. In epidemiological studies, disease prevention has been considered at three levels: primary (avoiding the occurrence of disease), secondary (early detection and reversal), and tertiary (prevention or delay of complications). The major purpose of physical exercise for primary prevention and treatment of lifestyle-related diseases is to improve insulin sensitivity. It is known that, during physical exercise, glucose uptake by the working muscles rises 7 to 20 times over the basal level, depending on the intensity of the work performed. However, intense exercise provokes the release of insulin-counter regulatory hormones such as glucagons and catecholamines, which ultimately cause a reduction in the insulin action. Continued physical training improves the reduced peripheral tissue sensitivity to insulin in impaired glucose tolerance and Type II diabetes, along with regularization of abnormal lipid metabolism. Furthermore, combination of salt intake restriction and physical training ameliorates hypertension. In practical terms, before diabetic patients undertake any program of physical exercise, various medical examinations are needed to determine whether they have good glycemic control and are without progressive complications. Because the effect of exercise that is manifested in improved insulin sensitivity decreases within 3 days after exercise and is no longer apparent after 1 week, a continued program is needed. For a safety practice, moderate- or low-intensity exercise is preferable. In conclusion, we have found sufficient evidences that support the theory that, combined with other forms of therapy, mild exercise training increases insulin action despite no influence on body mass index or maximal oxygen uptake. Along with evident benefits in health promotion, moderate-intensity exercise might play an important role in facilitating treatment of various diseases.

Diabetes is caused by impaired insulin secretion in pancreatic β-cells and peripheral insulin resistance. Overload of pancreatic β-cells leads to β-cell exhaustion and finally to the development of diabetes. Reduced β-cell mass is evident in type 2 diabetes, and apoptosis is implicated in this process. One characteristic feature of β-cells is highly developed endoplasmic reticulum (ER) due to a heavy engagement in insulin secretion. The ER serves several important functions, including post-translational modification, folding, and assembly of newly synthesized secretory proteins, and its proper function is essential to cell survival. Various conditions can interfere with ER function and these conditions are called ER stress. Recently, we found that nitric oxide (NO)-induced apoptosis in β-cells is mediated by the ER-stress pathway. NO causes ER stress and leads to apoptosis through induction of ER stress-associated apoptosis factor CHOP. The Akita mouse with a missense mutation (Cys96Tyr) in the insulin 2 gene has hyperglycemia and a reduced β-cell mass. This mutation disrupts a disulfide bond between A and B chains of insulin and may induce its conformational change. In the development of diabetes in Akita mice, mRNAs for an ER chaperone Bip and CHOP were Induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 β-cells induced CHOP expression and led to apoptosis. Targeted disruption of the CHOP gene did not delay the onset of diabetes in the homozygous Akita mice, but it protected islet cells from apoptosis and delayed the onset of diabetes in the heterozygous Akita mice. We conclude that ER overload in β-cells causes ER stress and leads to apoptosis via CHOP induction. These results highlight the importance of chronic ER stress in β-cell apoptosis in type 2 diabetes, and suggest a new target to the management of the disease.

This study investigated mechanisms by which glucose increases readily releasable secretory granules via acting on preexocytotic steps, i.e., intracellular granule movement and granule access to the plasma membrane using a pancreatic β-cell line, MIN6. Glucose-induced activation of the movement occurred at a substimulatory concentration with regard to insulin output. Glucose activation of the movement was inhibited by pretreatment with thapsigargin plus acetylcholine to suppress intracellular Ca²⁺ mobilization. Inhibitors of calmodulin and myosin light chain kinase also suppressed glucose activation of the movement. Simultaneous addition of glucose with Ca²⁺ channel blockers or the ATP-sensitive K⁺ channel opener diazoxide failed to suppress the traffic activation, and addition of these substances on top of glucose stimulation resulted in a further increase. Although stimulatory glucose had minimal changes in the intracellular granule distribution, inhibition of Ca²⁺ influx revealed increases by glucose of the granules in the cell periphery. In contrast, high K⁺ depolarization decreased the peripheral granules. Glucose-induced granule margination was abolished when the protein kinase C activity was downregulated. These findings indicate that preexocytotic control of insulin release is regulated by distinct mechanisms from Ca²⁺ influx, which triggers insulin exocytosis. The nature of the regulation by glucose may explain a part of potentiating effects of the hexose independent of the closure of the ATP-sensitive K⁺ channel.

The endocrine cells in the pancreatic islet have cellular communication between the heterotypic cells as well as the homotypic cells. The present study was conducted to elucidate the cellular interaction between pancreatic α cells and β cells utilizing differentiated mouse cell lines (i.e., αTC clone 6 and βTC cells). Co-culture of these two cell lines on a gyratory shaker generated numerous cellular aggregates of homogenous size within 48 h. Immunohistochemical staining for insulin and glucagon demonstrated that βTC cells were located in the central core of each aggregate, while αTC cells formed a mantle layer surrounding the βTC cells. This segregation was observed regardless of the ratios of the two cell types employed. Although glucagon at concentrations of 10⁻⁸ M or higher stimulated Insulin secretion from βTC cells in both monolayer and aggregates, an increase in the ratio of αTC/βTC cells in aggregate cultures was accompanied by a decrease in secreted insulin and a rise in intracellular insulin content of the βTC component. The inhibitory effect of αTC cells on βTC insulin secretion was not limited to aggregate culture, since insulin secretion from βTC cells was also suppressed, and intracellular insulin content increased, by co-culture of αTC with βTC cells in monolayer. On the other hand, the secreted and intracellular insulin of βTC cells was not affected by αTC cells in a Transwell™ co-culture system in which direct cell-to-cell contacts were prevented by a semipermeable membrane that permitted chemical communication via medium metabolites. These data suggest that the insulin secretion from βTC cells may be inhibited possibly as a result of the contact with αTC cells.

This study investigated the effects of long-term treatment with glibenclamide (GLIB) on the susceptibility of streptozotocin (STZ)-Induced diabetic heart to ischemia/reperfusion insults. Starting 4 weeks after the injection of STZ, rats were treated with GLIB (0.1 mg/kg, ip, three times a week, STZ-GLIB group) or vehicle (STZ-VEH group) for 8 weeks. The recovery of cardiac performance, released creatine kinase (CK), and incidence of ventricular arrhythmias were studied during the reperfusion period in isolated hearts from rats in STZ-GLIB (n = 14) and in STZ-VEH groups (n = 13) and from age-matched control rats (CNT group, n = 14). Each heart was subjected to 5 min of global low-flow ischemia followed by 25 min of no-flow ischemia, with a subsequent 30 min of reperfusion. Plasma glucose level was not significantly different between the STZ-GLIB and STZ-VEH groups. The recovery of cardiac performance and the released CK during reperfusion period were significantly lower in both STZ-VEH and STZ-GLIB groups than in the CNT group (P < 0.01 and P < 0.05, respectively). Reperfusion resulted in an incidence of ventricular fibrillation in 23% and 21% in STZ-VEH and STZ-GLIB groups, respectively (P = ns). These values were significantly lower than that of the CNT group (100%, P < 0.001 for both). More importantly, the incidence of ventricular tachycardia in the STZ-GLIB group was significantly higher than that in the STZ-VEH group (93% vs 54%, P < 0.05) and was not significantly different from that in the CNT group (93% vs 100%, P = ns). The results suggest that STZ-induced protection against reperfusion-induced ventricular arrhythmias in diabetic heart may be partially abrogated by long-term treatment with GLIB.

The localization of leptin and leptin receptors in the stomach and small intestine has been reported. Their function is still unknown, although leptin is a hormone that regulates appetite and fat-related metabolism. The small intestine is one of the important organs for regulating metabolism. The purpose of the present study was to investigate whether leptin regulates apoptosis in the small intestinal mucosa. Intestinal apoptosis was evaluated by percent fragmented DNA, electrophoresis, TUNEL staining, and western blotting analysis of caspase-3. Mucosal apoptosis in the rat jejunum and Ileum was evaluated at 0, 3, 6, 12, and 24 hrs after injection. Rats were tested after ad libitum feeding and 24-hr fasting to exclude the anorectic effect of leptin. Leptin was injected intraperitoneally (ip) at a dose of 200 μg/rat and infused into the rat third cerebroventricle (icv) at a dose of 8 μg/rat. Leptin at a dose of 8 μg/rat significantly induced intestinal apoptosis in the small intestine at 3 and 6 hrs after icv administration in both ad libitum feeding and 24-hr fasted rats. This increase in apoptosis was not attenuated by vagotomy. Intestinal apoptosis increased 12 and 24 hrs after ip injection of leptin at a dose of 200 μg/rat. The peak of the increase in apoptosis in icv rats appeared earlier than that in ip rats. Leptin induced jejunal and ileal mucosal apoptosis in the rat, indicating that leptin might control intestinal function through the regulation of intestinal apoptosis.

Systemic thermal therapy, such as taking a warm-water bath and sauna, induces systemic vasodilation. It was found that repeated sauna therapy (60°C for 15 min) improved hemodynamic parameters, clinical symptoms, cardiac function, and vascular endothelial function in patients with congestive heart failure. Vascular endothelial function is impaired in subjects with lifestyle-related diseases, such as hypertension, hyperlipidemla, diabetes mellitus, obesity, and smoking. Sauna therapy also improved endothelial dysfunction in these subjects, suggesting a preventive role for atherosclerosis. In animal experiments, sauna therapy increases mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in aortas. In normalweight patients with appetite loss, repeated sauna therapy increased plasma ghrelin concentrations and daily caloric intake and Improved feeding behavior. In obese patients, the body weight and body fat significantly decreased after 2 weeks of sauna therapy without increase of plasma ghrelin concentrations. On the basis of these data, sauna therapy may be a promising therapy for patients with lifestyle-related diseases.

As a biochemist, I have been studying lipolytic and lipogenic pathways in fat cells since 1963. In 1966, I proposed a hormone-sensitive substrate theory in which catecholamines might not act on lipase but on substrate during their lipolytic processes. The lipolytic and lipogenic pathways are negative and positive processes in triglyceride content of fat cells. Insulin inhibits, the negative process (lipolysis) and stimulates the positive process (lipogenesis from glucose). On the other hand, catecholamine stimulates the negative process and inhibits the positive one. These hormones discriminate the negative and positive rules and regulate opposite ways.

We tried to find these hormone-like substances in various natural products. We isolated tea saponins, chitosan, and others as insulin-like substances and dimethyl-xanthine as a catecholamine-like one. It is well known that extracellular fluid pH changes from 7.4 to 6.8. Reduction of the pH from 7.4 causes insulin resistance. Insulin failed to stimulate glucose uptake at pH 7.0 of the extracellular fluid. We found minus ions, which stimulated lipogenesis from glucose by raising extracellular fluid pH to 7.4. These are our approaches to find functional substances that prevent lifestyle-related diseases.