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Older adults with prediabetes or obesity (i.e., those at risk for diabetes) exhibit impaired structural brain networks. Given findings that resistance training (RT) can combat brain impairments in many populations, this study aimed to test the effects of this type of exercise on white matter microstructure in older adults at risk for diabetes. Seventeen community-dwelling older adults (mean age 67.8 ± 5.7, 52.9 % female) with prediabetes or obesity were randomly allocated to thrice weekly RT or balance and tone training (BAT; control group) for six months. Diffusion weighted imaging via a 3T scanner was used to assess changes in white matter parameters –fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD) – over time. Participants in the RT group showed no significant changes in FA but had increased MD and RD in various regions related to cognitive function including the cingulate gyrus. Participants in the control group had both increased and decreased FA depending on the specific white matter tracts; increased FA was seen in areas related to motor coordination such as the middle cerebellar peduncle. The control group also exhibited decreased MD and RD in areas responsible for motor function (e.g., left anterior limb of the internal capsule). We conclude that both resistance and balance exercises result in changes in white matter microstructure albeit in divergent tracts that may be linked to the specific exercises performed.

Objective: Veterans are at high risk for health morbidities linked to premature mortality. Recently developed "epigenetic clock" algorithms, which compute intra-individual differences between biological and chronological aging, can help inform prediction of accelerated biological aging and mortality risk. To date, however, scarce research has examined potentially modifiable correlates of GrimAge, a novel epigenetic clock comprised of DNA methylation surrogates of plasma proteins and smoking pack-years associated with various morbidities and time-to-death. The objective of the study was to examine psychosocial correlates of this novel epigenetic clock., Design: Cross-sectional study., Setting: U.S. veteran population., Participants: Participants were male, European American (EA), and derived from a nationally representative sample of U.S. veterans (N = 1,135, mean age = 63.3, standard deviation [SD] = 13.0)., Measurements: We examined the prevalence of accelerated GrimAge and its association with a broad range of health, lifestyle, and psychosocial variables., Results: A total 18.3% of veterans had accelerated GrimAge (≥5 years greater GrimAge than chronological age; mean = 8.4 years acceleration, SD = 2.2). Fewer days of weekly physical exercise (relative variance explained [RVE] = 27%), history of lifetime substance use disorder (RVE = 21%), greater number of lifetime traumas (RVE = 19%), lower gratitude (RVE = 13%), reduced sleep quality (RVE = 7%), lower openness to experience (RVE = 7%), and unmarried/partnered status (RVE = 6%) were independently associated with increased odds of accelerated GrimAge. Increasing numbers of these risk factors were associated with greater odds of accelerated GrimAge, with greatest likelihood of acceleration for veterans with ≥3 risk factors (weighted 21.5%)., Conclusions: These results suggest that nearly 1-of-5 EA male U.S. veterans have accelerated GrimAge, and highlight a broad range of health, lifestyle, and psychosocial variables associated with accelerated GrimAge. Given that many of these factors are modifiable, these findings provide promising leads for risk stratification models of accelerated biological aging and precision medicine-based targets for interventions to mitigate risk for premature mortality in this population., (Copyright © 2022 American Association for Geriatric Psychiatry. All rights reserved.)

Introduction: For successful lymphaticovenous anastomosis (LVA), it is important to create anastomoses with high flow to maintain patency. To ensure that this can be achieved, we compared the efficacy of a modified intraoperative distal compression (IDC) technique with the conventional no compression (NC) method for lower limb lymphedema., Patients and Methods: In the IDC group, compression was applied to an area of the foot distal to the first LVA site. After completion of the first LVA, the distal compression was extended over the first LVA site to the distal end of the second LVA site., Results: There was no significant difference between the IDC (n = 25) and NC (n = 25) groups in detection rate. However, significant differences were observed in lymphatic vessel diameter and LVA success rate. No intraoperative anastomotic obstruction was seen at the conclusion of surgery. Intraoperative congestion with blood was detected in lymphatic vessels in 8 of 79 anastomoses (10.1%) in the NC group, but not in any cases in the IDC group (p = 0.002). There was a significant between-group difference in the rate of improvement in lymphedema between the IDC (16.1±3.6) and NC groups (14.0±3.4; p = 0.03)., Discussion: IDC during LVA is thought to increase lymph flow in larger caliber lymphatics, leading to a high success rate and a low rate of venous reflux. IDC is beneficial when performing LVA., Competing Interests: Declaration of Competing Interest None declared, (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Although pressure equalization of the sensor-tipped guidewire and systemic pressure is mandatory in measuring fractional flow reserve (FFR), pressure in the distal artery (Pd) with wire advancement can be influenced by hydrostatic pressure related to the height difference between the catheter tip and the distal pressure sensor. We therefore analyzed the impact of hydrostatic pressure on FFR in vivo by modification of the height difference., Methods: To reveal the anatomical height difference in human coronary arteries, measurement was performed during computed tomography angiography (CTA) of five consecutive patients. Utilizing the healthy coronary arteries of female swine, height difference diversity was reproduced by body rotation and vertical inclination. FFR measurements were performed during maximum hyperemia with adenosine. The height difference was calculated fluoroscopically with a contrast medium-filled balloon for reference., Results: In human coronary CTA, height averages from the ostium in the left anterior descending artery (34.6 mm) were significantly higher than in the left circumflex (-15.5 mm, p = 0.008) and right coronary arteries (-2.3 mm, p = 0.008). In our swine model, reproduced height variation ranged from -7.2 cm to +6.5 cm. Mean FFR was significantly lower in positive sensor height and higher in negative sensor height compared to the reference height. Linear regression analyses revealed significant correlations between height difference and FFR, observed among all coronary arteries, as well as between the height difference and Pd-aortic pressure mismatch. Subtracting 0.622 mmHg/cm height difference from Pd could correct the expected hydrostatic pressure influence., Conclusion: Hydrostatic pressure variation resulting from sensor height influenced FFR values might affect interpretation during FFR assessment., (Copyright © 2020. Published by Elsevier Ltd.)

Background: A drawback of multiple lymphaticovenular anastomoses (LVAs) is the need for at least two microsurgeons and the same number of microscopes. In practice, many hospitals find it difficult to access such resources. We have developed a novel line production system (LPS) to address this problem. We assessed whether or not the LPS is better than the conventional dual microscope (DM) system when performing multiple LVAs., Methods: An LPS group, wherein a novice microsurgeon used loupes to dissect lymphatics and an expert microsurgeon used a microscope to perform the LVAs, and a DM (control) group, wherein the surgeons used microscopes to perform the LVAs. We recorded the lymphatic detection rate through the loupes and the diameter of the detected lymphatics. We also investigated the impact of using the LPS by comparing the number and quality of LVAs and improvement in lymphedema between the study groups., Results: The mean lymphatic detection rate was 81%±15.60%, and the mean size of lymphatics was 0.44 ± 0.12 mm in the LPS. The number of LVAs/h in LPS was significantly higher than that in DM (2.15 ± 0.20 vs. 1.38 ± 0.17; p < 0.01). The number of successful LVAs/h in LPS was significantly higher than that in the DM (2.08 ± 0.22 vs. 0.84 ± 0.14; P < 0.01). Mean rate of improvement in LEL index was significantly higher than that in DM (9.36 ± 1.85 vs. 6.93 ± 1.73; P < 0.01)., Discussion: The number and quality of the LVAs increase using the LPS, which leads to further improvement in lymphedema, with fewer microscopes and microsurgeons and a shorter operating time., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Chronic kidney disease (CKD) deteriorates the prognosis of patients undergoing percutaneous coronary intervention (PCI). Because coronary artery disease (CAD) is the major cause of death in CKD patients, cardiovascular risk reduction has been clinically important in CKD. We hypothesized intensive lipid-lowering with statin/ezetimibe attenuated coronary atherosclerotic development even in patients with CKD., Methods: In the prospective, randomized, controlled, multicenter PRECISE-IVUS trial, 246 patients undergoing intravascular ultrasound (IVUS)-guided PCI were randomly assigned to receive atorvastatin/ezetimibe combination or atorvastatin alone (the dosage of atorvastatin was up-titrated to achieve the level of low-density lipoprotein cholesterol < 70 mg/dL). Serial volumetric IVUS findings obtained at baseline and 9-12 month follow-up to quantify the coronary plaque response in 202 patients were compared stratified by the presence or absence of CKD., Results: CKD was observed in 52 patients (26%) among 202 enrolled patients. Compared with the non-CKD group, the CKD group was significantly older (71.5 ± 8.6 years vs. 64.4 ± 9.6 years, P < 0.001) with similar prevalence of comorbid coronary risk factors and lipid profiles. Similar to the non-CKD group (-1.4 [-2.8 to -0.1]% vs. -0.2 [-1.7 to 1.0]%, P = 0.002), the atorvastatin/ezetimibe combination significantly reduced ∆PAV compared with atorvastatin alone even in the CKD group (-2.6 [-5.6 to -0.4]% vs. -0.9 [-2.4 to 0.2]%, P = 0.04)., Conclusions: As with non-CKD, intensive lipid-lowering therapy with atorvastatin/ezetimibe demonstrated stronger coronary plaque regression effect even in patients with CKD compared with atorvastatin monotherapy., Trial Registration: NCT01043380 (ClinicalTrials.gov)., (Published by Elsevier B.V.)

Ancylostoma (A.) ceylanicum, one of the most common species of hookworms infecting dogs and cats, also causes patent infections in humans and is now considered to be the second most common hookworm species infecting populations in southeast Asia. A Japanese patient who returned from a visit to Thailand and Lao People's Democratic Republic (PDR) was presented with intermittent watery diarrhea with eosinophilia. Hookworm eggs were found in feces samples, and adult worms were confirmed to be present in the jejunum with capsule endoscopy and double balloon enteroscopy. A diagnosis of A. ceylanicum infection was made based on the morphology of the adult worms along with findings of a PCR-based molecular study using larvae obtained from a fecal sample culture. The infection was considered likely to have been obtained during a 1-month stay in a Laotian village, where the patient had eaten local food, worn sandals on bare feet, and lived as a local native villager, though he had stayed in modern hotels during the visit to Thailand., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Limb amputation has historically been the first choice of treatment for patients with bone or soft-tissue sarcomas involving major blood vessels. However, recent advances in surgical technique have allowed limb-salvage surgery. We reviewed our experiences with limb-salvage surgery and immediate vascular reconstruction following en bloc resection of bone or soft-tissue sarcomas of the lower extremity., Materials and Method: We reviewed 23 patients (15 male and eight female; mean age, 43.6 years) who underwent limb-salvage surgery and immediate vascular reconstruction. Details of surgical factors and postoperative complications were evaluated., Results: Reconstructed vessels remained patent in 21 cases. The rate of limb oedema was higher in patients who underwent only arterial reconstruction after arteriovenous resection. Twenty patients could walk well without crutches a few months after reconstructive surgery. All patients avoided amputation. Two patients died of disseminated disease within 3 years after surgery., Conclusion: The high rate of limb oedema suggests that venous reconstruction is necessary after arteriovenous resection. Vascular reconstruction and musculocutaneous flap techniques are useful in limb-salvage surgery and are indicated for patients who have achieved good disease control. Evidence Rating Scale for Therapeutic Studies: Level III., (Copyright © 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background: The fibular flap and the scapular flap are widely used for immediate reconstruction after segmental mandibulectomy. The aim of this study was to compare perioperative complications between the fibular flap and the scapular flap in immediate mandibular reconstruction., Methods: Data were retrospectively collected on 56 patients who had undergone immediate mandibular reconstruction with a fibular flap (38 patients) or a scapular flap (18 patients) after segmental mandibulectomy from 2005 to 2011. The rates of perioperative recipient-site and donor-site complications were compared between the groups., Results: The overall rate of recipient-site complications did not differ significantly between the fibula group and the scapula flap. However, the rate of donor-site complications was significantly higher in the fibula group than in the scapula group. Partial skin-graft loss in the fibula group occurred in as high as 13 out of 38 patients., Discussion: For immediate mandibular reconstruction, a scapular flap provides short-term results equivalent to those with a fibular flap but with less donor-site morbidity. The major drawbacks of the fibular flap include prolonged healing of the donor site and the delayed mobilisation of patients. Although our first choice of vascularised bone graft is the fibular flap, the scapular flap in an alternative for those patients, especially elderly patients, in whom fibula harvest can result in significant morbidity., (Copyright © 2012 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Background: The reconstruction of large, complex defects of the abdominal wall after the ablation of malignant tumours can be challenging. The transfer of an anterolateral thigh (ALT) flap is an attractive option. This study compared free ALT flaps and pedicled ALT flaps for abdominal wall reconstruction., Methods: From 1996 through 2011, 20 patients underwent abdominal wall reconstruction with ALT flaps. The flaps were pedicled in 12 patients and free in eight patients. Medical records were reviewed for complications and clinical and demographic data. Abdominal wall defects were classified into the following four groups: upper midline, lower midline, upper quadrants and lower quadrants., Results: Pedicled flaps were transferred to the upper midline region in one patient, the lower midline region in six patients and lower quadrants in five patients. Free flaps were transferred to the lower midline region in two patients, upper quadrants in four patients and lower quadrants in two patients. Mean reconstructive time was significantly longer with free flaps (6 h 32 min) than with pedicled flaps (4 h 55 min, p = 0.035). Although free flaps (mean size, 360 cm(2)) were larger than pedicled flaps (mean size, 289 cm(2)), the difference was not significant (p = 0.218). The rates of complications did not differ between free flaps and pedicled flaps. No total flap loss occurred, and there was partial loss of only a single pedicled flap, which was the flap furthest from the pivot point. Infections developed of two pedicled flaps and three free flaps., Conclusion: This study suggests that complication rates do not differ between free and pedicled ALT flaps. The choice of flap depends on the size and location of the defect and the length of the vascular pedicle., (Copyright © 2012 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Objective: Hepatic artery (HA) reconstruction is an important part of resective surgery for advanced hepatobiliary and pancreatic malignancies, but few reports have been published. To identify indications for HA reconstruction, we retrospectively analyzed our surgical procedures and outcomes., Methods: En-bloc resection of advanced hepatobiliary and pancreatic malignancies followed by HA reconstruction was performed in 35 patients. Patients ranged in age from 27 to 81 years and included 18 men and 17 women. The primary site of cancer included the bile duct in 22 patients, the pancreas in 7, and others in 6. Reconstruction of the HA was necessitated by HA resection due to direct cancer invasion in 29 patients and by accidental arterial injury during surgical procedure in 6 patients., Results: The HA was reconstructed with end-to-end anastomosis between hepatic arteries in 17 patients. Transposition of an intra-abdominal artery, such as the gastroepiploic artery, was required in 14 patients, and arterial grafting was required in 4 patients. Although the HA patency was achieved in 30 patients, 4 cases of arterial thrombosis and 1 case of arterial rupture developed postoperatively. The overall RFS time was analyzed in all patients, and mean and median RFS times were 18 and 9 months, respectively., Conclusion: Although oncologic outcomes remain poor, HA resection and reconstruction can be performed in selected patients. We believe that the method of first choice for HA reconstruction is end-to-end anastomosis between HAs. A vascular autograft should be used only in selected cases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Incretin promotes insulin secretion acutely. Recently, orally-administered DPP-4 inhibitors represent a new class of anti-hyperglycemic agents. Indeed, inhibitors of dipeptidyl peptidase-IV (DPP-4), sitagliptin, has just begun to be widely used as therapeutics for type 2 diabetes. However, the effects of sitagliptin-treatment on insulin exocytosis from single β-cells are yet unknown. We therefore investigated how sitagliptin-treatment in db/db mice affects insulin exocytosis by treating db/db mice with des-F-sitagliptin for 2 weeks. Perfusion studies showed that 2 weeks-sitagliptin treatment potentiated insulin secretion. We then analyzed insulin granule motion and SNARE protein, syntaxin 1, by TIRF imaging system. TIRF imaging of insulin exocytosis showed the increased number of docked insulin granules and increased fusion events from them during first-phase release. In accord with insulin exocytosis data, des-F-sitagliptin-treatment increased the number of syntaxin 1 clusters on the plasma membrane. Thus, our data demonstrated that 2-weeks des-F-sitagliptin-treatment increased the fusion events of insulin granules, probably via increased number of docked insulin granules and that of syntaxin 1 clusters., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Osteocutaneous flaps are one of the best options for one-stage mandible reconstruction. However, the challenge remains to achieve optimal functional and cosmetic results. A new novel approach involving the preoperative prefabrication of a reconstructive plate through a calcium-sulphate three-dimensional (3D) model facilitates the contouring of vascularised bone grafts. We herein report our preparations and results using this technique., Methods: A total of 17 mandibular defects were reconstructed by this novel approach. A calcium-sulphate 3D model was constructed from computed tomography (CT) data. After the oncologist designed the cut line on the model, the mandibular arc was ground to the neo-mandible shape, which consisted of several linear planes according to the osteotomy of the bone graft. The reconstruction plate was shaped to fit this. After tumour resection, the prefabricated plate was placed to the remaining mandible and revealed the defect to be reconstructed, just as a mould. Rubber sticks were used as a template to shape the bone graft. The preoperative information, and functional and aesthetic results were retrospectively analysed., Results: As many as 12 fibular and 5 scapular flaps were applied. Postoperative complications included two salivary fistulae, one abscess and one partial skin loss, all of which were resolved after conservative treatment. Postoperatively, all patients could speak clearly, 12 had a normal diet and 12 had excellent cosmetic results., Conclusions: This is the first report using models made by calcium-sulphate. The largest advantage of this model is that the neo-mandible shape can be demonstrated preoperatively. The refinement of mandible reconstruction after tumour ablative surgery can be achieved with a prefabricated plate through the use of a calcium-sulphate 3D model. It enables more accurate, faster and simplified fabrication of reconstruction plates, thus leading to satisfactory functional and cosmetic results., (Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

The 320-row multidetector computed tomography (MDCT) is now used by both cardiologists and neurosurgeons. It enables dynamic 3D-CT angiography, because the wide-area detector eliminates helical scanning, thus achieving very fast scanning times for single 3D-CT volume data. Some microvascular surgeons are familiar with 64-row MDCT for perforator studies, but there are few reports of studies using 320-row MDCT. This MDCT system was used to follow the dynamic blood flow of small vessels. It is considered to have a great potential in the clinical field of microvascular surgery., (Copyright 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

We report the case of a 39-year-old man with a dermatofibrosarcoma protuberans (DFSP) on the right shoulder. A wide surgical excision of the tumour was performed, creating a 12-cm-wide defect. An anterolateral thigh flap created from two semicircular skin paddles was harvested and the two skin paddles were slid towards each other to cover the circular defect. The sliding technique is a useful design that preserves the suprafascial plexus and enables a single perforator to supply two split-skin paddles. Using this design, the donor site can be closed primarily without requiring a skin graft. This technique can be applied to other free flaps to reconstruct wide defects after the resection of cancers., (Copyright 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic beta cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+](PM)) with the Ca2+ indicator Fura Red in rat beta cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+](PM) caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+](PM) induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+](PM) rise directly determines the types of fusing granules.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions, but its physiological role in pancreatic islet cells remains to be investigated. In this study, we confirmed the presence of PPAR-gamma in rat isolated islets and examined its role on insulin and glucagon secretion by using PPAR-gamma-overexpressed islets. PPAR-gamma overexpression significantly suppressed insulin secretion induced by stimulatory concentration of glucose (p<0.05). In addition, insulin secretion evoked by high potassium depolarization also was significantly decreased from PPAR-gamma-overexpressed islets (p<0.05). On the other hand, no significant change in glucagon release was observed after high potassium depolarization between PPAR-gamma-overexpressed and control islets. Insulin and glucagon content in islets was not statistically different between the two groups. In addition, the expression of uncoupling protein-2 (UCP-2) was found to be induced in PPAR-gamma-overexpressed islets. This result clearly indicates that the deteriorative effect of PPAR-gamma overexpression on the secretory machinery is selective for pancreatic beta-cells. And it is possible that its site of action can be located in the energy-consuming exocytotic process of insulin secretory granules, and that the reduction of ATP production through increased UCP-2 reduces insulin exocytosis.

We previously reported that mice lacking bombesin receptor subtype-3 (BRS-3) exhibit mild late-onset obesity and glucose intolerance [Nature 390 (1997) 160]. To examine the mechanism by which glucose intolerance is developed in these mice, we studied insulin release and proinsulin biosynthesis in isolated pancreatic islets and glucose uptake and facilitative glucose transporter (GLUT)-4 translocation in adipose tissues. Although islet insulin contents and the size and number of islets of Langerhans in BRS-3-deficient mice decreased, there was no difference in glucose-stimulated insulin release and proinsulin biosynthesis between BRS-3-deficient and wild-type control mice. In contrast, adipose tissues exhibited a marked difference: the uptake of [(14)C]2-deoxy-D-glucose by adipocytes isolated from BRS-3-deficient mice was not stimulated by 10(-7)M insulin addition, and membrane fractionation analysis showed that GLUT4 was barely detected in the fraction of plasma membrane in BRS-3-deficient mice in the presence of 10(-7)M insulin. Quantitative reverse transcription-PCR (RT-PCR) showed that mRNA levels of GLUT4, insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2, syntaxin 4, SNAP23, and VAMP-2 in adipose tissues of BRS-3-deficient mice were unchanged compared with those in wild-type control mice. We concluded that impaired glucose metabolism observed in BRS-3-deficient mice was mainly caused by impaired GLUT4 translocation in adipocytes.

To investigate the cellular mechanism of insulinotropic effect of glutamate in pancreatic beta cells, we utilized patch-clamp technique to monitor directly the activities of ATP-sensitive potassium channels (K(ATP) channels). Dimethylglutamate (5mM), a membrane-permeable analog of glutamate, augmented the insulin release induced by the stimulatory concentrations of glucose (p<0.05-0.01). In the cell-attached configurations, dimethylglutamate reversibly and significantly suppressed the K(ATP) channel activities (p<0.01). On the other hand, no significant effect was observed when glutamate itself was applied to the inside-out patches, whereas the prompt and reversible suppression was recorded in the case of ATP (p<0.01). These results indicate that the insulinotropic action of glutamate in beta cells could be derived from the inhibition of K(ATP) channel activities, probably due to generation of messengers via intracellular metabolism such as ATP.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) regulates several cellular functions; however, its physiological role in pancreatic beta cell functions remains to be determined. In the present study, we investigated the synergistic effect of PPAR-gamma and its agonist, pioglitazone, on proinsulin biosynthesis and insulin release in a glucose-responsible insulinoma cell line, MIN6 cells. Expression of PPAR-gamma in MIN6 cells was not detectable by RT-PCR and immunoblot analysis. When PPAR-gamma-1 was overexpressed adenovirally in MIN6 cells, glucose-stimulated proinsulin biosynthesis and insulin release were inhibited. Pioglitazone treatment alone had no effects on these parameters of beta cell function in control MIN6 cells, although pioglitazone synergistically augmented the inhibitory effect of PPAR-gamma on proinsulin biosynthesis and insulin release under the condition of PPAR-gamma overexpression. Our results demonstrate that PPAR-gamma plays a negative role in pancreatic beta cells.

We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.

The effects of fatty acids on pancreatic beta cell are still controversial. Here, in order to determine whether free fatty acids acutely affect beta cell functions, we studied the effect of palmitic acid (PA) on proinsulin biosynthesis and insulin secretion using rat islets in vitro. Exposure of islets to PA for 1 h reduced glucose-stimulated proinsulin biosynthesis in a dose-dependent manner; in contrast, no change in insulin secretion was observed after 1 h incubation with PA. Furthermore, PA treatment did not cause any change of preproinsulin mRNA level during 1-h incubation period. Thus, our data indicate that PA primarily suppresses glucose-induced proinsulin biosynthesis within 1 h at the translational level., (Copyright 2001 Academic Press.)

To explore alpha-SNAP function in insulin exocytosis from either immature or mature secretory granules in pancreatic beta cells, we studied the effects of overexpression of adenovirus-mediated wild-type alpha-SNAP and C-terminally deleted alpha-SNAP mutant (1-285) on newly synthesized proinsulin and insulin release by rat islets and MIN6 cells. Rat islets overexpressing alpha-SNAP and mutant alpha-SNAP were pulse-chased. Exocytosis from immature and mature insulin secretory granules was measured as fractional (%) labeled-proinsulin release immediately after the pulse-labeling and percentage labeled-insulin release after a 3-h chase period, respectively. There was no difference in percentage labeled-proinsulin release between the control and alpha-SNAP or mutant alpha-SNAP-overexpressed islets. Although percentage labeled-insulin release after a 3-h chase period was significantly increased in alpha-SNAP-overexpressed islets, it was decreased in mutant alpha-SNAP-overexpressed islets. Thus, the results demonstrated that alpha-SNAP overexpression in rat islets primarily increased exocytosis from mature, but not immature insulin secretory granules. On the other hand, in MIN6 cells, alpha-SNAP overexpression scarcely affected glucose-stimulated insulin release; therefore, we examined the effect of mutant alpha-SNAP overexpression as the dominant-negative inhibitor on the newly synthesized proinsulin/insulin release using the same protocol as in the rat islet experiments. alpha-SNAP mutant (1-285) overexpression in MIN6 cells decreased the percentage labeled insulin release from mature secretory granules, but not percentage labeled proinsulin release from immature secretory granules. Thus, our data demonstrate that alpha-SNAP functions mainly in the mature insulin secretory granules in pancreatic beta cells., (Copyright 1999 Academic Press.)

Toward elucidating the functional aspects ofGLUT3, a primary neuronal glucose transporter isoform in the vertebrate central nervous system, this study examined its expression in cholinergic amacrine cells made identifiable by the presence of acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), in the rat retina. Double-immunofluorescence staining of adult rat retinal tissue with anti-GLUT3 and anti-ChAT antibodies revealed characteristic stratified GLUT3 immunoreactivity (GLUT3-IR) in the inner plexiform layer (IPL) that was identical to the arborization pattern of ChAT-positive neuronal processes there. In addition, approximately 30-50% of intensely GLUT3-immunoreactive cell bodies in the inner nuclear layer and ganglion cell layer showed ChAT-IR, while the majority of ChAT-positive cell bodies were also intensely GLUT3 immunoreactive. Analysis at the cellular level using retinal cells in culture revealed similar findings. These results collectively indicate that cholinergic amacrine cells constitute the major component of GLUT3-expressing cells in the rat retina. It is expected that the link demonstrated here between GLUT3 expression and cholinergic amacrine cell population will provide clues for further analyzing GLUT3 function in the retina., (Copyright 1999 Academic Press.)

We have previously established a stable beta TC3 cell line that overexpresses syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin. beta TC3 and beta TC-hpc1 cells were simultaneously pulse-labeled with 3H-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from beta TC-hpc1 cells was lower than that from beta TC3 cells. The secretion of newly synthesized proinsulin/insulin from both cell types, which in beta TC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that syntaxin 1A preferentially functions in the regulated insulin release pathway in beta TC3 cells. In order to clarify the effect of overexpressed syntaxin 1A on glucose metabolism and intracellular Ca2+ we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+ ([Ca2+]i). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of beta TC-hpc1 cells were not different from those of beta TC3 cells. Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between beta TC3 and beta TC-hpc1 cells. [Ca2+]i measured by using fura 2 demonstrated that there was no difference in [Ca2+]i between beta TC3 and beta TC-hpc 1 cells under glucose-stimulated conditions. The present experiments indicate that syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+]i in beta TC3 cells.

The developmental change of both GLUT1 and GLUT3 protein in rat embryonal and fetal brain was examined using Western blot analysis and immunohistochemistry. The brains were collected from fetuses (gestational days 10 to 20), newborn, and adult rats. On day 10, the levels of GLUT1 and GLUT3 expressions were twofold higher than those of adult levels, but thereafter decreased rapidly as the gestation progressed. The tissue distribution of GLUT1 and GLUT3 in embryo was apparently distinct. On day 10, GLUT1 was expressed in the neural tube, gut, heart and optic vesicle, while GLUT3 was expressed in the surface ectoderm and gut. Thus, high affinity glucose transporters may be required in the early organogenesis period because their energy requirement is completely dependent upon anaerobic glycolysis. GLUT3 may facilitate glucose transfer from amniotic fluid to the embryo and GLUT1 may supply glucose for use as an embryonal fuel.

We examined whether arachidonoyl CoA (AA-CoA) can be a possible supplier of arachidonic acid (AA) for prostaglandin (PG) synthesis in rabbit kidney medulla. AA-CoA was preincubated with or without the 105,000 g supernatant (cytosol) fraction from the kidney medulla for 5 min at 37 degrees C followed by the incubation with the microsomal fraction (0.5 mg protein) (a rich source of PG synthesizing enzymes) in the presence of reduced glutathione and hydroquinone for 5 min at 37 degrees C, and the formed PGE2, F2 alpha and D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of AA-CoA (20 nmol) alone changed the total PG formation (the sum of PGE2, F2 alpha and D2) from 0.14 to 1.55 ng. When the cytosolic fraction (10-100 microliters) was added together with 20 nmol-AA-CoA in the preincubation mixture, total PG formation was further increased 2- to 3-fold compared to AA-CoA alone. Experiments utilizing AA, instead of AA-CoA and boiled cytosolic fraction revealed that the stimulatory action of the cytosolic fraction on the AA-CoA-induced PG synthesis may not be due to the effect on cyclooxygenase activity and due to long-chain acyl CoA hydrolase. These results show that long-chain acyl CoA hydrolase which hydrolyzes AA-CoA to AA is present in the cytosol of rabbit kidney medulla and suggest that this enzyme activity is a potential mediator of supply of AA for PG synthesis in this region.

We examined the mechanism by which prior glucose exposure accelerates conversion of proinsulin to insulin in pancreatic islets. Using actinomycin A, introduced at different times after a 3H-leucine pulse to block the intracellular transport of proinsulin, transport was found to be slightly accelerated after prolonged glucose. However, computer simulation of pulse-chase experiments showed that activation of conversion is mostly due to increased converting activity. Measurement of islet procathepsin B by immunoprecipitation indicated synthesis of this enzyme was stimulated about two fold after glucose. Pre-exposure with tunicamycin inhibited the acceleration of conversion, suggesting that the converting system requires glycosylation.