Stimulatory effect of imeglimin on incretin secretion.

AIMS/INTRODUCTION: Imeglimin is a new antidiabetic drug structurally related to metformin. Despite this structural similarity, only imeglimin augments glucose-stimulated insulin secretion (GSIS), with the mechanism underlying this effect remaining unclear. Given that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) also enhance GSIS, we examined whether these incretin hormones might contribute to the pharmacological actions of imeglimin. MATERIALS AND METHODS: Blood glucose and plasma insulin, GIP, and GLP-1 concentrations were measured during an oral glucose tolerance test (OGTT) performed in C57BL/6JJcl (C57BL/6) or KK-Ay/TaJcl (KK-Ay) mice after administration of a single dose of imeglimin with or without the dipeptidyl peptidase-4 inhibitor sitagliptin or the GLP-1 receptor antagonist exendin-9. The effects of imeglimin, with or without GIP or GLP-1, on GSIS were examined in C57BL/6 mouse islets. RESULTS: Imeglimin lowered blood glucose and increased plasma insulin levels during an OGTT in both C57BL/6 and KK-Ay mice, whereas it also increased the plasma levels of GIP and GLP-1 in KK-Ay mice and the GLP-1 levels in C57BL/6 mice. The combination of imeglimin and sitagliptin increased plasma insulin and GLP-1 levels during the OGTT in KK-Ay mice to a markedly greater extent than did either drug alone. Imeglimin enhanced GSIS in an additive manner with GLP-1, but not with GIP, in mouse islets. Exendin-9 had only a minor inhibitory effect on the glucose-lowering action of imeglimin during the OGTT in KK-Ay mice. CONCLUSIONS: Our data suggest that the imeglimin-induced increase in plasma GLP-1 levels likely contributes at least in part to its stimulatory effect on insulin secretion.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in ß-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in ß-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled ß-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects ß-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes.

O-GlcNAcylation of myocyte-specific enhancer factor 2D negatively regulates insulin secretion from pancreatic ß-cells.

Patients with type 2 diabetes often exhibit impairments in both glucose-induced insulin secretion (GIIS) and incretin-induced insulin secretion (IIIS). These phenotypes are associated with altered glucose metabolism in pancreatic ß-cells, although the molecular mechanisms remain unclear. Here, we used MIN6-K8 pancreatic ß-cell lines as a model to examine the effect of O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation), a glucose-induced protein posttranslational modification, on insulin secretion. O-GlcNAcylation was enhanced in high-glucose-treated MIN6-K8 cells, and high levels of O-GlcNAcylation attenuated PKA-dependent phosphorylation, suggesting that the two protein modifications may compete with each other. Immunoprecipitation proteomic analysis identified six candidate proteins that were O-GlcNAcylated by high-glucose treatment, whereas the O-GlcNAcylations were removed by treatment with an incretin mimetic, exendin-4. Among these proteins, knockdown of myocyte enhancer factor 2D (Mef2d) enhanced insulin secretion, and high-glucose treatment increased the level of O-GlcNAcylation of Mef2d in MIN6-K8 cells. Furthermore, knockout of Mef2d promoted GIIS in MIN6-K8 cells, whereas adenovirus-mediated rescue of Mef2d decreased GIIS in the knockout cells. These results suggest that Mef2d negatively regulates insulin secretion through O-GlcNAcylation.

Increased glycolysis affects ß-cell function and identity in aging and diabetes.

OBJECTIVE: Age is a risk factor for type 2 diabetes (T2D). We aimed to elucidate whether ß-cell glucose metabolism is altered with aging and contributes to T2D. METHODS: We used senescence-accelerated mice (SAM), C57BL/6J (B6) mice, and ob/ob mice as aging models. As a diabetes model, we used db/db mice. The glucose responsiveness of insulin secretion and the [U-13C]-glucose metabolic flux were examined in isolated islets. We analyzed the expression of ß-cell-specific genes in isolated islets and pancreatic sections as molecular signatures of ß-cell identity. ß cells defective in the malate-aspartate (MA) shuttle were previously generated from MIN6-K8 cells by the knockout of Got1, a component of the shuttle. We analyzed Got1 KO ß cells as a model of increased glycolysis. RESULTS: We identified hyperresponsiveness to glucose and compromised cellular identity as dysfunctional phenotypes shared in common between aged and diabetic mouse ß cells. We also observed a metabolic commonality between aged and diabetic ß cells: hyperactive glycolysis through the increased expression of nicotinamide mononucleotide adenylyl transferase 2 (Nmnat2), a cytosolic nicotinamide adenine dinucleotide (NAD)-synthesizing enzyme. Got1 KO ß cells showed increased glycolysis, ß-cell dysfunction, and impaired cellular identity, phenocopying aging and diabetes. Using Got1 KO ß cells, we show that attenuation of glycolysis or Nmnat2 activity can restore ß-cell function and identity. CONCLUSIONS: Our study demonstrates that hyperactive glycolysis is a metabolic signature of aged and diabetic ß cells, which may underlie age-related ß-cell dysfunction and loss of cellular identity. We suggest Nmnat2 suppression as an approach to counteract age-related T2D.

Glutamate is an essential mediator in glutamine-amplified insulin secretion.

AIMS/INTRODUCTION: Glutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine. MATERIALS AND METHODS: Clonal pancreatic ß-cells MIN6-K8, wild-type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal ß-cells (Glud1KOßCL), and glutamate-oxaloacetate transaminase 1 (GOT1) knockout clonal ß-cells (Got1KOßCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca2+ concentration ([Ca2+ ]i ) was also measured. RESULTS: Glutamine dose-dependently amplified insulin secretion in the presence of high glucose in both MIN6-K8 cells and Glud1KOßCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine-amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca2+ ]i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm-Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca2+ ]i as well as induced insulin secretion at high glucose. These effects of glutamine and dm-Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal ß-cells MIN6-m14, which otherwise exhibit no insulin secretory response to glucose. CONCLUSIONS: Glutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine-amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose-induced insulin secretion.

Proposal of a prediction model describing the metabolic interaction between warfarin and sorafenib using a population pharmacokinetic approach.

OBJECTIVE: We attempted to examine the applicability of a population pharmacokinetic-pharmacodynamic (PK-PD) model describing the metabolic interaction between warfarin and sorafenib due to CYP2C9 inhibition and to predict the plasma concentrations of sorafenib and S-warfarin, the international normalized ratio (INR), and the optimal maintenance dose of warfarin in the presence of sorafenib in vivo. MATERIALS AND METHODS: The sorafenib inhibition constant for S-warfarin metabolism was determined in vitro, and the unbound fraction in the liver was estimated using the published equations. A population PK-PD model describing the interaction between warfarin and sorafenib assuming competitive metabolic inhibition of S-warfarin by sorafenib was developed using NONMEM. The model was evaluated using clinical data and INR collected from the literature. RESULTS: The observed time courses of INR retrieved from Japanese and Caucasian patients given warfarin and sorafenib were mostly within the 90% range of the predicted values. Then, we predicted the plasma sorafenib and S-warfarin concentrations and INR after administration of warfarin (3 mg/day) alone and warfarin + sorafenib (800 mg/day). The predicted mean plasma S-warfarin concentration and INR at steady state were almost 6 and 2 times greater, respectively, in the presence of sorafenib than those for warfarin alone. The predicted S-warfarin concentrations and INR after reduction of the warfarin dose (0.5 mg/day) in the presence of sorafenib were comparable to those after 3 mg/day warfarin alone. CONCLUSION: The proposed population PK-PD model has the potential to predict an increase in INR quantitatively after concurrent administration of warfarin and sorafenib.

Gs/Gq signaling switch in ß cells defines incretin effectiveness in diabetes.

By restoring glucose-regulated insulin secretion, glucagon-like peptide-1-based (GLP-1-based) therapies are becoming increasingly important in diabetes care. Normally, the incretins GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) jointly maintain normal blood glucose levels by stimulation of insulin secretion in pancreatic ß cells. However, the reason why only GLP-1-based drugs are effective in improving insulin secretion after presentation of diabetes has not been resolved. ATP-sensitive K+ (KATP) channels play a crucial role in coupling the systemic metabolic status to ß cell electrical activity for insulin secretion. Here, we have shown that persistent membrane depolarization of ß cells due to genetic (ß cell-specific Kcnj11-/- mice) or pharmacological (long-term exposure to sulfonylureas) inhibition of the KATP channel led to a switch from Gs to Gq in a major amplifying pathway of insulin secretion. The switch determined the relative insulinotropic effectiveness of GLP-1 and GIP, as GLP-1 can activate both Gq and Gs, while GIP only activates Gs. The findings were corroborated in other models of persistent depolarization: a spontaneous diabetic KK-Ay mouse and nondiabetic human and mouse ß cells of pancreatic islets chronically treated with high glucose. Thus, a Gs/Gq signaling switch in ß cells exposed to chronic hyperglycemia underlies the differential insulinotropic potential of incretins in diabetes.

Prediction of the impact of CYP2C9 and VKORC1 genotypes on warfarin-sorafenib interactions in whites and Asians.

Aim: To predict the impact of the different CYP2C9 and VKORC1 genotypes on warfarin-sorafenib interactions in whites and Asians. Materials & methods: The influences of the CYP2C9*1/*3 and VKORC1 -1639 A/A genotypes on increases in anticoagulation responses (international normalized ratio [INR]) in the presence of sorafenib were predicted using the population pharmacokinetic/pharmacodynamic (PK/PD) model in whites and Asians. Results: INRs were predicted to be 2.0-2.1- versus 1.8-1.9-times higher in the presence of sorafenib in the CYP2C9 (*1/*1 vs *1/*3) groups than those for warfarin alone in both whites and Asians. INRs were also predicted to be 2.1-2.2- versus 1.9-2.1-times higher in the VKORC1 (GG or GA vs AA) groups. Conclusion: Warfarin-sorafenib interactions might be similar irrespective of CYP2C9 and VKORC1 genotypes or ethnicity.

Tumor-like features of gene expression and metabolic profiles in enlarged pancreatic islets are associated with impaired incretin-induced insulin secretion in obese diabetes: A study of Zucker fatty diabetes mellitus rat.

AIMS/INTRODUCTION: Pancreatic islets are heterogenous. To clarify the relationship between islet heterogeneity and incretin action in the islets, we studied gene expression and metabolic profiles of non-large and enlarged islets of the Zucker fatty diabetes mellitus rat, an obese diabetes model, as well as incretin-induced insulin secretion (IIIS) in these islets. MATERIALS AND METHODS: Pancreatic islets of control (fa/+) and fatty (fa/fa) rats at 8 and 12 weeks-of-age were isolated. The islets of fa/fa rats at 12 weeks-of-age were separated into non-large islets (≤200 µm in diameter) and enlarged islets (>300 µm in diameter). Morphological analyses, insulin secretion experiments, transcriptome analysis, metabolome analysis and oxygen consumption analysis were carried out on these islets. RESULTS: The number of enlarged islets was increased with age in fatty rats, and IIIS was significantly reduced in the enlarged islets. Markers for ß-cell differentiation were markedly decreased in the enlarged islets, but those for cell proliferation were increased. Glycolysis was enhanced in the enlarged islets, whereas the tricarboxylic acid cycle was suppressed. The oxygen consumption rate under glucose stimulation was reduced in the enlarged islets. Production of glutamate, a key signal for IIIS, was decreased in the enlarged islets. CONCLUSIONS: The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for IIIS, show tumor cell-like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age-dependent increase in such islets could contribute to the pathophysiology of obese diabetes.

Identification of risk factors for venous thromboembolism and evaluation of Khorana venous thromboembolism risk assessment in Japanese lung cancer patients.

BACKGROUND: The reported incidence of venous thromboembolism (VTE) in cancer patients is 4-20%. The Khorana VTE risk score (KRS) and the Vienna VTE risk score (VRS) have been proposed as scoring models for evaluation of cancer-associated VTE. However, the risk factors of VTE in Japanese lung cancer patients have not been clarified. METHODS: This retrospective study included 682 hospitalized Japanese patients with newly diagnosed lung cancer who were examined for VTE on admission between January 2014 and December 2016. RESULTS: Seventy-one (10.4%) of the 682 patients were diagnosed with VTE. Multivariate logistic regression analysis showed that body mass index (BMI) ≥25 kg/m2 (OR, 2.02; 95% CI, 1.06-3.72), white blood cell (WBC) count >11 × 109/L (OR, 2.31; 95% CI, 1.11-4.61), pre-chemotherapy serum D-dimer concentration ≥1.44 µg/mL (OR, 2.73; 95% CI, 1.49-4.99), and non-small cell lung cancer (OR, 3.13; 95% CI, 1.32-9.23) were significantly associated with VTE in these patients. The cut-off values for BMI, WBC count, and D-dimer concentration determined using receiver operating characteristic curves were 25.4 kg/m2, 11.2 × 109/L, and 1.95 µg/mL, respectively. CONCLUSIONS: In this study, we were able to identify four independent risk factors for cancer-associated VTE in Japanese lung cancer patients for the first time. Moreover, we showed that a cut-off level of ≥25 kg/m2 for BMI was a risk factor for VTE in this cohort.

[Effects of Seven Tyrosine Kinase Inhibitors on the Anticoagulation Activity of Warfarin].

Several studies have reported increased anticoagulation effect of warfarin(WF)when combined with tyrosine kinase inhibitors(TKIs), such as gefitinib and erlotinib. However, effects of TKIs other than gefitinib and erlotinib on the anticoagulation effect of WF have not been clarified. To assess the degree and onset of prothrombin time-international normalized ratio (PT-INR)elevation and changes in WF daily doses in patients additionally receiving TKIs, this retrospective, single-center observational study compared PT-INR values and WF daily doses during WF treatment in the absence and presence of TKIs. Seven different TKIs(afatinib, alectinib, axitinib, crizotinib, pazopanib, regorafenib, and vandetanib)were prescribed during treatment with WF of venous thromboembolism in 10 cancer patients. Compared to baseline PT-INR, significant PT-INR elevations were observed in all patients during the combination therapy. The median PT-INR increased 1.6-fold from the baseline in the presence of TKIs(p<0.01), and the onset of PT-INR elevation was observed at a median of 18 days. As all patients receiving WF with the 7 TKIs showed PT-INR elevation, enhancement of the anticoagulation effect of WF in the presence of TKIs appears to be highly frequent. PT-INR should be carefully monitored, and adjusting the WF dosage may become necessary during the WF and TKI combination therapy.

[Effects of Tyrosine Kinase Inhibitors on Control of PT-INR in Patients Receiving Warfarin].

Few studies have evaluated the influence of anticancer drugs on the anticoagulation response to warfarin(WF). This retrospective, single-center, observationalstudy evaluated the changes in prothrombin time-internationalnormal ized ratio (PT-INR)in patients receiving a combination of WF and anticancer drugs. We compared(a)PT-INR changes between groups receiving WF and concomitantly started on either tyrosine kinase inhibitors(TKI)(WF+TKI group: n=14)or anticancer drugs other than TKI(WF+non-TKI group: n=20)and(b)PT-INR changes between groups that were started on WF concomitantly while receiving either TKI(TKI+WF group: n=16)or anticancer drugs other than TKI(non-TKI+WF group: n=13). (a)PT-INR changes were significantly larger in the WF+TKI group than in the WF+non-TKI group(2.23 vs 0.42, p<0.001). In the WF+TKI group, the WF dose was reduced after all 14 patients(100.0%)showed increased PT-INR.(b)PT-INR changes during the WF induction period were significantly larger in the TKI+WF group than in the non-TKI+WF group(2.18 vs 0.68, p<0.001). In the TKI+WF group, the WF dose was reduced after 12 patients(75.0%)showed increased PT-INR. It might be necessary to consider a reduction in WF dose when WF is administered in combination with TKIs.

Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release.

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

Glutamate as intracellular and extracellular signals in pancreatic islet functions.

l-Glutamate is one of the most abundant amino acids in the body and is a constituent of proteins and a substrate in metabolism. It is well known that glutamate serves as a primary excitatory neurotransmitter and a critical neuromodulator in the brain. Recent studies have shown that in addition to its pivotal role in neural functions, glutamate plays many important roles in a variety of cellular functions, including those as intracellular and extracellular signals. In pancreatic islets, glutamate is now known to be required for the normal regulation of insulin secretion, such as incretin-induced insulin secretion. In this review, we primarily discuss the physiological and pathophysiological roles of glutamate as intracellular and extracellular signals in the functions of pancreatic islets.

Differences in Warfarin Pharmacodynamics and Predictors of Response Among Three Racial Populations.

BACKGROUND: Population differences in warfarin dosing requirement have been reported; however, unlike the pharmacokinetics (PK) of warfarin, the quantitative influences of pharmacodynamic (PD) factors on the anticoagulation response to warfarin in different ethnic populations are totally unknown. METHODS: Using population PK/PD analysis, we attempted to identify predictors of S-warfarin clearance [CL(S)] and half maximal effective concentration (EC50) to quantify racial differences in both PK and PD parameters, and to assess the contribution of these parameters to the international normalized ratio (INR) and over-anticoagulation response (INR ≥ 4) in a cohort of 309 White, Asian and African American patients. RESULTS: Similar to our previous findings, the median CL(S) was 30% lower in African American patients than Asian and White patients (169 vs. 243 and 234 mL/h, p < 0.01). EC50 showed a greater racial difference than CL(S) [1.03, 1.70 and 2.76 µg/mL for Asian, White and African American patients, respectively, p < 0.01). Significant predictors of INR included demographic/clinical (age, body weight, creatinine clearance and sex) and genotypic (CYP2C9*3,*8 and VKORC1 -1639G>A) factors, as well as African American ethnicity. In all three racial groups, genetic predictors of INR appeared to have greater influence than demographic/clinical predictors. Both CL(S) and EC50 contributed to the over-anticoagulation response to warfarin. Patients having VKORC1 -1639 G>A and/or factors associated with reduced CYP2C9 activity were more likely to have an INR ≥ 4. CONCLUSIONS: Although there were contrasting racial differences in CL(S) and EC50 that impacted on the INR, the racial difference in EC50 was greater than that for CL(S), thus explaining the higher warfarin requirement for African American patients.

Arsenic modifies serotonin metabolism through glucuronidation in pancreatic ß-cells

In arsenic-endemic regions of the world, arsenic exposure correlates with diabetes mellitus. Multiple animal models of inorganic arsenic (iAs, as As3+) exposure have revealed that iAs-induced glucose intolerance manifests as a result of pancreatic ß-cell dysfunction. To define the mechanisms responsible for this ß-cell defect, the MIN6-K8 mouse ß-cell line was exposed to environmentally relevant doses of iAs. Exposure to 0.1-1 µM iAs for 3 days significantly decreased glucose-induced insulin secretion (GIIS). Serotonin and its precursor, 5-hydroxytryptophan (5-HTP), were both decreased. Supplementation with 5-HTP, which loads the system with bioavailable 5-HTP and serotonin, rescued GIIS, suggesting that recovery of this pathway was sufficient to restore function. Exposure to iAs was accompanied by an increase in mRNA expression of UDP-glucuronosyltransferase 1 family, polypeptide a6a (Ugt1a6a), a phase-II detoxification enzyme that facilitates the disposal of cyclic amines, including serotonin, via glucuronidation. Elevated Ugt1a6a and UGT1A6 expression levels were observed in mouse and human islets, respectively, following 3 days of iAs exposure. Consistent with this finding, the enzymatic rate of serotonin glucuronidation was increased in iAs-exposed cells. Knockdown by siRNA of Ugt1a6a during iAs exposure restored GIIS in MIN6-K8 cells. This effect was prevented by blockade of serotonin biosynthesis, suggesting that the observed iAs-induced increase in Ugt1a6a affects GIIS by targeting serotonin or serotonin-related metabolites. Although it is not yet clear exactly which element(s) of the serotonin pathway is/are most responsible for iAs-induced GIIS dysfunction, this study provides evidence that UGT1A6A, acting on the serotonin pathway, regulates GIIS under both normal and pathological conditions.

Drug interactions between tyrosine kinase inhibitors (gefitinib and erlotinib) and warfarin: Assessment of international normalized ratio elevation characteristics and in vitro CYP2C9 activity.

BACKGROUND: Elevation of the international normalized ratio and bleeding complications has been reported in patients taking warfarin concomitantly with tyrosine kinase inhibitors such as gefitinib and erlotinib. OBJECTIVE: To assess the frequency, degree, and onset of international normalized ratio elevation in patients receiving warfarin with gefitinib or erlotinib, and changes in vitro cytochrome P450 2C9 activity. METHODS: This retrospective, single-center, observational study compared international normalized ratio values during the treatment with warfarin in the absence and presence of the tyrosine kinase inhibitors, gefitinib, and erlotinib. In addition, the inhibitory effect of tyrosine kinase inhibitors on cytochrome P450 2C9 activity was screened in an in vitro study. RESULTS: Compared with international normalized ratio at the baseline significant (P < 0.05) international normalized ratio elevations were observed in the majority of the patients (5/6 patients with gefitinib, 83.3%; 6/7 patients with erlotinib, 85.7%) during concurrent therapy. The international normalized ratio was increased 1.8- and 1.6-fold relative to the baseline value, on median, in the presence of gefitinib and erlotinib, respectively, and the onset of international normalized ratio elevation was observed at a median of seven days and nine days, respectively. In vitro (S)-warfarin 7-hydroxylation activity was inhibited by 36% in the presence of 1 µM gefitinib and 27% by 10 µM erlotinib, which are comparable to the steady-state plasma levels of these tyrosine kinase inhibitors after standard dosing. CONCLUSION: In most patients, international normalized ratio elevation was observed within two weeks of the start of concomitant therapy with warfarin and gefitinib or erlotinib. To avoid excessive anticoagulant response by warfarin, international normalized ratio should be carefully monitored weekly and dosage adjustment of warfarin might be recommended during the first month after the start of concurrent tyrosine kinase inhibitor therapy.

Activation of GLP-1 receptor signalling alleviates cellular stresses and improves beta cell function in a mouse model of Wolfram syndrome.

AIMS/HYPOTHESIS: Loss of functional beta cells results in a gradual progression of insulin insufficiency in Wolfram syndrome caused by recessive WFS1 mutations. However, beta cell dysfunction in Wolfram syndrome has yet to be fully characterised, and there are also no specific treatment recommendations. In this study, we aimed to characterise beta cell secretory defects and to examine the potential effects of a glucagon-like peptide-1 (GLP-1) receptor agonist on diabetes in Wolfram syndrome. METHODS: Insulin secretory function was assessed by the pancreatic perfusion method in mice used as a model of Wolfram syndrome. In addition, granule dynamics in living beta cells were examined using total internal reflection fluorescence microscopy. Acute and chronic effects of exendin-4 (Ex-4) on glucose tolerance and insulin secretion were examined in young Wfs1-/- mice without hyperglycaemia. Molecular events associated with Ex-4 treatment were investigated using pancreatic sections and isolated islets. In addition, we retrospectively observed a woman with Wolfram syndrome who had been treated with liraglutide for 24 weeks. RESULTS: Treatment with liraglutide ameliorated our patient's glycaemic control and resulted in a 20% reduction of daily insulin dose along with an off-drug elevation of fasting C-peptide immunoreactivity. Glucose-stimulated first-phase insulin secretion and potassium-stimulated insulin secretion decreased by 53% and 59%, respectively, in perfused pancreases of 10-week-old Wfs1-/- mice compared with wild-type (WT) mice. The number of insulin granule fusion events in the first phase decreased by 41% in Wfs1-/- beta cells compared with WT beta cells. Perfusion with Ex-4 increased insulin release in the first and second phases by 3.9-fold and 5.6-fold, respectively, in Wfs1-/- mice compared with perfusion with saline as a control. The physiological relevance of the effects of Ex-4 was shown by the fact that a single administration potentiated glucose-stimulated insulin secretion and improved glucose tolerance in Wfs1-/- mice. Four weeks of administration of Ex-4 resulted in an off-drug amelioration of glucose excursions after glucose loading in Wfs1-/- mice, with insulin secretory dynamics that were indistinguishable from those in WT mice, despite the fact that there was no alteration in beta cell mass. In association with the functional improvements, Ex-4 treatment reversed the increases in phosphorylated eukaryotic initiation factor (EIF2α) and thioredoxin interacting protein (TXNIP), and the decrease in phosphorylated AMP-activated kinase (AMPK), in the beta cells of the Wfs1-/- mice. Furthermore, Ex-4 treatment modulated the transcription of oxidative and endoplasmic reticulum stress-related markers in isolated islets, implying that it was able to mitigate the cellular stresses resulting from Wfs1 deficiency. CONCLUSIONS/INTERPRETATION: Our study provides deeper insights into the pathophysiology of beta cell dysfunction caused by WFS1 deficiency and implies that activation of the GLP-1 receptor signal may alleviate insulin insufficiency and aid glycaemic control in Wolfram syndrome.

Inhibition of SNAT5 Induces Incretin-Responsive State From Incretin-Unresponsive State in Pancreatic ß-Cells: Study of ß-Cell Spheroid Clusters as a Model.

ß-Cell-ß-cell interactions are required for normal regulation of insulin secretion. We previously found that formation of spheroid clusters (called K20-SC) from MIN6-K20 clonal ß-cells lacking incretin-induced insulin secretion (IIIS) under monolayer culture (called K20-MC) drastically induced incretin responsiveness. Here we investigated the mechanism by which an incretin-unresponsive state transforms to an incretin-responsive state using K20-SC as a model. Glutamate production by glucose through the malate-aspartate shuttle and cAMP signaling, both of which are critical for IIIS, were enhanced in K20-SC. SC formed from ß-cells deficient for aspartate aminotransferase 1, a critical enzyme in the malate-aspartate shuttle, exhibited reduced IIIS. Expression of the sodium-coupled neutral amino acid transporter 5 (SNAT5), which is involved in glutamine transport, was downregulated in K20-SC and pancreatic islets of normal mice but was upregulated in K20-MC and islets of rodent models of obesity and diabetes, both of which exhibit impaired IIIS. Inhibition of SNAT5 significantly increased cellular glutamate content and improved IIIS in islets of these models and in K20-MC. These results suggest that suppression of SNAT5 activity, which results in increased glutamate production, and enhancement of cAMP signaling endows incretin-unresponsive ß-cells with incretin responsiveness.

Characteristics of repaglinide effects on insulin secretion.

The dynamics of insulin secretion stimulated by repaglinide, a glinide, and the combinatorial effects of repaglinide and incretin were investigated. At 4.4 mM glucose, repaglinide induced insulin secretion with a gradually increasing first phase, showing different dynamics from that induced by glimepiride, a sulfonylurea. In the presence of glucagon-like peptide-1 (GLP-1), insulin secretion by repaglinide was augmented significantly but to lesser extent and showed different dynamics from that by glimepiride. At 4.4 mM glucose, the intracellular Ca2+ level was gradually increased by repaglinide alone or repaglinide plus GLP-1, which differs from the Ca2+ dynamics by glimepiride alone or glimepiride plus GLP-1, suggesting that the difference in Ca2+ dynamics contributes to the difference in the dynamics of insulin secretion. At a higher concentration (8.8 mM) of glucose, the dynamics of insulin secretion stimulated by repaglinide was similar to that by glimepiride. Combination of repaglinide and GLP-1 significantly augmented insulin secretion, the amount of which was comparable to that by the combination of glimepiride and GLP-1. The Ca2+ dynamics was similar for repaglinide and glimepiride at 8.8 mM glucose. Our data indicate that repaglinide has characteristic properties in its effects on the dynamics of insulin secretion and intracellular Ca2+ and that the combination of repaglinide and GLP-1 stimulates insulin secretion more effectively than the combination of glimepiride and GLP-1 at a high concentration of glucose, providing a basis for its use in clinical settings.