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Pituitary adenomas are benign tumours that can cause an individual various clinical manifestations including tumour mass effects and/or the diverse effects of abnormal pituitary hormone secretion. Given the morbidity and limited treatment options for pituitary adenomas, there is a need for better biomarkers and treatment options. One molecule that is of specific interest is the signal transducer and activator of transcription 3 (STAT3), a transcription factor that plays a critical role in mediating cytokine-induced changes in gene expression. In addition, STAT3 controls cell proliferation by regulating mitochondrial activity. Not only does activation of STAT3 play a crucial role in tumorigenesis, including pituitary tumorigenesis, but a number of studies also demonstrate pharmacological STAT3 inhibition as a promising treatment approach for many types of tumours, including pituitary tumours. This review will focus on the role of STAT3 in different pituitary adenomas, in particular, growth hormone-producing adenomas and null cell adenomas. Furthermore, how STAT3 is involved in the cell proliferation and hormone regulation in pituitary adenomas and its potential role as a molecular therapeutic target in pituitary adenomas will be summarized.

Corticotroph carcinomas and aggressive corticotroph tumors can be resistant to conventional therapy, including surgery, radiotherapy, and medical treatment. Recent evidence suggests that temozolomide (an oral alkylating agent) administered with capecitabine (pro-drug of 5-fluorouracil) may improve progression-free survival in patients with high-risk corticotroph tumors and carcinomas. This led to the use of capecitabine and temozolomide (CAPTEM) in two patients, one with a corticotroph carcinoma and the other with an aggressive corticotroph tumor, as well the in vitro analysis of capecitabine and 5-fluorouracil on cell growth and hormone production. Both patients had previous surgical and radiation therapy. The first patient developed leptomeningeal spread 2 years after his radiation treatment. He had 12 cycles of CAPTEM, which resulted in tumor control associated with clinical and radiological improvement. Twenty-seven months later, CAPTEM was restarted for disease recurrence with ongoing tumor response. The second patient had a rapid tumor regrowth 2 years after his third surgical resection. He was treated with 12 cycles of CAPTEM, which led to tumor shrinkage with no tumor regrowth 22 months after cessation of therapy. Experiments using mouse ACTH-producing pituitary tumor AtT20 cells demonstrated that treatment with 5-fluorouracil in combination with temozolomide had an additive effect in reducing cell viability and ACTH production in the culture medium. Our patients and experimental data in AtT20 cells support CAPTEM as a potential treatment option for aggressive corticotroph tumors and carcinomas. However, a prospective clinical trial is required to determine whether CAPTEM is superior to temozolomide in the treatment of these tumors.

Introduction Pituitary tumors (PTs) occur in almost 17% of the population and they represent about 10% of all intracranial tumors. At present, many patients with pituitary tumors suffer from symptoms and complications caused by abnormal hormone production and/or mass effects due to limited treatment options. Therefore, the development of novel therapies is an urgent issue. The inositol requiring enzyme 1a (IRE1a)-X-box binding protein 1 (XBP1) pathway is an important component for the endoplasmic reticulum (ER) stress response. This pathway is activated by immature protein overload in the ER and elimination or repair of such protein can reduce ER stress. This pathway has been reported to regulate cancer cell growth and secretory protein production. The detection of potential XBP1 binding sites in the promoter region of rat GH and mouse POMC similar to those of human genes suggests the involvement of the IRE1a-XBP1 pathway in the production of these hormones and pituitary tumor cell growth. In this study, we aimed to clarify the role of the IRE1a-XBP1 pathway in functional pituitary tumor cells. Methods and Results We conducted in vitro experiments by using rat GH-producing pituitary GH4 cells and mouse ACTH-producing AtT-20 cells as the models of GH- and ACTH-producing pituitary adenomas, respectively. Cell proliferation was measured by WST-8 assay, gene expression levels by qPCR, and protein expression by western blotting, respectively. In GH4 cells, STF08310 (40µM), an IRE1a inhibitor, reduced levels of GH and XBP1 spliced form (XBP1s) mRNA and GH protein with suppressed cell proliferation. In AtT-20 cells, STF08310 (40µM) also suppressed protein expression of proopiomelanocortin (POMC) and cell proliferation. Gene suppression of IRE1a by siRNA and over-expression of XBP1s showed marked reduction and increase of POMC protein, respectively. Conclusion Our findings suggest that the IRE1a-XBP1s pathway would be a possible therapeutic target that has both anti-tumoral and hormone control effects against functional pituitary tumor. Presentation: No date and time listed

The papillary subtype of craniopharyngioma (CP) rarely occurs in children and commonly presents as a suprasellar lesion. Patients with papillary CPs frequently harbor the BRAF-V600E mutation, and treatment with a BRAF inhibitor results in tumor shrinkage in several patients. Herein, we report a patient with childhood-onset papillary CP treated with vemurafenib for 40 months after multiple surgeries. At age 10, he presented with growth failure secondary to an intrasellar cystic lesion. He had 3 transsphenoidal surgeries before age 12 and a 4th surgery 25 years later for massive tumor recurrence. Pathology showed a papillary CP with positive BRAF-V600E mutation. Rapid tumor regrowth 4 months after surgery led to treatment with vemurafenib that resulted in tumor reduction within 6 weeks. Gradual tumor regrowth occurred after a dose reduction of vemurafenib because of elevated liver enzymes. He had further surgeries and within 7 weeks after stopping vemurafenib, there was massive tumor recurrence. He resumed treatment with vemurafenib before radiation therapy and similar tumor shrinkage occurred within 16 days. In this patient with childhood-onset papillary CP that was refractory to multiple surgeries, the use of vemurafenib resulted in significant tumor shrinkage that allowed for the completion of radiation therapy and tumor control.

Corticotroph carcinomas and aggressive corticotroph tumors can be resistant to conventional therapy, including surgery, radiotherapy, and medical treatment. Recent evidence suggests that temozolomide (an oral alkylating agent) administered with capecitabine (pro-drug of 5-fluorouracil) may improve progression-free survival in patients with high-risk corticotroph tumors and carcinomas. This led to the use of capecitabine and temozolomide (CAPTEM) in two patients, one with a corticotroph carcinoma and the other with an aggressive corticotroph tumor, as well the in vitro analysis of capecitabine and 5-fluorouracil on cell growth and hormone production. Both patients had previous surgical and radiation therapy. The first patient developed leptomeningeal spread 2 years after his radiation treatment. He had 12 cycles of CAPTEM, which resulted in tumor control associated with clinical and radiological improvement. Twenty-seven months later, CAPTEM was restarted for disease recurrence with ongoing tumor response. The second patient had a rapid tumor regrowth 2 years after his third surgical resection. He was treated with 12 cycles of CAPTEM, which led to tumor shrinkage with no tumor regrowth 22 months after cessation of therapy. Experiments using mouse ACTH-producing pituitary tumor AtT20 cells demonstrated that treatment with 5-fluorouracil in combination with temozolomide had an additive effect in reducing cell viability and ACTH production in the culture medium. Our patients and experimental data in AtT20 cells support CAPTEM as a potential treatment option for aggressive corticotroph tumors and carcinomas. However, a prospective clinical trial is required to determine whether CAPTEM is superior to temozolomide in the treatment of these tumors.

Pituitary tumors (PTs) can cause significant mortality and morbidity due to limited therapeutic options. L-type amino acid transporters (LATs), in particular, the LAT1 isoform, is expressed in a variety of tumor cells. Pharmacological inhibition or genetic ablation of LAT1 can suppress leucine transport into cancer cells, resulting in suppression of cancer cell growth. However, roles of LAT1 in PTs have not been elucidated. Therefore, we assessed LAT1 expression in PTs and evaluated a LAT1-specific inhibitor, JPH203, on rat somatomammotroph tumor cells, GH4 cells. GH4 cells dominantly express LAT1 mRNA rather than other LAT isoforms, whereas LAT2 transcripts were most abundant in normal rat pituitary tissues. JPH203 inhibited leucine uptake and cell growth in GH4 cells in a concentration-dependent manner, and appeared to be independent of the mechanistic target, the rapamycin pathway. Although JPH203 did not induce apoptosis, it suppressed growth hormone production in GH4 cells. Also, genetic downregulation of LAT1 showed similar effects on cell growth and hormone production. These results indicated that restriction of LAT1 substrates by JPH203 modulated both cell growth and hormone production. In conclusion, LAT1 may be a new therapeutic target for PTs because its inhibition leads to suppression of cell growth as well as hormone production. JPH203 may represent a promising drug for clinical use in patients with PTs, with the potential of hormonal control and tumor suppression.

To determine the benefits of diabetes nurse practitioner (DNP) intervention on glycemic control, quality of life and diabetes treatment satisfaction in patients with type 2 diabetes (T2DM) admitted to cardiology inpatient services at a tertiary centre.Patients admitted to the cardiology service with T2DM who had suboptimal control (HbA1c6.5%) were approached for the study. Diabetes care was optimized by the DNP through medication review, patient education and discharge care planning. Glycemic control was evaluated with 3-month post-intervention HbA1c. Secondary outcomes of lipid profiles, quality of life and treatment satisfaction were evaluated at baseline and at 3 months with fasting lipids, Audit of Diabetes-Dependent Quality of Life questionnaires (ADDQoL) and Diabetes Treatment Satisfaction Questionnaires (DTSQ) respectively.With almost 49% of patients admitted to the Mazankowski Alberta Heart Institute having HbA1c6.5%, only 23 patients completed the study over a 12-month period. We found a significant decrease in HbA1c values at 3 months post-intervention from 8.0% (SD=1. 2) to 6.9% (SD=0.7), p=0.002. LDL showed a significant decrease at 3 months from 1.7 mmol/L (SD=0.7) to 1.1 mmol /L (SD=0.6), p=0.011. Overall median ADDQoL impact scores improved at follow up, from -1.4 to -0.4, p = 0.0003. Overall no significant changes in DTSQ scores were seen.Short-term DNP intervention in T2DM patients admitted to the inpatient cardiology service was associated with benefits in areas of glycemic control and various domains of QoL. Our study provides support for the involvement of DNP in the care of cardiology inpatients at tertiary centres.

Introduction: Aggressive corticotroph tumors exhibit continued growth despite multimodal therapy, including surgeries, radiotherapy and/or medical treatment. Temozolomide (TMZ), an oral alkylating agent, has been used to treat aggressive pituitary tumors; however, most patients only showed a partial response. Recent evidence suggests that TMZ administered with capecitabine (CAP) is effective and safe in treating patients with aggressive corticotroph tumors resistant to conventional therapy. Here, we present two patients with recurrence of aggressive corticotroph pituitary tumors after surgery and radiation therapy and good response to treatment with CAP and TMZ (CAPTEM). Clinical presentation: Case 1: A 54-year-old man presented with right-sided headache and dizziness. Examination revealed palsies of the right 3rd, 4th, 5th and 6th cranial nerves. MRI confirmed a 3.6 x 3.7 x 3.5 cm sellar/suprasellar mass with right cavernous sinus invasion, chiasmal compression and clival replacement. He had transsphenoidal surgery and pathology showed a sparsely granulated ACTH adenoma with necrosis. With evidence of hypopituitarism, he was placed on prednisone, levothyroxine and testosterone. He also received stereotactic radiotherapy to manage his residual adenoma. Three years later, he presented with new onset neck pain and imaging studies showed leptomeningeal disease and biopsy confirmed ACTH adenoma. He was treated with CAPTEM for one year which resulted in clinical remission with marked radiological improvement, and persistent tumor control 20 months after cessation of therapy. Case 2: A 48 year-old man seen post transsphenoidal surgery for management of his recurrent silent corticotroph adenoma. He presented at age 21 with vision loss and had his first craniotomy. At age 31, he had repeat craniotomy for tumor recurrence that was followed by radiation therapy. With documented hypopituitarism, he has been on hydrocortisone, levothyroxine and testosterone. Although he has been legally blind, he did notice further vision loss prior to his transsphenoidal surgery. MR sella showed a 3.0 x 3.6 x 2.5 cm sellar mass infiltrating the clivus, sphenoid sinus, both cavernous sinuses and extending and compressing the optic chiasm. Even though there was good debulkment of the sellar mass, rapid tumor regrowth was noted on follow up MRI 18 months later. As the neurosurgeon did not feel that the patient would benefit from further surgery, he was treated with a year of CAPTEM. This led to significant tumor shrinkage with no tumor regrowth four months after completion of therapy. Conclusion: Our cases represent the sixth and seventh cases of CAPTEM-treated aggressive corticotroph tumors with good response to therapy. TMZ in combination with CAP seems to be promising in the management of patients with aggressive corticotroph pituitary tumors, and potentially other aggressive pituitary adenomas.

Objective: To clarify the selection of medical therapy following transsphenoidal surgery in patients with acromegaly, based on growth hormone (GH)/insulin-like growth factor 1 (IGF-1) response and glucometabolic control. Methods: We carried out a systematic literature review on three of the best studied and most practical predictive markers of the response to somatostatin analogues (SSAs): somatostatin receptor (SSTR) expression, tumor morphologic classification, and T2-weighted magnetic resonance imaging (MRI) signal intensity. Additional analyses focused on glucose metabolism in treated patients. Results: The literature survey confirmed significant associations of all three factors with SSA responsiveness. SSTR expression appears necessary for the SSA response; however, it is not sufficient, as approximately half of SSTR2-positive tumors failed to respond clinically to first-generation SSAs. MRI findings (T2-hypo-intensity) and a densely granulated phenotype also correlate with SSA efficacy, and are advantageous as predictive markers relative to SSTR expression alone. Glucometabolic control declines with SSA monotherapy, whereas GH receptor antagonist (GHRA) monotherapy may restore normoglycemia. Conclusion: We propose a decision tree to guide selection among SSAs, dopamine agonists (DAs), and GHRA for medical treatment of acromegaly in the postsurgical setting. This decision tree employs three validated predictive markers and other clinical considerations, to determine whether SSAs are appropriate first-line medical therapy in the postsurgical setting. DA treatment is favored in patients with modest IGF-1 elevation. GHRA treatment should be considered for patients with T2-hyperintense tumors with a sparsely granulated phenotype and/or low SSTR2 staining, and may also be favored for individuals with diabetes. Prospective analyses are required to test the utility of this therapeutic paradigm. Abbreviations: DA = dopamine agonist; DG = densely granulated; GH = growth hormone; GHRA = growth hormone receptor antagonist; HbA1c = glycated hemoglobin; IGF-1 = insulin-like growth factor-1; MRI = magnetic resonance imaging; SG = sparsely granulated; SSA = somatostatin analogue; SSTR = somatostatin receptor.

Previous studies have linked systemic glucocorticoid use with intestinal perforation. However, the association between intestinal perforation and endogenous hypercortisolism has not been well described, with only 14 previously published case reports. In this study, we investigated if intestinal perforation occurred more frequently in patients with ectopic ACTH syndrome and in those with a greater than 10-fold elevation of 24-hour urinary free cortisol level. Of 110 patients with ACTH-dependent Cushing’s syndrome followed in two clinics in Canada, six cases with intestinal perforation were identified over 15 years. Age of patients ranged from 52 to 72, five females and one male, four with Cushing’s disease and two with ectopic ACTH production, one from a pancreatic neuroendocrine tumor and one from medullary carcinoma of the thyroid. Five had diverticular perforation and one had intestinal perforation from a stercoral ulcer. All cases had their lower intestinal perforation when the cortisol production was high, and one patient had diverticular perforation 15 months prior to the diagnosis of Cushing’s disease. As in previously reported cases, most had hypokalemia and abdominal pain with minimal or no peritoneal symptoms and this occurred during the active phase of Cushing’s syndrome. Whereas all previously reported cases occurred in patients with 24-hour urinary free cortisol levels greater than 10-fold the upper limit of normal when measured and 11 of 14 patients had ectopic ACTH production, only one of our patients had this degree of hypercortisolism and four of our six patients had Cushing’s disease. Similar to exogenous steroid use, patients with endogenous hypercortisolism also have a higher risk of intestinal, in particular diverticular, perforation and should be monitored closely for its occurrence with a low threshold for investigation and surgical intervention. Elective colonoscopy probably should be deferred until Cushing’s syndrome is under control.

Cushing’s disease (CD) is characterized by cortisol overproduction due to ACTH hypersecretion from a pituitary tumour (PT). With an incidence of approximately 1.2 to 2.4 cases per million per year, CD patients have higher rates of morbidity and mortality than the general population. Surgical management is currently the first therapeutic option. However, remission rates vary between studies, and patients may suffer from complications caused by hormonal abnormalities from remnant PT tissues, the surgery itself, as medical treatment options are limited. Macrophage migratory inhibitory factor (MIF) is a cytokine expressed in various tumors, including ACTH-producing PTs, and has been found to play a crucial role in tumorigenesis. Previous studies demonstrate that MIF regulates cell growth via the signal transducer and activator of transcription 3 (STAT3) pathway, the mammalian target of rapamycin (mTOR) pathway, and autophagy. Together, these indicate MIF as a potential therapeutic target for PTs. However, the role of MIF in ACTH-producing PTs remains unknown. Using mouse ACTH-producing PT cells, AtT-20 cells as a model, we established that MIF overexpression led to increased cell growth. In contrast, pharmacological MIF inhibition by 4-iodo-6-phenylpyrimidine (4-IPP) and (S,R)-3-(4-Hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1) and genetic MIF downregulation by siRNA both suppressed cell viability and induced apoptosis, suggesting an anti-apoptotic role of MIF. Genetic MIF downregulation also increased the expression of apoptosis-inducible genes such as activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and reduced ACTH production. However, pharmacological MIF inhibition had no effect on ACTH production, which suggests that the mechanism of pharmacological MIF inhibition may be different from MIF downregulation. Neither MIF upregulation nor downregulation affected cell signalling pathways such as the STAT3 pathway, the mTOR pathway, or autophagy. Our findings suggest that MIF inhibition can be a viable therapeutic approach for ACTH-producing PTs.

Previous gestational diabetes (GDM) is a risk factor for type 2 diabetes and increased metabolic risk, but the link with vascular dysfunction is not clear. This study examined vascular function in women 4-10 years after a diagnosis of GDM who had a normal oral glucose tolerance test (OGTT) in the first postpartum year.We studied 90 women with a history of GDM and 59 age-matched controls, examining differences in insulin resistance as measured by the Homeostatic Model Assessment (HOMA-IR) and glucose responses during an OGTT, adiposity, lipid profile and C-reactive protein (CRP). Using pulse wave analysis, we also measured cardiac function, vascular compliance, and systemic vascular resistance.Women with a history of GDM had higher measures of adiposity (body mass index 28.9 ± 6.5 vs. 26.6 ± 6.9 kg/m(2), P=0.04, waist-hip ratio 0.85 ± 0.06 vs. 0.79 ± 0.07, P0.001), dyslipidemia (LDL cholesterol 2.78 ± 0.64 vs. 2.41 ± 0.56 mmol/L, P0.001, total cholesterol: HDL cholesterol 3.93 ± 1.2 vs. 3.21 ± 0.82 mmol/L, P0.001) and abnormal glucose metabolism (50% vs. 12%, P0.001). However, there was no difference in CRP, HOMA-IR, or measures of cardiovascular function including pulse rate, pulse pressure, mean arterial pressure, cardiac output, systemic vascular resistance, small and large artery elasticity index. After controlling for adiposity, blood pressure, lipids and CRP, glycemic status did not contribute to vascular function.Despite a higher incidence of adiposity, dyslipidemia, and impaired glycemia, women with a history of GDM who had a normal postpartum OGTT did not have impaired vascular function 4-10 years postpartum, when compared to healthy controls.

Tumors of hypothalamic neurons that produce vasopressin are rare. We retrieved all cases of vasopressin-positive tumors in the sellar region from the database of the Department of Pathology. Five cases fulfilled the selection criteria, representing the first series of such tumors. Clinical, radiologic, and pathologic features were reviewed. Four tumors classified as neurocytomas were identified in 3 females and 1 male patient; the ages at onset of symptoms ranged from 17 to 40 years. All were large sellar masses with suprasellar extension and/or invasion of the parasellar sinuses. Three patients had the syndrome of inappropriate antidiuresis; in one of these, a 6-year history was initially considered to be idiopathic. One patient died of progressive disease; 3 had incomplete resections and are being followed. In contrast to these patients with neurocytoma, a 65-year-old woman had Cushing disease and a 0.8 cm mass that was completely resected at transsphenoidal surgery; this tumor was a gangliocytoma producing vasopressin associated with corticotroph hyperplasia. We postulate that the small amount of vasopressin secreted by this mature gangliocytic tumor was locally bound to corticotrophs, resulting in hyperplasia and Cushing disease, without sufficient overproduction to cause systemic effects of vasopressin excess. Hypothalamic neurocytoma is a tumor that can mimic pituitary neuroendocrine tumors and olfactory neuroblastoma but is distinguished by positivity for neurofilaments, NeuN, and TTF-1 and negative staining for adenohypophysial biomarkers. Our cases illustrate that neurocytoma and gangliocytoma are 2 variants of tumors of hypothalamic neurons that can produce vasopressin. The morphologic and proliferative features of these 2 tumor types represent 2 ends of a spectrum; their function also can result in divergent clinical manifestations, one characterized by reduced urine output and the other by the more insidious features of glucocorticoid excess.

Background and Objective Gestational diabetes (GDM) occurs more often in women from certain ethnic groups and is also associated with fetal macrosomia. In this study, we investigated the ability of a gestational diabetes screening test (GDS), the 2 h 75 g-Oral Glucose Tolerance Test (OGTT), and glycated hemoglobin (HbA1c) in predicting postpartum dysglycemia and fetal macrosomia in women of Caucasian, Filipino, Chinese and South-Asian descent. Methods 848 women diagnosed with carbohydrate intolerance in pregnancy who completed a 2 h 75 g- OGTT within 6 months postpartum, were included in the study. Receiver Operating Characteristic curve analysis was used to test the ability of antepartum GDS, HbA1c and OGTT in predicting postpartum hyperglycemia, type 2 diabetes (T2D) and neonatal macrosomia (birth weight >4000 g). Results 20.2% had postpartum hyperglycemia while 3.8% had T2D. Those with postpartum dysglycemia were more likely to be non-Caucasian (South-Asian > Filipino > Chinese), have higher antepartum glucose values, require insulin during pregnancy and have cesarean births. Of HbA1c and the antepartum glucose values, a fasting glucose of ≥5.25 mmol/L was predictive of fetal macrosomia in Caucasians. 1 h glucose of ≥11.05 mmol/L was predictive of postpartum hyperglycemia, while 2 h glucose of ≥9.75 mmol/L was predictive of T2D; ethnicity influenced the predictive ability of these tests. Conclusions Ethnicity influences the ability of antepartum glucose and HbA1c to predict the risk of macrosomia and postpartum dysglycemia. This information will help detect those most at risk of T2D.

Salt inducible kinase 1 (SIK1) has been reported to repress cAMP-response element binding protein (CREB)-mediated gene transcription by causing the nuclear export of CREB-regulated transcription coactivators (CRTCs) through phosphorylation. Although the repressor role of SIK1 in suppressing the expression of arylalkylamine N-acetyltransferase, the enzyme that controls the daily rhythm in melatonin production in the rat pineal gland, has been established, whether SIK1 regulates the phosphorylation and localization of CRTC1 and CRTC2 in this tissue remains unclear. The present study found that overexpressing SIK1 in NE-stimulated rat pinealocytes could increase the phosphorylation of CRTC1 and CRTC2, reduced selectively the nuclear level of CRTC2 (but not that of CRTC1), and elevated the cytosolic levels of both CRTC1 and CRTC2. In contrast, transient knockdown of endogenous SIK1 had no effect on the phosphorylation or distribution of CRTC1 and CRTC2 in norepinephrine (NE)-stimulated pinealocytes. Our results also showed that adrenergic blockade during NE stimulation led to a rapid rephosphorylation and decline in the nucleus levels of CRTC1 and CRTC2; however SIK1 knockdown had no effect on this rapid rephosphorylation. Moreover, studies with kinase inhibitors revealed that kinase(s) sensitive to KT5823 appeared to be involved in this rapid rephosphorylation. Together, these results indicate that although overexpressing SIK1 can phosphorylate CRTC1 and CRTC2 in the NE-stimulated pinealocyte, the endogenous SIK1, in spite of its induction by NE, does not appear to be the main regulator of the phosphorylation and intracellular localization of these two coactivators.

OBJECTIVE Depressive symptoms are common and, when coexisting with diabetes, worsen outcomes and increase health care costs. We evaluated a nurse case-manager–based collaborative primary care team model to improve depressive symptoms in diabetic patients. RESEARCH DESIGN AND METHODS We conducted a controlled implementation trial in four nonmetropolitan primary care networks. Eligible patients had type 2 diabetes and screened positive for depressive symptoms, based on a Patient Health Questionnaire (PHQ) score of ≥10. Patients were allocated using an “on-off” monthly time series. Intervention consisted of case-managers working 1:1 with patients to deliver individualized care. The main outcome was improvement in PHQ scores at 12 months. A concurrent cohort of 71 comparable patients was used as nonscreened usual care control subjects. RESULTS Of 1,924 patients screened, 476 (25%) had a PHQ score >10. Of these, 95 were allocated to intervention and 62 to active control. There were no baseline differences between groups: mean age was 57.8 years, 55% were women, and the mean PHQ score was 14.5 (SD 3.7). Intervention patients had greater 12-month improvements in PHQ (7.3 [SD 5.6]) compared with active-control subjects (5.2 [SD 5.7], P = 0.015). Recovery of depressive symptoms (i.e., PHQ reduced by 50%) was greater among intervention patients (61% vs. 44%, P = 0.03). Compared with trial patients, nonscreened control subjects had significantly less improvement at 12 months in the PHQ score (3.2 [SD 4.9]) and lower rates of recovery (24%, P < 0.05 for both). CONCLUSIONS In patients with type 2 diabetes who screened positive for depressive symptoms, collaborative care improved depressive symptoms, but physician notification and follow-up was also a clinically effective initial strategy compared with usual care.

The incidence of gestational diabetes mellitus (GDM) is increasing. However, less is known about the incidence of preeclampsia (PE) and whether it is affected by the presence of GDM. We sought to document the population-level incidence of GDM and PE during the last decade and examine the association between GDM and PE after accounting for established risk factors.We selected a population-based cohort retrospectively using data from the Alberta Perinatal Health Program registry. Logistic regression was used to examine the association between GDM and PE after adjusting for baseline characteristics.Of 426 296 deliveries between 2000 and 2009, 422 672 were in women without pre-existing diabetes. Among these women, the incidence of GDM increased from 3.1% in 2000 to 4.6% in 2009 (P0.01), while the incidence of PE remained stable at approximately 1.3% per year. The incidence of PE was significantly higher in women with GDM than in those without GDM (2.6% vs. 1.2%; P0.01). After adjustment, women with GDM had a 90% higher risk of PE than those without GDM (OR 1.9; 95% CI 1.7 to 2.1). Other significant risk factors for PE were age, obesity, nulliparity, multifetal gestation, pre-existing hypertension, and chronic kidney disease.In this contemporary population-based study spanning 10 years, there was a significant increase in the incidence of GDM over time. The higher incidence of PE in women with GDM than in normoglycemic women suggests a need for heightened surveillance and monitoring of women with GDM for the development of PE.

Vascular dysfunction has been described in women with a history of gestational diabetes mellitus. Furthermore, previous gestational diabetes mellitus increases the risk of developing Type 2 diabetes mellitus, a risk factor for cardiovascular disease. Factors contributing to vascular changes remain uncertain. The aim of this review was to summarize vascular structure and function changes found to occur in women with previous gestational diabetes mellitus and to identify factors that contribute to vascular dysfunction. A systematic search of electronic databases yielded 15 publications from 1998 to March 2014 that met the inclusion criteria. Our review confirmed that previous gestational diabetes mellitus contributes to vascular dysfunction, and the most consistent risk factor associated with previous gestational diabetes mellitus and vascular dysfunction was elevated body mass index. Heterogeneity existed across studies in determining the relationship of glycaemic levels and insulin resistance to vascular dysfunction.

The distribution of transducers of regulated cAMP-response element-binding protein activity (TORC) between the cytoplasm and the nucleus is tightly regulated and represents one of the main mechanisms whereby the cAMP response element activation activities of TORC are controlled. Whereas both cAMP and Ca2+ pathways can cause translocation of TORC, the relative importance of these two pathways in regulating different TORC within the same cell is unclear. In this study, we determined the mechanism that regulated TORC1 translocation and compared it with that of TORC2 in rat pinealocytes. Stimulation of pinealocytes with norepinephrine (NE), although having no effect on Torc1 transcription, caused rapid dephosphorylation of TORC1. Although NE also caused rapid dephosphorylation of TORC2, pharmacological studies revealed that TORC1 dephosphorylation could be induced by both β-adrenoceptor/cAMP and α-adrenoceptor/intracellular Ca2+ pathways contrasting with TORC2 dephosphorylation being induced mainly through the β-adrenoceptor/cAMP pathway. PhosTag gel indicated a different pattern of TORC1 desphosphorylation resulting from the selective activation of α- or β-adrenoceptors. Interestingly, only the α-adrenoceptor/intracellular Ca2+-mediated dephosphorylation could translocate TORC1 to the nucleus, whereas the β-adrenoceptor/cAMP-mediated dephosphorylation of TORC1 was ineffective. In comparison, translocation of TORC2 was induced predominantly by the β-adrenoceptor/cAMP pathway. Studies with different protein phosphatase (PP) inhibitors indicated that the NE-mediated translocation of TORC1 was blocked by cyclosporine A, a PP2B inhibitor, but that of TORC2 was blocked by okadaic acid, a PP2A inhibitor. Together these results highlight different intracellular signaling pathways that are involved in the NE-stimulated dephosphorylation and translocation of TORC1 and TORC2 in rat pinealocytes.

Histone modifications have been shown to play an important role in regulating gene expression. In this study, we investigated the impact of histone modifications on the adrenergic-regulated transcription of type 2 deiodinase (Dio2), a CREB-target gene in the rat pinealocyte. Treatment of pinealocytes with inhibitors of aurora C, a histone kinase, resulted in an inhibitory effect on the adrenergic-stimulated histone H3 Ser10 phosphorylation and Dio2 transcription. Given the established link between histone phosphorylation and acetylation, the role of histone acetylation on the adrenergic-induced Dio2 transcription was investigated. Treatment of pinealocytes with histone deacetylase inhibitors inhibited the adrenergic-induced Dio2 transcription. Chromatin immunoprecipitation with antibodies against acetylated Lys14 of H3 showed an increase in DNA recovery of the promoter region of Dio2 following treatment with trichostatin A. Together, our results indicate that, beside activation of CREB, epigenetic factors such as histone modifications also play an important role in regulating Dio2 transcription.

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (

Although the use of supplemental oxygen to resuscitate asphyxiated neonates remains controversial, the effects of hypoxia and reoxygenation (room air versus pure oxygen) on the hypothalamo-pituitary-adrenal axis are unknown. We aimed to evaluate the effect of hypoxia and reoxygenation with either 21% or 100% oxygen on plasma cortisol before and after an adrenocorticotrophin (ACTH) challenge in newborn piglets. Thirty-five piglets (aged 1-3 days, weighing 1.5-2.4 kg) were instrumented to measure heart rate, MAP, and cardiac output. After 2 h of normocapnic hypoxia (PaO2, 20-30 mmHg; pH

An inverted circadian rhythm of melatonin (MT) likely contributes to the sleep disturbance in patients with Smith–Magenis syndrome (SMS). Plasma MT levels have documented this altered rhythm, but daytime levels of salivary MT has not been determined. Daytime measures of salivary MT might have utility in home/outpatient settings for assessing MT levels in undiagnosed patients with clinical features of SMS. The objective of this study was to determine the utility of daytime salivary MT as a diagnostic test in SMS. Thirty individuals with confirmed SMS [28 with del 17p11.2 and 2 with the retinoic acid induced 1 (RAI1) gene mutation] and five controls were studied. Single or serial daytime salivary MT levels were measured. The mean midday salivary MT level was 79.0 pg/ml in SMS patients, compared with 16.3 pg/ml in controls, with nine patients having values similar to controls. The median MT level in SMS patients was 49.0 pg/ml (first and third quartile values = 15.5 and 106.8 pg/ml). Twenty-six (90%) of 29 patients had at least one MT value >15.5 pg/ml, including 70 (78%) of 90 samples from patients with del 17p11.2 and one (20%) of five samples from the two patients with the RAI1 mutation. Neither the pattern of medication use nor age had an effect on daytime salivary MT levels. Although most SMS patients had elevated daytime salivary MT levels, multiple sampling appears necessary to distinguish patients with SMS from other conditions. © 2009 Wiley-Liss, Inc.

The recognition of the basic leucine zipper domain in the regulation of transcriptional activity of cAMP response element-binding protein by salt-inducible kinase (SIK) prompted our investigation of the regulatory role of this kinase in the induction of Aa-nat and other cAMP-regulated genes in the rat pineal gland. Here we report Sik1 expression was induced by norepinephrine (NE) in rat pinealocytes primarily through activation of β-adrenergic receptors, with a minor contribution from activation of α-adrenergic receptors. Treatments with dibutyryl cAMP, and to a lesser extent, agents that elevate intracellular Ca2+ mimicked the effect of NE on Sik1 expression. In parallel to the results of the pineal cell culture studies, a marked nocturnal induction of Sik1 transcription was found in whole-animal studies. Knockdown of Sik1 by short hairpin RNA amplified the NE-stimulated Aa-nat transcription and other adrenergic-regulated genes, including Mapk phosphatase 1, inducible cAMP repressor, and type 2 iodothyronine deiodinase in a time-dependent manner. In contrast, overexpressing Sik1 had an inhibitory effect on the NE induction of Aa-nat and other adrenergic-regulated genes. Together, our results indicate that the adrenergic induction of Sik1 in the rat pineal gland is primarily through the β-adrenergic receptor → protein kinase A pathway. SIK1 appears to function as part of an endogenous repressive mechanism that regulates the peak and indirectly the duration of expression of Aa-nat and other cAMP-regulated genes. These findings support a role for SIK1 in framing the temporal expression profile of Aa-nat and other adrenergic-regulated genes in the rat pineal gland.

We have shown previously that Ser10 phosphorylation of histone H3 occurs in rat pinealocytes after stimulation with norepinephrine (NE) and that histone modifications such as acetylation appear to play an important role in pineal gene transcription. Here we report the nocturnal phosphorylation of a Ser10 histone H3 kinase, Aurora C, in the rat pineal gland. The time profile of this phosphorylation parallels the increase in the level of phospho-Ser10 histone H3. Studies with cultured pinealocytes indicate that Aurora C phosphorylation is induced by NE and this induction can be blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor. Moreover, only treatment with dibutyryl cAMP, but not other kinase activators, mimics the effect of NE on Aurora C phosphorylation. These results indicate that Aurora C is phosphorylated primarily by a β-adrenergic/protein kinase A-mediated mechanism. Treatment with an Aurora C inhibitor reduces the NE-induced histone H3 phosphorylation and suppresses the NE-stimulated induction of arylalkylamine N-acetyltransferase (AA-NAT), the rhythm-controlling enzyme of melatonin synthesis, and melatonin production. The effects of Aurora C inhibitors on adrenergic-induced genes in rat pinealocytes are gene specific: inhibitory for Aa-nat and inducible cAMP repressor but stimulatory for c-fos. Together our results support a role for the NE-stimulated phosphorylation of Aurora C and the subsequent remodeling of chromatin in NE-stimulated Aa-nat transcription. This phenomenon suggests that activation of this mitotic kinase can be induced by extracellular signals to participate in the transcriptional induction of a subset of genes in the rat pineal gland.

In the present study, we investigated the effect of ceramide on the outward K(+) current in rat pinealocytes using whole cell and single channel recordings. Three components of the whole cell outward K(+) current were separated, an iberiotoxin (IBTX)-sensitive K(+) current (I(KCa)), a transient A current (I(A)) and a delayed rectifier current (I(K)). C6-ceramide reduced all three components of the outward K(+) current. C6-ceramide (30 microM) caused a 53% inhibition of I(KCa) [a component that is generated by the IBTX-sensitive K(+) channel (BK channel)], a 27% inhibition of I(A) and a 17% inhibition of I(K). Additional studies showed that the BK channel was not inhibited by dihydroC6-ceramide, the inactive analog of C6-ceramide, but mimicked by sphingomyelinase which increased intracellular ceramide. The ceramide inhibition of the BK channel was only partly dependent on its inhibition of the L-type Ca(2+) channel. Studies using specific kinase inhibitors showed that calphostin C (a protein kinase C inhibitor) and to a lesser degree lavendustin A (a tyrosine kinase inhibitor) were effective in reducing the ceramide inhibition of I(KCa). Taken together, our results show that, in rat pinealocytes, ceramide reduces the outward K(+) current predominantly by inhibiting I(KCa). Moreover, protein kinase C appears to be the main kinase involved in the ceramide inhibition of I(KCa).

In this study, we investigated the role of two inducible repressor proteins, inducible cAMP early repressor (ICER) and Fos-related antigen 2 (Fra-2) in the adrenergic induction of MAPK phosphatase-1 (MKP-1) as compared with their roles in the induction of arylalkylamine-N-acetyltransferase (AA-NAT) in rat pinealocytes. Treatment of pinealocytes with norepinephrine (NE) caused an increase in the mRNA and protein levels of MKP-1 and AA-NAT, as well as in the AA-NAT activity and melatonin production. NE stimulation also caused a simultaneous increase in the mRNA and protein levels of ICER and Fra-2. Transient knockdown of icer using adenovirus expressing small interfering RNA (siRNA) abolished the NE induction of icer expression but had little effect on the NE induction of mkp-1 or aa-nat expression. In contrast, pretreatment with adenovirus overexpressing icer was effective in reducing the NE induction of mkp-1 and aa-nat. The inhibitory effect of overexpressing icer was reversed by cotreatment with siRNA against icer. siRNA against fra-2 also abolished the NE-stimulated expression of fra-2 but had little effect on the NE induction of mkp-1 and aa-nat expression. Proteasomal inhibition, which reduced the NE-stimulated induction of aa-nat, caused a reduction of ICER and Fra-2. Together, these results indicate that whereas overexpression of ICER can suppress the NE induction of aa-nat and mkp-1, the amount of the repressors, ICER and Fra-2, present during NE induction appears insufficient to exert a significant effect in controlling the expression of these genes.

Tumor immune microenvironment has been gradually recognized as a key contributor to tumor development, progression, and control. Immune cell infiltrates in brain tumors have been increasingly studied, but few have published on immune cell infiltrates in pituitary adenomas. We quantitatively assessed the infiltration of macrophages and lymphocytes in 35 pituitary adenomas, including 9 densely granulated growth hormone (DG-GH), 9 sparsely granulated growth hormone (SG-GH), 9 null cell (NC), and 8 adrenocorticotropic hormone (ACTH) adenomas. All the adenomas showed varying degrees of CD68+ macrophage infiltration. While SG-GH adenomas were significantly larger in size than DG-GH and ACTH adenomas, the infiltration of CD68+ macrophages was significantly greater in SG-GH than in DG-GH and ACTH adenomas. Similarly, NC adenomas that were significantly larger than DG-GH and ACTH adenomas had significantly greater infiltration of CD68+ macrophages than DG-GH and ACTH adenomas. The numbers of CD68+ macrophages were positively correlated with the tumor sizes and Knosp classification grades for tumor invasiveness. The infiltration of CD4+ and CD8+ T cells was relatively scant in these adenomas, but GH adenomas exhibited significantly more CD4+ and CD8+ T cells than non-GH adenomas. Both DG-GH and SG-GH adenomas had significantly more CD4+ cells than ACTH adenomas and significantly more CD8+ cells than NC adenomas. These results suggest an association of CD68+ macrophage infiltration with an increase in the pituitary adenoma size and invasiveness. Our observation contributes to understanding the growth environment of pituitary adenomas, for which adjuvant immunotherapy may help to constrain the tumor enlargement and invasiveness.

Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE → cGMP activation of p42/44MAPK is rapid and transient, the NE → cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine-N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.

In the rat pineal gland, the steady-state level of arylalkylamine N-acetyltransferase (AANAT) protein is controlled by transcriptional and translational mechanisms as well as by proteasome-mediated degradation. Studies with proteasome inhibitors, MG132 and clasto-lactacystin beta-lactone (c-lact), show two opposite effects of proteasomal inhibition on norepinephrine (NE)-induction of Aanat. Addition of MG132 or c-lact following NE stimulation causes an increase in AANAT protein level and enzyme activity without affecting the level of Aanat mRNA. In contrast, addition of inhibitors prior to NE stimulation reduces the NE-stimulated Aanat mRNA, AANAT protein, and enzyme activity. The inhibitory effect of proteasomal inhibition on adrenergic-induced Aanat transcription appears specific for Aanat because it has no effect on the adrenergic induction of mitogen-activated protein kinase phosphatase-1 (mkp-1). The effects of the proteasome inhibitors on NE-stimulated Aanat induction appear to be mediated by accumulation of a protein repressor.

Differences in the time profiles of activation between p38MAPK and p42/44MAPK by norepinephrine (NE) in rat pinealocytes suggest involvement of mechanisms other than the phosphorylation cascades in their activation. In the present study we investigated whether protein turnover played a role in regulating p38MAPK activation in the rat pineal gland. NE stimulation caused an increase in MAPK kinase3/6 (MKK 3/6) and p38MAPK phosphorylation that occurred in the absence of changes in the mRNA or protein levels of p38MAPK or MKK3/6. The stimulatory effect of NE on phosphorylated MKK3/6 and p38MAPK, but not phosphorylated p42/44MAPK, was blocked by treatment with actinomycin or cycloheximide, indicating a requirement of transcription and translation in activation of the p38MAPK but not the p42/44MAPK pathway. Moreover, inhibition of proteasomes by clasto-lactacystin β-lactone or Z-Leu-Leu-Leu-CHO (MG132) selectively increased basal and NE-stimulated phosphorylated MKK3/6 and p38MAPK levels without affecting the mRNA or protein levels of MKK3 or p38MAPK. In contrast, the effect of proteasomal inhibition on NE-stimulated p42/44MAPK phosphorylation was inhibitory. Treatment with MG132 also reduced the decline in the phosphorylated levels of NE-stimulated MKK3/6 and p38MAPK that normally follows β-adrenergic blockade. Together, our results indicate that p38MAPK but not p42/44MAPK activation in the rat pineal gland is tightly coupled to protein synthesis and degradation. The synthesis of an activator upstream of MKK3/6 is required for the NE-activation of p38MAPK.

In this study, we investigated the mechanisms through which norepinephrine (NE) regulates MAPK phosphatase-1 (MKP-1) expression in rat pinealocytes. Stimulation with NE (a mixed alpha- and beta-adrenergic agonist) caused a rapid increase in MKP-1 mRNA and protein that peaked around 1 h post stimulation, and the response was sustained for at least 4 h. Selective activation of beta-adrenergic receptors with isoproterenol for 1 h caused a similar increase in MKP-1 mRNA and protein as observed with NE, but at 3 h, the isoproterenol response was much lower relative to NE. In contrast, selective activation of alpha-adrenergic receptors caused only small increases in MKP-1 mRNA and protein and appeared to function primarily in prolonging the beta-adrenergic-stimulated responses. In NE-stimulated pinealocytes, blockade of beta-adrenergic receptors caused a rapid reduction in MKP-1 mRNA, but it had a minimal effect on MKP-1 protein. In contrast, blockade of alpha-adrenergic receptors specifically reduced NE-induced MKP-1 protein but not mRNA. At the postreceptor level, treatment with dibutyryl cAMP caused parallel increases in MKP-1 mRNA and protein. However, treatment with a protein kinase C activator caused a significant increase in MKP-1 protein but had little effect on MKP-1 mRNA. Together, these results suggest that, in rat pinealocytes, NE activates the beta-adrenergic receptor --protein kinase A pathway to induce transcription and translation of MKP-1 expression and the alpha-adrenergic receptor --protein kinase C pathway to prolong the stimulated responses through increased stability of the MKP-1 protein.

In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both α- and β-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4β-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca2+/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a β-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca2+ signaling pathways are involved in NE regulation of p38MAPK.

In this study, we investigated whether there was a diurnal difference in mitogen-activated protein kinase (p42/44(MAPK)) phosphorylation in the rat pineal gland. Under a lighting regimen with 12h of darkness, there was a two- to four-fold increase in phosphorylated levels of MAPK kinase 1/2 (MEK1/2) and p42/44(MAPK) 2h after onset of darkness, an increase that was sustained for 8h. The increases in phosphorylated levels of MEK1/2 and p42/44(MAPK) occurred without increases in MEK1/2 and p42/44(MAPK) proteins. When rats were treated with propranolol 1h before onset of darkness or subjected to continuous light exposure during the dark phase, the nocturnal increase in MEK1/2 and p42/44(MAPK) phosphorylation was reduced. Acute light exposure during darkness caused a decline in MEK1/2 and p42/44(MAPK) phosphorylation within 30 min of light exposure. These results indicate the presence of a diurnal difference in MEK1/2 and p42/44(MAPK) phosphorylation in the rat pineal gland that is under adrenergic control.

In this study the subunits of the dihydropyridine-sensitive L-type Ca2+ channels (L-channels) expressed in rat pinealocytes were characterized using reverse transcription (RT)-PCR analysis, and the modulation of these channels by adrenergic agonists and by pituitary adenylate cyclase-activating polypeptide (PACAP) was studied using the patch-clamp technique. RT-PCR analysis showed that rat pinealocytes expressed alpha 1D, alpha 2b, beta 2, and beta 4 Ca(2+)-channel subunit mRNAs. Other alpha 1 subunit transcripts were either not expressed or present at very low levels, indicating that the pinealocytes express predominantly alpha 1D L-channels. Electrophysiological studies confirmed that the pineal expressed a single population of L-channels. The L-channel currents were inhibited by two agonists that elevate cyclic AMP: the beta-adrenergic agonist isoproterenol and PACAP. Similar inhibition was observed with a cyclic AMP analogue, 8-bromo-cyclic AMP. The presence of a cyclic AMP antagonist, Rp-adenosine 3',5'-cyclic monophosphorothioate, blocked the inhibition by isoproterenol and PACAP. Norepinephrine, a mixed alpha- and beta-adrenergic agonist, also inhibited the L-channel currents, but the inhibition was smaller. The smaller inhibition by norepinephrine was secondary to the simultaneous activation of alpha- and beta-adrenergic receptors. These results indicate that (a) pinealocytes express predominantly alpha 1D L-channels, and (b) the beta-adrenergic agonist isoproterenol and PACAP inhibit the L-channel currents through elevation of cyclic AMP. However, an alpha-adrenergic-mediated mechanism also appears to be involved in the effect of norepinephrine on the L-channel currents.

The effects of p38 mitogen-activated protein kinase (p38MAPK) inhibitors on the adrenergic-stimulated cyclic nucleotide production in rat pinealocytes were investigated. Treatment with SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)IH-imidazole] and SB203580 [4-(4-fluoropheny)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole] (1–100 μM), two pyridinyl imidazole compounds that inhibit p38MAPK, as well as SB202474 [4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)IH-imidazole], an inactive analog, was effective in potentiating norepinephrine- and isoproterenol-stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) accumulation in a concentration-dependent manner. All three compounds caused a greater increase in the cGMP than the cAMP response, with SB202474 being substantially more potent than the two active analogs. At 100 μM, SB202474 potentiated the isoproterenol-stimulated cAMP and cGMP accumulation by 65 and 500%, respectively. Pharmacological studies indicated that the potentiating effect of SB202474 was independent of protein kinase C activation, intracellular calcium elevation, or serine/threonine phosphatase inhibition, three pathways known to potentiate the β-adrenergic-stimulated cyclic nucleotide responses in rat pinealocytes. In contrast, the potentiating effect of SB202474 was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine. At 100 μM, all three compounds inhibited cAMP- and cGMP-phosphodiesterase activities by 50 and 80%, respectively. These results suggest that the commonly used p38MAPK inhibitors can modulate cyclic nucleotide responses through phosphodiesterase inhibition, a mechanism that appears to be independent of p38MAPK inhibition.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a vasoactive peptide, modulates the L-type Ca(2+) channel current (L channel current) in vascular smooth muscle cells (VSMC) through activation and integration of two intracellular pathways, protein kinase A and protein kinase C (PKC). In the present study we compared the effects of PACAP on the L channel current in VSMC from the spontaneously hypertensive rats (SHR) and normotensive controls, Wistar Kyoto rats (WKY). We found that compared with WKY, VSMC from SHR had a higher L channel current density. Stimulation by PACAP (10 nM) caused an increase in the amplitude of the whole cell current and prolonged open time in VSMC from SHR and WKY, with the increase greater in SHR. These effects of PACAP on the L channel current was mimicked by an activator of PKC. In contrast, PACAP caused a smaller increase in cAMP accumulation in VSMC from SHR than WKY, and there was no difference in the inhibitory effect of 8-bromo-cAMP on the L channel current from both type of cells. The greater increase in amplitude of the L channel current by PACAP in VSMC from SHR persisted in the presence of adenosine cyclic 3',5'-monophosphothioate, Rp-isomer, a cAMP antagonist, but not calphostin C, a PKC inhibitor. Taken together, our results show an increase in L channel current density and an enhanced PACAP effect on the L channel current in VSMC from SHR compared with WKY. This difference in PACAP response appears to be predominately secondary to an increased PKC sensitivity.

The role of adrenergic stimulation in the regulation of mitogen-activated protein kinase (MAPK) in rat pinealocytes was investigated by measuring phosphorylated MAPK using Western blot analysis and a MAPK enzymatic assay. Stimulation with the endogenous neurotransmitter, norepinephrine (NE; a mixed α- and β-adrenergic agonist), concentration dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of NE was blocked by PD98059 and UO126 (two inhibitors of MEK). Treatment with prazosin or propranolol significantly reduced the effect of NE on MAPK phosphorylation, suggesting the involvement of both α- andβ -adrenergic receptors. Investigation into the intracellular mechanisms of NE action revealed that the increase in MAPK phosphorylation was blocked by KT5823 (a protein kinase G inhibitor), but was enhanced by H89 (a protein kinase A inhibitor). Calphostin C (a protein kinase C inhibitor) and KN93 (a Ca2+/calmodulin-dependent protein kinase inhibitor) also attenuated NE-mediated MAPK activation, but to a lesser degree. Furthermore, inhibition of MAPK phosphorylation by (Bu)2cAMP was effective in reducing MAPK activation by (Bu)2cGMP, an active phorbol ester or ionomycin. These results indicate that the effect of NE on MAPK phosphorylation represents mainly the integration of two signaling mechanisms, protein kinase A and protein kinase G, each having an opposite effect on MAPK phosphorylation.

Interaction between p38MAPK and p42/44MAPK in rat pinealocytes was examined by determining the effects of p38MAPK inhibitors on the phosphorylation of p42/44MAPK using Western blot analysis. Treatment with SB202190, a specific inhibitor of p38MAPK, increased p42/44MAPK phosphorylation in a concentration-dependent manner. SB202190 also enhanced the magnitude and the duration of norepinephrine-activated p42/44MAPK phosphorylation. The effect of SB202190 on p42/44MAPK phosphorylation was abolished by PD98059 or UO126, inhibitors of MEK, suggesting that SB202190 is acting upstream of MEK in activating p42/44MAPK. The SB202190-induced phosphorylation of p42/44MAPK was not blocked by inhibitors of cGMP-dependent kinase (KT5823), protein kinase C (calphostin C) or Ca2+/calmodulin dependent kinase (KN93) suggesting that these pathways may not be involved in the effect of SB202190. SB202190 further increased p42/44MAPK phosphorylation that was stimulated by 8-bromo-cGMP, 4β phorbol 12-myristate 13-acetate, or iono...

OBJECTIVE To determine the rate of adherence to postpartum glycemic testing in women with gestational diabetes mellitus (GDM) and the performance of fasting plasma glucose (FPG) versus the 75-g oral glucose tolerance test (OGTT) in detecting postpartum glucose intolerance. RESEARCH DESIGN AND METHODS The study was a retrospective cohort of 1,006 women with GDM attending a pregnancy diabetes clinic. RESULTS Postpartum screening was completed in 438 (48%) women. Women nonadherent to testing had higher parity (1.10 vs. 0.87) and were less likely to require insulin for management of their GDM. Among women who were tested, 89 (21%) had an abnormal result, only 25 (28%) of whom were identified by FPG. Factors associated with abnormal postpartum diabetes screening include non-Caucasian ethnicity, previous GDM, higher A1C, and OGTT values during pregnancy and treatment with insulin. CONCLUSIONS The rate of postpartum diabetes screening is low, and FPG lacks sensitivity as a screening test in comparison with OGTT.

In rat pinealocytes, ceramide can inhibit the KCl- and BayK 8644-mediated potentiation of cAMP and cGMP accumulation, suggesting that the L-type Ca2+ channel is a target of ceramide action. This was examined in the present study using intracellular Ca2+ measurement and patch-clamp studies. In fura-2-loaded pinealocytes, C2- and C6-ceramide inhibited the Ca2+ increase caused by BayK 8644 and KCl, but not that caused by norepinephrine, suggesting an inhibitory effect of ceramide on the L-type Ca2+ channels. Patch-clamp analysis confirmed that C2- and C6-ceramide, but not C2-dihydroceramide (the inactive analog) inhibited the L-type Ca2+ channel current. Furthermore, treatments known to increase cellular ceramide levels, including a glucosylceramide synthase inhibitor and sphingomyelinase, also inhibited this current. The inhibitory effect of ceramide on the current was attenuated by lavendustin A, a tyrosine kinase inhibitor, but not by H7, a serine/threonine kinase inhibitor. The effect of ceramide was mim...

We describe the changes in calcium homeostasis seen in a hypoparathyroid woman during the third trimester and with lactation following her second pregnancy. During lactation her need for supplemental calcium and calcitriol abated, and in fact she was transiently hypercalcemic and hypophosphatemic. This change was associated with a rise of serum parathyroid hormone-related peptide (PTHrP) released systemically during lactation. This is the first documentation of the time course of serum PTHrP levels from the late third trimester throughout lactation in a hypoparathyroid woman. In this context PTHrP may have sufficient biological activity to compensate for parathyroid hormone deficiency.

Interaction between sphingomyelin metabolism and cyclic nucleotide synthesis in rat pinealocytes was investigated by determining the effect of ceramide on adrenergic-stimulated cAMP and cGMP accumulation. Although C2-, C6-, and C8-ceramide had no effect on basal, isoproterenol-, or norepinephrine-stimulated cAMP and cGMP accumulation, they inhibited the potentiation caused by depolarising concentrations of K+ or BayK 8644. Similar inhibition was observed when ceramide metabolism was inhibited by a glucosylceramide synthase inhibitor. In contrast, the potentiation of cAMP and cGMP accumulation caused by other intracellular Ca(2+)-elevating agents such as ionomycin or thapsigargin or by an activator of protein kinase C was not affected by ceramide. Taken together, our results suggest that ceramide selectively inhibits cyclic nucleotide synthesis when the nucleotide synthesis is potentiated by an increase in intracellular Ca2+ through L-type Ca2+ channels and that the sphingomyelin cycle probably plays an important role in the regulation of these channels.

Tyrosine phosphorylation has recently been shown to modulate ion channel activity. In the present study, the effect of growth factors on the L-type Ca2+ channel current in rat pinealocytes was investigated using the whole cell version of the patch clamp technique. Both insulin and insulin-like growth factor-I (IGF-I) inhibited the L-type Ca2+ channel current. This inhibition was dependent on concentration, with median effective concentration (EC50) values of 60 nm for insulin and 0.14 nm for IGF-I. Heat-inactivated insulin or IGF-I had no effect on the L-type Ca2+ channel current. The presence of anti-IGF-I receptor antibodies blocked the inhibitory effect of IGF-I on the L-type Ca2+ channel current. Two other growth factors, nerve growth factor and epidermal growth factor, had no effect on this current. The effects of insulin and IGF-I were blocked by lavendustin A, a tyrosine kinase inhibitor. Calphostin C, a protein kinase C inhibitor, attenuated the effect of insulin and IGF-I, whereas wortmannin, a p...

Inhibition of tyrosine kinase activities elevates cyclic GMP (cGMP) levels in rat pinealocytes. Since protein kinase C (PKC) and intracellular Ca2+ both interact with the agonist-stimulated cGMP accumulation, in this study their interactions with the tyrosine kinase inhibitor-mediated cGMP response were investigated. Two tyrosine kinase inhibitors, genistein and tyrphostin B42, increased basal cGMP accumulation concentration dose-dependently. This increase in cGMP accumulation was potentiated by 4β-phorbol 12-myristate 13-acetate (PMA), an activator of PKC, and blocked by calphostin C, a specific PKC inhibitor. The tyrosine kinase inhibitors had no effect on the in vitro or PMA-mediated translocation of PKC activity. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine (IBMX), neither the tyrosine kinase inhibitors alone nor in combination with PMA had an effect on cGMP accumulation, suggesting that phosphodiesterase is a probable site of action of the inhibitors. In comparison, elevation of intracellular Ca2+ by BayK 8644, ionomycin, or KCl inhibited the genistein- or tyrphostin B42-mediated increase in cGMP accumulation. This inhibition persisted in the presence of IBMX and was partly reversed by a Ca2+/calmodulin inhibitor. These results suggest that PKC modulates the rate of cGMP degradation through signalling pathways involving tyrosine phosphorylation. However, the inhibitory effect of the Ca2+-elevating agents on the tyrosine kinase inhibitor stimulated cGMP accumulation appears to be independent of phosphodiesterase inhibition.

The process involved in relocation of the coactivator, transducer of regulated cAMP-regulated element-binding protein (TORC) to the cytoplasm, unlike its activation, is not well understood. Using cultured pineal cells prepared from male rats, we found that although both α- and β-adrenergic stimulation could cause TORC1 dephosphorylation, only α-adrenergic stimulation was effective in the norepinephrine (NE)-mediated translocation of TORC1 into the nucleus. In contrast, blockade of either the α- or the β-adrenergic receptor after NE stimulation was effective in causing the rephosphorylation and rapid relocation of TORC1 into the cytoplasm. Studies with phosphoprotein phosphatase (PP) inhibitors indicated that although both PP2A and PP2B could dephosphorylate TORC1, only PP2B could cause translocation into the nucleus. However, after NE stimulation, treatment with either PP2A or PP2B inhibitors could cause the rephosphorylation and cytoplasmic relocation of TORC1. These results indicate a requirement of continuous activation of both α- and β-adrenergic receptors as well as PP2A and PP2B activities for the nuclear retention of TORC1 during NE stimulation. Knockdown of salt-inducible kinase 1 (SIK1) had no effect on the phosphorylation or localization of TORC1. Although overexpressing SIK1 could induce TORC1 phosphorylation in the nucleus, it did not reduce TORC1 level in the nucleus, indicating that SIK1-mediated TORC1 phosphorylation may not be sufficient for its relocation into the cytoplasm. Together, these results demonstrate that, in the rat pineal gland, different mechanisms are involved in regulating the nuclear entry and exit of TORC1 and that the SIK1-mediated phosphorylation of TORC1 may not lead to its nuclear exit.

Background Although allergic reactions to insulin are uncommon, they can be difficult to diagnose and management may be very difficult in subjects with Type 1 diabetes with severe allergy. Access to allergists and specialist diagnostic tests is limited and few diabetes specialists are familiar with desensitization as a means of treating allergy. People with diabetes may develop symptoms which mimic insulin allergy but are attributable to other conditions. Case reports Here we describe three cases of insulin allergy. One patient presented with severe, albeit localized, urticarial reactions at injection sites. The most severe case was a woman with recent-onset Type 1 diabetes who presented with grade 2 anaphylaxis. The third patient presented with generalized urticaria and angioedema. Insulin allergy was confirmed in all three cases. Methods Assessment involved measurement of immunoglobulin and anti-insulin antibody levels. Skin testing was performed in two cases. Treatments included desensitization in one case, alternative insulin preparations, antihistamines and continuous subcutaneous insulin infusion. In all three cases of insulin allergy there has been successful resolution of symptoms. Conclusions The clinical assessment and investigation in cases of suspected insulin allergy is described, along with detailed algorithms for skin testing and desensitization. This case series demonstrates an approach to challenging cases of suspected insulin allergy which will be helpful for diabetes specialists.