Pituitary Apoplexy in Pregnancy: What do We Know?

Pituitary apoplexy refers to a rare clinical syndrome consisting of signs and symptoms that occur due to rapid expansion of the contents of the sella turcica. It can occur spontaneously or associated with pituitary tumors. It can have a broad clinical spectrum, but usually presents with severe headache, visual impairment and hypopituitarism. Sudden onset of symptoms associated to imagiologic confirmation makes the diagnosis. Surgical treatment is advised when there is important compression of the optic tract. We present a case report and a review of the literature on pituitary apoplexy in pregnancy. The cases were reviewed to obtain information on maternal characteristics, clinical presentation, diagnostic studies, therapeutic modalities and maternal and fetal outcomes. Our review found 36 cases of pituitary apoplexy in pregnancy. Most of the cases occurred in the second trimester of pregnancy and headache was the most frequent symptom at presentation. Surgical therapy was required in more than half of the patients. In what respect maternal and fetal outcomes, there were 3 cases of preterm delivery and one case of maternal death. Our clinical case and literature review reinforces the importance of an early diagnosis to avoid potential adverse consequences.

Pituitary Apoplexy in Pregnancy: What do We Know? / Apoplexia hipofisária na gravidez: o que sabemos?

Abstract Pituitary apoplexy refers to a rare clinical syndrome consisting of signs and symptoms that occur due to rapid expansion of the contents of the sella turcica. It can occur spontaneously or associated with pituitary tumors. It can have a broad clinical spectrum, but usually presents with severe headache, visual impairment and hypopituitarism. Sudden onset of symptoms associated to imagiologic confirmation makes the diagnosis. Surgical treatment is advised when there is important compression of the optic tract. We present a case report and a review of the literature on pituitary apoplexy in pregnancy. The cases were reviewed to obtain information on maternal characteristics, clinical presentation, diagnostic studies, therapeutic modalities and maternal and fetal outcomes. Our review found 36 cases of pituitary apoplexy in pregnancy. Most of the cases occurred in the second trimester of pregnancy and headache was the most frequent symptom at presentation. Surgical therapy was required in more than half of the patients. In what respect maternal and fetal outcomes, there were 3 cases of preterm delivery and one case of maternal death. Our clinical case and literature review reinforces the importance of an early diagnosis to avoid potential adverse consequences.

The Role of Beta Cell Recovery in Type 2 Diabetes Remission.

Type 2 diabetes (T2D) has been considered a relentlessly worsening disease, due to the progressive deterioration of the pancreatic beta cell functional mass. Recent evidence indicates, however, that remission of T2D may occur in variable proportions of patients after specific treatments that are associated with recovery of beta cell function. Here we review the available information on the recovery of beta cells in (a) non-diabetic individuals previously exposed to metabolic stress; (b) T2D patients following low-calorie diets, pharmacological therapies or bariatric surgery; (c) human islets isolated from non-diabetic organ donors that recover from "lipo-glucotoxic" conditions; and (d) human islets isolated from T2D organ donors and exposed to specific treatments. The improvement of insulin secretion reported by these studies and the associated molecular traits unveil the possibility to promote T2D remission by directly targeting pancreatic beta cells.

Non-alpine primary thyroid angiosarcoma

SUMMARY Thyroid angiosarcoma is an extremely rare malignancy, which occurs more frequently in the alpine region, likely associated with iodine deficiency and endemic goiter. This is an aggressive neoplasm that usually harbors a poor prognosis. We report the case of a 49-year-old Portuguese female patient presenting with a large nodule in the anterior neck region, with rapid growth and associated dysphonia. The neck ultrasound showed a hypoechogenic and heterogeneous thyroid nodule, with a larger axis of 44 mm. The fine needle aspiration cytology was not conclusive, and a biopsy of the lesion was performed. The result was suggestive of a mesenchymal tumor constituted by spindle cells and vascular clefts, showing positivity for endothelial markers and negativity for thyroglobulin, calcitonin and TTF1. The chest CT scan performed before surgery showed multiple pulmonary nodules suggestive of secondary lesions. The patient was submitted to total thyroidectomy and lymph node dissection in order to relieve compressive symptoms. A diagnosis of thyroid angiosarcoma was made after histologic examination of the surgical specimen. Despite undergoing multiple lines of palliative chemotherapy, the pulmonary lesions increased in size and number. The patient died due to respiratory failure 29 months after the diagnosis. Thyroid angiosarcoma is a rare malignancy, generally with poor prognosis. In our case, the patient presented with pulmonary metastases at diagnosis, which is a negative prognostic factor. Due to its rarity, data regarding management and treatment of this disease are scarce.

Non-alpine primary thyroid angiosarcoma.

Thyroid angiosarcoma is an extremely rare malignancy, which occurs more frequently in the alpine region, likely associated with iodine deficiency and endemic goiter. This is an aggressive neoplasm that usually harbors a poor prognosis. We report the case of a 49-year-old Portuguese female patient presenting with a large nodule in the anterior neck region, with rapid growth and associated dysphonia. The neck ultrasound showed a hypoechogenic and heterogeneous thyroid nodule, with a larger axis of 44 mm. The fine needle aspiration cytology was not conclusive, and a biopsy of the lesion was performed. The result was suggestive of a mesenchymal tumor constituted by spindle cells and vascular clefts, showing positivity for endothelial markers and negativity for thyroglobulin, calcitonin and TTF1. The chest CT scan performed before surgery showed multiple pulmonary nodules suggestive of secondary lesions. The patient was submitted to total thyroidectomy and lymph node dissection in order to relieve compressive symptoms. A diagnosis of thyroid angiosarcoma was made after histologic examination of the surgical specimen. Despite undergoing multiple lines of palliative chemotherapy, the pulmonary lesions increased in size and number. The patient died due to respiratory failure 29 months after the diagnosis. Thyroid angiosarcoma is a rare malignancy, generally with poor prognosis. In our case, the patient presented with pulmonary metastases at diagnosis, which is a negative prognostic factor. Due to its rarity, data regarding management and treatment of this disease are scarce.

Parathyroid Carcinoma: A Rare Endocrine Malignancy.

BACKGROUND Parathyroid carcinoma (PC) is an extremely rare endocrine malignancy, with a reported increase in incidence in the past decade. PC generally presents in an indolent fashion, featuring nonspecific symptoms associated with hypercalcemia. CASE REPORT Case 1: A 30-year-old man was admitted for symptoms associated with hypercalcemia and elevated parathyroid hormone (PTH). Imaging examinations showed the presence of a cervical nodular lesion. The patient underwent surgery, and the pathological diagnosis was PC. Case 2: A 45-year-old man with a history of hypothyroidism was referred to our Endocrinology Department for a cervical nodular lesion. A fine-needle aspiration was performed, and the result was suggestive of papillary carcinoma. Blood testing showed only mild hypercalcemia and PTH elevation, with no associated symptoms. The patient underwent surgery, and the histological examination confirmed the diagnosis of PC. Case 3: A 38-year-old man presented with diffuse bone pain and muscle weakness, severe hypercalcemia, high levels of PTH, and a cervical mass. The patient underwent surgery. Diagnostic pathology confirmed the diagnosis of PC. Five years later, the patient presented with a cutaneous metastasis, followed 1 year later by pulmonary metastases. CONCLUSIONS Most PCs are slow-growing tumors. Some of these tumors are diagnosed in association with hereditary syndromes. A clear distinction between benign and malignant lesions is not always simple because there is a lack of specific clinical distinguishing features of malignant lesions. Currently, surgical resection is the preferred approach; however, owing to the rarity of this condition, there is a void of high-quality data.

Ovarian Leydig Cell Tumor: Cause of Virilization in a Postmenopausal Woman.

BACKGROUND Only 0.5% of all ovarian tumors are Leydig cell tumors and they are generally benign and unilateral. These androgen-secreting tumors lead to virilizing symptoms, most often in postmenopausal women. Because Leydig cell tumors are typically small, diagnosing them accurately can be challenging. CASE REPORT We report the case of a 77-year-old woman who was referred to our Endocrinology Clinic because of a 5-year history of hirsutism (Ferriman-Gallwey score of 11) with no discernible cause. The patient had high levels of serum testosterone and a normal level of dehydroepiandrosterone sulfate. Imaging, including transvaginal ultrasound and pelvic magnetic resonance, revealed a 16-mm uterine nodule, which was suspected to be a submucous leiomyoma, but no adrenal or ovarian lesions. Despite the lack of findings on imaging and because of the high suspicion for an androgen-secreting ovarian tumor, bilateral laparoscopic oophorectomy was performed. Histological examination of the specimen revealed a non-hilar Leydig cell tumor that measured 8 mm in its largest axis. After the surgery, the patient had significant clinical improvement and her laboratory test results normalized. Her sister had the same symptoms and laboratory findings at a similar age, which raised the suspicion of a possible familial genetic syndrome. No genetic testing was performed, however, because the patient's sister declined further diagnostic investigation. CONCLUSIONS Leydig cell tumors are rare, and even when they are small, they can cause symptoms related to androgen excess. As a result, diagnosing them often is challenging.

Hypogonadotropic hypogonadism in human immunodeficiency virus-infected men: uncommonly low testosterone levels.

Hypogonadotropic hypogonadism (HH) is common and occurs prematurely in HIV-infected men. However, HH with very low testosterone has not been described. Three men with normal pubertal development and HIV1 diagnosis at the ages of 22, 34 and 35 years. All complained of decreased libido, anejaculation and erectile dysfunction thirteen years, six months and one year after HIV diagnosis, respectively. Two had depressive syndrome and two were treated with antiretroviral therapy. Laboratory tests revealed isolated HH in all. Sellar and head CT scans were normal and all had normal CD4 count. They started testosterone replacement therapy, with symptoms improvement. Causes of HH in HIV-infected men include undernutrition, severe illness, drugs, pituitary dysfunction and comorbidities. Despite having none of these conditions (except two that were treated with low-dose psychotropics), our patients had HH with uncommonly low testosterone. This suggests that a different mechanism contributes to severe HH in HIV-infected men. LEARNING POINTS: The pathogenesis of hypogonadotropic hypogonadism in HIV-infected men is multifactorial and androgen deficiency is more often a consequence of secondary hypogonadism than primary hypogonadism.Causes of hypogonadotropic hypogonadism in HIV-infected men include undernutrition, severe illness, drugs (psychotropics, opiates, megestrol acetate or steroids), pituitary dysfunction (tumor, hyperprolactinemia), an AIDS-related lesion (very rarely) and comorbid conditions, such as antibody to hepatitis C virus seropositivity and injection drug use.Highly active antiretroviral therapy (HAART), particularly protease inhibitor therapy has been associated with sexual dysfunction in men, but the causal nature of this relation has not been clearly established.Hypogonadotropic hypogonadism with uncommonly low testosterone levels are not usually associated with the conditions referred and this suggests that a different mechanism could contribute to severe hypogonadotropic hypogonadism in HIV-infected men.Screening for hypogonadism in all HIV-infected men might help to understand its etiology.

Online continuing interprofessional education on hospital-acquired infections for Latin America

Abstract Introduction: Latin America is a large and diverse region, comprising more than 600 million inhabitants and one million physicians in over 20 countries. Resistance to antibacterial drugs is particularly important in the region. This paper describes the design, implementation and results of an international bi-lingual (Spanish and Portuguese) online continuing interprofessional interactive educational program on hospital-acquired infections and antimicrobial resistance for Latin America, supported by the American Society for Microbiology. Methods: Participation, satisfaction and knowledge gain (through pre and post tests) were used. Moreover, commitment to change statements were requested from participants at the end of the course and three months later. Results: There were 1169 participants from 19 Latin American countries who registered: 57% were physicians and 43% were other health care professionals. Of those, 1126 participated in the course, 46% received a certificate of completion and 54% a certificate of participation. There was a significant increase in knowledge between before and after the course. Of 535 participants who took both tests, the grade increased from 59 to 81%. Commitments to change were aligned with course objectives. Discussion: Implementation of this educational program showed the feasibility of a continent-wide interprofessional massive course on hospital acquired-infections in Latin America, in the two main languages spoken in the region. Next steps included a new edition of this course and a "New Challenges" course on hospital-acquired infections, which were successfully implemented in the second semester of 2015 by the same institutions.

Management of hidradenitis suppurativa with biological therapy: report of four cases and review of the literature.

Several studies report the usefulness of different biological therapies in the management of a difficult-to-treat pathology such as hidradenitis suppurativa (HS). However, there are little data on this, which shows the great difficulty in the management of this disease. We will report herein our experience of the treatment of four complex cases of HS. We will also review previous cases published in the literature in order to further assess the results obtained with different biological drugs in terms of efficacy and safety. We conclude that, in the near future, biological therapy could become an essential tool in the management of cases of HS who have not previously responded to classical treatment.

Primary cutaneous adenoid cystic carcinoma: an unusual case.

Primary cutaneous adenoid cystic carcinoma (PCACC) is an uncommon adnexal skin tumor. It is usually located on the head or neck of people over 50 years of age, affecting men and women equally. The etiology is unclear; the source used to be considered the eccrine glands, but more recent hypotheses defend this carcinoma as originating in the apocrine glands or modified apocrine glands. The tumor may clinically appear as a normal skin-colored nodule with ill-defined edges and slow growth. Histologically, a proliferation of neoplastic cells appears, which could present in a tubular, cribriform, or solid pattern. Perineural invasion is characteristic for this tumor. For diagnosis of PCACC it is essential to exclude metastasis, especially in the salivary glands. Treatment involves surgical excision. We present a case of primary adenoid cystic carcinoma in a young woman located on the skin of the upper limb, which is an unusual place.

ENPP1 affects insulin action and secretion: evidences from in vitro studies.

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.

Calpain-10 expression is elevated in pancreatic islets from patients with type 2 diabetes.

BACKGROUND: Calpain-10 was the first gene to be identified influencing the risk of type 2 diabetes (T2D) by positioning cloning. Studies in beta-cell lines and rodent islets suggest that calpain-10 may act as a regulator of insulin secretion. However, its role in human pancreatic islets remains unclear. The aim of this study was to examine if calpain-10 expression is altered in islets from patients with T2D and if the transcript level correlates with insulin release. We also tested if polymorphisms in the CAPN10 gene are associated with gene expression and insulin secretion in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Calpain-10 mRNA expression was analysed in human pancreatic islets from 34 non-diabetic and 10 T2D multi-organ donors. CAPN10 SNP-43 and SNP-44 were genotyped and related to gene expression and insulin release in response to glucose, arginine and glibenclamide. The mRNA level of calpain-10 was elevated by 64% in pancreatic islets from patients with T2D compared with non-diabetic donors (P = 0.01). Moreover, the calpain-10 expression correlated positively with arginine-stimulated insulin release in islets from non-diabetic donors (r = 0.45, P = 0.015). However, this correlation was lost in islets from patients with T2D (r = 0.09; P = 0.8). The G/G variant of SNP-43 was associated with reduced insulin release in response to glucose (P

The non-peptidyl low molecular weight radical scavenger IAC protects human pancreatic islets from lipotoxicity.

BACKGROUND: Chronic exposure to high free fatty acids (FFA) can lead to irreversible damage of beta-cell accounting for impaired insulin secretion. Multiple mechanisms concur in generating the damage, but activation of oxidative stress may contribute to the final toxic effect. To better understand the phenomenon of lipotoxicity in human beta-cells, we evaluated the effects of 24-h pre-culture with 1.0 mmol/l FFA on the function, survival and mRNA expression of several enzymes involved in the generation and scavenging of reactive oxygen species (ROS). MATERIAL AND METHODS: Human islets, prepared by collagenase digestion and density gradient purification from 9 pancreases of multiorgan donors, were incubated for 24-h in the presence 1.0 mmol/l long-chain mixture (oleate:palmitate, 2:1) FFA, with or without 100 micromol/l IAC, a non-peptidyl low molecular weight radical scavenger. At the end of incubation period, insulin secretion was measured by static incubation, and mRNA expression of insulin, Cu/Zn-SOD, Mn-SOD, Catalase, Glutathione peroxidase (GSH-px) and HO-1 by quantitative Real-Time RT-PCR. Nitrotyrosine levels were determined by an ELISA technique. RESULTS: As compared to control incubation (Ctrl, no FFA), exposure to FFA was associated with impaired insulin release and reduced insulin mRNA expression. The presence of IAC in the incubation medium increased insulin release significantly and prevented changes in mRNA expression. Exposure to FFA was associated with oxidative stress as indicated by a significant accumulation of nitrotyrosine and IAC restrained such an increase. mRNA expression of Cu/Zn-SOD, Mn-SOD, Catalase, GSH-Px, and HO-1 were all modified after FFA exposure. These changes were partially prevented in the presence of IAC. CONCLUSIONS: In human islets 24-h exposure to high FFA causes oxidative stress associated with changes of several enzymes involved in ROS scavenging. These effects were prevented by the use of an antioxidant molecule.

Effects of C-peptide on isolated human pancreatic islet cells.

BACKGROUND: Recent data have demonstrated that pro-insulin-derived C-peptide can affect the function of several different cell types. We hypothesized that C-peptide might have an influence on the function and survival of isolated human islets. METHODS: Islets were prepared by combining enzymatic digestion and density gradient centrifugation, and the effects of human C-peptide were evaluated acutely and after 24-h incubation. Insulin secretion, apoptosis, quantitative RT-PCR and western-blotting experiments were then performed. RESULTS: Glucose-stimulated insulin secretion was not affected by C-peptide and, accordingly, mRNA expression of glucose transporter 2 and glucokinase did not differ between islets pre-cultured or not with the hormone. However, apoptosis was significantly lower in islets exposed to C-peptide than in control islets. This was accompanied by a significant increase of mRNA and protein expression of Bcl2, an anti-apoptotic molecule, with no change in the expression of Bax, a pro-apoptotic molecule. CONCLUSION: These results show that in human islets pro-insulin C-peptide has no direct effects on insulin secretion, but it decreases islet cell apoptosis. A direct role of C-peptide on beta-cell mass regulation is therefore suggested.

The E23K variant of KCNJ11 encoding the pancreatic beta-cell adenosine 5'-triphosphate-sensitive potassium channel subunit Kir6.2 is associated with an increased risk of secondary failure to sulfonylurea in patients with type 2 diabetes.

CONTEXT: Several studies suggest that genetic factors may play a role in the different responses to antidiabetic therapy; however, conclusive evidence is still lacking. OBJECTIVE: The objective of the study was to investigate whether diabetic patients carrying the E23K variant in KCNJ11 are at increased risk for secondary sulfonylurea failure. DESIGN: Secondary sulfonylurea failure was defined as fasting plasma glucose greater than 300 mg/dl despite sulfonylurea-metformin combined therapy and appropriate diet, in the absence of other conditions causing hyperglycemia. SETTING: The study was conducted in an ambulatory care facility. PATIENTS: A total of 525 Caucasian type 2 diabetic patients were enrolled in the study. INTERVENTION: Sulfonylurea treatment was followed by sulfonylurea-metformin combined therapy and then insulin treatment. MAIN OUTCOME MEASURE: Secondary failure was the main outcome measure. RESULTS: Of the diabetic patients enrolled in the study, 38.5% were E23E homozygous, 51.4% were E23K heterozygous, and 10.1% were K23K homozygous. The frequency of carriers of the K allele was 58 and 66.8% among patients treated with oral therapy or secondary sulfonylurea failure, respectively (odds ratio, 1.45; 95% confidence interval, 1.01-2.09; P = 0.04). Adjustment for age, gender, fasting glycemia, glycosylated hemoglobin, age at diagnosis, and duration of diabetes in a logistic regression analysis did not change this association (odds ratio, 1.69; 95% confidence interval, 1.02-2.78; P = 0.04). Islets isolated from carriers of the K allele showed no differences in glucose-stimulated insulin secretion and a tendency toward reduced response upon glibenclamide stimulation (P = 0.09). After 24-h exposure to high (16.7 mmol/liter) glucose concentration, impairment of glibenclamide-induced insulin release was significantly (P = 0.01) worse with the E23K variant. CONCLUSIONS: These data suggest that the E23K variant in KCNJ11 may influence the variability in the response of patients to sulfonylureas, thus representing an example of pharmacogenetics in type 2 diabetes.

The direct effects of the angiotensin-converting enzyme inhibitors, zofenoprilat and enalaprilat, on isolated human pancreatic islets.

OBJECTIVE: Data from prospective studies suggest a significant reduction in the risk of new diabetes from drug therapies containing angiotensin-converting enzyme (ACE) inhibitors. Since the renin-angiotensin system (RAS) has been found locally in several tissues and cells, including pancreatic islets, we hypothesized that the positive metabolic effects of ACE inhibitors may be due to a beneficial action of these compounds on insulin-secreting beta-cells. DESIGN AND METHODS: Isolated human pancreatic islets were studied after 24 h of incubation with 22.2 mmol/l glucose, with or without the presence in the incubation medium of 0.5-6.0 mmol/l zofenoprilat or enalaprilat, ACE inhibitor drugs which differ by the presence of a sulphydryl or a carboxyl group in their structural formula. Functional and molecular studies were then performed to assess insulin secretion, redox balance, mRNA and protein expression. RESULTS: Angiotensinogen, ACE and angiotensin type 1 receptor mRNA expression increased in islets cultured in high glucose; this was similarly prevented by the presence of either ACE inhibitor. As expected, preculture of human islets in high glucose determined a marked reduction in insulin secretion which was associated with enhanced oxidative stress, as shown by increased nitrotyrosine concentrations, and enhanced expression of protein kinase C beta and NADPH oxidase. The presence of either of the ACE inhibitors counteracted several of the deleterious effects of high glucose exposure, including reduction of insulin secretion and increased oxidative stress; zofenoprilat showed significantly more marked effects. CONCLUSIONS: These results showed that: (a) RAS molecules are present in human islets and their expression is sensitive to glucose concentration, (b) ACE inhibitors, and in particular zofenoprilat, protect human islets from glucotoxicity and (c) the effects of ACE inhibition are associated with decreased oxidative stress. Together, these findings provide evidence that the possible beneficial effects of ACE inhibitors in human diabetes are due, at least in part, to a protective action on pancreatic beta-cells.

Functional and molecular defects of pancreatic islets in human type 2 diabetes.

To shed further light on the primary alterations of insulin secretion in type 2 diabetes and the possible mechanisms involved, we studied several functional and molecular properties of islets isolated from the pancreata of 13 type 2 diabetic and 13 matched nondiabetic cadaveric organ donors. Glucose-stimulated insulin secretion from type 2 diabetic islets was significantly lower than from control islets, whereas arginine- and glibenclamide-stimulated insulin release was less markedly affected. The defects were accompanied by reduced mRNA expression of GLUT1 and -2 and glucokinase and by diminished glucose oxidation. In addition, AMP-activated protein kinase activation was reduced. Furthermore, the expression of insulin was decreased, and that of pancreatic duodenal homeobox-1 (PDX-1) and forkhead box O1 (Foxo-1) was increased. Nitrotyrosine and 8-hydroxy-2'-deoxyguanosine concentrations, markers of oxidative stress, were significantly higher in type 2 diabetic than control islets, and they were correlated with the degree of glucose-stimulated insulin release impairment. Accordingly, 24-h exposure to glutathione significantly improved glucose-stimulated insulin release and decreased nitrotyrosine concentration, with partial recovery of insulin mRNA expression. These results provide direct evidence that the defects of insulin secretion in type 2 diabetic islets are associated with multiple islet cell alterations. Most importantly, the current study shows that the functional impairment of type 2 diabetic islets can be, at least in part, reversible. In this regard, it is suggested that reducing islet cell oxidative stress is a potential target of human type 2 diabetes therapy.

The role of peripheral benzodiazepine receptors on the function and survival of isolated human pancreatic islets.

OBJECTIVE: Peripheral benzodiazepine receptors (PBRs) are part of the mitochondrial permeability transition pore, and their activation may induce cell death. PBRs are expressed in human pancreatic islets, and cytokine-induced damage is accompanied by changes in their properties. We hypothesized that PBRs can have a role in human islet physiopathology, and evaluated the effects of prolonged exposure to two specific PBR ligands, PK11195 and Ro5-4864 on the function and survival of isolated human islets. DESIGN: Isolated human islets were prepared from the pancreas of 25 multiorgan cadaveric donors and incubated for 12 h in the presence of PK11195 or Ro5-4864. Insulin secretion studies and apoptosis experiments were then performed, together with assessment of intracellular pathways involved in islet cell function and survival. METHODS: Islets were prepared by enzymatic digestion and density gradient purification. Insulin secretion was assessed by the batch incubation method, and glucose oxidation was evaluated by the use of D-[U-(14)C]glucose. Apoptosis was studied using the TUNEL technique, ELISA methods, and electron microscopy evaluation. PCR experiments were performed by the use of specific primers. RESULTS: Glucose-stimulated insulin release was significantly lower after exposure to PK11195 than after exposure to Ro5-4864. This was accompanied by reduced glucose oxidation and no major change of insulin or GLUT-1 mRNA expression. Apoptosis was higher in PK11195-exposed islets, and electron microscopy demonstrated the involvement of beta-cells. The apoptotic effects were prevented by bongkrekic acid and low-dose cyclosporin A, which stabilize the mitochondrial membrane, and were associated with no evident change of inducible nitric oxide synthase (iNOS), B-cell leukemia/lymphoma-2 (Bcl-2) or Bcl-2-associated X protein (Bax) expression. Caspase inhibition markedly reduced the amount of apoptosis, and the role of these proteases was confirmed by the increased activity of caspase-3 and caspase-9. CONCLUSIONS: Prolonged binding to PBRs may cause human beta-cells functional damage and apoptosis, a phenomenon which is prevented by stabilizing the mitochondrial membrane; occurs without changes of iNOS, Bax and Bcl-2 mRNA expression; and involves caspase activation. These results suggest an involvement of PBRs in human pancreatic beta-cell function and survival.

Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells.

Because adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP), we inquired whether HBP activation affects pancreatic beta-cell survival. Exposure of human islets to high glucose resulted in increased apoptosis of beta-cells upon serum deprivation that was reversed by azaserine. Also, glucosamine, a direct precursor of the downstream product of the HBP, increased human beta-cells apoptosis upon serum deprivation, which was reversed by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADGP), an inhibitor of protein O-glycosylation. These results were reproduced in RIN rat beta-cells. Glucosamine treatment resulted in inhibition of tyrosine-phosphorylation of the insulin receptor (IR), IRS-1, and IRS-2, which was associated with increased O-glycosylation. These changes caused impaired activation of the PI 3-kinase/Akt survival signaling that resulted in reduced GSK-3 and FOXO1a inactivation. BADGP reversed the glucosamine-induced reduction in insulin-stimulated phosphorylation of IR, IRS-1, IRS-2, Akt, GSK-3, and FOXO1a. Impaired FOXO1a inactivation sustained expression of the pro-apoptotic protein Bim, without affecting Bad, Bcl-XL, or Bcl-2 expression. These results indicate that hyperglycemia may increase susceptibility to apoptosis of human and rat beta-cell through activation of the HBP. Increased routing of glucose through this metabolic pathway results in impaired activation of the IR/IRSs/PI3-kinase/Akt survival pathway by induction of O-glycosylation of signaling molecules.