Efficacy of multivitamin support following bariatric surgery in patients with obesity: a prospective observational study.

PURPOSE: Bariatric surgery (BS), an effective treatment for severe obesity and its comorbidities, may result in micronutrient and vitamin deficiencies. This monocentric prospective observational study aimed at evaluating the efficacy of a specifically designed vitamin/mineral formula (Bariatrifast, BIOITALIA S.r.l., Italy) for preventing and treating micronutrient deficiencies in patients submitted to BS. METHODS: Twenty patients with severe obesity (mean weight and BMI: 123.5 kg (range 88-174) and 43.3 kg/m2 (range 37-54) respectively) underwent BS (10 vertical sleeve gastrectomy VSG, 10 Roux-en-Y gastric bypass, RYGB). The mean age was 49.9 years (range 27-68). After a presurgical visit (V0), follow-up visits were performed at 1, 3, 6 and 12 months after surgery (V1-V4). Recorded data included weight, height and BMI. A complete blood count, measurement of ferritin, folic acid, vitamin B12, ionized calcium, 25 OH vitamin D, parathyroid hormone (PTH) were obtained. Following BS, patients started the daily oral multivitamin and mineral supplement. RESULTS: All patients achieved a significant weight loss (mean - 34.7 ± 11.8 kg). No deficiencies of various vitamins/micronutrients were detected during the entire study period. The serum concentrations of vitamin B12, 25-OH Vitamin D and folic acid increased over the follow-up period compared with V0 (mean increase 243 ng/L, 23 µg /L, 8 µg/L, respectively). Compared to RYGB, patients who underwent sleeve gastrectomy showed higher levels of 25-OH vitamin D at V2, V3 and V4 (all p < 0.05), and higher levels of Vitamin B12 and folic acid at V4 (p < 0.05 and p < 0.005, respectively). No adverse events were reported. CONCLUSION: Following VSG or RYGB, Bariatrifast administration was associated with normal values of essential micronutrients, and it was well-tolerated without evidence of gastrointestinal side effects. Clinical Trial Registration ClinicalTrials.gov, identifiers NCT06152965.

Characterization and expression analysis of KIT and MITF-M genes in llamas and their relation to white coat color.

The llama (Lama glama) is a fiber-producing species that presents a wide range of coat colors, among which white is one of the most important for the textile industry. However, there is little information about the molecular mechanisms that control the white phenotype in this species. In domestic mammals, a white coat is usually produced by mutations in the KIT proto-oncogene receptor tyrosine kinase (KIT) and microphthalmia-associated transcription factor (MITF) genes. In this work we have sequenced and described the coding regions of KIT and MITF-M, the melanocyte-specific isoform, and the two transcriptional variants MITF-M(-) and MITF-M(+). Moreover, we studied the expression of these genes in the skin of white and colored llamas. Although no variants were revealed to be associated with white coat color, significant differences between phenotypes were observed in the expression levels of KIT and MITF-M. Interestingly, white llamas expressed less MITF-M(+) than did colored ones, which is consistent with a consequent reduction in the synthesis of melanin. Even though our results indicate that downregulation of KIT and MITF-M expression is involved in white phenotype production in llamas, the causative gene of white coat color remains unknown.

Genetic diversity and conservation status of managed vicuña (Vicugna vicugna) populations in Argentina.

The vicuña (Vicugna vicugna) was indiscriminately hunted for more than 400 years and, by the end of 1960s, it was seriously endangered. At that time, a captive breeding program was initiated in Argentina by the National Institute of Agricultural Technology (INTA) with the aim of preserving the species. Nowadays, vicuñas are managed in captivity and in the wild to obtain their valuable fiber. The current genetic status of Argentinean vicuña populations is virtually unknown. Using mitochondrial DNA and microsatellite markers, we assessed levels of genetic diversity of vicuña populations managed in the wild and compared it with a captive population from INTA. Furthermore, we examined levels of genetic structure and evidence for historical bottlenecks. Overall, all populations revealed high genetic variability with no signs of inbreeding. Levels of genetic diversity between captive and wild populations were not significantly different, although the captive population showed the lowest estimates of allelic richness, number of mitochondrial haplotypes, and haplotype diversity. Significant genetic differentiation at microsatellite markers was found between free-living populations from Jujuy and Catamarca provinces. Moreover, microsatellite data also revealed genetic structure within the Catamarca management area. Genetic signatures of past bottlenecks were detected in wild populations by the Garza Williamson test. Results from this study are discussed in relation to the conservation and management of the species.

Prevalence of Differentiated High-Grade Thyroid Carcinoma Among Well-Differentiated Tumors: A Systematic Review and Meta-Analysis.

Background: The current edition of the World Health Organization (WHO) classification of endocrine tumors introduced grading for follicular cell-derived thyroid cancer. Tumors with necrosis and/or high mitotic count but not fulfilling the Turin criteria for poorly differentiated carcinoma will be reclassified as differentiated high-grade thyroid carcinoma (DHGTC). However, the impact of this reclassification has not been evaluated. In this study, we performed a systematic review and meta-analysis to estimate the prevalence of this new entry across thyroid tumor subtypes. Methods: In this systematic review and meta-analysis, studies reporting data on necrosis and/or mitoses in well-differentiated thyroid carcinoma (WDTC) were used to estimate the prevalence of DHGTC. Heterogeneity and potential publication bias were also evaluated. Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines were followed, and quality assessment was performed using a modification of the Newcastle-Ottawa scale. The study has been registered in the International Prospective Register of Systematic Reviews (PROSPERO, ID: CRD42022378716). Results: In clinically unselected patients, the prevalence of DHGTC in WDTC was 0.072 [95% confidence interval, CI, = 0.045-0.113]. The proportion of high-grade tumors greatly varied across growth patterns and subtypes. Overall, the prevalence of DHGTC was higher in follicular thyroid carcinoma (FTC; 0.146 [CI = 0.101-0.205]) than in papillary thyroid carcinoma (PTC; 0.059 [CI = 0.036-0.097]). Diffuse sclerosing, follicular, and classic subtype PTC had the lowest rates of high-grade features (i.e., 0.018 [CI = 0.004-0.084]; 0.036 [CI = 0.010-0.124]; and 0.042 [CI = 0.027-0.066], respectively), while a greater proportion of solid trabecular and histologically aggressive PTC could be reclassified as DHGTC (i.e., 0.154 [CI = 0.067-0.314] and 0.168 [CI = 0.108-0.252], respectively). Similar proportions were obtained for minimally and widely invasive FTC (i.e., 0.136 [CI = 0.058-0.287] and 0.152 [CI = 0.086-0.254], respectively). Finally, in a cohort of patients with poor prognosis (i.e., fatal cases, metastatic and radioiodine resistant tumors, cases with biochemical recurrence), the proportion of DHGTC was 0.287 [CI = 0.155-0.469]. Conclusions: Following the current WHO indications, some tumors will be reclassified as DHGTC. The proportion of tumors with high-grade features is relevant in FTC, solid trabecular, and histologically aggressive PTC subtypes. A remarkable enrichment in DHGTC among patients with poor prognosis confirms the negative impact of high-grade features on outcome.

Prevalence, Molecular Landscape, and Clinical Impact of DICER1 and DGCR8 Mutated Follicular-Patterned Thyroid Nodules.

BACKGROUND: Mutations in micro-RNA (miRNA) regulators DICER1 and DGCR8 have recently been uncovered, revealing a potential novel mechanism driving thyroid tumor development. However, the true frequency of these hotspot mutations in follicular-patterned thyroid tumors (FTs) and their relation to established driver gene events remain elusive. METHODS: A total of 440 FTs from 2 institutions were interrogated for DICER1, DGCR8, and RAS family hotspot mutations using Sanger sequencing. Whole-exome sequencing was also performed to identify additional driver gene aberrations in DICER1/DGCR8-mutant cases. Subsets of cases were further analyzed using miRNA expression profiling, and key dysregulated miRNAs were validated as markers of DICER1 mutations using quantitative RT-PCR analysis. The Cancer Genome Atlas (TCGA) database was also probed for DICER1/DGCR8 mutations and miRNA dysregulation. RESULTS: Fourteen (3.2%) and 4 (1%) FTs harbored DICER1 and DGCR8 hotspot mutations, respectively, in the combined cohort, and no cases with normal tissue available were found to exhibit a constitutional variant. Two DGCR8-mutant cases also harbored oncogenic RAS mutations. Whole-exome sequencing analysis did not identify additional driver gene events in DICER1/DGCR8-positive cases. Comprehensive miRNA expression profiling revealed a unique pattern of dysregulated miRNAs in DICER1/DGCR8-mutant cases compared with wild-type lesions. Moreover, DICER1-mutant cases showed a remarkable reduction of 5' arm miRNAs, findings corroborated in the TCGA cohort. CONCLUSION: DICER1 and DGCR8 hotspot mutations are rare in unselected cohorts of FTs, and mutated cases exhibit a specific miRNA profile. Although DGCR8 mutations may coexist with established RAS gene alterations, FTs with DICER1 variants were devoid of other driver gene events.

The ASIP gene in the llama (Lama glama): Alternative transcripts, expression and relation with color phenotypes.

The Agouti gene (ASIP) is one of the most important genes for coat color determination in mammals. It has a complex structure with several promoters and alternative non-coding first exons that are transcribed into mRNAs with different 5'UTR. These mRNA isoforms regulate the temporal and spatial expression of the gene, producing diverse pigmentation patterns. Here, we studied ASIP transcriptional variants and their expression in the skin of llamas with different coat color phenotypes. We also described the ASIP locus, including promoter usage and the splicing events that originate each transcript variant. Using 5'RACE-PCR we isolated seven ASIP transcripts with alternative 5'UTR, where exons 1A, 1A', 1C, 1D, and a novel non-coding exon 1A" were identified. Additionally, new alternative spliced forms were found. The diversity of ASIP 5'UTRs is originated by a complex pattern of alternative promoter usage, multiple transcription start sites and splicing events that include exon skipping and alternative 3' splicing site selection. We found that ASIP was highly expressed in llamas with white and brown phenotypes while black animals presented very low expression. The main responsible for this difference was a fusion transcript between ASIP and NCOA6 genes, which was present in the skin of white and brown llamas but not in the black ones. The rest of ASIP transcripts presented very low expression in the skin, indicating that the main regulation point for ASIP gene expression is at the transcriptional level. Nevertheless, the characteristics of the 5'UTRs sequences suggest that alternative transcripts could be regulated differently at the protein synthesis level.

Exploring clinical and gene expression markers of benefit from FOLFOXIRI/bevacizumab in patients with BRAF-mutated metastatic colorectal cancer: Subgroup analyses of the TRIBE2 study.

BACKGROUND: Recent data from the TRIBE2 study have failed to suggest a higher magnitude of benefit from upfront FOLFOXIRI/bevacizumab in patients with BRAF-mutant metastatic colorectal cancer (mCRC) as previously reported in the TRIBE study. PATIENTS AND METHODS: Clinical characteristics and gene expression signatures of patients with BRAF-mutant mCRC enrolled in the TRIBE2 study were evaluated with the aim of understanding that patients may derive benefit from the intensification of the upfront chemotherapy. RESULTS: Of 46 BRAF-mutant tumour samples analysed, 24 (52%) and 22 (48%) were classified as BM1 and BM2, respectively, and 27 (59%) and 19 (41%) were assigned to ligand-independent (LI) and ligand-dependent (LD) Wnt pathway subgroups, respectively. No prognostic impact was shown for both BM1/BM2 and LI/LD subtypes. No interaction was evident between BM1/BM2 or LI/LD signatures and the benefit provided by FOLFOXIRI/bevacizumab. Significant interaction effect was evident in terms of progression-free survival between treatment arm and primary tumour sidedness (P = 0.05) and Eastern Cooperative Oncology Group performance status (ECOG-PS; P < 0.001). CONCLUSIONS: Gene expression analysis failed to identify patients with BRAF-mutant mCRC candidate to upfront FOLFOXIRI/bevacizumab. ECOG-PS >0 and left-sidedness seem associated with no benefit from the intensified treatment.

Digital Gene Expression Analysis on Cytology Smears Can Rule Out Malignancy in Follicular-Patterned Thyroid Tumors.

Patients with indeterminate thyroid nodules (Bethesda III and IV) are often treated with diagnostic lobectomy, which in most cases represents an overtreatment. A reliable rule-out molecular test could spare patients unnecessary surgery. Stained smears of 88 indeterminate thyroid nodules with histologic diagnosis of follicular-patterned tumors were selected: 34 follicular adenomas (FAs), 34 follicular variant papillary thyroid carcinomas (FVPTCs), and 20 noninvasive follicular neoplasms with papillary-like nuclear features (NIFTPs). The expression level of 126 genes was measured by digital counting. Mutation testing was performed for the main gene mutations and fusions. Performance of gene expression and mutation tests was calculated by receiver operating characteristic analysis. The gene expression model showed an area under the curve (AUC) of 88%, with 91% negative predictive value in FAs and FVPTCs only. Part of NIFTPs was labeled as benign, and part was labeled as malignant; thus, the classifier performance worsened. Two FAs (5.9%), eight NIFTPs (40%), and 22 FVPTCs (64.7%) were mutation positive. Mutation testing AUC was 79% in FAs and FVPTCs, and decreased by including NIFTPs. This gene expression-based test was feasible in thyroid-stained smears, showed higher AUC than mutation test, and had a high negative predictive value-making it a good candidate as a rule-out test for indeterminate thyroid cytology. NIFTPs have a heterogeneous phenotype, and their preoperative diagnosis requires further investigation.

Differential Diagnosis of Malignant Pleural Mesothelioma on Cytology: A Gene Expression Panel versus BRCA1-Associated Protein 1 and p16 Tests.

Pleural effusions are among the first clinical manifestations of malignant pleural mesothelioma (MPM) and often constitute the only available material for diagnosis. Although an MPM diagnosis can be reliable on cytology, the reported sensitivity is low (30% to 75%). Particularly, it can be hard to discriminate epithelioid MPM, the most common histotype, from reactive mesothelial hyperplasia (MH). Currently, BRCA1-associated protein 1 (BAP1) and CDKN2A (p16), evaluated by immunohistochemistry and fluorescent in situ hybridization, respectively, are the most valuable markers to discriminate MPM and MH. Both markers have a high specificity, but their sensitivity is not always satisfying, even when used together. We have recently developed a 117-gene expression panel, based on Nanostring technology, able to differentiate epithelioid MPM from MH pleural tissues better than BAP1 and p16. Herein, we evaluated the efficacy of the same panel on an independent retrospective cohort of 23 MPM and 11 MH pleural effusions (cell blocks and smears). The overall sensitivity and specificity of the panel were equal to 0.9565 and 1, respectively. Moreover, the panel performance was compared with BAP1 and p16 on 25 cell blocks. Sensitivity levels of gene panel, BAP1 alone, p16 alone, and BAP1 plus p16 were 1, 0.5882, 0.4706, and 0.7647, respectively. Specificity was always 1. Although further validation is needed, this gene panel could really facilitate patients' management, allowing a definitive MPM diagnosis directly on pleural effusions.

Clinical phenotype and beta-cell autoimmunity in Italian patients with adult-onset diabetes.

OBJECTIVE: To characterize the phenotype of a large population of Italian patients with adult onset (> or =40 years) diabetes who were attending outpatient clinics and who were screened for glutamic acid decarboxylase 65 autoantibodies (GADA), protein tyrosine phosphatase IA-2 (IA-2A) and IA-2beta/phogrin (IA-2betaA). DESIGN AND METHODS: This was a cross-sectional study comprising a total of 881 patients, aged < or = 70 years, diagnosed with type 2 diabetes after the age of 40 years, and consecutively recruited in five clinics located in different geographic areas of Italy (Milan, Florence, Rome, Naples and Catania). Their mean disease duration was 8.1 (6.9; s.d.) years. GADA, IA-2A and IA-2betaA were measured with radiobinding assays with in vitro translated S-methionine-labelled glutamic acid decarboxylase 65 (GAD65) or IA-2 or IA-2beta. Anthropometric and clinical data were collected and compared amongst patients with or without autoantibodies. RESULTS: Sixty-three (7.1%) patients had one or more autoantibodies, 58 (6.6%) had GADA, 22 (2.5%) had IA-2A, six (0.7%) had IA-2betaA and 19 (2.15%) had two or more autoantibodies. IA-2A or IA-2betaA, in the absence of GADA, were found in only five patients. Autoantibody-positive patients were more often female (63.5 vs 36.5%; P < 0.009), had higher glycated haemoglobin (Hb A1c) (P < 0.001), lower body mass index (BMI; P < 0.0005) and waist/hip ratio (WHR; P < 0.01); female gender being the main contributor to BMI and WHR. We did not observe any differences in age at diagnosis or duration of disease with respect to the presence or absence of islet autoantibodies. The proportion of patients on insulin therapy was higher in patients with two or more antibodies, compared with those with one antibody only, and no antibodies (P for trend < 0.001), and among patients with GADA, in those with higher antibody titre (73.9% in those with > 10 units vs 42.0% in those with < or = 10 units; P < 0.007). CONCLUSIONS: Patients with adult onset diabetes characterized by autoimmunity to beta-cells showed a clinical phenotype with anthropometric features that differed from those classically observed in patients with type 2 diabetes. The number and titre of autoantibodies, which reflect the severity of autoimmunity and beta-cell impairment, amplified this difference. The usefulness of autoantibody screening in adult-onset diabetes is further emphasized by these findings.

Hexokinase shift to mitochondria is associated with an increased sensitivity to glucose in rat pancreatic islets.

When rat pancreatic islets are incubated in 5.5 or 16.7 mmol/l glucose for 3 h, an increased sensitivity is observed in islets pre-exposed to high glucose, as indicated by a shift to the left of the glucose dose-response curve (EC50 7.1 +/- 0.9 and 11.5 +/- 1.2 in high- and low-glucose-exposed islets, respectively; n = 5, P < 0.05). To investigate the mechanism(s) responsible for this effect, we measured hexokinase and glucokinase activity both in the cytosolic fraction and in a mitochondrion-enriched fraction, since binding to the outer mitochondrial membrane has been reported to result in an increased enzyme activity. In islets cultured at 16.7 mmol/l glucose, the cytosolic hexokinase activity was similar to control islets, but mitochondrial enzyme activity was significantly increased (124 +/- 7 vs. 51 +/- 9 nmol x microg(-1) x 90 min(-1), P < 0.01). As a consequence, the cytosolic:mitochondrial fraction ratio was altered in comparison with control islets. In contrast, glucokinase activity in the two groups of islets was similar in the cytosolic fraction and undetectable in the mitochondrial fraction. Hexokinase I quantitation by Western blot confirmed the enzyme translocation from the free cytosolic to the mitochondria-bound form in islets cultured at 16.7 mmol/l glucose. Glucose-induced alterations were reversible after 1 h exposure to 5.5 mmol/l glucose. Moreover, in islets exposed to 16.7 mmol/l glucose, inhibition of hexokinase binding to mitochondria by the addition of 20 nmol/l dicyclohexylcarbodiimide resulted in no increase of glucose sensitivity (EC50 10.9 +/- 0.4, n = 3, similar to that of control islets). These data indicate that after chronic exposure to high glucose, the beta-cell becomes more sensitive to glucose before eventually getting desensitized. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity secondary to a subcellular shift of the enzyme from the free cytosolic to the mitochondria-bound, more active form.

Metformin restores insulin secretion altered by chronic exposure to free fatty acids or high glucose: a direct metformin effect on pancreatic beta-cells.

Because metformin affects glucose and free fatty acid (FFA) metabolism in peripheral insulin target tissues, we investigated the effect of this drug in restoring a normal secretory pattern in rat pancreatic islets whose function has been impaired by chronic exposure to elevated FFA or glucose concentrations. We cultured rat pancreatic islets with or without FFA (2 mmol/l oleate/palmitate 2:1) or high glucose (16.7 mmol/l) concentrations in the presence or absence of metformin (0.25-12.5 microg/ml) and then measured insulin release, glucose utilization, glucose, and FFA oxidation. When compared with control islets, islets exposed to high FFA or glucose concentrations showed an increased basal and a decreased glucose-induced insulin release. In islets cultured for an additional 24 h with FFA or glucose in the presence of metformin (2.5 microg/ml), both basal and glucose-induced insulin secretions were restored. Both glucose utilization and glucose oxidation were altered in islets pre-exposed to high FFA or glucose concentrations. In particular, regarding control islets, glucose utilization was increased at 2.8 mmol/l glucose and decreased at 16.7 mmol/l glucose; glucose oxidation was similar to control islets at 2.8 mmol/l glucose but decreased at 16.7 mmol/l glucose. In contrast, oleate oxidation was increased in islets pre-exposed to FFA. All of these abnormalities were reversed in islets cultured for an additional 24 h with high FFA or glucose concentrations in the presence of metformin (2.5 microg/ml). In conclusion, our data show that metformin is able to restore the intracellular abnormalities of glucose and FFA metabolism and to restore a normal secretory pattern in rat pancreatic islets whose secretory function has been impaired by chronic exposure to elevated FFA or glucose levels. These data raise the possibility that, in diabetic patients, metformin (in addition to its peripheral effects) may have a direct beneficial effect on the beta-cell secretory function.

Fast reversibility of glucose-induced desensitization in rat pancreatic islets. Evidence for an involvement of ionic fluxes.

The present study was done to achieve a better understanding of the role of ionic flux alterations in glucose-induced desensitization of pancreatic beta-cells. Moreover, we investigated the reversibility of glucose-induced desensitization after different times of exposure to high glucose to ascertain the time necessary for desensitization reversal and to determine whether it depends on the length of high glucose exposure. Glucose desensitization was obtained by incubating rat pancreatic islets for 6 h in CMRL medium containing 16.7 mmol/l glucose. At the end of this period, insulin release, 86Rb efflux, and 45Ca uptake were measured in parallel experiments. In islets cultured at 16.7 mmol/l glucose, maximal glucose-induced insulin release was reduced (848 +/- 97 pg x islet-1 x 30 min-1) in comparison to islets incubated at 5.5 mmol/l glucose (1,436 +/- 144, n = 7, P < 0.01). In contrast, insulin content was similar in the two groups, being 41.0 +/- 2.7 and 47.8 +/- 2.2 ng/islet in islets exposed to 16.7 or 5.5 mmol/l glucose, respectively (P = 0.167). The effect of glucose on both 86Rb efflux and 45Ca uptake was also significantly reduced in 16.7 mmol/l glucose-cultured islets. 86Rb efflux was inhibited only 19 +/- 4% in islets cultured at high glucose with respect to 56 +/- 7% in control islets (n = 5, P < 0.001). 45Ca uptake was 10.5 +/- 1.7 pmol/islet (mean +/- SE, n = 9) in islets cultured at high glucose with respect to 19.7 +/- 2.4 pmol/islet in control islets (P < 0.001). In contrast, the effect of glyburide (10 micromol/l) on insulin release, 86Rb efflux, and 45Ca uptake was similar in islets exposed to 5.5 or 16.7 mmol/l glucose. All the abnormalities observed in islets cultured at 16.7 mmol/l glucose were promptly and simultaneously reversible after islets were transferred in culture medium at 5.5 mmol/l glucose; both insulin secretion and glucose effects on 86Rb efflux and 45Ca uptake returned to values similar to control islets within 5 min. Also, islets exposed to high glucose for a longer period (24 h) recovered from both secretory and ionic abnormalities after 5 min of incubation in CMRL medium at 5.5 mmol/l glucose. Reversal from glucose desensitization was slower (45 - 60 min) when islets were incubated at 5.5 mmol/l glucose in Krebs-Ringer HEPES buffer instead of CMRL medium. The present data suggest that ion flux and consequent membrane-potential changes play a key role in the mechanism leading to glucose-induced desensitization of pancreatic beta-cells. Because a normal response to glyburide was observed in islets exposed to high glucose, a proximal signal defect for closure of K+ channels rather than an intrinsic defect in the channel is likely.

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells.

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.

Alterations of insulin secretion from mouse islets treated with sulphonylureas: perturbations of Ca2+ regulation prevail over changes in insulin content.

1. To determine how pretreatment with sulphonylureas alters the beta cell function, mouse islets were cultured (18 - 20 h) without (controls) or with (test) 0.01 microM glibenclamide. Acute responses to glucose were then determined in the absence of glibenclamide. 2. Test islets were insensitive to drugs (sulphonylureas and diazoxide) acting on K+-ATP channels, and their [Ca2+]i was already elevated in the absence of stimulation. 3. Insulin secretion was increased in the absence of glucose, and mainly stimulated between 0 - 10 instead of 7 - 20 mM glucose in controls. The maximum response was halved, but this difference disappeared after correction for the 45% decrease in the islet insulin content. 4. The first phase of glucose-induced insulin secretion was abrogated because of a paradoxical decrease of the high basal [Ca2+]i in beta cells. The second phase was preserved but occurred with little rise of [Ca2+]i. These abnormalities did not result from alterations of glucose metabolism (NADPH fluorescence). 5. In islets cultured with 50 microM tolbutamide, glucose induced biphasic increases in [Ca2+]i and insulin secretion. The decrease in the secretory response was matched by the decrease in insulin content (45%) except at maximal glucose concentrations. Islets pretreated with tolbutamide, however, behaved like those cultured with glibenclamide if tolbutamide was also present during the acute functional tests. 6. In conclusion, treatment with a low glibenclamide concentration causes long-lasting blockade of K+-ATP channels and rise of [Ca2+]i in beta cells. Glucose-induced insulin secretion occurs at lower concentrations, is delayed and is largely mediated by a modulation of Ca2+ action on exocytosis. It is suggested that glucose regulation of insulin secretion mainly depends on a K+-ATP channel-independent pathway during in vivo sulphonylurea treatment.

Exposure to glibenclamide increases rat beta cells sensitivity to glucose.

An increased sensitivity to glucose was observed in islets pre-exposed for 1 h to glibenclamide (0.1 micromol 1(-1)), but not to tolbutamide (100 micromol l(-1)), as indicated by a shift to the left of the dose-response curve (EC(50) at 5.8+/-0.3 mmol l(-1) glucose vs 10.6+/-0.8 in control islets; n=11, P<0.005). According to this secretory pattern also glucose utilization at 2.5 and 5.0 mmol l(-1) glucose was higher in islets exposed to glibenclamide. Since binding to mitochondria results in an increased enzyme activity, we measured hexokinase (HK) and glucokinase (GK) activity both in a cytosolic and in a mitochondrion-enriched fractions. Cytosolic hexokinase activity was similar in islets exposed to glibenclamide and in control islets but mitochondrial hexokinase activity was significantly increased after exposure to glibenclamide (124+/-7 vs 51+/-9 nmol microgram prot(-1) 90 min(-1), P<0.01), with no change in the enzyme protein content. In contrast, glucokinase activity in the two groups of islets was similar. When in islets < exposed to glibenclamide hexokinase binding to mitochondria was inhibited by the addition of 20 nmol l(-1) dicyclohexylcarbodiimide (DCC), no increase of glucose sensitivity was observed (EC(50) 10.9+/-1.3 mmol l(-1) glucose, n=3, similar to that of control islets). These data indicate that a 1 h exposure to glibenclamide causes the beta cell to become more sensitive to glucose. This increased sensitivity is associated with (and may be due to) an increased hexokinase activity, in particular the mitochondrial-bound, more active, form. This mechanism may contribute to the hypoglycemic action of this drug.

Effects of prolonged glucose stimulation on pancreatic beta cells: from increased sensitivity to desensitization.

The prolonged exposure of pancreatic islets and isolated beta cells to elevated glucose concentrations induces a state of unresponsiveness to glucose (desensitization). However, an increased sensitivity to glucose (detected by a shift to the left of the dose-response curve of glucose-induced insulin release) has been also reported after chronic exposure to glucose, making the overall response less comprehensible. In vitro models have many theoretical and practical advantages in better understanding the effects of the prolonged glucose stimulation; moreover, they are also suitable for studying the mechanisms responsible of the observed alterations. We have performed a time-course study of the effect of the exposure to glucose at high concentration on the secretory behaviour of beta cells. Rat pancreatic islets exposed for 30 min to high glucose (300 mg/dl) showed increased basal insulin secretion (175 +/- 29 vs 44 +/- 8 pg/islet (per 30 min; n = 5, P < 0.002) was the only difference from control islets (exposed to 100 mg/dl). After 3 h exposure to high glucose, also increased sensitivity to glucose was observed, as indicated by a shift to the left of the glucose dose-response curve (EC50 123 +/- 10 and 177 +/- 11 mg/dl, respectively; n = 5, P < 0.05). After 6 h exposure to high glucose, besides the two alterations already described, also a decrease in glucose-induced insulin release was observed (688 +/- 104 vs 1184 +/- 34 pg/islet per 30 min; n = 5, P < 0.01). We studied the mechanism responsible for these alterations and we found that the "supersensitivity" to glucose may be related to alterations in the "glucose-sensing" mechanism of beta cells, in particular in glucose phosphorylation. In contrast, in islets desensitized to glucose our data suggest that ion flux and consequent membrane potential changes play a key role in determining the secretory defect. Since a normal response to glyburide was observed, a proximal signal defect for closure of potassium channels is more likely than an intrinsic defect in the channel. In conclusion, our data show what the prolonged stimulation of beta cells with glucose at high concentration induces a series of distinct secretory abnormalities, with a pattern of response that leads first to increased sensitivity and then to decreased responsiveness to glucose.

Prevention by metformin of alterations induced by chronic exposure to high glucose in human islet beta cells is associated with preserved ATP/ADP ratio.

AIM: We have explored whether the insulin secretory defects induced by glucotoxicity in human pancreatic islets could be prevented by metformin and investigated some of the possible mechanisms involved. METHODS: Human pancreatic islets and INS-1E cells were cultured for 24h with or without high glucose (16.7mM) concentration in the presence or absence of therapeutical concentration of metformin and then glucose-stimulated insulin release, adenine nucleotide levels and mitochondrial complex I and II activities were measured. Islet ultrastructure was analyzed by electron microscopy. RESULTS: Compared to control islets, human islets cultured with high glucose showed a reduced glucose-stimulated insulin secretion that was associated with lower ATP levels and a lower ATP/ADP ratio. These functional and biochemical defects were significantly prevented by the presence of metformin in the culture medium, that was also able to significantly inhibit the activity of mitochondrial complex I especially in beta cells exposed to high glucose. Ultrastructural observations showed that mitochondrial volume density was significantly increased in high glucose cultured islets. The critical involvement of mitochondria was further supported by the observation of remarkably swollen organelles with dispersed matrix and fragmented cristae. Metformin was able to efficiently prevent the appearance of all these ultrastructural alterations in human islets exposed to high glucose. CONCLUSIONS: Our results show that the functional, biochemical and ultrastructural abnormalities observed in human islet cells exposed to glucotoxic condition can be significantly prevented by metformin, further highlighting a direct beneficial effect of this drug on the insulin secreting human pancreatic beta cells.

Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.