Improvement in Serum Magnesium Levels With Sodium-Glucose Cotransporter 2 Inhibitors.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are associated with a modest but significant increase in serum magnesium levels. This report describes improvement in serum magnesium and associated symptoms after initiating SGLT2i therapy in a patient with refractory hypomagnesemia. A 58-year-old woman presented with persistent hypomagnesemia refractory to oral magnesium supplements. She had history of type 2 diabetes mellitus, hypothyroidism, fibromyalgia, and degenerative disk disease. The cause of hypomagnesemia was attributed to excessive renal losses. Laboratory investigations revealed serum magnesium of 1.2 mg/dL with fractional excretion of magnesium of 8.9%. She was started on empagliflozin 10 mg daily. Within 4 weeks of therapy, her serum magnesium level corrected with symptomatic improvement, which was sustained a few weeks later. Subsequently, her oral magnesium supplements dose was reduced. SGLT2i has been shown to improve magnesium levels in patients with urinary magnesium wasting. Several mechanisms have been postulated, but the exact physiology remains unknown. SGLT2i have been efficacious for glycemic control, renal protection, decreasing the risk of atherosclerotic cardiovascular disease events, and cardiac mortality in patients with diabetes. In addition, renal and cardiac benefits are also demonstrated in patients without diabetes. This observation demonstrates that SGLT2i can improve the management of patients with otherwise intractable hypomagnesemia.

Assessment of the Role of Serum 25-Hydroxy Vitamin D Level on Coronary Heart Disease Risk With Stratification Among Patients With Type 2 Diabetes Mellitus.

We investigated the role of vitamin D on glycemic regulation and cardiac complications in patients with type 2 diabetes mellitus (T2DM). A total of 1139 patients (49.3% males vs 50.7% females) were included. Information on sociodemographic lifestyle, family history, blood pressure (BP), and coronary heart disease (CHD) complications was collected. Significant differences were found between males and females regarding age-groups (P = .002), body mass index (BMI; P = .008), physical activity (P = .010), sheesha smoking (P = .016), cigarette smoking (P = .002), hypertension (P = .050), metabolic syndrome (P = .026), and CHD (P = .020). There were significant differences between vitamin D deficiency, insufficiency, and sufficiency in relation to age-group (P = .002), income (P = .002), waist circumference (P = .002), hip circumference (P = .028), waist-hip ratio (P = .002), and BMI (P = .002). Further, mean values of hemoglobin, magnesium, creatinine, hemoglobin A1c (HbA1c), total cholesterol, uric acid, and diastolic BP were significantly higher among patients with vitamin D deficiency compared with those with insufficiency and sufficiency. Multiple logistic regression analysis revealed that 25-hydroxy vitamin D, 25(OH)D, HbA1c, waist circumference, uric acid, duration of T2DM, total cholesterol, systolic and diastolic BP, and BMI were strong predictor risk factors for CHD among patients with T2DM. The present study supports that 25(OH)D may have a direct effect on CHD and on its risk factors.

Sleeping disturbances and predictor risk factors among type 2 diabetic mellitus patients.

Background: Sleep disturbance is a major health issue among people with type 2 diabetes mellitus (T2DM). The Pittsburgh Sleep Quality Index (PSQI) has been the most widely used instrument to measure subjective sleep disturbance. Aim: The aim of this study was to determine the impact of sleeping factor structure of the PSQI as potential predictor for glycosylated hemoglobin A1c (HbA1C) among people living with T2DM in the Turkish community to facilitate its use in the clinical practice and research. Subjects and Methods: This is a cross-sectional study and participants were between the age group of 25 and 65 years old who visited the diabetes and endocrinology department of Mega Medipol University Teaching Hospital, Istanbul. The PSQI was conducted on 871 patients with T2DM. Good sleep quality was defined as PSQI score <5. Multivariate logistic regression analysis was used to estimate the associated risk factors for the T2DM. Results: The current study showed significant differences between male and female patients with respect to their age in years, body mass index (BMI) (kg/m2), physical activity, smoking habit, sheesha smoking, income, family history of metabolic syndrome, coronary heart disease (CHD), and PSQI. The results revealed significant differences between HbA1c ≤7 and females and HbA1c >7 T2DM patients with respect to gender, BMI (kg/m2), CHD, and PSQI. The study demonstrated significant differences between sleeping categories PSQI as good, average, and poor sleeping among T2DM patients with respect to age and gender. Meanwhile, significant differences were reported between sleeping categories among T2DM patients with respect to their: number of sleeping hours, wake-up time, sleeping time, HbA1c, fasting blood glucose, uric acid, and systolic and diastolic blood pressure. This study showed very strong statistically significant correlations between low HbA1c and poor sleep quality in patients with T2DM patients, including subjective sleep quality r = 0.763, sleep latency r = 0.327, sleep duration r = 0.472, habitual sleep efficiency r = 0.575, sleep disturbances r = 0.564, use of sleep medication r = 0.728, and daytime dysfunction r = 0.734. Multivariate stepwise logistic regression analysis revealed that Vitamin D (mmol/L) (P < 0.001), HbA1c (P < 0.001), duration of DM (P < 0.001), uric acid (mmol/L) (P < 0.001), systolic blood pressure mmHg (P = 0.006), diastolic blood pressure mmHg (P = 0.015), and BMI (P = 0.024) were considered at higher risk as the predictors for sleeping quality among T2DM patients. Conclusion: The results suggest a strong positive correlation between PSQI with HbA1c levels, systolic and diastolic blood pressure, age, BMI, among type 2 diabetic patients. This study ascertains that poor sleep quality may be due to elevated level of HbA1c, metabolic syndrome, diabetes, obesity, and/or hypertension.

RésuméObjectif: Le but de cette étude était de déterminer l'impact de la structure du facteur de sommeil du PSQI en tant que prédicteur potentiel de l'HbA1C chez les personnes vivant avec le DT2 dans la communauté turque afin de faciliter son utilisation dans la pratique clinique et la recherche. Méthodes: Il s'agit d'une étude transversale et les participants étaient âgés de 25 à 65 ans et ont visité le service de diabète et d'endocrinologie du Mega Medipol University Teaching Hospital d'Istanbul. Le PSQI a été mené sur 871 patients atteints de DT2. Une bonne qualité de sommeil a été définie comme un score PSQI <5. Une analyse de régression logistique multivariée a été utilisée pour estimer les facteurs de risque associés au T2DM. Résultats: la présente étude a montré des différences significatives entre les hommes et les femmes en ce qui concerne leur âge en années, l'IMC (kg / m2), l'activité physique, le tabagisme, le tabagisme, le revenu, les antécédents familiaux de syndrome métabolique, les maladies coronariennes ( CHD) et PSQI. Les résultats ont révélé des différences significatives entre l'HbA1C≤7 et les femmes et l'HbA1C> 7 patients T2DM en termes de sexe, d'IMC (kg/m2), de maladie coronarienne (CHD) et de PSQI. L'étude a démontré des différences significatives entre les catégories de sommeil PSQI comme bon, moyen et mauvais sommeil chez les patients T2DM en ce qui concerne l'âge et le sexe.. Une analyse de régression logistique par étapes multivariée a révélé que la vitamine D (mmol / L) (P <0,001), l'HbA1c (P <0,001), la durée de la DM (P <0,001), l'acide urique (mmol / L) (P <0,001), la systolique La pression artérielle mmHg (P = 0,006), la pression artérielle diastolique mmHg (P = 0,015) et l'IMC (P = 0,024) étaient considérées comme présentant un risque plus élevé comme prédicteurs de la qualité du sommeil chez les patients atteints de DT2.Conclusion: Les résultats suggèrent une forte corrélation positive entre le PSQI avec les niveaux d'HbA1C, la pression artérielle systolique et diastolique, l'âge, l'IMC, chez les patients diabétiques de type 2. Cette étude établit qu'une mauvaise qualité de sommeil peut être due à un niveau élevé d'HbA1C, au syndrome métabolique, au diabète, à l'obésité et / ou à l'hypertension.

Prevalence of Helicobacter pylori Infection among Type 2 Diabetes Mellitus.

BACKGROUND: Several conducted studies have reported a higher and more frequent Helicobacter pylori infection rate in type 2 diabetes mellitus (T2DM). The aim of this study was to investigate the prevalence and its association between H. pylori infection and T2DM. MATERIALS AND METHODS: A case and control study was conducted based on 529 T2DM patients and 529 control. H. pylori was assessed by Serum anti-H. pylori immunoglobulin G (IgG) and IgA. Furthermore, patients were investigated for fasting blood glucose (FBG) levels, glycosylated hemoglobin (HbA1c), serum cholesterol, and other biochemistry parameters. RESULTS: The findings showed a positive significantly higher antibody titer for H. pylori infection (IgA > 250) in diabetic patients (50.7%) compared to controls (38.2%) (P < 0.001). Similarly, H. pylori infection for IgG > 300 titer was higher in T2DM patients (73.5%) compared to controls 61.8%) (P < 0.001). Further, the mean values were statistically significant diabetes with H. pylori infection for IgG > 300 titer and IgA > 250 titer, regarding Vitamin D, HbA1C (P < 0.001), FBG, calcium, creatinine, total cholesterol, LHDL, triglyceride levels, uric acid, bilirubin, thyroid-stimulating hormone (TSH), and systolic and diastolic blood pressure. The diabetic patients showed higher prevalence rate of symptoms than controls included: hypertension (14.3%), vomiting (15.5%), muscular symptoms (35.2%), bloating/distension (13.2%), abdominal pain (17%), nausea (9.6%), anemia (17%), kidneys (20.8%), chronic bronchitis (14.7%), gastrointestinal (23.8%), and diarrhea (20.4%). CONCLUSIONS: The current study revealed that H. pylori infections were significantly higher in diabetic patients compared to controls. Furthermore, T2DM patients infected with H. pylori positive reported a higher prevalence rate of symptoms than H. pylori negative.

The prevalence of restless legs syndrome and comorbid condition among patient with type 2 diabetic mellitus visiting primary healthcare.

AIM: The aim of this study was to determine the prevalence of restless legs syndrome (RLS) and Pittsburgh Sleep Quality Index (PSQI) in patients with type 2 diabetes mellitus (T2DM) attending primary healthcare. SUBJECTS AND METHODS: This is a cross-sectional study and participants were between 25 and 70 years old who visited the diabetes and endocrinology department of Mega Medipol University Teaching Hospital, Istanbul. The diagnosis of RLS was performed according to the International Restless Legs Syndrome Study Group consensus criteria. The RLS and PSQI instruments were conducted on 871 patients with T2DM. Good sleep quality was defined as PSQI score <5. RLS severity was assessed by the Restless Legs Syndrome-6 Scales (RLS-6). The scale development and validation was carried out using Rasch measurement model. RESULTS: The prevalence of RLS was 22.8% including 60.3% of females and 39.7% of males. This study showed significant differences between RLS and no RLS patients with respect to their age (years), body mass index (BMI) (kg/m2), physical activity, smoking habit, sheesha smoking, income, and sleeping quality with PSQI. Also, the analysis presented that statistically significant differences between both RLS and no RLS reported sleep complaints including difficulty falling asleep, inadequate sleep, anytime fatigue, and leg discomfort. There were statistically significant differences between RLS and no RLS patients regarding hypoglycemia, numbness in legs, retinopathy, neuropathy, nephropathy high blood pressure, depression, stroke, anemia, diabetic foot, ulcer, arthritis, respiratory disease, metabolic syndrome, and coronary heart disease. Furthermore, there were statistically significant differences between RLS and no RLS concerning the number of sleeping hours, wake-up time (AM), sleeping time (PM), BMI (kg/m2), HbA1c, vitamin D, calcium, creatinine, fasting blood glucose, low-density lipoprotein, triglyceride, uric acid, and systolic and diastolic blood pressure (mmHg). CONCLUSION: This study confirms positive relation and high prevalence of RLS among patients with T2DM visiting primary healthcare. The results suggest that physical activity is associated with a better perception of functional capacity and pain in diabetic patients with RLS, and thus a more active lifestyle should be encouraged.

Effect of ramadan fasting on glycemic control and other essential variables in diabetic patients.

Background: Fasting during the holy month of Ramadan is a religious obligation for all Muslims who represent 1.8 billion of the world population (24%). This study explores the effect of Ramadan fasting on the blood glucose, glycated hemoglobin (HbA1c), lipid profile, sleeping quality, and essential lifestyle parameters and also explores the safety of fasting for a whole month among diabetic patients. Aim: The aim of the present study was to assess the impact of Ramadan fasting on the blood glucose, HbA1c, lipid profile, sleeping quality, and lifestyle parameters among patients with type 2 diabetes mellitus (T2DM) in Turkey. Subjects and Methods: A total of 1780 diabetic patients were approached, and 1246 (70%) participated in this cross-sectional study carried out during the period from May 27, 2017, to June 24, 2017. Data analysis comprised sociodemographic features, lifestyle habits, blood pressure measurements, serum lipid profiles, serum calcium, Vitamin D 25-hydroxy, uric acid, and HbA1c at before 4 weeks and after 12 weeks from Ramadan. Results: Out of 1246 patients, 593 (47.6%) were male and 653 (52.4%) were female. The mean ± standard deviation age of the patients was 50.39 ± 15.3 years. Males were significantly older than females (51.53 ± 12.56 vs. 49.26 ± 14.4; P = 0.003, respectively). Significant differences were found in Vitamin D, blood glucose, HbA1c level, creatinine, bilirubin, albumin, total cholesterol, triglycerides, high-density lipoprotein-cholesterol (female), low-density lipoprotein-cholesterol (male), uric acid, and systolic and diastolic blood pressure after and before the holy month of Ramadan (P < 0.05 for each). HbA1c (P < 0.001), physical activity (P < 0.001), hours of sleeping (P < 0.001), systolic blood pressure (BP) (mmHg) (P = 0.007), BMI (P = 0.016), diastolic BP (mmHg) (P = 0.018), family history (P = 0.021), and smoking (P = 0.045) were identified as significantly associated with Ramadan fasting as contributing factors. Conclusion: In one of the largest studies of its kind, we show that Ramadan fasting has positive effects on T2DM patients as it reduces their blood pressure, blood glucose, HbA1C, and BMI. Furthermore, there are improvements in the duration of sleep and physical activity, the role of Ramadan fasting in diabetes therapy has been confirmed.

RésuméContexte: Le jeûne pendant le mois sacré du Ramadan est une obligation religieuse pour tous les musulmans qui représentent 1,8 milliard de personnes dans le monde population (24%). Cette étude explore l'effet du jeûne du Ramadan sur la glycémie, l'hémoglobine glyquée (HbA1c), le profil lipidique, qualité de sommeil, et les paramètres essentiels de style de vie et explore également la sécurité du jeûne pour un mois entier chez les patients diabétiques. But: Le but de la présente étude était d'évaluer l'impact du jeûne du Ramadan sur la glycémie, l'HbA1c, le profil lipidique, le sommeil paramètres de qualité et de style de vie chez les patients atteints de diabète sucré de type 2 (DT2) en Turquie. Sujets et méthodes: Un total de 1780 patients diabétiques ont été approchés, et 1246 (70%) ont participé à cette étude transversale réalisée au cours de la période Du 27 mai 2017 au 24 juin 2017. L'analyse des données comprenait des caractéristiques sociodémographiques, des habitudes de vie, des mesures de la tension artérielle, les profils sériques des lipides, le calcium sérique, la vitamine D 25-hydroxy, l'acide urique et l'HbA1c avant 4 semaines et après 12 semaines de Ramadan. Résultats: Sur 1246 patients, 593 (47,6%) étaient des hommes et 653 (52,4%) étaient des femmes. L'âge moyen ± écart-type des patients était de 50,39 ± 15,3 ans. Les mâles étaient significativement plus âgés que les femelles (51,53 ± 12,56 contre 49,26 ± 14,4, P = 0,003, respectivement). Important différences ont été trouvées dans la vitamine D, la glycémie, le taux d'HbA1c, la créatinine, la bilirubine, l'albumine, le cholestérol total, les triglycérides, la densité lipoprotéine-cholestérol (femelle), lipoprotéine-cholestérol de basse densité (mâle), acide urique et tension artérielle systolique et diastolique après et avant le mois sacré du Ramadan (P <0,05 pour chacun). HbA1c (P < 0,001), activité physique (P <0,001), heures de sommeil (P <0,001), tension artérielle systolique (TA) (mmHg) (P = 0,007), IMC (P = 0,016), TA diastolique (mmHg) (P = 0,018), antécédents familiaux (P = 0,021), et le tabagisme (P = 0,045) a été identifié comme étant significativement associé au jeûne du Ramadan en tant que facteurs contributifs. Conclusion: Dans l'un des les plus grandes études de son genre, nous montrons que le jeûne du Ramadan a des effets positifs sur les patients atteints de DT2 car il réduit leur tension artérielle, le sang glucose, HbA1C et BMI. En outre, il y a des améliorations dans la durée du sommeil et de l'activité physique, le rôle du jeûne du Ramadan dans la thérapie du diabète a été confi rmée. Mots-clés: Indice de masse corporelle, diabète sucré, hémoglobine glyquée, jeûne du Ramadan, qualité de sommeil.

Adrenal incidentalomas: are they being worked up appropriately?

INTRODUCTION: Adrenal incidentalomas are defined as masses picked up on imaging studies that were done for apparently different reasons. With frequent use of imaging modalities, incidental adrenal masses are commonly encountered in clinical practice. Guidelines are currently available for the diagnosis and management of adrenal incidentalomas, but the appropriateness of initial work-up and subsequent follow-up of incidental adrenal masses in the community hospital setting is unknown. OBJECTIVE: We studied the appropriateness of initial work-up and follow-up of incidental adrenal masses discovered on abdominal computerized tomography (CT). METHODS: In our retrospective study, we reviewed sequential CT scans of the abdomen performed in the month of January 2010 at a community hospital. Once patients with one or more adrenal masses were identified, outpatient charts for initial biochemical testing and follow-up imaging were obtained either through directly accessing the electronic medical records or through contacting primary care physician's offices. Patient charts were reviewed to assess the data for the next 2 years following the discovery of an adrenal abnormality. RESULTS: Twenty adrenal masses were incidentally discovered on 723 abdominal CTs performed within the month of January 2010 resulting in an overall incidence of 2.76%. Of the patients with incidentally discovered adrenal masses, appropriate biochemical and follow-up imaging were only performed in patients referred to an endocrinologist (2 of 20 patients). Thirty percent of patients with incidental masses received a repeat CT scan for non-adrenal reasons, and no change in the mass size was noted. CONCLUSION: Despite published guidelines, the initial work-up and follow-up of patients with an incidentally discovered adrenal mass is unsatisfactory. There is a desperate need for education of providers regarding appropriate work-up of incidental adrenal masses.

Insulin Autoimmune Syndrome: a rare cause of postprandial hypoglycemia.

A 65-year-old obese Caucasian woman presented with symptomatic postprandial hypoglycemic episodes, resolution of symptoms with carbohydrate intake and significantly elevated anti-insulin antibody levels. She did not have any evidence for the use of oral antidiabetic medications, insulin, herbal substances, performing strenuous exercise or history of bariatric surgery. Fingerstick blood glucose readings revealed blood sugar of 35 mg/dL and 48 mg/dL, when she had these symptoms. Her medical history was significant for morbid obesity, hypothyroidism and gastro esophageal reflux disease. Her home medications included levothyroxine, propranolol and omeprazole. A blood sample obtained during the symptoms revealed the following: fingerstick blood sugar 38 mg/dL, venous blood glucose 60 mg/dL (normal (n): 70-99 mg/dL), serum insulin 202 IU/mL (n: <21), proinsulin 31.3 pmol/L (n: <28.9), C-peptide 8 ng/mL (n: 0.9-7), beta-hydroxybutyrate 0.12 mmol/L (n: 0.02-0.27) anti-insulin antibody >45.4 U/mL (n: <0.4). The result obtained while screening for serum sulfonylurea and meglitinides was negative. The repeated episodes of postprandial hypoglycemia associated with significantly elevated anti-insulin antibodies led to a diagnosis of insulin antibody syndrome (IAS). Significant improvement of hypoglycemic symptoms and lower anti-insulin antibody levels (33 U/mL) was noted on nutritional management during the following 6 months. Based on a report of pantoprazole-related IAS cases, her omeprazole was switched to a H2 receptor blocker. She reported only two episodes of hypoglycemia, and anti-insulin antibody levels were significantly lower at 10 U/mL after the following 12-month follow-up. LEARNING POINTS: Initial assessment of the Whipple criteria is critical to establish the clinical diagnosis of hypoglycemia accurately.Blood sugar monitoring with fingerstick blood glucose method can provide important information during hypoglycemia workup.Autoimmune hypoglycemia is a rare cause of hypoglycemia, which can be diagnosed on high index of clinical suspicion and systematic evaluation.

Predicting remission of diabetes after RYGB surgery following intensive management to optimize preoperative glucose control.

BACKGROUND: The purpose of the study is to investigate the association of preoperative glucose optimization prior to a Roux-en-Y gastric bypass (RYGB) and diabetes remission. METHODS: The study is a retrospective review of 245 patients with a history of diabetes type II and a RYGB from 2008 to 2012 at UMass Memorial Hospital. RESULTS: Patients that benefited from glucose optimization prior to RYGB were more likely to achieve diabetes remission 1 year after surgery. The preoperative glucose optimization intervention demonstrated that when patients decreased their HbA1c prior to surgery by 1 %, these individuals were 68 % more likely to remit (p = 0.015). Duration of diabetes (p = 0.005) and insulin use (p < 0.001) were also significant predictors of remission, whereas age, race, and gender were not. CONCLUSIONS: Our study results indicate that a greater degree of glycemic improvement in response to presurgical medical intervention is associated with higher rates of diabetes remission post-operatively among obese adults with diabetes type II. Conversely, the lack of favorable glycemic response to intensification of medical management predicts a poor glycemic response to bariatric surgery. Further research is needed to determine if this difference is due to physiological factors or is simply an indicator of patient behavior.

TRAIL and DcR1 expressions are differentially regulated in the pancreatic islets of STZ- versus CY-applied NOD mice.

TNF-related apoptosis-inducing ligand (TRAIL) is an important component of the immune system. Although it is well acknowledged that it also has an important role in Type 1 Diabetes (T1D) development, this presumed role has not yet been clearly revealed. Streptozotocin (STZ) and Cyclophosphamide (CY) are frequently used agents for establishment or acceleration of T1D disease in experimental models, including the non-obese diabetic (NOD) mice. Although such disease models are very suitable for diabetes research, different expression patterns for various T1D-related molecules may be expected, depending on the action mechanism of the applied agent. We accelerated diabetes in female NOD mice using STZ or CY and analyzed the expression profiles of TRAIL ligand and receptors throughout disease development. TRAIL ligand expression followed a completely different pattern in STZ- versus CY-accelerated disease, displaying a prominent increase in the former, while appearing at reduced levels in the latter. Decoy receptor 1 (DcR1) expression also increased significantly in the pancreatic islets in STZ-induced disease. Specific increases observed in TRAIL ligand and DcR1 expressions may be part of a defensive strategy of the beta islets against the infiltrating leukocytes, while the immune-suppressive agent CY may partly hold down this defense, contributing further to diabetes development.

Quantitative assessment of islets of Langerhans encapsulated in alginate.

Improved methods have recently been developed for assessing islet viability and quantity in human islet preparations for transplantation, and these measurements have proven useful for predicting transplantation outcome. The objectives of this study were to adapt these methods for use with microencapsulated islets, to verify that they provide meaningful quantitative measurements, and to test them with two model systems: (1) barium alginate and (2) barium alginate containing a 70% (w/v) perfluorocarbon (PFC) emulsion, which presents challenges to use of these assays and is of interest in its own right as a means for reducing oxygen supply limitations to encapsulated tissue. Mitochondrial function was assessed by oxygen consumption rate measurements, and the analysis of data was modified to account for the increased solubility of oxygen in the PFC-alginate capsules. Capsules were dissolved and tissue recovered for nuclei counting to measure the number of cells. Capsule volume was determined from alginate or PFC content and used to normalize measurements. After low oxygen culture for 2 days, islets in normal alginate lost substantial viable tissue and displayed necrotic cores, whereas most of the original oxygen consumption rate was recovered with PFC alginate, and little necrosis was observed. All nuclei were recovered with normal alginate, but some nuclei from nonrespiring cells were lost with PFC alginate. Biocompatibility tests revealed toxicity at the islet periphery associated with the lipid emulsion used to provide surfactants during the emulsification process. We conclude that these new assay methods can be applied to islets encapsulated in materials as complex as PFC-alginate. Measurements made with these materials revealed that enhancement of oxygen permeability of the encapsulating material with a concentrated PFC emulsion improves survival of encapsulated islets under hypoxic conditions, but reformulation of the PFC emulsion is needed to reduce toxicity.

Enumeration of islets by nuclei counting and light microscopic analysis.

Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.

Quantitative analysis of cell composition and purity of human pancreatic islet preparations.

Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light microscopy (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed an accurate identification and quantification of cell types with measured cell number fractions (mean±s.e.m.) of 35.6±2.1% ß-cells, 12.6±1.0% non-ß-islet cells (48.3±2.6% total islet cells), 22.7±1.5% duct cells, and 25.3±1.8% acinar cells. Of the islet cells, 73.6±1.7% were ß-cells. For comparison with the literature, estimates of cell number fraction, cell volume, and extracellular volume were combined to convert number fraction data to volume fractions applicable to cells, islets, and the entire preparation. The mathematical framework for this conversion was developed. By volume, ß-cells were 86.5±1.1% of the total islet cell volume and 61.2±0.8% of intact islets (including the extracellular volume), which is similar to that of islets in the pancreas. Our estimates produced 1560±20 cells in an islet equivalent (volume of 150-µm diameter sphere), of which 1140±15 were ß-cells. To test whether LM analysis of the same tissue samples could provide reasonable estimates of purity of the islet preparations, volume fraction of the islet tissue was measured on thin sections available from 27 of the clinical preparations by point counting morphometrics. Islet purity (islet volume fraction) of individual preparations determined by LM and EM analyses correlated linearly with excellent agreement (R²=0.95). However, islet purity by conventional dithizone staining was substantially higher with a 20-30% overestimation. Thus, both EM and LM provide accurate methods to determine the cell composition of human islet preparations and can help us understand many of the discrepancies of islet composition in the literature.

Adenovirus-mediated TRAIL gene (Ad5hTRAIL) delivery into pancreatic islets prolongs normoglycemia in streptozotocin-induced diabetic rats.

Type 1 diabetes (T1D), characterized by permanent destruction of insulin-producing beta cells, is lethal unless conventional exogenous insulin therapy or whole-organ transplantation is employed. Although pancreatic islet transplantation is a safer and less invasive method compared with whole-organ transplant surgery, its treatment efficacy has been limited by islet graft malfunction and graft failure. Thus, ex vivo genetic engineering of beta cells is necessary to prolong islet graft survival. For this reason, a novel gene therapy approach involving adenovirus-mediated TRAIL gene delivery into pancreatic islets was tested to determine whether this approach would defy autoreactive T cell assault in streptozotocin (STZ)-induced diabetic rats. To test this, genetically modified rat pancreatic islets were transplanted under the kidney capsule of STZ-induced diabetic rats, and diabetic status (blood sugar and body weight) was monitored after islet transplantation. STZ-induced diabetic rats carrying Ad5hTRAIL-infected islets experienced prolonged normoglycemia compared with animals grafted with mock-infected or AdCMVLacZ-infected islets. In addition, severe insulitis was detected in animals transplanted with mock-infected or AdCMVLacZ-infected islets, whereas the severity of insulitis was reduced in animals engrafted with Ad5hTRAIL-infected islets. Thus, TRAIL overexpression in pancreatic islets extends allograft survival and function, leading to a therapeutic benefit in STZ-induced diabetic rats.

High levels of endogenous tumor necrosis factor-related apoptosis-inducing ligand expression correlate with increased cell death in human pancreas.

OBJECTIVES: Type 1 diabetes (T1D) has been characterized by the T cell-mediated destruction of pancreatic beta cells. Although various members of the tumor necrosis factor (TNF) family, such as Fas ligand or TNF, have recently been implicated in the development of T1D, the lack of TNF-related apoptosis-inducing ligand (TRAIL) expression or function facilitates the onset of T1D. Thus, the goal of the present study was to investigate the expression profiles of TRAIL and its receptors in human pancreas. METHODS: Pancreata of 31 patients were analyzed by immunohistochemistry using antibodies developed against TRAIL and its receptors. Apoptosis was confirmed by Annexin V-fluorescein isothiocyanate binding and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assays. RESULTS: Acinar cells displayed high levels of TRAIL and death receptor 4, but only low levels of death receptor 5. In contrast, only TRAIL and TRAIL decoy receptors (DcR1, DcR2) were detected in ductal cells. Similarly, Langerhans islets expressed only TRAIL and TRAIL decoy receptor. High levels of TRAIL expression in pancreas correlated with increased number of apoptotic cells. CONCLUSIONS: Although the expression of TRAIL decoy receptors might be necessary for defense from TRAIL-induced apoptosis, high levels of TRAIL may provide protection for Langerhans islets from the immunological attack of cytotoxic T cells.

Molecular mechanisms of death ligand-mediated immune modulation: a gene therapy model to prolong islet survival in type 1 diabetes.

Type 1 diabetes results from the T cell-mediated destruction of pancreatic beta cells. Islet transplantation has recently become a potential therapeutic approach for patients with type 1 diabetes. However, islet-graft failure appears to be a challenging issue to overcome. Thus, complementary gene therapy strategies are needed to improve the islet-graft survival following transplantation. Immune modulation through gene therapy represents a novel way of attacking cytotoxic T cells targeting pancreatic islets. Various death ligands of the TNF family such as FasL, TNF, and TNF-Related Apoptosis-Inducing Ligand (TRAIL) have been studied for this purpose. The over-expression of TNF or FasL in pancreatic islets exacerbates the onset of type 1 diabetes generating lymphocyte infiltrates responsible for the inflammation. Conversely, the lack of TRAIL expression results in higher degree of islet inflammation in the pancreas. In addition, blocking of TRAIL function using soluble TRAIL receptors facilitates the onset of diabetes. These results suggested that contrary to what was observed with TNF or FasL, adenovirus mediated TRAIL gene delivery into pancreatic islets is expected to be therapeutically beneficial in the setting of experimental models of type 1 diabetes. In conclusion; this study mainly reveals the fundamental principles of death ligand-mediated immune evasion in diabetes mellitus.

Gene expression of purified beta-cell tissue obtained from human pancreas with laser capture microdissection.

CONTEXT: Human beta-cell gene profiling is a powerful tool for understanding beta-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-beta-cells and the trauma of the isolation procedure. OBJECTIVE: The objective was to use laser capture microdissection (LCM) of human beta-cells from pancreases of cadaver donors and compare their gene expression with that of handpicked isolated islets. DESIGN: Endogenous autofluorescence of beta-cells facilitated procurement of purified beta-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected beta-cell samples and three isolated islet preparations were obtained. The array data were normalized using DNA-Chip Analyzer software (Harvard School of Public Health, Boston, MA), and the lower confidence bound evaluated differentially expressed genes. Real-time PCR was performed on selected acinar genes and on the duct cell markers, carbonic anhydrase II and keratin 19. RESULTS: Endogenous autofluorescence facilitates the microdissection of beta-cell rich tissue in human pancreas. When compared with array profiles of purified beta-cell tissue, with lower confidence bound set at 1.2, there were 4560 genes up-regulated and 1226 genes down-regulated in the isolated islets. Among the genes up-regulated in isolated islets were pancreatic acinar and duct genes, chemokine genes, and genes associated with hypoxia, apoptosis, and stress. Quantitative RT-PCR confirmed the differential expression of acinar gene transcripts and the duct marker carbonic anhydrase II in isolated islets. CONCLUSION: LCM makes it possible to obtain beta-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.

Differentiation of affinity-purified human pancreatic duct cells to beta-cells.

To test whether pancreatic duct cells are in vitro progenitors, they were purified from dispersed islet-depleted human pancreatic tissue using CA19-9 antibody. The purified fraction was almost entirely CK19+ with no insulin+ cells, whereas the unpurified cells (crude duct) were 56% CK19+ and 0.4% insulin+ of total cells (0.7% of CK19+ cells). These cells were expanded as monolayers, aggregated under serum-free conditions, and transplanted into normoglycemic NOD/SCID mice. In crude duct grafts, insulin+ cells increased to 6.1% of CK19+ cells. Purified duct cells had slow expansion and poor aggregation, as well as engraftment. The addition of 0.1% cultured stromal cells improved these parameters. These stromal cells contained no CK19+ cells and no insulin by either quantitative RT-PCR or immunohistochemistry; stromal cell aggregates and grafts contained no insulin+ cells. Aggregation of purified duct plus stromal preparations induced insulin+ cells (0.1% of CK19+ cells), with further increase to 1.1% in grafts. Insulin mRNA mirrored these changes. In these grafts, all insulin+ cells were in duct-like structures, while in crude duct grafts, 85% were. Some insulin+ cells coexpressed duct markers (CK19 and CA19-9) and heat shock protein (HSP)27, a marker of nonislet cells, suggesting the transition from duct. Thus, purified duct cells from adult human pancreas can differentiate to insulin-producing cells.

Long-term normoglycemia in rats receiving transplants with encapsulated islets.

BACKGROUND: To follow up on previously successful transplantation of encapsulated islets in mice, the present study was performed in rats to determine the effects of several factors, including alginate composition and concentration of cross-linking agent and capsule size on the effectiveness of encapsulated islets. METHODS: Highly purified alginate of either high guluronic acid or high mannuronic acid (M) with low endotoxin content was used. Regular-size (0.8-1.1 mm) or small microcapsules (0.5-0.7 mm) were produced by cross-linking with BaCl2 without additional poly-L-lysine coating and were transplanted into abdominal cavity of normoglycemic (empty capsules) or streptozotocin induced diabetic Lewis rats (islet containing capsules). RESULTS: Empty regular-size capsules made of different alginate compositions had similar biocompatibility and stability results. Compared with empty capsules, regular-size capsules made of high-M alginate containing syngeneic islets had inferior stability indicated with lower fractional volume retrieved. Islet-containing smaller-size microcapsules made of high-M alginate were more stable and had less cellular attachment compared with the regular-size capsules, although the normoglycemic period was comparable between two groups of rats receiving transplants with smaller-size microcapsules (48+/-8 days, n=8) or regular-size capsules (59+/-11 days, n=4) in allogeneic experiments. In syngeneic experiments, all of the rats (n=4) maintained normoglycemia up to 210 days after transplantation. CONCLUSION: These results indicate that regular-size alginate capsules do less well in rats than in our previous experiments with mice. Smaller capsules made of alginate cross-linked with barium appear to provide better stability and may be a useful strategy for use in larger recipients.