Roger Lehmann

Improved Human Islet Isolation Using a New Enzyme Blend, Liberase

Enzymatic digestion of donor pancreases is a vital step in human and large mammalian islet isolation. The variable enzymatic activities of different batches of commercially available collagenase is a major obstacle in achieving reproducibility in islet isolation procedures. In the present work, the effectiveness of Liberase, a standardized mixture of highly purified enzymes recently developed for the separation of human islets, was compared with that of a traditional collagenase preparation (type P). The results of 50 islet isolations using Liberase enzyme were compared with those of 36 isolations with collagenase, type P. No significant differences in donor age, cold ischemia time, digestion time, or weight of the pancreases were observed between the two groups. Islet yield was significantly higher in the group where the Liberase enzyme was used. All parameters examined (islet number, islet number per gram of tissue, islet equivalent number, and islet equivalent number per gram of tissue) were significantly improved when Liberase enzyme was used. Different lots of Liberase enzyme were tested, and no difference was observed. Islets isolated with Liberase enzyme were also of larger size and were much less fragmented, suggesting a gentler enzymatic action and better preservation of anatomical integrity. Islets isolated with Liberase enzyme, assessed both in vitro and in vivo, revealed a functional profile similar to that of islets separated with collagenase. Liberase enzyme appears, therefore, to represent a new powerful tool for improving the quality of human islet isolation.

Superiority of Small Islets in Human Islet Transplantation

Many factors influence the outcome of islet transplantation. As islets in the early posttransplant setting are supplied with oxygen by diffusion only and are in a hypoxic state in the portal system, we tested whether small human islets are superior to large islets both in vitro and in vivo. We assessed insulin secretion of large and small islets and quantified cell death during hypoxic conditions simulating the intraportal transplant environment. In the clinical setting, we analyzed the influence of transplanted islet size on insulin production in patients with type 1 diabetes. Our results provide evidence that small islets are superior to large islets with regard to in vitro insulin secretion and show a higher survival rate during both normoxic and hypoxic culture. Islet volume after 48 h of hypoxic culture decreased to 25% compared with normoxic culture at 24 h due to a preferential loss of large islets. In human islet transplantation, the isolation index (islet volume as expressed in islet equivalents/islet number), or more simply the islet number, proved to be more reliable to predict stimulated C-peptide response compared with islet volume. Thus, islet size seems to be a key factor determining human islet transplantation outcome.

Factors Associated with the Incidence of Type 2 Diabetes Mellitus in HIV-Infected Participants in the Swiss HIV Cohort Study

BACKGROUND Human immunodeficiency virus (HIV)-infected persons may be at increased risk for developing type 2 diabetes mellitus because of viral coinfection and adverse effects of treatment. METHODS We studied associations of new-onset diabetes mellitus with hepatitis B virus and hepatitis C virus coinfections and antiretroviral therapy in participants in the Swiss HIV Cohort Study, using Poisson regression. RESULTS A total of 123 of 6513 persons experienced diabetes mellitus during 27,798 person-years of follow-up (PYFU), resulting in an incidence of 4.4 cases per 1000 PYFU (95% confidence interval [CI], 3.7-5.3 cases per 1000 PYFU). An increased incidence rate ratio (IRR) was found for male subjects (IRR, 2.5; 95% CI, 1.5-4.2), older age (IRR for subjects >60 years old, 4.3; 95% CI, 2.3-8.2), black (IRR, 2.1; 95% CI, 1.1-4.0) and Asian (IRR, 4.9; 95% CI, 2.2-10.9) ethnicity, Centers for Disease Control and Prevention disease stage C (IRR, 1.6; 95% CI, 1.04-2.4), and obesity (IRR, 4.7; 95% CI, 3.1-7.0), but results for hepatitis C virus infection or active hepatitis B virus infection were inconclusive. Strong associations were found for current treatment with nucleoside reverse-transcriptase inhibitors (IRR, 2.22; 95% CI, 1.11-4.45), nucleoside reverse-transcriptase inhibitors plus protease inhibitors (IRR, 2.48; 95% CI, 1.42-4.31), and nucleoside reverse-transcriptase inhibitors plus protease inhibitors and nonnucleoside reverse-transcriptase inhibitors (IRR, 3.25; 95% CI, 1.59-6.67) but were not found for treatment with nucleoside reverse-transcriptase inhibitors plus nonnucleoside reverse-transcriptase inhibitors (IRR, 1.47; 95% CI, 0.77-2.82). CONCLUSIONS In addition to traditional risk factors, current treatment with protease inhibitor- and nucleoside reverse-transcriptase inhibitor-containing regimens was associated with the risk of developing type 2 diabetes mellitus. Our study did not find a significant association between viral hepatitis infection and risk of incident diabetes.

Apoptosis in hypoxic human pancreatic islets correlates with HIF-1α expression

To become insulin independent, patients with type 1 diabetes mellitus require transplantation of at least two donor pancreata because of massive β–cell loss in the early post‐transplantation period. Many studies describing the introduction of new immunosuppressive protocols have shown that this loss is due to not only immunological events but also nonimmunological factors. To test to what extent hypoxia may contribute to early graft loss, we analyzed the occurrence of apoptotic events and the expression of hypoxia‐inducible factor 1 (HIF‐1), a heterodimeric transcription factor consisting of an oxygen‐dependent α subunit and a constitutive β subunit. Histological analysis of human and rat islets revealed nuclear pyknosis as early as 6 h after hypoxic exposure (1% O2). Moreover, immunoreactivity to activated caspase‐3 was observed in the core region of isolated human islets. Of note, both of these markers of apoptosis topographically overlap with HIF‐1α immunoreactivity. HIF‐1α mRNA was detected in islets from human and rat as well as in several murine β–cell lines. When exposed to hypoxia, mouse insulinoma cells (MIN6) had an increased HIF‐1α protein level, whereas its mRNA level did not alter. In conclusion, our data provide convincing evidence that reduced oxygenation is an important cause of β–cell loss and suggest that HIF‐1α protein level is an indicator for hypoxic regions undergoing apoptotic cell death. These observations suggest that gene expression under the control of HIF‐1 represents a potential therapeutic tool for improving engraftment of transplanted islets.

Impact of Physical Activity on Cardiovascular Risk Factors in IDDM

OBJECTIVE To study the impact of physical activity on glycemic control and plasma lipids [HDL cholesterol (HDL-C), HDL-C subfractions, triglycerides, lipoprotein(a)], blood pressure, weight, and abdominal fat and to determine the necessary short-term adaptations in diabetes management during intensive endurance training in patients with IDDM. RESEARCH DESIGN AND METHODS Well-controlled subjects with IDDM (n = 20; HbA1c = 7.6%) engaged in a regular exercise program over a period of 3 months involving endurance sports such as biking, long-distance running, or hiking. Subjects were instructed to exercise at least 135 min per week. If baseline activity exceeded this level, subjects were to increase further their physical activity as much as possible and record the type and time of such activity. RESULTS During the 3-month intervention, physical activity increased from 195 ± 176 to 356 ± 164 min (mean ± SD) per week (P < 0.001). Physical fitness as assessed by VO2max increased from 2,914 ± 924 to 3,092 ± 905 ml/min (P < 0.001), and insulin sensitivity increased significantly (steady-state plasma glucose [SSPG] decreased from 10.5 ± 4.8 to 7.0 ± 3.3 mmol/l; P < 0.01). Subsequently, LDL cholesterol decreased by 14% (P < 0.05), and HDL and HDL3-C subfraction increased by 10 (P < 0.05) and 16% (P < 0.05), respectively. Systolic and diastolic blood pressure decreased significantly from 127 ± 9 to 124 ± 8 (P < 0.05) and from 80 ± 5 to 77 ± 5 mmHg (P < 0.01), respectively. Resting heart rate decreased from 63 ± 6 to 59 ± 7 bpm (P < 0.01). Waist-to-hip circumference ratio decreased from 0.882 ± 0.055 to 0.858 ± 0.053 (P < 0.001), body weight decreased from 70.7 ± 10.4 to 68.7 ± 10.2 kg (P = 0.003), with a consequent decrease in body fat from 21.9 ± 8.2 to 18.0 ± 6.3% (P < 0.001) and an increase in lean body mass from 54.9 ± 12.2 to 56.8 ± 11.0 kg. These effects occurred independently of glycemic control. The overall frequency of severe hypoglycemic episodes was reduced from 0.14 to 0.10 per patient-year during the study period. CONCLUSIONS This study shows that increasing physical activity is safe and does not result in more hypoglycemic episodes and that there is a linear dose-response between increased physical activity and loss of abdominal fat and a decrease in blood pressure and lipid-related cardiovascular risk factors, with a preferential increase in the HDL3-C subfraction.

FLIP switches Fas-mediated glucose signaling in human pancreatic β cells from apoptosis to cell replication

Type 2 diabetes mellitus results from an inadequate adaptation of the functional pancreatic β cell mass in the face of insulin resistance. Changes in the concentration of glucose play an essential role in the regulation of β cell turnover. In human islets, elevated glucose concentrations impair β cell proliferation and induce β cell apoptosis via up-regulation of the Fas receptor. Recently, it has been shown that the caspase-8 inhibitor FLIP may divert Fas-mediated death signals into those for cell proliferation in lymphatic cells. We observed expression of FLIP in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro exposure of islets from nondiabetic organ donors to high glucose levels decreased FLIP expression and increased the percentage of apoptotic terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL)-positive β cells; FLIP was no longer detectable in such TUNEL-positive β cells. Up-regulation of FLIP, by incubation with transforming growth factor β or by transfection with an expression vector coding for FLIP, protected β cells from glucose-induced apoptosis, restored β cell proliferation, and improved β cell function. The beneficial effects of FLIP overexpression were blocked by an antagonistic anti-Fas antibody, indicating their dependence on Fas receptor activation. The present data provide evidence for expression of FLIP in the human β cell and suggest a novel approach to prevent and treat diabetes by switching Fas signaling from apoptosis to proliferation.

Central necrosis in isolated hypoxic human pancreatic islets: evidence for postisolation ischemia.

A variety of explanations have been provided to elucidate the requirement of the large islet mass that is essential for a successful treatment of patients with type I diabetes by intrahepatic transplantation. The purpose of this study was to investigate islet cell survival under the effect of prolonged hypoxia and/or nutrient withdrawal, which mimics posttransplantation environment of transplanted islets in the liver. We studied the influence of 24 h of hypoxia (1% O2) in intact isolated human and rat islets as well as the effect of combined oxygen/nutrient deprivation in a mouse insulinoma cell line (MIN6). In intact human islets, 24 h of hypoxia led to central necrosis combined with apoptotic features such as nuclear pyknosis and DNA fragmentation. In the course of hypoxic treatment, ultrastructural analysis demonstrated a gradual transition from an apoptotic to a necrotic morphology particularly pronounced in central areas of large islets. In MIN6 cells, on the other hand, hypoxia led to a twofold (p < 0.01) increase in caspase-3 activity, an indicator of apoptosis, but not to necrosis, as determined by release of lactate dehydrogenase (LDH). Only in combination with nutrient/serum deprivation was a marked increase in LDH release observed (sixfold vs. control, p < 0.01). We therefore conclude that, similar to MIN6 cells, central necrosis in isolated hypoxic islets is the result of the combined effects of hypoxia and nutrient/serum deprivation, most likely due to limited diffusion. Provided that transplanted islets undergo a similar fate as shown in our in vitro study, future emphasis will require the development of strategies that protect the islet graft from early cell death and accelerate the revascularization process.

Using fasting plasma glucose concentrations to screen for gestational diabetes mellitus: prospective population based study

Abstract Objective: To evaluate whether measuring fasting plasma glucose concentration is an easier screening procedure for gestational diabetes mellitus than the 1 hour 50 g glucose challenge test. Design: Prospective population based study. Setting: Outpatient clinic in a university hospital. Participants: 520 pregnant women (328 (63%) white, 99 (19%) Asian, 31 (6%) African, 62 (12%) others) with mean age 28.4 (SD 0.2; range 17-45) years. All underwent a glucose challenge test between the 24th and 28th gestational week, followed by a diagnostic 3 hour 100 g oral glucose tolerance test within one week. This was done irrespective of the result of the challenge test. Main outcome measure: Receiver operating curves were used to determine the best cut off values for screening with fasting plasma glucose concentrations. Results: Fasting plasma glucose concentration at a threshold value of 4.8 mmol/l and the glucose challenge test with a threshold value of 7.8 mmol/l yielded sensitivities of 81% and 59% respectively and specificities of 76% and 91% respectively. Measuring fasting plasma glucose concentration as a screening procedure required a diagnostic test in 30%, compared with 14% when the challenge test was used. Conclusions: Measuring fasting plasma glucose concentrations using a cut off value of ≥4.8 mmol/l is an easier screening procedure for gestational diabetes than the 50 g glucose challenge test and allows 70% of women to avoid the challenge test. Key messages Identifying women susceptible to gestational diabetes is particularly important not only to prevent perinatal morbidity but also to improve long term outcomes for the mother and her child Measuring fasting plasma glucose concentration is an easier screening procedure for gestational diabetes than the 1 hour 50 g glucose challenge test With a threshold value of 4.8 mmol/l it yields a sensitivity of 81% and a specificity of 76% Measuring fasting plasma glucose concentration allows 70% of women to avoid a glucose challenge test

Is the ADA/EASD algorithm for the management of type 2 diabetes (January 2009) based on evidence or opinion? A critical analysis

The ADA and the EASD recently published a consensus statement for the medical management of hyperglycaemia in patients with type 2 diabetes. The authors advocate initial treatment with metformin monotherapy and lifestyle modification, followed by addition of basal insulin or a sulfonylurea if glycaemic goals are not met (tier 1 recommendations). All other glucose-lowering therapies are relegated to a secondary (tier 2) status and only recommended for selected clinical settings. In our view, this algorithm does not offer physicians and patients the appropriate selection of options to individualise and optimise care with a view to sustained control of blood glucose and reduction both of diabetes complications and cardiovascular risk. This paper critically assesses the basis of the ADA/EASD algorithm and the resulting tiers of treatment options.

The effects of maintenance doses of FK506 versus cyclosporin A on glucose and lipid metabolism after orthotopic liver transplantation

BACKGROUND Posttransplant diabetes mellitus (PTDM) has gained widespread attention due to the micro and macro-vascular complications that increase the morbidity and mortality of patients receiving solid organs. The higher incidence of PTDM has been mainly attributed to the immunosuppressive therapy. Therefore, this study compares the metabolic side effects of low dose maintenance therapy of FK-506 and Cyclosporin A (CsA) in 14 patients 1 year after orthotopic liver transplant and analyzes possible factors that contribute to the development of PTDM. METHODS Two groups (n=7) differing in their immunosuppressive regimen (FK506 or CsA) were matched to eight control subjects and compared to each other. The effects of in vivo insulin action were assessed by means of the euglycemic hyperinsulinemic clamp technique. Arginine stimulation tests at normo- (5.5 mM) and hyperglycemic (15 mM) levels were performed and the acute insulin, C-peptide, and glucagon response (2-5 min) to arginine were determined. RESULTS Insulin sensitivity (total glucose disposal) was statistically lower in patients treated with FK-506 and CsA (5.05+/-0.47 and 5.05+/-0.42 mg/kg/min) as compared to controls (6.62+/-0.38 mg/kg/min) (P<0.02), with a significantly higher nonoxidative glucose disposal for the control group (P<0.01), and lower free fatty acid levels (P<0.05). Absolute values for acute insulin response were higher but not significantly different for the transplanted groups. The lower percentage of increase of insulin release after arginine stimulation observed in the FK-506 and CsA groups as compared with controls (754%+/-100, 644%+/-102 vs. 1191%+/-174) (P<0.03 and 0.02, respectively), suggests a reduced beta cell secretory reserve in both treated groups. Also, the acute glucagon response to arginine during hyperglycemia declined less in the FK-506 (28%) and CsA groups (29%) compared with controls (48%) (P<0.05) indicating a defect in the pancreatic beta cell-alpha cell axis. CONCLUSIONS There are no major metabolic differences on low maintenance doses between FK-506 and CsA. Both immunosuppressant agents contribute to the development of PTDM at different levels.