Effects of cryopreservation on in vitro and in vivo long-term function of human islets.

BACKGROUND: The possibility of performing transplantation several days after explant seems to be a peculiarity of islet grafts, and the opportunity to cryopreserve human islets may permit an indefinite period for modulating the recipient immune system. The aim of the present study was the evaluation of in vitro and in vivo functional properties of cryopreserved human islets. METHODS: We used six consecutive human islet preparations not suitable for an immediate transplantation in diabetic patients because the limited islet mass separated. The in vitro function of cryo and fresh islets was studied by determination of insulin and glucagon secretion in response to such classical stimuli as glucose (16.7 mM), glucose (16.7 mM) + 3-isobutyl-1-methylxanthine (0.1 mM), arginine (10 mM), and tolbutamide (100 microM). In vivo islet function was assessed through intravenous glucose tolerance tests performed at 15, 30, 60, and 90 days after transplantation of 1000 hand-picked fresh or cryopreserved islets in nude mice. RESULTS: Basal secretion of true insulin was significantly higher in cryopreserved islets than in fresh ones. The response of cryopreserved islets to arginine and glucose + isobutyl-1-methylxanthine seemed partially impaired. Proinsulin-like molecule secretion seemed higher in cryopreserved than in fresh islets in response to all secretagogues used, and the difference was statistically significant for arginine. The capacity of human cryopreserved islets to maintain a correct metabolic control in diabetic nude mice was progressively lost in 3 months. CONCLUSIONS: These findings showed that cryopreservation affects the function of isolated human islets, maintaining in vivo function for a limited period of time.     

Mitochondrial reactive oxygen species are required for hypothalamic glucose sensing

The physiological signaling mechanisms that link glucose sensing to the electrical activity in metabolism-regulating hypothalamus are still controversial. Although ATP production was considered the main metabolic signal, recent studies show that the glucose-stimulated signaling in neurons is not totally dependent on this production. Here, we examined whether mitochondrial reactive oxygen species (mROS), which are physiologically generated depending on glucose metabolism, may act as physiological sensors to monitor the glucose-sensing response. Transient increase from 5 to 20 mmol/l glucose stimulates reactive oxygen species (ROS) generation on hypothalamic slices ex vivo, which is reversed by adding antioxidants, suggesting that hypothalamic cells generate ROS to rapidly increase glucose level. Furthermore, in vivo, data demonstrate that both the glucose-induced increased neuronal activity in arcuate nucleus and the subsequent nervous-mediated insulin release might be mimicked by the mitochondrial complex blockers antimycin and rotenone, which generate mROS. Adding antioxidants such as trolox and catalase or the uncoupler carbonyl cyanide m-chlorophenylhydrazone in order to lower mROS during glucose stimulation completely reverses both parameters. In conclusion, the results presented here clearly show that the brain glucose-sensing mechanism involved mROS signaling. We propose that this mROS production plays a key role in brain metabolic signaling.     

Mechanical stress and ATP synthesis are coupled by mitochondrial oxidants in articular cartilage

Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5 Hz with amplitude of 0.25 MPa for 1 h. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase‐based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 191–196, 2013     

Evidence of functional impairment of syngeneically transplanted mouse pancreatic islets retrieved from the liver

A drawback in pancreatic islet transplantation is the large number of islets needed to obtain insulin independence in patients with diabetes. This most likely reflects extensive posttransplantation islet cell death and functional impairment of the remaining endocrine cells. We aimed to develop an experimental method to retrieve transplanted islets from the mouse liver, which would enable comparisons of transplanted and endogenous islets and provide valuable information on functional changes induced by intraportal transplantation. Transplanted islets were obtained by retrograde perfusion of the liver with collagenase. The identity of retrieved tissue as transplanted islets was confirmed by intravital staining, immunohistochemistry, and electron microscopy. The retrieved islets, irrespective of whether they had resided in diabetic or nondiabetic recipients, had a markedly lower insulin content and glucose-stimulated insulin release when compared with isolated endogenous islets. The glucose oxidation rate was also markedly lower in the retrieved islets, suggesting mitochondrial dysfunction. These disturbances in insulin content, insulin release, and glucose oxidation rate were not reversed by a few days of culture after retrieval. The results implicate changes in islet function after intraportal transplantation. Such dysfunction may contribute to the high number of islets needed for successful transplantation in diabetic individuals.     

Antioxidant effects of the essential oil of Syzygium aromaticum on bovine epididymal spermatozoa

Focusing on its application in reproductive biotechnology, we evaluated the effects of the essential oil of Syzygium aromaticum (EOSA) on bovine epididymal sperm quality variables, including morphology, membrane functional integrity, membrane structural integrity, mitochondrial activity, metabolic activity, motility and oxidative stress by reactive oxygen species (ROS) levels. Bovine spermatozoa from eight males were incubated into the following groups: EOSA0 (without EOSA), EOSA10 (10 μg/ml of EOSA), EOSA15 (15 μg/ml of EOSA) and EOSA20 (20 μg/ml of EOSA); the incubation time with and without the EOSA was 1 or 6 hr. None of the sperm quality variables presented difference among the EOSA concentrations. However, the incubation time had a significant effect on the membrane functional integrity, membrane structural integrity, mitochondrial activity, progressive motility and some kinetic parameters. The effect of interaction among EOSA and incubation time was significant only on ROS levels. Spermatozoa incubated in the presence of 15 μg/ml of the EOSA for 1 hr had significantly reduced ROS levels compared with all other groups in the same time. In conclusion, the EOSA at a concentration of 15 µg/ml has antioxidant effects and protects bovine epididymal spermatozoa; hence, the EOSA may potentially be used in the field of reproductive biotechnology.     

Quantitative in vivo islet potency assay in normoglycemic nude mice correlates with primary graft function after clinical transplantation

Reliable assays are critically needed to monitor graft potency in islet transplantation (IT). We tested a quantitative in vivo islet potency assay (QIVIPA) based on human C-peptide (hCP) measurements in normoglycemic nude mice after IT under the kidney capsule. QIVIPA was initially tested by transplanting incremental doses of human islets. hCP levels in mice were correlated with the number of transplanted islet equivalents (r(2) = 0.6, P<0.01). We subsequently evaluated QIVIPA in eight islet preparations transplanted in type 1 diabetic patients. Conversely to standard criteria including islet mass, viability, purity, adenosine triphosphate content, or glucose stimulated insulin secretion, hCP in mice receiving 1% of the final islet product was correlated to primary graft function (hCP increase) after IT (r(2)=0.85, P<0.01). QIVIPA appears as a reliable test to monitor islet graft potency, applicable to validate new methods to produce primary islets or other human insulin secreting cells.     

OP-142 gene expression profile of nonfunctional human pancreatic islets: predictors of transplant failure?

Islet culture has become a standard part of most successful protocols for clinical islet transplantation. To date, however, islets are transplanted based on crude measures of viability, purity and in vitro insulin production without adequate prior assessment of the potential for in vivo function. The purpose of this study was to define the gene expression profiles of human islets associated with in vivo function using a nonimmune NOD-scid mouse model. Human islets from eight isolations were maintained in culture for 7 to 14 days in Memphis serum-free media until transplanted. The RNA was extracted from 10,000 IEQ using RNASTAT-60. The gene expression profiles were analyzed using high-density Affymetrix U133A GeneChips and Genespring software. An aliquot of 2000 IEQ from each islet preparation was also transplanted into NOD-scid animals (n = 5) for in vivo function assessments. Islet function was assessed by measurements of human C-peptide at days 7 and 14 posttransplant. Human C-peptide levels were determined by radioimmunoassay. Gene analysis of nonfunction islets (4 of 8 islet preparations) showed high relative levels of expression of proinflammatory genes and low relative levels of genes directed toward insulin processing and secretion as well as islet integrity. Overexpression of hypoxia and proinflammatory genes may result in reduced insulin secretion and lead to islet destruction posttransplantation. Identifying and validating those genes could allow the development of a potency assay for human transplantation that would be very useful for screening human islet preparations before clinical transplant.     

Adenovirus-mediated expression of the anticoagulant hirudin in human islets: a tool to make the islets biocompatible to blood

Human islets induce an injurious clotting reaction at the time of transplantation. A potential strategy to counteract this reaction would be to allow the islets to express hirudin, a protein with direct anticoagulative activity. Human islets were transduced with an adenoviral vector encoding hirudin, an empty corresponding vector, or left untreated. Islet culture supernatants were analyzed for hirudin using an ELISA, a chromogenic substrate assay based on the thrombin-binding properties of hirudin and in a whole blood viscosimetry assay. Immunohistochemical evaluation and determination of hirudin content revealed an abundant expression of hirudin after transduction. Hirudin content in transduced islets was in the range of the insulin content levels. A delay in human whole blood clotting time could be observed after addition of supernatants taken from islet cultures expressing hirudin. However, transduced islets showed an impaired glucose-stimulated insulin release, but could readily be retrieved 6 weeks after transplantation to athymic mice. A marked expression and secretion of hirudin with functional capacity can be induced in human islets using an adenoviral vector. The impairment in glucose-stimulated insulin release in hirudin-secreting islets, compared to controls, indicates that the additional protein synthesis affects the functional capacity of the islets.     

Survival of Microencapsulated Adult Pig Islets in Mice In Spite of an Antibody Response

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.     

The use of the BD oxygen biosensor system to assess isolated human islets of langerhans: oxygen consumption as a potential measure of islet potency

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding ("dO2 slope"). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized "dO2 slope" in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.     

Influence of a current style of culture on the quality of isolated pancreatic islets

OBJECTIVE: Comparable outcomes of islet transplantation with short periods of culture may be achieved with various culture media. To clarify the influence of a style of culture on isolated pancreatic islets, islet quality of fresh islets was compared with those cultured in several different fashions including not only for viability but also for inflammatory mediators. MATERIALS AND METHODS: Wistar rat islets were cultured for 48 hours with CMRL including 10% allogeneic serum; CMRL including 0.5% human serum albumin (HSA); and Miami medium including 0.5% HSA. The influence of culture conditions on islet integrity was evaluated by survival rate of islets during culture and visual scoring. The influence of culture conditions on islet function and viability was examined by ADP/ATP tests, insulin/DNA content, and glucose stimulation tests. RESULTS: Although the survival rates were similar for all groups, the visual scoring was lower in Miami medium. The stimulation index in glucose challenge tests was higher for fresh islets than the media (P = .02). Insulin/DNA ratios revealed the same tendency as glucose challenge tests (P = .0005). ADP/ATP ratio was lower in both the fresh and serum groups than in the others (P = .38), suggesting that apoptotic islets are relatively fewer in both fresh and serum groups. Most importantly, the expression of tissue factor (TF) on the islets was considerably lower in the fresh group, suggesting that a current style of culture could enhance TF-dependent instant blood-mediated inflammatory reactions after transplantation. CONCLUSION: In conclusion, Isolated islets without prior culture shows characteristics beneficial for transplantation using current modes of culture.     

Polymyxin B,scavenger of endotoxin,enhances isolation yield and in vivo function of islets

Collagenase purified from bacteria has been used to isolate islets for transplantation. However, collagenase is contaminated with small amounts of endotoxin, which induces dysfunction or apoptosis of islets. In this study, we investigated the effects of polymyxin B, endotoxin scavenger, on the yield and quality of isolated islets. It is revealed that polymyxin B neutralized endotoxin in vitro and inhibited endotoxin‐mediated decreases of the glucose stimulation index. Additionally, adenosine triphosphate (ATP) quantitation, islet regression assay, and caspase‐3 activation assay demonstrated that polymyxin B efficiently blocked the toxic effects induced by endotoxin. Thereafter, we isolated mouse islets both with and without polymyxin B and compared total islet equivalents (IEQs), glucose‐stimulated insulin release, and ATP content. Polymyxin B enhanced islet recovery, and ATP content of islets, and glucose stimulation index, and reduced TNF‐α expression of islets. Marginal transplantation (200 IEQs/mouse) under the kidney capsule of diabetic mice induced normoglycemia in 30% of the polymyxin B group, but not in any mouse of control group. This result suggests that islets isolated with polymyxin B more effectively lower blood glucose levels as compared with control islets. Thus, polymyxin B could serve as a useful agent in the protection of islets from endotoxin‐induced inflammation and apoptosis.     

Beneficial Effects of Desferrioxamine on Encapsulated Human Islets—In Vitro and In Vivo Study

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 μM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose‐induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF‐1α (Hypoxia inducible factor‐1 alpha), caspase‐3 and caspase‐8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase‐3 and caspase‐8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6‐ and 2.5‐fold at 24 and 72 h, respectively, with a concomitant increase in HIF‐1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF‐1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.     

In vivo bioluminescence imaging of transplanted islets and early detection of graft rejection

BACKGROUND: Bioluminescence imaging (BLI) modalities are being developed to monitor islet transplant mass and function in vivo. The aim of this study was to use the BLI system to determine how the change in functional islet mass correlated to metabolic abnormalities during the course of alloimmune rejection in a murine transplant model. METHODS: Islets obtained from a transgenic mouse strain (FVB/NJ-luc) that constitutively expressed firefly luciferase were transplanted to various implantation sites of syngeneic wild-type FVB/NJ or allogeneic Balb/C streptozotocin-induced diabetic recipients. In vivo graft luminescent signals were repeatedly measured after transplantation using the BLI system and related to blood glucose levels and graft site histologic findings. RESULTS: The BLI signals were detected in as few as 10 islets implanted in the renal subcapsular space, intrahepatic, intraabdominal, and subcutaneous locations. There was a linear relationship between the number of islets transplanted and luminescence intensity. In isografts, stable luminescence intensity signals occurred within 2 weeks of transplantation and remained consistent on a long-term basis (18 months) after transplantation. In allografts, after normoglycemia was achieved and stable luminescence intensity occurred, graft bioluminescent intensity progressively decreased several days before permanent recurrence of hyperglycemia as a result of histologically proven rejection ensued. CONCLUSIONS: Bioluminescence imaging is a sensitive method for tracking the fate of islets after transplantation and is a useful method to detect early loss of functional islet mass caused by host immune responses even before overt metabolic dysfunction is evident. Bioluminescence imaging holds promise for use in designing and testing interventions to prolong islet graft survival.     

Fresh islets are more effective for islet transplantation than cultured islets

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.     

A significant role for histocompatibility in human islet transplantation

BACKGROUND: In recent years, transplantation of islets and pancreas has become a viable option for patients debilitated with type I diabetes. The success of islet transplantation has been attributed to the ability to isolate high quality islets for transplantation and capacity to maintain the recipient's immunosuppressive levels within a specific target range following transplantation. The purpose of this study was to determine the role of pretransplant sensitization to human leukocyte antigen (HLA) in islet transplantation. METHODS: We retrospectively analyzed seven patients that were transplanted with islets under the auspices of the Juvenile Diabetes Research Foundation and Islet Cell Resource Center/National Institutes of Health. Humoral sensitization towards donor antigens both prior to and following islet transplantation was detected by FLOW panel reactive antibodies (PRA) and donor-specific cellular sensitization was detected by performing enzyme-linked immunospot assay analysis for cytokines interferon-gamma and interleukin-2. RESULTS: Our analysis demonstrates that humoral and cellular sensitization to histocompatibility antigens prior to and after islet transplantation are associated with the failure of transplanted islets CONCLUSION: Patient selection based on sensitization to donor HLA may be one of the factors crucial for the success of islet transplant. Further, in some patients, rejection of islets can be associated with sensitization to mismatched donor histocompatibility antigens.     

Mitochondrial Reactive Oxygen Species Are Obligatory Signals for Glucose-Induced Insulin Secretion

OBJECTIVE—Insulin secretion involves complex events in which the mitochondria play a pivotal role in the generation of signals that couple glucose detection to insulin secretion. Studies on the mitochondrial generation of reactive oxygen species (ROS) generally focus on chronic nutrient exposure. Here, we investigate whether transient mitochondrial ROS production linked to glucose-induced increased respiration might act as a signal for monitoring insulin secretion.RESEARCH DESIGN AND METHODS—ROS production in response to glucose was investigated in freshly isolated rat islets. ROS effects were studied using a pharmacological approach and calcium imaging.RESULTS—Transient glucose increase from 5.5 to 16.7 mmol/l stimulated ROS generation, which was reversed by antioxidants. Insulin secretion was dose dependently blunted by antioxidants and highly correlated with ROS levels. The incapacity of β-cells to secrete insulin in response to glucose with antioxidants was associated with a decrease in ROS production and in contrast to the maintenance of high levels of ATP and NADH. Then, we investigated the mitochondrial origin of ROS (mROS) as the triggering signal. Insulin release was mimicked by the mitochondrial-complex blockers, antimycin and rotenone, that generate mROS. The adding of antioxidants to mitochondrial blockers or to glucose was used to lower mROS reversed insulin secretion. Finally, calcium imaging on perifused islets using glucose stimulation or mitochondrial blockers revealed that calcium mobilization was completely reversed using the antioxidant trolox and that it was of extracellular origin. No toxic effects were present using these pharmacological approaches.CONCLUSIONS—Altogether, these complementary results demonstrate that mROS production is a necessary stimulus for glucose-induced insulin secretion.Elucidating the mechanisms by which pancreatic β-cells couple glucose sensing to insulin secretion, a vital process in energy homeostasis, is of prime importance. Although ATP production is considered the main mitochondrial signal, detailed studies show that insulin secretion cannot be restricted to ATP synthesis, and numerous experimental clues show that additional mitochondrial factors involved in glucose-secretion coupling are necessary, although not yet identified (1).Transient increases in glucose metabolism generate NADH and FADH₂, leading rapidly to increased superoxide anion (O₂^·) production; obligatorily associated with the respiratory chain function, superoxide anion will be converted into H₂O₂ (2). This production of mitochondrial reactive oxygen species (mROS)—transient because H₂O₂-inactivating enzymes rapidly quench it before a damage to the physiological conditions of the cell occurs—is now recognized as an intracellular messenger (3,4). These features make mROS a good candidate for rapidly regulating pathways that depend directly on metabolic fluxes. Based on such a view, we recently demonstrated that mROS production is required for hypothalamic glucose and lipid sensing (5,6). These results lead us to speculate that O₂^· might operate more generally in nutrient-sensitive cells and also to look for the role of mROS as a signal involved in glucose-stimulated insulin secretion (GSIS). Recently, a study revealed that H₂O₂ is effectively a signal of GSIS (7). Here, we provide clues that glucose-induced mitochondrial O₂^· production is an obligatory stimulus for insulin secretion.     

Content of CoA-esters in perifused rat islets stimulated by glucose and other fuels

Fluorometry and high-performance liquid chromatography were used to measure the content of free CoA and the esters of acetate, malonate, succinate, and long-chain fatty acids in isolated perifused rat pancreatic islets exposed to 25 mM glucose or a mixture of fuels (25 mM glucose plus 10 mM glutamine, 10 mM lactate, and 1 mM pyruvate) to assess the role of intermediates of lipid metabolism as candidate metabolic coupling factors in the mechanism of fuel-induced insulin secretion. Insulin secretion was stimulated in a biphasic manner with the fuel mixture, showing twice the potency compared with high glucose alone. Islets perifused for 3 min with high glucose alone or the fuel mixture compared with 2.5 mM glucose showed a significant increase in malonyl-CoA and succinyl-CoA and a decrease in acetyl-CoA. Free CoA and long-chain acyl-CoA levels were unaltered. Perifused islets stimulated with 25 mM glucose for 30 min showed a significant increase in succinyl-CoA and long-chain acyl-CoA and decrease in acetyl-CoA, whereas malonyl-CoA was not affected. However, when islets were stimulated by the fuel mixture for 30 min, malonyl-CoA was maintained at a high level, and the change in succinyl-CoA and long-chain acyl-CoA was similar to that observed in islets stimulated with 25 mM glucose alone. The acetyl-CoA concentration in the islets stimulated with the fuel mixture decreased slightly. These results confirm the viability of the hypothesis that malonyl-CoA and long-chain acyl-CoA serve as metabolic coupling factors in signal transduction when islets are stimulated by high glucose or glucose combined with other fuels.     

Progressive deterioration of endocrine function after intraportal but not kidney subcapsular rat islet transplantation

In inbred streptozocin-induced diabetic rats, the long-term function of different endocrine pancreatic isografts was compared. Isolated islets transplanted into the portal vein showed a progressive deterioration of function over time. In contrast, islets under the kidney capsule sustained a constant long-term function controlling all clinical signs of diabetes. Recipients of kidney subcapsular islets displayed normal growth rate, peripheral serum glucose and insulin levels, and metabolic parameters. However, their functional reserve was markedly reduced as revealed by diminished glucose tolerance and reduced insulin-secreting capacity after an intravenous glucose challenge. Vascularized whole-organ pancreatic grafts with portal venous drainage led to complete normalization of all parameters determined in this study. This study showed that the long-term function of islets transplanted under the kidney capsule is superior compared with islets transplanted into the portal vein.     

Integrity of mitochondrial membrane potential reflects human sperm quality

The aim of this work was to evaluate intracellular reactive oxygen species (ROS) levels, phosphatidylserine (PS) externalisation and mitochondrial membrane potential integrity in the spermatozoa of healthy donors and outpatients who consulted for infertility and to correlate the results with the classic sperm parameters. For the evaluation of intracellular ROS levels, PS externalisation and mitochondrial membrane potential integrity, the fluorescent compounds dihydroethidium, annexin V-FITC and JC-1, respectively, were used and analysed by using flow cytometry. Conventional seminal analysis, including motility, viability, morphology, sperm count and volume, was performed according to the WHO criteria. The mitochondrial membrane potential and ROS results showed significant differences between the spermatozoa of individuals with a normal semen analysis and those of the group presenting abnormality in at least one of the sperm parameters. Mitochondrial membrane potential showed a significant and direct correlation with all the sperm parameters analysed. ROS were inversely correlated with motility, viability and morphology. PS externalisation, however, did not show any differences between the two groups, nor was it correlated with the sperm parameters examined. The evaluation of mitochondrial membrane potential integrity is a test that reflects sperm quality, which makes it highly recommendable to be applied as a complement to routine sperm analyses.