Effect of dietary fat on diabetes-induced changes in liver microsomal fatty acid composition and glucose-6-phosphatase activity in rats

Experimental diabetes may manifest itself in a defect in liver microsomal fatty acid desaturation and increased activity of glucose-6-phosphatase (G-6-Pase). The present study was designed to determine whether these changes could be normalized by a change in the dietary fat consumed. Control and streptozotocin-induced diabetic rats were fed nutritionally adequate diets which varied in fatty acid composition. Fatty acid analysis of liver microsomal phospholipids revealed that non-diabetic control animals fed saturated fat (beef tallow) or a diet high in ω3 fatty acids (fish oil) exhibited a significantly higher level of 18∶2ω6 and a lower level of 20∶4ω6 in the phosphatidylcholine and phosphatidylethanolamine fractions compared with diabetic animals. Control and diabetic animals fed the high linoleic acid diet had similar levels of 18∶2ω6 in the microsomal phosphatidylcholine and phosphatidylserine fractions. Microsomal G-6-Pase activity was higher in diabetic than in control animals. Activity of G-6-Pase was lower in microsomes of control animals fed the soybean oil or the fish oil diet, but was not significantly reduced in diabetic animals fed high polyunsaturated fats. Blood glucose levels were similar in control groups fed the different diets, but the plasma hemoglobin A1c level was lower in diabetic animals fed the soybean oil diet. Cholesterol and triglyceride levels were lower in diabetic animals fed the fish oil-based diet. The results suggest that dietary fat manipulation has the potential to change at least some of the abnormalities in the microsomal membrane in experimental diabetes.     

Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression

We prepared single-cell suspensions of Lewis rat ?RT1(1/l)? testicular cells and cultured these in vitro for 48 h under conditions that promoted the formation of cellular aggregates. In the absence of systemic immunosuppression, the transplantation of a sufficient quantity of these aggregates (containing 11 x 10(6) cells, (75% Sertoli cells), together with 2,000 purified Lewis rat islets, reversed the diabetic state for >95 days in 100% (5/5) of the chemically diabetic Wistar-Furth ?RT1(u/u)? recipients. Similar grafts consisting of islets alone or islets plus 50% fewer testicular cell aggregates survived for only 10 days. Functioning composite allografts harvested from normoglycemic animals at approximately 100 days showed healthy beta-cells in close association with Fas ligand-expressing Sertoli cells. Because no gene therapy protocol is required, the transplantation of composite grafts consisting of purified human allogeneic islets plus human allogeneic testicular cell aggregates can be applied in clinical islet transplantation as soon as it has been proven in a large animal model.     

Interleukin-4 or interleukin-10 expressed from adenovirus-transduced syngeneic islet grafts fails to prevent beta cell destruction in diabetic NOD mice

BACKGROUND: We performed ex vivo adenoviral gene transfer in a mouse pancreatic islet transplant model to test the efficacy of this expression system. We then determined whether adenoviral-mediated expression of mouse interleukin (IL) 4 or IL-10 from transduced syngeneic islet grafts could prevent disease recurrence in diabetic nonobese diabetic (NOD) mice. METHODS: An adenoviral vector expressing beta-galactosidase (AdCMV betaGal) was used to transduce BALB/c islets (2.5 x 10(3) plaque-forming units/islet), which were analyzed for glucose responsiveness, islet cell recovery, and efficiency of gene transfer. In vivo function and reporter gene expression were examined with AdCMV betaGal-transduced islet grafts in alloxan-induced diabetic syngeneic recipients. Adenoviruses expressing either IL-4 or IL-10 were used in a similar fashion to infect NOD islets, which were characterized in vitro, as well as transplanted into diabetic syngeneic recipients. RESULTS: In vitro functional studies showed no significant difference between control or transduced islets, with 50+/-4% of AdCMV betaGal-infected islet cells staining positive for beta-galactosidase. Transplant recipients became nomoglycemic within 48 hr after transplant, and, although beta-galactosidase expression decreased over time, it was detectable in the graft for up to 8 weeks. Despite the nanogram quantities of IL-4 or IL-10 produced/day from each graft equivalent in vitro, transduced and transplanted NOD islets failed to prevent disease recurrence. CONCLUSIONS: These results suggest that adenoviruses are efficient for at least medium term gene expression from islets in vivo, but neither IL-4 nor IL-10 alone can prevent autoimmune disease recurrence in NOD mice.     

Cryopreservation of rat pancreatic islets: effect of ethylene glycol on islet function and cellular composition

BACKGROUND: Inasmuch as cryopreservation can facilitate clinical islet transplantation by providing a means of storing supplemental islets in order to augment marginally adequate grafts, protocols are needed to allow for a minimal loss in viable beta cells. By replacing the cryoprotectant dimethyl sulfoxide (DMSO) with ethylene glycol (EG), a more simplified cryopreservation protocol was developed, which resulted in improved survival and function of rat pancreatic islets. METHODS: Nonfrozen islets, islets cryopreserved in DMSO, and EG-cryopreserved islets were compared for percent recovery, cellular composition, in vitro viability, and metabolic function after transplantation. RESULTS: After cryopreservation in DMSO or EG, islet yield was similar to that of nonfrozen controls; however, islets cryopreserved in DMSO exhibited lower cellular DNA, insulin, and glucagon content, as well as an impaired insulin secretory capacity in vitro than the nonfrozen controls. When compared with controls, islets cryopreserved in DMSO contained a higher proportion of beta cells but a lower number of glucagon-positive cells, whereas cryopreservation with EG resulted in similar DNA/hormone contents, in vitro viability, and cellular composition. Transplantation of islet grafts composed of comparable numbers of beta cells (2.1-2.3 million) corrected diabetes in 100% (6/6; nonfrozen controls), 92% (10/11; DMSO), and 100% (14/14; EG) of the recipients; however, those who received DMSO-treated islets took longer to achieve euglycemia and remained glucose-intolerant. CONCLUSIONS: These results demonstrate that EG allows for the successful cryopreservation of rat islet beta and a cells with the same yield and quality as nonfrozen islets. The observation that alpha-cell survival was better after cryopreservation with EG may explain the improved functional viability of these grafts. Further studies are needed to assess whether this protocol provides any advantage for cryopreserving large numbers of human islets.     

Endogenous immune response to glutamic acid decarboxylase (GAD67) in NOD mice is modulated by adjuvant immunotherapy

We have shown that immunization of non-obese diabetic (NOD) mice with adjuvants (CFA or BCG) prevents the onset of diabetes by induction of regulatory cells. Since autoimmune responses to glutamic acid decarboxylase (GAD) are up-regulated in insulin-dependent diabetes mellitus (IDDM), in this study GAD67-specific antibody, T cell proliferation and lymphokine production patterns were analysed in the adjuvant-treated mice to characterize the regulatory mechanisms underlying the protection. We used both spontaneous diabetes and syngeneic islet transplantation models in NOD mice. Protection against spontaneous diabetes and prevention of syngeneic islet graft rejection by CFA or BCG treatment was found to be accompanied by the production of long lasting and high titre anti-GAD67 antibody of IgG1 isotype in the sera. Upon in vitro stimulation with GAD67, draining lymph node and spleen cells from CFA-immunized NOD mice or syngeneic islet-grafted and BCG-protected NOD mice produced much more IL-4, whereas there was no significant change in IFN-gamma production. The strong early T cell proliferative response to GAD67 in CFA or BCG-immunized NOD mice was followed by a low or unresponsiveness state. Taken together, these results suggest a shift in Th1/Th2 balance in the GAD67-specific endogenous immune response to a change in Th2 levels after adjuvant treatment. We postulate that the protective effect of CFA or BCG is due to the diversion of GAD-specific endogenous cellular immune response to a non-pathogenic humoral response.     

Portal vein insulin flux and arterial glucose fluctuations in response to an oral meal test in islet cell-transplanted dogs

Insulin flux was determined in the portal vein and simultaneously arterial blood glucose was measured before and during an oral glucose meal in conscious normal and pancreatic islet cell-autotransplanted dogs to test their insulinogenic reserve. These dogs had previously been chronically instrumented with blood flow probes on the portal vein and carotid artery, and blood sampling catheters in the portal vein, hepatic vein, carotid artery, and right external jugular vein. Such a model permits quantitative portal-peripheral comparisons and assessment of hepatic extraction. Sixteen dogs, 10 normal (N) and six long-term (2 months to 2 yrs) islet cell-transplanted dogs (IT) were fed an oral glucose meal as a test (OMT). Baseline portal vein insulin fluxes (PVF) were similar in both groups (25.6 +/- 0.04 pmol/min in N and 24.7 +/- 19.4 pmol/min in IT). Immediately after OMT, PVF rose to 248.2 +/- 40.9 pmol/min in N, but only to 55.9 +/- 17.9 pmol/min in IT. After 30 min PVF peaked for the second time in N at 156 +/- 35.9 pmol/min, declining slowly to baseline after 3 h. In IT, a similar peak at 30 min was seen (143.7 +/- 22.1 pmol/min), declining to a value not different from baseline after 3 h. However, cumulative insulin PV fluxes in the two groups over 3 h were not different. Differences were also seen in postprandial glucose fluctuations, which reached a maximum excursion of 11.8 +/- 0.45 mM in IT, while never rising above 7.8 +/- 0.33 mM in N. After 3 h both groups had similar glucose values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of CTLA4-Ig by biolistically transfected mouse islets promotes islet allograft survival

BACKGROUND: Localized delivery of immunosuppressive molecules, limited to the graft site, may allow transplantation of tissue in the absence of systemic immunosuppressive agents. We tested whether purified mouse islets that had been engineered to produce human CTLA4-Ig locally at the graft site could survive in allogeneic recipients receiving no systemic immunosuppression. METHODS: CBA (H2(k)) islets were subjected to biolistic (gene gun) transfection with a cDNA encoding human CTLA4-Ig under control of the human cytomegalovirus immediate early promoter. After 40-48 hr of culture, the transfected islets (500 per recipient) were transplanted beneath the renal capsule of alloxan-induced diabetic BALB/c (H2(d)) recipients. RESULTS: Control grafts (n=10) consisting of islets biolistically transfected with the expression plasmid alone (i.e., no gene inserted) survived for 12.8+/-3.6 (mean +/- SD) days. In contrast, islets transfected with CTLA4-Ig (n=12) survived 66.8+/-61.5 days (P=0.01), with 50% demonstrating functional survival until follow-up was concluded at 50 (n=2), 130 (n=2), or 165 (n=2) days. Immunohistochemistry on grafts that survived long term showed well-granulated, insulin-positive islets lying adjacent to, but not infiltrated by, dense aggregates of mononuclear cells. CONCLUSIONS: Transfection of allogeneic mouse islets with human CTLA4-Ig results in prolonged allograft survival. Although on histology mononuclear cells are present in the area of the transfected graft, they do not appear to infiltrate or destroy the islet graft.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase

Determining the way in which deleterious mutations interact in their effects on fitness is crucial to numerous areas in population genetics and evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last (synergistic epistasis), then the evolution of sex and recombination may be favored to facilitate the elimination of deleterious mutations. However, there is a severe shortage of relevant data. Three relatively simple experimental methods to test for epistasis between deleterious mutations in haploid species have recently been proposed. These methods involve crossing individuals and examining the mean and/or skew in log fitness of the offspring and parents. The main aim of this paper is to formalize these methods, and determine the most effective way in which tests for epistasis could be carried out. We show that only one of these methods is likely to give useful results: crossing individuals that have very different numbers of deleterious mutations, and comparing the mean log fitness of the parents with that of their offspring. We also reconsider experimental data collected on Chlamydomonas moewussi using two of the three methods. Finally, we suggest how the test could be applied to diploid species.     

TNF-alpha down-regulates type 1 cytokines and prolongs survival of syngeneic islet grafts in nonobese diabetic mice

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.     

Field tests of a circuit breaker synchronous control

A circuit breaker synchronous control interface which controls the point-on-wave at which shunt reactor circuit breakers open or close has been developed and tested on Hydro-Quebec's 735-kV power system. It takes into account the influence of outdoor temperature on the breaker closing and opening times. It is also equipped with a reignition and a high-inrush-current detection system. Opening tests at different preset arcing times were conducted and the arcing time range where there are no re-ignitions in air-blast breakers was established. The tests showed that the interface is a valuable device for the elimination of re-ignitions associated with the interruption of small inductive currents. Closing tests have shown that the interface is also useful for the limitation of high inrush currents by selecting an appropriate point-on-wave for circuit breaker closing