Carina Carlsson

Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rat pancreatic islets in vitro.

The aim of the present investigation was to study whether prolonged exposure of isolated rat islets to the long chain fatty acid sodium palmitate leads to uncoupling of respiration. It was found that culture of islets in the presence of palmitate abolished glucose-sensitive insulin release and decreased insulin contents. This was paralleled by decreased ATP contents, increased respiration, and decreased islet cell mitochondrial membrane potential. Using electron microscopy, an increase in the β-cell mitochondrial volume in islets exposed to palmitate was observed. The addition of the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, at a concentration that decreased mitochondrial membrane potential to a similar extent as palmitate, diminished the glucose-induced insulin release. In addition, islet generation of reactive oxygen species, but not of nitric oxide, was increased in response to a long-term palmitate exposure. It is concluded that long-term exposure to a long chain fatty acid induc...

Growth Hormone and Prolactin Stimulate the Expression of Rat Preadipocyte Factor-1/Δ-Like Protein in Pancreatic Islets: Molecular Cloning and Expression Pattern during Development and Growth of the Endocrine Pancreas1

GH and PRL have been shown to stimulate proliferation and insulin production in islets of Langerhans. To identify genes regulated by GH/PRL in islets, we performed differential screening of a complementary DNA library from neonatal rat islets cultured for 24 h with human GH (hGH). One hGH-induced clone had 96% identity with mouse preadipocyte factor-1 (Pref-1, or δ-like protein (Dlk)]. The size of Pref-1 messenger RNA (mRNA) in islets was 1.6 kilobases, with two less abundant mRNAs of 3.7 and 6.2 kilobases. The Pref-1 mRNA content of islets from adult rats was only 1% of that in neonatal islets. Pref-1 mRNA was markedly up-regulated in islets from pregnant rats from day 12 to term compared with those from age-matched female rats. Two peaks in mRNA expression were observed during gestation, one on day 14 and the other at term, whereafter it decreased to nonpregnant levels. Pref-1 mRNA was up-regulated 3- to 4-fold in neonatal rat islets of Langerhans after 48-h culture with hGH, as found also with bovine G...

Implantation Site–Dependent Dysfunction of Transplanted Pancreatic Islets

OBJECTIVE—Clinical islet transplantations are performed through infusion of islets via the portal vein into the liver. This study aimed at characterizing the influence of the implantation microenvironment on islet graft metabolism and function. RESEARCH DESIGN AND METHODS—Islets were transplanted into their normal environment, i.e., the pancreas, or intraportally into the liver of mice. One month posttransplantation, the transplanted islets were retrieved and investigated for changes in function and gene expression. RESULTS—Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis, and glucose oxidation rate were markedly decreased in islets retrieved from the liver, both when compared with islets transplanted into the pancreas and endogenous islets. Islets transplanted into the pancreas showed normal insulin content, (pro)insulin biosynthesis, and glucose oxidation rate but increased basal insulin secretion and impaired glucose stimulation index. Gene expression data for retrieved islets showed downregulation of pancreatic and duodenal homeobox gene-1, GLUT-2, glucokinase, mitochondrial glycerol-phosphate dehydrogenase, and pyruvate carboxylase, preferentially in intraportally transplanted islets. CONCLUSIONS—Islets transplanted into their normal microenvironment, i.e., the pancreas, display gene expression changes when compared with endogenous islets but only moderate changes in metabolic functions. In contrast, site-specific properties of the liver markedly impaired the metabolic functions of intraportally transplanted islets.

Expression, biosynthesis and release of preadipocyte factor-1/ delta-like protein/fetal antigen-1 in pancreatic beta-cells: possible physiological implications

Preadipocyte factor-1 (Pref-1)/delta-like protein/fetal antigen-1 (FA1) is a member of the epidermal growth factor-like family. It is widely expressed in embryonic tissues, whereas in adults it is confined to the adrenal gland, the anterior pituitary, the endocrine pancreas, the testis and the ovaries. We have previously cloned Pref-1 from neonatal rat islets stimulated by GH. The aim of the present study was to elucidate the biosynthesis and release of Pref-1/FA1 in beta-cells and to determine if Pref-1/FA1 is mediating the mitogenic effect of GH in insulin-producing cells. First we studied the biosynthesis and processing of Pref-1 to the soluble form, FA1, in pancreatic islets and insulinoma cells transfected with Pref-1 cDNA. We measured the release of FA1 by ELISA and the possible effect of FA1 in GH-stimulated beta-cell proliferation by incorporation of bromodeoxyuridine (BrdU) in insulin-positive islet cells. We found that Pref-1 was synthesized in normal islets and in RINm5F insulinoma cells and released into the medium in two forms, of which one corresponded to FA1. Both the expression of the mRNA for Pref-1 and the release of the soluble form(s) were stimulated by GH and prolactin (PRL). Whereas 2 h exposure to high glucose or 3-isobutyl-1-methylxanthine stimulated insulin release, only a small change was seen in FA1 release, suggesting that the FA1 is released by a different pathway than insulin. However, long-term exposure (48 h) to high glucose increased FA1 secretion, indicating that FA1 is regulated by glucose. Neither FA1 nor conditioned medium from GH-stimulated islets depleted for GH was able to increase beta-cell replication and overexpression of Pref-1 resulted in attenuated proliferation of the RINm5F cells. By immunocytochemistry of GH-stimulated islet cells no correlation between high Pref-1 expression and BrdU incorporation was observed and there was an inverse relationship between the levels of insulin and Pref-1. These results indicate that Pref-1/FA1 is not mediating the mitogenic effect of GH and PRL. Therefore the function of Pref-1 in the beta-cell remains unknown.

Antioxidant enzyme activity and mRNA expression in the islets of Langerhans from the BB/S rat model of type 1 diabetes and an insulin-producing cell line

It has been proposed that low activities of antioxidant enzymes in pancreatic beta cells may increase their susceptibility to autoimmune attack. We have therefore used the spontaneously diabetic BB/S rat model of type 1 diabetes to compare islet catalase and superoxide dismutase activities in diabetes-prone and diabetes-resistant animals. In parallel studies, we employed the RINm5F beta cell line as a model system (previously validated) to investigate whether regulation of antioxidant enzyme activity by inflammatory mediators (cytokines, nitric oxide) occurs at the gene or protein expression level. Diabetes-prone rat islets had high insulin content at the age used (58–65 days) but showed increased amounts of DNA damage when subjected to cytokine or hydrogen peroxide treatments. There was clear evidence of oxidative damage in freshly isolated rat islets from diabetes-prone animals and significantly lower catalase and superoxide dismutase activities than in islets from age-matched diabetes-resistant BB/S and control Wistar rats. The mRNA expression of antioxidant enzymes in islets from diabetes-prone and diabetes-resistant BB/S rats and in RINm5F cells, treated with a combination of cytokines or a nitric oxide donor, DETA-NO, was analysed semi-quantitatively by real time PCR. The mRNA expression of catalase was lower, whereas MnSOD expression was higher, in diabetes-prone compared to diabetes-resistant BB/S rat islets, suggesting regulation at the level of gene expression as well as of the activities of these enzymes in diabetes. The protein expression of catalase, CuZnSOD and MnSOD was assessed by Western blotting and found to be unchanged in DETA-NO treated cells. Protein expression of MnSOD was increased by cytokines in RINm5F cells whereas the expression of CuZnSOD was slightly decreased and the level of catalase protein was unchanged. We conclude that there are some changes, mostly upregulation, in protein expression but no decreases in the mRNA expression of catalase, CuZnSOD or MnSOD enzymes in beta cells treated with either cytokines or DETA-NO. The lower antioxidant enzyme activities observed in islets from diabetes-prone BB/S rats could be a factor in the development of disease and in susceptibility to DNA damage in vitro and could reflect islet alterations prior to immune attack or inherent differences in the islets of diabetes-prone animals, but are not likely to result from cytokine or nitric oxide exposure in vivo at that stage.

7-Ethoxyresorufin O-deethylase induction in cultured gill epithelial cells from rainbow trout

Abstract Induction of the cytochrome P4501A (CYP1A)-mediated enzyme activity 7-ethoxyresorufin O-deethylase (EROD) was measured in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. Monolayers of adherent cells were exposed to the inducers β-naphthoflavone (β-NF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB#126) and benzo[k]fluoranthene (B[k]F). EROD activity was either measured directly in the adherent cells after exposure to inducer, or the exposed adherent cells were dislodged by trypsination and EROD activity was measured in suspended cells. A time course study of the induction caused by β-NF showed that the EROD activity remained at a similar level from 24 to 72 h after addition of the compound. A second time course study was performed using all four substances. For β-NF, B[k]F and TCDD the induction was similar throughout the time period and for PCB#126 maximal induction was reached at 24 h, followed by a decrease. Concentration–response relationships for EROD induction were established for the two different methods by exposing the cells to 0.036, 0.36 and 3.6×10−6 M β-NF. When measured in suspended cells, EROD activity increased at the two lowest concentrations. At the highest concentration no further increase was detected. The adherent cells showed a different concentration–response to the inducer, with an increase at the low concentration, a maximum at 0.36×10−6 M, and a decrease at the highest concentration. The maximal EROD activity was about two times higher in adherent cells than in suspended cells. All further measurements were therefore performed on adherent intact cells. The potencies and efficacies of the different compounds to induce EROD were compared in a set of experiments using concentrations ranging from 10−11 to 10−6 M. In all cases, EROD activity increased with increasing concentrations up to a maximal level, and thereafter the activity decreased. TCDD caused maximal EROD activity at a concentration of 10−9 M, and all other inducers caused maximal EROD activity at a concentration of 10−7 M. The maximal EROD activities obtained with B[k]F, β-NF and PCB#126 were about 80, 80 and 40%, respectively, of the maximal activity obtained with TCDD. The relative potencies compared with TCDD were 0.008 for B[k]F, 0.0035 for PCB#126 and 0.002 for β-NF, based on EC50 values determined from the concentration–response curves.

Engraftment and growth of transplanted pancreatic islets

Abstract Transplantation of pancreatic islets may provide a cure for type 1 diabetes. How—ever, this treatment can currently be offered only to very few patients. To improve transplantation success we need to understand better the mechanisms of how the implanted islets survive, grow and/or maintain adequate function. We herein report on our studies to evaluate the factors responsible for the engraftment, i.e. revascularization, reinnervation etc., of transplanted islets and relate these factors to the metabolism and growth of the islets. Graft metabolism can be monitored by microdialysis probes that allow for the measurement of minute amounts of islet metabolites and hormonal products. Growth of the endocrine cells can be stimulated both in vitro before implantation and in vivo post-transplantation. Another problem is rejection of transplanted islets, which may be overcome by the microencapsulation of islets. The knowledge gained by the present studies will enable us to elucidate the optimal treatment of islets to ensure a maximal survival of the transplanted islets, and may be applied also to clinical islet transplantation.

Pref-1 and adipokine expression in adipose tissues of GK and Zucker rats

In view of the central role of preadipocyte factor-1, adiponectin and leptin in white adipose tissue function, the aim of the present study was to analyze the mRNA expression of these proteins and of the inflammatory markers interleukin-6 and tumor necrosis factor-alpha in visceral and subcutaneous fat pads of rats with different metabolic disorders. We demonstrated highly divergent expression of preadipocyte factor-1, upregulated expression of adiponectin, interleukin-6 and TNF-alpha mRNA in adipose tissues of the diabetic Goto Kakizaki rat compared to the obese Zucker rat. This was correlated to an increased number of large adipocytes and serum levels of adiponectin. Furthermore, in all four strains studied (as above plus Wistar Furth and Zucker Lean), significant heterogeneity was evident in adipokine expression within specific adipose tissues previously defined as belonging to the visceral or subcutaneous fat depots. These results suggest that significantly increased levels of inflammation and redistribution of adipocyte size are mechanisms contributing to the development of type 2 diabetes in the GK rat.

Cytokine-induced PGE2 formation is reduced from iNOS deficient murine islets

Cytokines may be involved in islet destruction during Type 1 diabetes. Exposure to interleukin-1beta (IL-1beta) or IL-1beta plus interferon-gamma (IFN-gamma) of rodent islets induces expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent formation of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) may impair beta-cell function. Using iNOS deficient (iNOS -/-) islets, we have further investigated the relation between NO formation and PGE(2) induction. We found that iNOS -/- islets responded with a reduced PGE(2) formation following IL-1beta or (IL-1beta + IFN-gamma) treatment compared to wild-type (wt) islets, while COX-2 mRNA or protein content were unchanged. By the addition of an NO donor together with IL-1beta, PGE(2) formation could be stimulated from iNOS -/- islets. We conclude that the lowered capacity of PGE(2) formation observed from cytokine exposed iNOS -/- islets is due to a decreased stimulation of PGE(2) formation by the COX-2 enzyme in the absence of NO, rather then differences in expressed COX-2 protein.

Isomer-specific bioactivation and toxicity of dichlorophenyl methylsulphone in rat olfactory mucosa.

This study aimed to explain the isomer- and site-specific toxic effects of dichlorophenyl methylsulphone in the olfactory mucosa of rats. A single ip dose of the 2,6-chlorinated isomer (16 or 65 mg/kg) induced necrosis preferentially in the Bowmans glands and neuroepithelium in the dorsomedial part of the olfactory region. Only minor damage occurred at this site in rats dosed with the 2,5-chlorinated isomer (65 mg/kg). A strong concentration-and time-dependent covalent binding of the 14C-labeled 2,6-isomer to rat olfactory microsomes was demonstrated. In contrast, no significant covalent binding of the 14C-labeled 2,5-isomer was observed. The cytochrome P450 (CYP) inhibitors metyrapone, tranylcypromine and acetonitrile inhibited covalent binding of the 2,6-isomer to olfactory microsomes. Glutathione (GSH) appeared to play a protective role as a scavenger of a reactive intermediate whereas methyl-GSH did not alter covalent binding to olfactory microsomes. As determined by microautoradiography, binding of the 2,6-chlorinated isomer in the olfactory mucosa was confined to the Bowmans glands. Both isomers showed a low binding to liver microsomes and caused no liver injury. We suggest that a CYP2A-catalyzed activation of the 2,6-chlorinated dichlorophenyl methylsulphone to a reactive intermediate and adduct formation in the Bowmans glands will initiate a site-specific toxicity of this isomer in the olfactory mucosa.