Redundancy of interleukin-6 in the differentiation of T cell and monocyte subsets during cutaneous leishmaniasis

Leishmania (L.) major is a protozoan parasite that infects mammalian hosts and causes a spectrum of disease manifestations that is strongly associated with the genetic background of the host. Interleukin (IL)-6 is an acute phase proinflammatory cytokine, known in vitro to be involved in the inhibition of the generation of regulatory T cells. IL-6-deficient mice were infected with L. major, and T cell and monocyte subsets were analyzed with flow cytometry. Our data show that at the site of infection in the footpad and in the draining popliteal lymph node, numbers of regulatory T cells remain unchanged between WT and IL-6-deficient mice. However, the spleens of IL-6^−/− mice contained fewer regulatory T cells after infection with L. major. The development of cutaneous lesions is similar between WT and IL-6-deficient mice, while parasite burden in IL-6^−/− mice is reduced compared to WT. The development of IFN-γ or IL-10 producing T cells is similar in IL-6^−/− mice. Despite a comparable adaptive T cell response, IL-6-deficient mice develop an earlier peak of some inflammatory cytokines than WT mice. This data indicate that the role of IL-6 in the differentiation of regulatory T cells is complex in vivo, and the effect of an absence of this cytokine can be counter-intuitive.     

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo

Keratan sulfate (KS) proteoglycan side chains are abundant in the human cartilage matrix, but these chains have been said to be absent in murine skeletal tissues. We previously showed that KS suppresses cartilage damage and ameliorates inflammation in mice arthritis model. Because mice deficient of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) (KS biosynthesis enzyme) are now available, we decided to do further examinations.We examined, in culture, the difference between GlcNAc6ST-1^−/− and wild-type (WT) mice for interleukin (IL)-1α-induced glycosaminoglycan (GAG) release from the articular cartilage. Arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail and subsequent intraperitoneal injection of lipopolysaccharide. We examined the differences in arthritis severities in the two genotypes. After intraperitoneal KS administration in phosphate-buffered saline (PBS) or PBS alone, we evaluated the potential of KS in ameliorating arthritis and protecting against cartilage damage in deficient mice.GAG release induced by IL-1α in the explants, and severity of arthritis were greater in GlcNAc6ST-1^−/− mice than their WT littermates. Intraperitoneal KS administration effectively suppressed arthritis induction in GlcNAc6ST-1^−/− mice. Thus, GlcNAc6ST-1^−/− mice cartilage is more fragile than WT mice cartilage, and exogenous KS can suppress arthritis induction in GlcNAc6ST-1^−/− mice. Vestigial KS chain or altered glycosylation in articular cartilage in GlcNAc6ST-1^−/− mice may be protective against arthritis and associated cartilage damage as well as cartilage damage in culture. KS may offer therapeutic opportunities for chondroprotection and suppression of joint damage in inflammatory arthritis and may become a therapeutic agent for treating rheumatoid arthritis.     

Lipopolysaccharide-induced hepatic oxidative injury is not potentiated by knockout of GPX1 and SOD1 in mice

Knockout of copper, zinc-superoxide dismutase (SOD1) and (or) cellular glutathione peroxidase (GPX1) has been reported to have dual impacts on coping with free radical-induced oxidative injury. Because bacterial endotoxin lipopolysaccharide (LPS) triggers inflammatory responses involving the release of cytokines, nitric oxide and superoxide in targeted organs such as liver, in this study we used SOD1 knockout (SOD1−/−), GPX1 knockout (GPX1−/−), GPX1 and SOD1 double-knockout (DKO) and their wild-type (WT) mice to investigate the role of these two antioxidant enzymes in LPS-induced oxidative injury in liver. Mice of the four genotypes (2 month old) were killed at 0, 3, 6 or 12 h after an i.p. injection of saline or 5 mg LPS/kg body weight. The LPS injection caused similar increase in plasma alanine aminotransferase among the four genotypes. Hepatic total glutathione (GSH) was decreased (P < 0.05) compared with the initial values by the LPS injection at all time points in the WT mice, but only at 6 and 12 h in the other three genotypes. The GSH level in the DKO mice was higher (P < 0.05) than in the WT at 6 h. Although the LPS injection resulted in substantial increases in plasma NO in a time-dependent manner in all genotypes, the NO level in the DKO mice was lower (P < 0.05) at 3, 6 and 12 h than in the WT. The level in the GPX1−/− and SOD1−/− mice was also lower (P < 0.05) than in the WT at 3 h. The LPS-mediated hepatic protein nitration was detected in the WT and GPX1−/− mice at 3, 6 or 12 h, but not in the SOD1−/−. In conclusion, knockout of SOD1 and (or) GPX1 did not potentiate the LPS-induced liver injury, but delayed the induced hepatic GSH depletion and plasma NO production.     

Effects of atypical antipsychotic drugs on body weight and food intake in dopamine D2 receptor knockout mice

Many atypical antipsychotic drugs cause weight gain, but the mechanism of this weight gain is unclear. To dissect the role of the dopamine D2 receptor (D2R), an important receptor in the pharmacology of antipsychotic drugs, we analyzed the effect of olanzapine, risperidone, and ziprasidone on changes in body weight and food intake in male wild-type (WT) and D2R knockout (D2R^−/−) mice. The oral delivery of atypical antipsychotics, olanzapine (5 and 10 mg/kg), risperidone (0.1 and 1.0 mg/kg) and ziprasidone (10 and 20 mg/kg) in both strains mice for 2 weeks suppressed body weight gain, except for olanzapine treatment in D2R^−/− mice. Olanzapine treatment suppressed body weight gain and decreased food intake in WT mice, but also reduced fat body mass and locomotor activity, whereas D2R^−/− mice did not show these changes. Ziprasidone and risperidone treatment produced similar responses in WT and D2R^−/− mice. These data suggest the involvement of D2R in the effect of olanzapine on metabolic regulation. Further studies are required to explore the implications of D2R activity in antipsychotic-mediated metabolic complications.     

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr^−/−) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr^−/− and α7BX2-mdx/utr^−/− mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr^−/− muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr^−/− mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2-mdx/utr^−/− compared to mdx/utr^−/− mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL⁺ cells observed in α7BX2-mdx/utr^−/− mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.     

Reactive oxygen species on bone mineral density and mechanics in Cu,Zn superoxide dismutase (Sod1) knockout mice

Reactive oxygen species (ROS) play a role in a number of degenerative conditions including osteoporosis. Mice deficient in Cu,Zn-superoxide dismutase (Sod1) (Sod1^−/− mice) have elevated oxidative stress and decreased muscle mass and strength compared to wild-type mice (WT) and appear to have an accelerated muscular aging phenotype. Thus, Sod1^−/− mice may be a good model for evaluating the effects of free radical generation on diseases associated with aging. In this experiment, we tested the hypothesis that the structural integrity of bone as measured by bending stiffness (EI; N/mm²) and strength (MPa) is diminished in Sod1^−/− compared to WT mice. Femurs were obtained from male and female WT and Sod1^−/− mice at 8 months of age and three-point bending tests were used to determine bending stiffness and strength. Bones were also analyzed for bone mineral density (BMD; mg/cc) using micro-computed tomography. Femurs were approximately equal in length across all groups, and there were no significant differences in BMD or EI with respect to gender in either genotype. Although male and female mice demonstrated similar properties within each genotype, Sod1^−/− mice exhibited lower BMD and EI of femurs from both males and females compared with gender matched WT mice. Strength of femurs was also lower in Sod1^−/− mice compared to WT as well as between genders. These data indicate that increased oxidative stress, due to the deficiency of Sod1 is associated with decreased bone stiffness and strength and Sod1^−/− mice may represent an appropriate model for studying disease processes in aging bone.     

The effects of point of substitution on the electrochemical behavior of new manganese phthalocyanines, tetra-substituted with diethylaminoethanethiol

The syntheses and comparative studies of the spectral, voltammetry and spectroelectrochemical properties of new manganese phthalocyanine complexes, tetra-substituted with diethylaminoethanethio at the peripheral (complex 3a) and non-peripheral positions (complex 3b) are reported. Solution electrochemistry of complex 3a showed quasi-reversible metal-based (Mn^IIIPc⁻²/Mn^IIPc⁻², E_1/2 = −0.07 V vs. Ag|AgCl) and ring-based (Mn^IIPc⁻²/Mn^IIPc⁻³, E_1/2 = −0.78 V vs. Ag|AgCl) reductions, but no ring-based oxidation. However, complex 3b showed weak irreversible ring-oxidation signal (E_p = +0.86 vs. Ag|AgCl). Reversible metal-based (Mn^IIIPc⁻²/Mn^IIPc⁻², E_1/2 = −0.04 V vs. Ag|AgCl) and ring-based (Mn^IIPc⁻²/Mn^IIPc⁻³, E_1/2 = −0.68 V vs. Ag|AgCl) reductions were also observed for complex 3b. Spectroelectrochemistry was used to confirm these processes. Reduction process involving the metal (Mn^IIIPc⁻²/Mn^IIPc⁻²) was associated with the formation of manganese μ-oxo complex in complex 3a.     

Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties

We report for the first time abnormalities in cardiac ventricular electrophysiology in a genetically modified murine model lacking the Scn3b gene (Scn3b^−/−). Scn3b^−/− mice were created by homologous recombination in embryonic stem (ES) cells. RT-PCR analysis confirmed that Scn3b mRNA was expressed in the ventricles of wild-type (WT) hearts but was absent in the Scn3b^−/− hearts. These hearts also showed increased expression levels of Scn1b mRNA in both ventricles and Scn5a mRNA in the right ventricles compared to findings in WT hearts. Scn1b and Scn5a mRNA was expressed at higher levels in the left than in the right ventricles of both Scn3b^−/− and WT hearts. Bipolar electrogram and monophasic action potential recordings from the ventricles of Langendorff-perfused Scn3b^−/− hearts demonstrated significantly shorter ventricular effective refractory periods (VERPs), larger ratios of electrogram duration obtained at the shortest and longest S₁–S₂ intervals, and ventricular tachycardias (VTs) induced by programmed electrical stimulation. Such arrhythmogenesis took the form of either monomorphic or polymorphic VT. Despite shorter action potential durations (APDs) in both the endocardium and epicardium, Scn3b^−/− hearts showed ΔAPD₉₀ values that remained similar to those shown in WT hearts. The whole-cell patch-clamp technique applied to ventricular myocytes isolated from Scn3b^−/− hearts demonstrated reduced peak Na⁺ current densities and inactivation curves that were shifted in the negative direction, relative to those shown in WT myocytes. Together, these findings associate the lack of the Scn3b gene with arrhythmic tendencies in intact perfused hearts and electrophysiological features similar to those in Scn5a^+/− hearts.     

Dietary isoflavone increases insulin-like growth factor-I production, thereby promoting hair growth in mice

Sensory neurons release calcitonin gene-related peptide (CGRP) upon activation. We previously demonstrated that CGRP increases insulin-like growth factor-I (IGF-I) production in various tissues of mice including the skin. We demonstrated that isoflavone increases the CGRP synthesis in the dorsal root ganglion (DRG) neurons in rats. Since IGF-I plays a critical role in hair growth, we hypothesized that isoflavones may promote hair growth by increasing the IGF-I production in hair follicles. We examined this hypothesis using wild-type (WT) and CGRP-knockout (CGRP^−/−) mice. Isoflavone significantly increased the CGRP mRNA levels in DRG neurons isolated from WT mice (P<.01). Administration of isoflavone for 3 weeks increased the dermal levels of CGRP, IGF-I and IGF-I mRNA in WT mice, but not in CGRP^−/− mice. Isoflavone administration increased the immunohistochemical expression of IGF-I in hair follicle dermal papilla cells in WT mice. Significant enhancements of hair follicle morphogenesis, hair regrowth, and hair pigmentation were also observed in WT mice administered isoflavone. However, none of these effects in WT mice were observed in CGRP^−/− mice.These observations strongly suggest that isoflavone might increase IGF-I production in the hair follicle dermal papilla cells in mice through increasing CGRP production in the sensory neurons, thereby promoting hair growth associated with melanogenesis in mice.     

Temporary upregulation of anti-inflammatory cytokine IL-13 expression in the brains of CD14 deficient mice in the early stage of prion infection

CD14 deficient (CD14^−/−) mice survived longer than wild-type (WT) C57BL/6J mice when inoculated with prions intracerebrally, accompanied by increased expression of anti-inflammatory cytokine IL-10 by microglia in the early stage of infection. To assess the immune regulatory effects of CD14 in detail, we compared the gene expression of pro- and anti-inflammatory cytokines in the brains of WT and CD14^−/− mice infected with the Chandler strain. Gene expression of the anti-inflammatory cytokine IL-13 in prion-infected CD14^−/− mice was temporarily upregulated at 75 dpi, whereas IL-13 gene expression was not upregulated in prion-infected WT mice. Immunofluorescence staining showed that IL-13 was mainly expressed in neurons of the thalamus at 75 dpi. These results suggest that CD14 can suppress IL-13 expression in neurons during the early stage of prion infection.     

Genetic interactions and dosage effects of Polycomb group genes of Drosophila

The Polycomb (Pc) group of genes are required for maintenance of cell determination in Drosophila melanogaster. At least 11 Pc group genes have been described and there may be up to 40; all are required for normal regulation of homeotic genes, but as a group, their phenotypes are rather diverse. It has been suggested that the products of Pc group genes might be members of a heteromeric complex that acts to regulate the chromatin structure of target loci. We examined the phenotypes of adult flies heterozygous for every pairwise combination of Pc group genes in an attempt to subdivide the Pc group functionally. The results support the idea that Additional sex combs (Asx), Pc, Polycomblike (Pcl), Posterior sex combs (Psc), Sex combs on midleg (Scm), and Sex combs extra (Sce) have similar functions in some imaginal tissues. We show genetic interactions among extra sex combs (esc) and Asx, Enhancer of Pc, Pcl, Enhancer of zeste E(z), and super sex combs and reassess the idea that most Pc group genes function independently of esc. Most duplications of Pc group genes neither exhibit anterior transformations nor suppress the extra sex comb phenotype of Pc group mutations, suggesting that not all Pc group genes behave as predicted by the mass-action model. Surprisingly, duplications of E(z) enhance homeotic phenotypes of esc mutants. Flies with increasing doses of esc ⁺ exhibit anterior transformations, but these are not enhanced by mutations in trithorax group genes. The results are discussed with respect to current models of Pc group function.     

Dual roles of CagA protein in Helicobacterpylori-induced chronic gastritis in mice

Cytotoxin-associated gene A (CagA) acts directly on gastric epithelial cells. However, the roles of CagA in host adaptive immunity against Helicobacter pylori (H. pylori) infection are not fully understood. In this study, to investigate the roles of CagA in the development of H. pylori-induced chronic gastritis, we used an adoptive-transfer model in which spleen cells from C57BL/6 mice with or without H. pylori infection were transferred into RAG2^−/− mice, with gastric colonization of either CagA⁺H. pylori or CagA⁻H. pylori. Colonization of CagA⁺H. pylori but not CagA⁻H. pylori in the host gastric mucosa induced severe chronic gastritis in RAG2^−/− mice transferred with spleen cells from H. pylori-uninfected mice. In addition, when CagA⁺H. pylori-primed spleen cells were transferred into RAG2^−/− mice, CD4⁺ T cell infiltration in the host gastric mucosa were observed only in RAG2^−/− mice infected with CagA⁺H. pylori but not CagA⁻H. pylori, suggesting that colonization of CagA⁺H. pylori in the host gastric mucosa is essential for the migration of H. pylori-primed CD4⁺ T cells. On the other hand, transfer of CagA⁻H. pylori-primed spleen cells into CagA⁺H. pylori-infected RAG2^−/− mice induced more severe chronic gastritis with less Foxp3⁺ regulatory T-cell infiltration as compared to transfer of CagA⁺H. pylori-primed spleen cells. In conclusion, CagA in the stomach plays an important role in the migration of H. pylori-primed CD4⁺ T cells in the gastric mucosa, whereas CagA-dependent T-cell priming induces regulatory T-cell differentiation, suggesting dual roles for CagA in the pathophysiology of H. pylori-induced chronic gastritis.     

Sclerostin deficient mice rapidly heal bone defects by activating β-catenin and increasing intramembranous ossification

We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost^−/−; KO) and sclerostin wild-type (Sost^+/+; WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2^−/− mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost^−/− mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.     

Ascorbate promotes carbon tetrachloride-induced hepatic injury in senescence marker protein 30-deficient mice by enhancing inflammation

The genetic deletion of the senescence marker protein 30 (SMP30) gene results in ascorbate deficiency and the premature aging processes in mice. Apparent liver injury of SMP30^−/− mice was less severe than those of wild type (WT) mice, upon chronic CCl₄ injection. The purpose of this study was to investigate the pathophysiology underlying the mild CCl₄ toxicity in SMP30^−/− mice. Along with the lower level of serum alanine aminotransferase, the livers of SMP30^−/− mice revealed a lesser glycogen depletion, a decrease in c-Jun N-terminal kinase (JNK)-mediated inflammatory signaling in parallel with tumor necrosis factor-alpha and interleukin-1 beta, inducible nitric oxide synthase and glutathione peroxidase, and the lower lipid peroxidation as compared to those of WT mice. CCl₄-induced proliferation, measured by the expression of proliferating cell nuclear antigen, was low in SMP30^−/− mice as compared with that of WT mice whereas the levels of p21 and Bax were comparable to those of the CCl₄-treated WT mice. Moreover, CCl₄ toxicity in ascorbate-fed SMP30^−/− mice was comparable to that of the CCl₄-alone treated WT mice, accompanied by an increase in the above mentioned factors. Conversely, ascorbate partly compensated for the CCl₄-induced oxidative stress in WT mice, indicating that sufficient ascorbate may be required for an antioxidant function under severe levels of oxidative stress. Our data suggest that the restoration of ascorbate-deficiency reverses a sluggish immune system into an activated condition by an increase in JNK-mediated inflammation and free radical cascade; thus leading to accelerated hepatic damage in SMP30^−/− mice.     

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR^−/−) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR^−/− mice fed MCD diet (FXR^−/−/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR^−/−/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR^−/−/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR^−/−/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.     

Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1^−/− renal cells displayed aberrant cell–cell contacts and tubulomorphogenesis. The Pkhd1^−/− cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1^−/− mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1^−/− kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.     

Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice

Aims/hypothesis

Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR^−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca^2 + handling in these islets.

Methods

Isolated islets from both LDLR^−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca^2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).

Results

The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR^−/− than in WT islets, paralleled by an impairment of Ca^2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR^−/− compared with WT islets. Removal of excess cholesterol from LDLR^−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca^2 + handling was also normalized in cholesterol-depleted LDLR^−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca^2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.

Conclusion

Abnormally high (LDLR^−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca^2 + handling. Normalization of cholesterol improves Ca^2 + handling and insulin secretion in LDLR^−/− islets.