The role of DNA methylation in the pathogenesis of type 2 diabetes mellitus.

Diabetes mellitus (DM) is a chronic condition characterised by ß cell dysfunction and persistent hyperglycaemia. The disorder can be due to the absence of adequate pancreatic insulin production or a weak cellular response to insulin signalling. Among the three types of DM, namely, type 1 DM (T1DM), type 2 DM (T2DM), and gestational DM (GDM); T2DM accounts for almost 90% of diabetes cases worldwide.Epigenetic traits are stably heritable phenotypes that result from certain changes that affect gene function without altering the gene sequence. While epigenetic traits are considered reversible modifications, they can be inherited mitotically and meiotically. In addition, epigenetic traits can randomly arise in response to environmental factors or certain genetic mutations or lesions, such as those affecting the enzymes that catalyse the epigenetic modification. In this review, we focus on the role of DNA methylation, a type of epigenetic modification, in the pathogenesis of T2DM.

The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex.

The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.

Identification of early indicators of altered metabolism in normal development using a rodent model system.

Although the existence of a close relationship between the early maternal developmental environment, fetal size at birth and the risk of developing disease in adulthood has been suggested, most studies, however, employed experimentally induced intrauterine growth restriction as a model to link this with later adult disease. Because embryonic size variation also occurs under normal growth and differentiation, elucidating the molecular mechanisms underlying these changes and their relevance to later adult disease risk becomes important. The birth weight of rat pups vary according to the uterine horn positions. Using birth weight as a marker, we compared two groups of rat pups - lower birth weight (LBW, 5th to 25th percentile) and average birth weight (ABW, 50th to 75th percentile) - using morphological, biochemical and molecular biology, and genetic techniques. Our results show that insulin metabolism, Pi3k/Akt and Pparγ signaling and the genes regulating growth and metabolism are significantly different in these groups. Methylation at the promoter of the InsII (Ins2) gene and DNA methyltransferase 1 in LBW pups are both increased. Additionally, the Dnmt1 repressor complex, which includes Hdac1, Rb (Rb1) and E2f1, was also upregulated in LBW pups. We conclude that the Dnmt1 repressor complex, which regulates the restriction point of the cell cycle, retards the rate at which cells traverse the G1 or G0 phase of the cell cycle in LBW pups, thereby slowing down growth. This regulatory mechanism mediated by Dnmt1 might contribute to the production of small-size pups and altered physiology and pathology in adult life.

Metallic nanoparticles to eradicate bacterial bone infection.

Treatment of osteomyelitis by conventional antibiotics has proven to be challenging due to limited accessibility to this unique location. Inorganic routes against bacterial infection have been reported for external and topical applications, however in vivo application of these antimicrobials has not been fully explored. Targeted delivery of metallic nanoparticles with inherent antimicrobial activity represents an alternative means of overcoming the challenges posed by multidrug-resistant bacteria and may potentially reduce overall morbidity. In this study we utilized silver-copper-boron composite nanoparticles in an attempt to eradicate S. aureus bone infection in mice. Our results demonstrate effective response when nanoparticles were administered via i.v. or i.m. route (1mg/kg dose) where 99% of bacteria were eliminated in an induced osteomyelitis mouse model. The 1mg/kg dose was neither toxic nor produced any adverse immune response, hence it is believed that metallic nanoparticles present an alternative to antibiotics for the treatment of bone infection.

Higher O-GlcNAc Levels Are Associated with Defects in Progenitor Proliferation and Premature Neuronal Differentiation during in-Vitro Human Embryonic Cortical Neurogenesis.

The nutrient responsive O-GlcNAcylation is a dynamic post-translational protein modification found on several nucleocytoplasmic proteins. Previous studies have suggested that hyperglycemia induces the levels of total O-GlcNAcylation inside the cells. Hyperglycemia mediated increase in protein O-GlcNAcylation has been shown to be responsible for various pathologies including insulin resistance and Alzheimer's disease. Since maternal hyperglycemia during pregnancy is associated with adverse neurodevelopmental outcomes in the offspring, it is intriguing to identify the effect of increased protein O-GlcNAcylation on embryonic neurogenesis. Herein using human embryonic stem cells (hESCs) as model, we show that increased levels of total O-GlcNAc is associated with decreased neural progenitor proliferation and premature differentiation of cortical neurons, reduced AKT phosphorylation, increased apoptosis and defects in the expression of various regulators of embryonic corticogenesis. As defects in proliferation and differentiation during neurodevelopment are common features of various neurodevelopmental disorders, increased O-GlcNAcylation could be one mechanism responsible for defective neurodevelopmental outcomes in metabolically compromised pregnancies such as diabetes.

Bioenergetics of the spinal cord in experimental autoimmune encephalitis of rats.

BACKGROUND: Mitochondrial dysregulation is important in axonal damage and demyelination in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). There is however, no evidence in the literature of any study that has examined cellular bioenergetics of the central nervous system (CNS) during the early development and clinical course of EAE. EAE, a rodent model of relapsing/remitting MS, is a CD4(+) T cell-mediated disease of the CNS. We hypothesize that CNS bioenergetics might predict prognosis, and that preserved bioenergetics might underlie the remission from disease. The study aims therefore, to determine whether the clinical history of EAE is influenced by cellular respiration of the CNS in susceptible Dark Agouti (DA) and resistant Albino Oxford (AO) rats. METHODS: Experimental autoimmune encephalomyelitis was induced by myelin basic protein in complete Freud Adjuvant in the footpads of DA and AO rats. A phosphorescence analyzer that determines cellular respiration was used to monitor oxygen consumption and ATP concentration was measured using the Enliten ATP assay system. Disease pathology was demonstrated by H&E and Luxol fast blue staining of sections of the lumbar regions of the spinal cord. Mitochondrial size in relation to axonal size was determined by electron microscopy. Apoptosis was studied by HPLC measurement of intracellular caspase-3 activity and caspase immunohistochemistry. Role and source of caspase 1 was studied by double immunofluorescence with antibodies for caspase-1, microglia (anti-Iba1) and astrocytes (anti-GFAP). RESULTS: The cellular respiration of the CNS did not vary between diseased and normal rats. We also demonstrate here, that at the peak of disease, inflammation as shown by caspase-1, produced by activated microglia and infiltrating cells, was significant in susceptible DA rats. The mitochondrial:axonal size ratio did not vary in the different groups although mitochondria were smaller in spinal cords of diseased DA rats. Demyelination, observed only in areas of mononuclear infiltration of the spinal cord of diseased DA rats, was demonstrated by light microscopy and electron microscopy. CONCLUSION: We conclude that EAE at this early stage does not significantly affect CNS cellular respiration and this might underlie the reason for the recovery of diseased rats.

The cytotoxicity of aflatoxin b1 in human lymphocytes.

OBJECTIVES: Aflatoxin B1 (AFB1) is a naturally occurring carcinogenic and immunosuppressive compound. This study was designed to measure its toxic effects on human peripheral blood mononuclear cells (PBMC). METHODS: The study recruited 7 healthy volunteers. PBMC were isolated and cellular respiration was monitored using a phosphorescence oxygen analyser. The intracellular caspase activity was measured by the caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin. Phosphatidylserine exposure and membrane permeability to propidium iodide (PI) were measured by flow cytometry. RESULTS: Cellular oxygen consumption was inhibited by 2.5 µM and 25 µM of AFB1. Intracellular caspase activity was noted after two hours of incubation with 100 µM of AFB1. The number of Annexin V-positive cells increased as a function of AFB1 concentration and incubation time. At 50 µM, a significant number of cells became necrotic after 24 hours (Annexin V-positive and PI-positive). CONCLUSION: The results show AFB1 is toxic to human lymphocytes and that its cytotoxicity is mediated by apoptosis and necrosis.

Pam3CSK(4) enhanced beta cell loss and diabetogenesis: the roles of IFN-gamma and IL-17.

Toll like receptors are primary sensors of both innate and adaptive immune systems. They activate APCs and influence T-cell function in inflammatory autoimmune response. Studies have shown that TLR manipulation may lead to either tolerance or trigger autoimmunity. Using diabetogenic and subdiabetogenic multiple low doses of streptozotocin, we demonstrate here that Pam3 CYS-CK4 a TLR-2 agonist, enhances and promotes diabetes in C57BL/6 male mice following increased apoptosis of ß islet cells. FACS analysis of isolated pancreatic lymph node cells revealed significant increased number of macrophages, dendritic cells, CD4(+) TNF-α(+), CD4(+) IFN-γ(+) and most significantly, CD4(+) IL-17(+) and reduced number of CD25(+)Fox p3(+) T cells after Pam3CSK4 treatment. Genetic deletion of IFN-γ prevents whereas deletion of IL-17 reduced severity of Pam3CSK4-induced enhancement of diabetes. TLR-2 agonist-enhanced diabetogenesis is also influenced by enhanced influx of antigen presenting cells and suppression of regulatory T cell activity.

Derangements of liver tissue bioenergetics in concanavalin A-induced hepatitis.

BACKGROUND: A novel in vitro system was employed to investigate liver tissue respiration (mitochondrial O2 consumption) in mice treated with concanavalin A (Con A). This study aimed to investigate hepatocyte bioenergetics in this well-studied hepatitis model. METHODS: C57Bl/6 and C57Bl/6 IFN-γ-/- mice were injected intravenously with 12 mg ConA/kg. Liver specimens were collected at various timepoints after injection and analyzed for cellular respiration and caspase activation. Serum was analyzed for interferon-gamma (IFN-γ) and aminotransferases. Fluorescence activated cell sorting analysis was used to determine the phenotype of infiltrating cells, and light and electron microscopy were used to monitor morphological changes. Phosphorescence analyzer that measured dissolved O2 as function of time was used to evaluate respiration. RESULTS: In sealed vials, O2 concentrations in solutions containing liver specimen and glucose declined linearly with time, confirming zero-order kinetics of hepatocyte respiration. O2 consumption was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Enhanced liver respiration (by ≈68%, p<0.02) was noted 3 hr after ConA treatment, and occurred in conjunction with limited cellular infiltrations around the blood vessels. Diminished respiration (by ≈30%, p=0.005) was noted 12 hr after ConA treatment, and occurred in conjunction with deranged mitochondria, areas of necrosis, and prominent infiltrations with immune cells, most significantly, CD3+NKT+ cells. Increases in intracellular caspase activity and serum IFN-γ and aminotransferase levels were noted 3 hr after ConA treatment and progressed with time. The above-noted changes were less pronounced in C57Bl/6 IFN-γ-/- mice treated with ConA. CONCLUSIONS: Based on these results, liver tissue bioenergetics is increased 3 hr after ConA exposure. This effect is driven by the pathogenesis of the disease, in which IFN-γ and other cytokines contribute to. Subsequent declines in liver bioenergetics appear to be a result of necrosis and active caspases targeting the mitochondria within hepatocytes.

IFN-γ deficiency exacerbates experimental autoimmune neuritis in mice despite a mitigated systemic Th1 immune response.

Previous studies have shown that interferon-gamma (IFN-γ) is a proinflammatory cytokine that contributes to the pathogenesis of Guillain-Barré syndrome and its animal model, experimental autoimmune neuritis (EAN). Treatments with anti-IFN-γ antibodies improve clinical outcome in GBS patients and EAN animals and administration of IFN-γ markedly worsens EAN. Paradoxically, the mice deficient in IFN-γ remain susceptible to experimental autoimmune encephalomyelitis, an analogous disease in the central nervous system. These observations raise a question whether IFN-γ might be protective in autoimmune demyelinating diseases. To clarify the role of IFN-γ in the pathogenesis of autoimmune demyelinating diseases, we used P0 protein peptide 180-199 to induce EAN in IFN-γ knockout (KO) mice. After the acute phase of EAN, the clinical signs of IFN-γ KO mice were significantly more severe than those of wild type (WT) controls. After antigenic stimulation, the proliferation of splenic mononuclear cells was significantly higher in IFN-γ KO than in WT mice with EAN. At the peak of EAN, the proportion of interleukin (IL)-17A expressing cells in cauda equina (CE) infiltrating cells, and the levels of IL-17A in sera were elevated in IFN-γ KO mice when compared with their WT counterparts. The proportions of major histocompatibility complex (MHC) II, macrosialin, and IL-12/IL-23p40 expressing cells, relative to total CE infiltrating cells were correspondingly higher in IFN-γ KO than in WT mice with EAN. However, IFN-γ deficiency reduced the production of NO by cultured macrophages in response to proinflammatory stimuli and induced a systemic Th2-oriented immune response. In conclusion, IFN-γ deficiency exacerbates EAN via upregulating Th17 cells despite a mitigated systemic Th1 immune response.

Early influx of macrophages determines susceptibility to experimental allergic encephalomyelitis in Dark Agouti (DA) rats.

Experimental allergic encephalomyelitis (EAE) is characterized by inflammatory infiltrates of myelin antigen(s) specific T cells and consecutive demyelination. Injection of encephalitogen into the footpads induces disease in genetically susceptible Dark Agouti rats (DA) but not in Albino Oxford (AO) rats although mild inflammatory infiltrates are observed in both strains early after disease induction. In addition, only DA rats develop disease when cells from (AO×DA) F(1) hybrids are passively transferred into sub-lethally radiated AO and DA parent hosts. The aim of the study was therefore to examine the participation of accessory cells, macrophages, dendritic cells and microglia in EAE development at the level of the target tissue in these two strains using specific membrane markers. We demonstrate here that in the induction phase of EAE in DA rats, macrophages (CD68(+); CD45(hi)CD11b(+)) are the first detectable infiltrating cells in the subpial regions of the spinal cord but were not found in AO rats. During the same period, resident microglial cells which are of the ramified variety are observed in both DA and AO rats. In DA rats at the peak of disease, when profuse influx of T cells is seen, macrophages and dendritic cells appear in the parenchyma of the CNS. In addition, at that time, microglial cells are activated. FACS analyses also reveal a significant increase in CD45(hi)CD11c(+) dendritic cells and CD45(hi)D11b(+) macrophages compared with levels in naïve and immunized AO rats. During resolution of disease in DA rats, the expression of microglia and macrophage markers is comparable with those in naïve non-immunized DA and immunized AO rats. We conclude that an initial influx of macrophages is indispensible for the development of EAE in DA rats. The presence of dendritic cells and myeloid dendritic cells at the peak of disease supports the role of these cells in EAE especially in relapses and chronicity. The activation pattern of microglia in DA rats does not indicate their role as antigen presenting cells in disease induction since they are ramified at the induction phase and only become activated after the overwhelming influx of T cells.

Regulatory T cells and ST2 signaling control diabetes induction with multiple low doses of streptozotocin.

Several peripheral mechanisms appear to be operational in limiting autoimmune damage of the islets of Langerhans and organ-specific T cell-mediated autoimmunity in general. These include cyclophosphamide sensitive T regulatory cells (Treg cells) and Th2 derived cytokine downregulation. We used the model of multiple low doses of streptozotocin (MLD-STZ) induced diabetes in susceptible C57BL/6 mice and resistant BALB/c mice to study these regulatory mechanisms. We show that low dose cyclophosphamide (CY) sensitive CD4(+)CD25(+)FoxP3(+) Treg cell-dependent mechanisms can be demonstrated in C57Bl/6 mice susceptible to MLD-STZ diabetes induction. CY pretreatment decreased Foxp3(+) cell count, glycemia, glycosuria and insulitis. In contrast, CY did not overcome resistance to diabetes induction in BALB/c mice. However, in BALB/c mice, deletion of ST2, an orphan member of the IL-1R family responsible for Th2 cell signaling leads to enhanced susceptibility to diabetes induction as evaluated by level of glycemia and glycosuria, number of infiltrating cells and beta cell loss. RT-PCR analysis of mRNA transcripts of diabetogenic cytokines revealed that the expression of TNF-alpha, and IFN-gamma was significantly enhanced in pancreatic lymph nodes by day 10 after diabetes induction in ST2-deficient mice in comparison with wild type BALB/c mice while IL-17 was detected only in ST2(-/-) mice by day 21. Our results are compatible with the notion that Treg cells are involved in MLD-STZ diabetes in susceptible mice and demonstrate that ST2-mediated signaling may also be involved in limiting Th1/Th17-mediated autoimmune pathology in diabetes resistant strain.

Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis.

Galectin-3 (Gal-3) is a member of the beta-galactoside-binding lectin family and plays an important role in inflammation. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Gal-3 deficient (Gal-3-/-) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3-/- mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3-/- mice produced less IL-17 and IFN-gamma than did those of the WT mice. In contrast, Gal-3-/- mice produced more serum IL-10, IL-5, and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3-/- mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3-/- dendritic cells induced Ag-specific T cells to produce more IL-10, IL-5, and IL-12, but less IL-17, than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 production.

Sensory and autonomic nerve changes in the monosodium glutamate-treated rat: a model of type II diabetes.

Rats that had been injected with monosodium glutamate (MSG) neonatally were studied for up to 70 weeks and compared with age-matched control rats to study changes in glucose tolerance and in sympathetic and sensory nerves. At 61 and 65 weeks of age, there were significant differences in glucose tolerance between the MSG and control groups, and the MSG group had raised fasting blood glucose. These changes were not associated with changes in the number of beta-cells in the islets of Langerhans. In addition, the diabetic MSG-treated rats had central obesity and cataracts. Hypoalgesia to thermal stimuli was present in MSG-treated rats as early as 6 weeks and persisted at 70 weeks. However, no differences were observed in the distribution of substance P, the neurokinin-1 receptor or calcitonin gene-related peptide in the dorsal horn of L3-L5 at this age (70 weeks). Diabetic MSG-treated animals at 65 and 70 weeks of age had significantly reduced noradrenaline concentrations in the heart, tail artery and ileum, while concentrations in the adrenal gland and corpus cavernosum were significantly increased. There was also a significant increase in adrenal adrenaline, dopamine and serotonin, largely attributable to changes in weight of the adrenal gland in the MSG-treated animals. The results indicate that MSG-treated animals develop a form of type II diabetes by about 60 weeks of age, and that there are significant changes in amine levels in various tissues associated with these developments.

The effect of streptozotocin-induced diabetes on the rat seminal vesicle: A possible pathophysiological basis for disorders of ejaculation.

In the streptozotocin (STZ)-diabetic rat major increases in noradrenaline concentration and content of the seminal vesicles were evident as early as 7 weeks following induction of hyperglycemia and returned toward normal after 34 weeks of hyperglycemia. There were significant reductions in the concentration and content of dopamine at 19-42 weeks of diabetes, and small occasionally significant reductions in the content of serotonin and adrenaline, particularly around 19-26 weeks after STZ treatment. The uptake of tritiated noradrenaline in the diabetics was increased at 12 weeks compared to the controls, and decreased to control levels with increasing age. Release of tritiated noradrenline was increased in response to electrical field stimulation and high potassium solutions, and raising calcium concentration caused increased release at rest and during electrical stimulation. Immunohistochemical demonstration of tyrosine hydroxylase was increased during the period when the noradrenaline concentration and content were elevated. It is concluded that there are significant changes in the sympathetic innervation of the seminal vesicle during the course of STZ diabetes, and that alterations in the reuptake, release, and synthesis of the neurotransmitter noradrenaline may contribute to changes in the concentration of the amine in the tissue. It is possible that the changes observed are related to the remodeling and regrowth of sympathetic nerve endings damaged in the early stages of hyperglycemia. These changes may also contribute to disorders of ejaculation in diabetes.

IL-23 leads to diabetes induction after subdiabetogenic treatment with multiple low doses of streptozotocin.

IL-23, a proximal regulator of IL-17, may be a major driving force in the induction of autoimmune inflammation. We have used a model of subdiabetogenic treatment with multiple low doses of streptozotocin (MLD-STZ; 4 x 40 mg/kg body weight) in male C57BL/6 mice to study the effect of IL-23 on immune-mediated beta cell damage and the development of diabetes, as evaluated by blood glucose, quantitative histology, immunohistochemistry and expression of relevant cytokines in the islets. Ten daily injections of 400 ng IL-23, starting on the first day of MLD-STZ administration led to significant and sustained hyperglycemia along with weight loss compared with controls (no IL-23), and a significant increase in the number of infiltrating cells, a lower insulin content, enhanced apoptosis, expression of IFN-gamma and IL-17 (not seen in the controls) and a significant increase in the expression of TNF-alpha and IL-18 in the pancreatic islets. IL-23 treatment started 5 days prior to MLD-STZ administration had no effect on diabetogenesis or cytokines expression in the pancreatic islets. We provide the first evidence in an animal model that IL-23 is involved in the development of type-1 diabetes, by inducing IL-17 and possibly IFN-gamma production in the target tissue.

Effects of ovariectomy and hormone replacement on collagen and blood vessels of the urethral submucosa of rats.

Collagen and blood vessels of the urethral submucosa of ovariectomized rats were studied following 28 daily subcutaneous injections of 17-beta estradiol (n=6, group 1), medroxy-progesterone acetate (n=6, group 2), both drugs (n=6, group 3) or vehicle (n= 6, control) and after sham surgery without castration or injection (n=6). Investigations included the immunohistochemistry of estrogen and progesterone receptors and collagen fibres, Western blot analysis of collagen types I and III and counting periurethral vessels by light microscopy. Our results showed positive immunostaining with estrogen, progesterone and collagen types I and III in all samples. Collagen type I and III levels were lower in the controls than in the sham group. The other groups showed increases (2>3>1) over the controls with a relatively higher increase in type III. The type I/III collagen ratio progressively decreased (control>1>2>3) below sham levels. The mean vessel count was significantly lower in control than in sham animals (P<0.00001). However, only estrogen treatment significantly increased the vessel number compared to controls (P=0.04). Our results indicate that estrogen and progesterone, alone or in combination, have an effect on collagen types I and III, and that estrogen has an effect on blood vessels of the urethral submucosa in female rats.