transforming-growth-factor-beta and Prediabetic-State

Alcohol consumption exacerbates liver abnormalities in animal models, but whether it increases the severity of liver disease in early diabetic or prediabetic rats is unclear. To investigate the molecular mechanisms underlying alcohol-induced liver steatosis or hepatitis, we used a prediabetic animal model. Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Fatty (LETO) rats were pair-fed with an ethanol-containing liquid diet for 6 weeks. Compared with controls, OLETF and LETO rats displayed more pronounced liver steatosis and higher plasma levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamate pyruvate transaminase (SPGT), indicating liver injury. Ethanol-fed LETO (Pd-L-E) rats showed mild liver steatosis and no inflammation compared with ethanol-fed OLETF (Pd-O-E) rats. Although precursor and active SREBP-1 levels in the liver of ethanol-fed OLETF rats significantly increased compared with control diet-fed OLETF rats (Pd-O-C), those of Pd-L-E rats did not. Bone morphogenetic protein (BMP) and TGF-β1 balance in Pd-O-E rats was significantly altered because BMP signaling was upregulated by inducing BMP2, BMP4, BMP7, BMP9, Smad1, and Smad4, whereas TGF-β1, Smad3, and Erk were downregulated. Activation of TGF-β/Smad signaling inhibited BMP2 and BMP9 expression and increased epithelial-mesenchymal transition (EMT) marker levels (Hepcidin, Snail, and Twist) in the liver of LETO rats. Livers of ethanol-fed OLETF rats showed increased levels of vimentin, FSP-1, α-SMA, MMP1, MMP13, and collagen III compared with rats of other groups, whereas EMT marker levels did not change. Thus, BMP exerted anti- and/or pro-fibrotic effects in ethanol-fed rats. Therefore, BMP and TGF-β, two key members of the TGF-β superfamily, play important but diverse roles in liver steatosis in young LETO and OLETF rats.

Topics: Animals; Diabetes Mellitus, Type 2; Disease Models, Animal; Ethanol; Male; Non-alcoholic Fatty Liver Disease; Prediabetic State; Rats; Rats, Inbred OLETF; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study evaluated early renal functional, structural, and biochemical changes in high-calorie/high-fat diet fed mice, a model of prediabetes and alimentary obesity. Male C57BL6/J mice were fed normal (11 kcal% fat) or high-fat (58 kcal% fat) diets for 16 wk. Renal changes were evaluated by histochemistry and immunohistochemistry, Western blot analysis, ELISA, enzymatic assays, and chemiluminometry. High-fat diet consumption led to increased body and kidney weights, impaired glucose tolerance, hyperinsulinemia, polyuria, a 2.7-fold increase in 24-h urinary albumin excretion, 20% increase in renal glomerular volume, 18% increase in renal collagen deposition, and 8% drop of glomerular podocytes. It also resulted in a 5.3-fold increase in urinary 8-isoprostane excretion and a 38% increase in renal cortex 4-hydroxynonenal adduct accumulation. 4-hydroxynonenal adduct level and immunoreactivity or Sirtuin 1 expression in renal medulla were not affected. Studies of potential mechanisms of the high-fat diet induced renal cortex oxidative injury revealed that whereas nicotinamide adenine dinucleotide phosphate reduced form oxidase activity only tended to increase, 12/15-lipoxygenase was significantly up-regulated, with approximately 12% increase in the enzyme protein expression and approximately 2-fold accumulation of 12(S)-hydroxyeicosatetraenoic acid, a marker of 12/15-lipoxygenase activity. Accumulation of periodic acid-Schiff -positive material, concentrations of TGF-β, sorbitol pathway intermediates, and expression of nephrin, CAAT/enhancer-binding protein homologous protein, phosphoeukaryotic initiation factor-α, and total eukaryotic initiation factor-α in the renal cortex were indistinguishable between experimental groups. Vascular endothelial growth factor concentrations were reduced in high-fat diet fed mice. In conclusion, systemic and renal cortex oxidative stress associated with 12/15-lipoxygenase overexpression and activation is an early phenomenon caused by high-calorie/high-fat diet consumption and a likely contributor to kidney disease associated with prediabetes and alimentary obesity.

Topics: Animal Feed; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Diabetic Nephropathies; Diet; Diet, High-Fat; Kidney Cortex; Male; Mice; Mice, Inbred C57BL; Models, Biological; Obesity; Oxidative Stress; Podocytes; Prediabetic State; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-β (TGF-β) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-β, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ-stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting β-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.

Topics: Animals; Cell Proliferation; Cell Tracking; Cells, Cultured; Diabetes Mellitus, Type 1; Female; Gene Expression Regulation; Immunotherapy, Adoptive; Insulin-Secreting Cells; Interferon-gamma Release Tests; Interleukin-10; Interleukin-4; Lymph Nodes; Macrophage Activation; Macrophages; Mice; Mice, Inbred NOD; Mice, Transgenic; Pancreas; Prediabetic State; RNA, Messenger; Transforming Growth Factor beta

Signaling through TLR2 promotes inflammation and modulates CD4(+) CD25(+) Tregs. We assessed mechanistically how this molecule would alter immunoregulation in type 1 diabetes (T1D). We also asked whether TLR2 may be involved in our recent discovery that viral infection can protect from autoimmune diabetes by expanding and invigorating Tregs. Treatment of prediabetic mice with a synthetic TLR2 agonist diminished T1D and increased the number and function of CD4(+) CD25(+) Tregs, also conferring DCs with tolerogenic properties. TLR2 ligation also promoted the expansion of Tregs upon culture with DCs and ameliorated their capacity to prevent the disease. Protection from T1D by lymphocytic choriomeningitis virus (LCMV) infection depended on TLR2. LCMV increased the frequency of CD4(+) CD25(+) Tregs and their production of TGF-β more significantly in WT than TLR2-deficient mice. Furthermore, LCMV infection in vivo or LCMV-infected DCs in vitro rendered, via TLR2, CD4(+) CD25(+) Tregs capable of diminishing T1D. We identify novel mechanisms by which TLR2 promotes immunoregulation and controls autoimmune diabetes in naïve or infected hosts. This work should help understand T1D etiology and develop novel immune-based therapeutic interventions.

Topics: Animals; Arenaviridae Infections; CD4 Antigens; Cells, Cultured; Dendritic Cells; Diabetes Mellitus, Type 1; Flow Cytometry; Immunity, Innate; Inflammation; Interleukin-2 Receptor alpha Subunit; Lymphocytic choriomeningitis virus; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Prediabetic State; Signal Transduction; T-Lymphocytes, Regulatory; Toll-Like Receptor 2; Transforming Growth Factor beta

Periodontitis was shown to have an impact on glucose levels in prediabetic and diabetic rats. The Zucker fatty rat (ZFR) is a well-characterized model of prediabetes presenting with impaired glucose tolerance, hyperinsulinemia, dyslipidemia, and moderate hypertension. The aim of the present study was to investigate whether periodontitis influences kidney changes in ZFRs.. Male adult ZFRs (N = 19) and their lean littermates (N = 18) were studied. Periodontitis was induced with ligatures in half of the ZFRs and lean rats, whereas the other half served as controls. After 4 weeks, the rats were sacrificed, and the kidneys, liver, and heart were removed and weighed. Kidneys were evaluated histologically for glomerular volume and renal mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptor 2, transforming growth factor-beta, connective tissue growth factor, collagen IValpha1, fibronectin, and nephrin. Urinary albumin excretion and creatinine clearance were also evaluated.. In prediabetic ZFRs, periodontitis was associated with kidney hypertrophy (P = 0.03) and a tendency for increased glomerular volume (P = 0.06). In lean littermates, elevated fibronectin mRNA levels (P = 0.03) were noted in the presence of periodontitis.. Our findings suggest the participation of periodontitis in the development of early renal changes in ZFRs.

Topics: Albuminuria; Animals; Collagen Type IV; Connective Tissue Growth Factor; Creatinine; Fibronectins; Hypertrophy; Immediate-Early Proteins; Insulin-Like Growth Factor Binding Proteins; Intercellular Signaling Peptides and Proteins; Kidney; Kidney Diseases; Kidney Glomerulus; Male; Membrane Proteins; Organ Size; Periodontitis; Prediabetic State; Random Allocation; Rats; Rats, Zucker; RNA, Messenger; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Whether temporary angiotensin II (AngII) blockade at the prediabetic stage attenuates renal injury in type 2 diabetic OLETF rats later in life was investigated. OLETF rats were treated with an AT(1) receptor antagonist (olmesartan, 0.01% in food), angiotensin-converting enzyme inhibitor (temocapril, 0.01% in food), a combination of the two, or hydralazine (25 mg/kg per d) at the prediabetic stage (4 to 11 wk of age) and then monitored without further treatment until 50 wk of age. At 11 wk of age, blood glucose levels and urinary protein excretion (U(protein)V) were similar between OLETF and control LETO rats. However, OLETF rats showed higher kidney AngII contents and type IV collagen mRNA expression than LETO rats at this age. These decreased with olmesartan, temocapril, and a combination of these but not with hydralazine. At 50 wk of age, diabetic OLETF rats showed higher BP, U(protein)V, and intrarenal AngII levels than LETO rats. Temporary AngII blockade did not affect glucose metabolism or the development of hypertension in OLETF rats but significantly suppressed proteinuria and ameliorated glomerular injury. However, no parameters were affected by temporary hydralazine treatment. The present study demonstrated that intrarenal AngII and type IV collagen expression are already augmented long before diabetes becomes apparent in OLETF rats. Furthermore, temporary AngII blockade at the prediabetic stage attenuates the progression of renal injury in these animals. These data suggest that early AngII blockade could be an effective strategy for preventing the development of type 2 diabetic renal injury later in life.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Blood Glucose; Blood Pressure; Body Weight; Collagen; Connective Tissue Growth Factor; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Drug Therapy, Combination; Hydralazine; Imidazoles; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; NADPH Oxidases; Olmesartan Medoxomil; Prediabetic State; Rats; Rats, Inbred OLETF; Receptors, Angiotensin; Renin; Tetrazoles; Thiazepines; Thiobarbituric Acid Reactive Substances; Transforming Growth Factor beta

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8(+) T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8(+) T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4(+)CD25(-)CD62L(+) but not CD4(+)CD25(+)CD62L(+) T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8(+) T cell AICD and increasing the number of CD4(+)CD25(+)CD62L(+) T cells in the periphery.

Topics: Aging; Animals; Antibodies, Monoclonal; Apoptosis; CD3 Complex; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Death; Diabetes Mellitus, Type 1; Female; Genetic Predisposition to Disease; Homeostasis; Humans; Interferon-gamma; Interleukin-10; Interleukin-4; L-Selectin; Lymphocyte Activation; Lymphocyte Depletion; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Prediabetic State; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Interleukin-2; Spleen; T-Lymphocyte Subsets; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Activation-induced cell death (AICD) represents a major means of peripheral tolerance induction, eliminating effector cells. NOD mice, a widely used model for autoimmune diabetes, are characterized by high levels of circulating T lymphocytes and by resistance to several apoptosis-inducing signals. The aim of this study was to analyse AICD in peripheral NOD T lymphocytes. First, we demonstrated in an in vitro AICD model that NOD T lymphocytes are more resistant to AICD (64+/-2%) compared to non-autoimmune C57BL/6 T lymphocytes (73+/-2%), but also diabetes-resistant NOR T lymphocytes (76+/-3%, P<0.05). Moreover, both CD4(+)and CD8(+)subsets were affected. Analysis of the cellular and molecular pathways revealed lower caspase 8 levels, a central caspase proximally involved in the AICD-pathway (fluorescence of 258+/-47 in NOD vs. 441+/-16 in NOR and 414+/-61 in C57BL/6 T lymphocytes, P<0.05). Gene expression analysis using real-time RT-PCR additionally revealed low expression of Fas and FasL, the death receptor system activating caspase 8 and contributing to AICD. Additionally, low IL-2 levels, together with high TGFbeta and Bclx-L levels, confirm the presence of a NOD-specific AICD-resistance profile. In conclusion, we present cellular and molecular evidence for disturbed AICD mechanisms in NOD T lymphocytes. This resistance in AICD may contribute to defective tolerance induction to autoantigens in NOD mice.

Topics: Animals; Apoptosis; bcl-X Protein; Caspase 8; Caspases; Cell Survival; Diabetes Mellitus, Type 1; Fas Ligand Protein; fas Receptor; Female; Gene Expression; In Vitro Techniques; Interleukin-2; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Prediabetic State; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; T-Lymphocytes; Transforming Growth Factor beta

B cells can serve dual roles in modulating T cell immunity through their potent capacity to present Ag and induce regulatory tolerance. Although B cells are necessary components for the initiation of spontaneous T cell autoimmunity to beta cell Ags in nonobese diabetic (NOD) mice, the role of activated B cells in the autoimmune process is poorly understood. In this study, we show that LPS-activated B cells, but not control B cells, express Fas ligand and secrete TGF-beta. Coincubation of diabetogenic T cells with activated B cells in vitro leads to the apoptosis of both T and B lymphocytes. Transfusion of activated B cells, but not control B cells, into prediabetic NOD mice inhibited spontaneous Th1 autoimmunity, but did not promote Th2 responses to beta cell autoantigens. Furthermore, this treatment induced mononuclear cell apoptosis predominantly in the spleen and temporarily impaired the activity of APCs. Cotransfer of activated B cells with diabetogenic splenic T cells prevented the adoptive transfer of type I diabetes mellitus (T1DM) to NOD/scid mice. Importantly, whereas 90% of NOD mice treated with control B cells developed T1DM within 27 wk, <20% of the NOD mice treated with activated B cells became hyperglycemic up to 1 year of age. Our data suggest that activated B cells can down-regulate pathogenic Th1 immunity through triggering the apoptosis of Th1 cells and/or inhibition of APC activity by the secretion of TGF-beta. These findings provide new insights into T-B cell interactions and may aid in the design of new therapies for human T1DM.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Apoptosis; Autoantigens; B-Lymphocyte Subsets; Cell Communication; Cell Separation; Diabetes Mellitus, Type 1; Down-Regulation; Fas Ligand Protein; fas Receptor; Female; Islets of Langerhans; Leukocytes, Mononuclear; Ligands; Lipopolysaccharides; Lymphocyte Activation; Lymphocyte Transfusion; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mice, SCID; Prediabetic State; Spleen; T-Lymphocyte Subsets; Th1 Cells; Transforming Growth Factor beta

The alterations of TGF-beta1 production are believed to contribute to the development of insulin-dependent diabetes mellitus (IDDM) in animal models as well as in humans. There is also increasing evidence about the role of this cytokine in the pathogenesis of diabetic vascular complications. The aim of our study was to evaluate in vitro TGF-beta1 production by peripheral blood of newly diagnosed type 1 diabetes patients and subjects in the pre-clinical stage of the disease in comparison to healthy controls and relatives of IDDM patients with low genetic risk for diabetes development. The study was carried out in three groups of subjects: 22 patients with a recently diagnosed type 1 diabetes, their 24 first degree relatives with a different genetic risk of IDDM development and 18 healthy volunteers (control group). In all studied groups whole blood was taken for morphology parameters. HbA1C and for 72 h cultures with PHA stimulation for the estimation of TGF-beta1 in vitro production. TGF-beta1 concentration in supernatants were quantified by ELISA. In the first degree relatives HLA typing (for DR3, DR4 and DQB1*0602 alleles), measurements of anti-pancreatic antibodies (ICA, GADA, IA-2A, IAA) and intravenous glucose tolerance tests were performed. The levels of TGF-beta1 in the supernatants were significantly higher in diabetic patients (P < 0.0002) and in their first degree relatives (P < 0.05) in comparison to the control group. In the group of first degree relatives TGF-beta1 levels were highest in subjects with the presence of two or more pancreatic autoantibodies and/or with impaired insulin release in IVGTT, but lowest in relatives with protective DQB1*0602 alleles (P < 0.01). There was also a significant positive correlation between the TGF-beta1 levels and HbA1C in the IDDM subjects and first degree relatives (P < 0.03). Our study suggests that the alterations of TGF-beta1 levels could be associated with the activity of autoimmune process leading to pancreatic B cells destruction and may have a role in the pathogenesis of diabetic complications, but further studies in humans are needed.

Topics: Adolescent; Adult; Animals; Autoantibodies; Case-Control Studies; Diabetes Mellitus, Type 1; Female; Glucose Tolerance Test; HLA-D Antigens; Humans; In Vitro Techniques; Male; Pancreas; Prediabetic State; Risk Factors; Transforming Growth Factor beta

Subcutaneous insulin treatment of young diabetes prone BB rats has been shown previously to suppress the development of autoimmune diabetes. In this study the hypothesis was tested that exogenous insulin may deviate the autoimmune process by acting on the Th1/Th2 cytokine balance in the pancreas. BB rats were implanted with pellets which continuously released insulin, at 50 d of age. Three weeks later cytokine mRNA expression in the pancreas and insulitis score were determined. While in control BB rats high levels of IFNgamma mRNA were detectable by RT-PCR, insulin treatment almost completely suppressed IFNgamma mRNA levels without concomitant upregulation of counterregulatory IL-10 and TGFbeta gene expression. Insulin also suppressed gene expression of inducible nitric oxide synthase. Mean insulitis scores were decreased after insulin treatment. We conclude that the protective effects of insulin treatment may not be due to the induction of protective Th2 immune reactivity but to general downregulation of immune activation in the pancreas, and hence also of Th1 autoimmunity.

Topics: Animals; Autoimmune Diseases; Gene Expression; Insulin; Interferon-gamma; Interleukin-10; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pancreas; Prediabetic State; Rats; Rats, Inbred BB; Rats, Wistar; Th1 Cells; Th2 Cells; Transforming Growth Factor beta