Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

Active insulin receptors (IR) phosphorylate insulin receptor substrate (IRS), but it is not clear whether IRS is phosphorylated mainly by IR at the plasma membrane or by internalized IR in the cytosol. In this thesis, structural identifiability analysis and parameter sensitivity analysis is performed for models of the first steps in the metabolic insulin signaling pathway. In particular, the identifiability of the kinetic parameters governing IRS phosphorylation are investigated. Given measurements of the relative increase in phosphorylation degree of IR and IRS, the structural identifiability analysis revealed that the parameters governing IRS phosphorylation are non-identifiable, but their ratio is identifiable. This is sufficient to study whether phosphorylation of IRS proceeds more rapidly by IR at the plasma membrane or by internalized IR in the cytosol. In the examined model structure, internalization of insulin receptors is shown to be necessary to reproduce the experimental data. Sensitivity analysis of the parameters governing IRS phosphorylation showed that many parameters need to be known in order to obtain ``practical identifiability'' of the interesting parameters.

Methylglyoxal (MG) is a reactive metabolite presents in all biological systems. The accumulation of MG in diabetic patients and animals has been long recognized. Recently, studies have shown that MG levels are elevated in hypertensive rats. However, the pathological effects of MG in diabetes and related insulin resistance syndrome such as obesity are currently unknown. In the present study, the role of MG in the pathogenesis of insulin resistance was investigated.<p> First, it was observed that MG induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG-insulin adducts, as evidenced by additional peaks observed upon mass spectrometric (MS) analysis. Tandem MS analysis of insulin B-chain adducts confirmed attachment of MG at an arginine residue. [3H]-2-deoxyglucose uptake ([3H]-2-DOG) by 3T3-L1 adipocytes was significantly and concentration-dependently decreased after treatment with MG-insulin adducts, in comparison with the effect of native insulin at the same concentration. A significant decrease of glucose uptake induced by MG-insulin adducts was also observed in L8 skeletal muscle cells. Unlike native insulin, MG-insulin adducts did not inhibit insulin release from pancreatic â-cells. The degradation of MG-insulin by cultured liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in the â-chain of insulin. The formation of this MG-insulin adduct decreases insulin-mediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of the insulin molecule may contribute to the pathogenesis of insulin resistance.<p> Second, the effects of MG on the insulin signaling pathway were investigated. After 9 weeks of fructose treatment, an insulin resistant state was developed in Sprague-Dawley (SD) rats, demonstrated as increased triglyceride and insulin levels, elevated blood pressure, and decreased insulin-stimulated glucose uptake by adipose tissue. A close correlation between insulin resistance and the elevated MG accumulation in adipose and skeletal muscle tissues was observed. The insulin resistant state and the elevated MG level were reversed by the MG scavenger, N-acetyl cysteine (NAC) and metformin. In cultured adipose cells, MG treatment impaired insulin signaling as measured by decreased tyrosine phosphorylation of insulin-receptor substrate-1 (IRS-1) and the decreased kinase activity of phosphatidylinositol 3-kinase (PI3K). The ability of NAC to block MG-impairment of PI3K activity and IRS-1 phosphorylation further confirmed the role of MG in the development of insulin resistance. In cultured skeletal muscle cells, MG treatment significantly reduced the expression of IRS-1 and PI3K at the mRNA level. Similar to adipose cells, MG also decreased tyrosine phosphorylation of IRS-1 and PI3K activity. We also examined the mechanism of metformin to inhibit AGEs. Using mass spectrometry, stable metformin-MG adducts were identified.<p> In addition, we investigated the causative effect of MG in the pathogenesis of obesity, another form of insulin resistance. This study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulating Akt signaling. In Zucker fatty rats, dramatically increased MG accumulations in serum and different tissues were identified. The serum MG level increased age. In 10 and 12 week-old obese rats, MG was 144±50% and 171±15% of the age-matched control Zucker rats; this value increased to 241±7 % and 329±10% by 14 and 16 weeks (P<0.05, n=4). Further study suggested that MG accumulation stimulates the phosphorylation of Akt and its effectors p21 and p27. The activated Akt pathway then increased the activity of Cdk2 and accelerates the cell cycle progression and proliferation of pre-adipocytes. The effects of MG were efficiently reversed by both alagebrium, and Akt inhibitor SH-6.<p> Overall, the current study investigated the effect of MG during the pathogenesis of insulin resistance syndrome. MG, as the most potent precursor of AGEs, impairs the activity of insulin signaling pathway by glycating the insulin molecule and other insulin signaling proteins. Moreover, this study observed a previously unrecognized causative effect of MG in the proliferation of adipocytes by up-regulating the Akt signaling pathway. The results from this study offer new mechanisms to explain the development of insulin resistance and to prevent the related diseases.

In Drosophila, neuropeptides have regulatory roles in development, growth, metabolism and reproduction. This study focused on GABA and the neuropeptides Drosophila tachykinin (DTK), short neuropeptide F (sNPF), adipokinetic hormone (AKH), corazonin (CRZ) and Drosophila insulin-like peptides (DILPs) as possible regulators of metabolic stress responses and homeostasis. We showed that metabotropic GABAB receptors (GBRs) are expressed on brain insulin producing cells (IPCs), suggesting an inhibitory regulation of these cells by GABA. Knockdown of GBR on IPCs shortened lifespan and stress resistance, altered carbohydrate and lipid metabolism at stress (paper I). We showed that three different neuropeptides; DTK, sNPF and ITP, are co-expressed in five pairs of adult neurosecretory cells (paper II). ITP-knock down was not studied yet, but sNPF- and DTK-knock down flies showed decreased stress resistance at desiccation and starvation and decreased water levels at desiccation, suggesting that these peptides are involved in water homeostasis during stress conditions. sNPF was previously shown to affect feeding, growth and DILP expression via the IPCs, but it was not known which sNPF-expressing neurons are responsible for these actions. We could identify a specific set of bilateral neurons (DLPs) that co-express sNPF and corazonin that target the IPCs. We showed that these peptides co-released from DLPs regulate DILP transcription and probably release in the adult Drosophila brain and thus have roles in regulation of stress resistance and metabolism (paper III). AKH signaling was previously shown to affect hemolymph carbohydrate levels and lipid stores in Drosophila. Insulin (DILP) signaling and AKH signaling are suggested to have opposing effects on lipid and sugar metabolism in Drosophila. We studied the possible functional relationship between these two systems; do they mutually regulate each other?  Our results suggest action of DILPs via the Insulin Receptor on the IPCs and the AKH producing cells, but we could not provide evidence for AKH action on IPCs or AKH cells (paper IV). / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Epub ahead of print. Paper 4: Manuscript.</p>

The present study revealed that FOXO1 is present in the nuclei of non-dividing pituitary cells and in a subset of differentiated cells with highest level of expression in somatotrophs, followed by corticotrophs, thyrotrophs and gonadotrophs throughout development and in adulthood stage. A significant difference in Foxo1 transcript between age-matched males and females at 8-9 weeks of age was demonstrated in the anterior pituitary for the first time. IHC data demonstrating (i) FOXO1 co-localization with p27kip1 (ii) an increase in FOXO1 immunopositive cells within anterior pituitary in p27KO embryos compared to WT (iii) absence of FOXO1 in the nucleus of BrdU positive cells suggested that in absence of p27Kip1 FOXO1 might be important for preventing unbridled cell proliferation. Data suggested that FOXO1 might not be important for initiating pituitary cell differentiation but might be involved with p27kip1 in maintaining pituitary cell quiescence. Increase in nuclear localization of FOXO1 in the pituitary of Foxp3 mutant (lacking insulin signaling) suggested that it might be a down-stream target of insulin/PI3K/PKB pathway in the pituitary as it is in several other tissues.

Orientador: Patrícia Monteiro Seraphim / Banca: Doris Hissako Sumida / Banca: Henrique Luiz Monteiro / Resumo: A obesidade é uma condição que afeta muitos indivíduos e está relacionada a vários tipos de disfunções,como a resistência insulínica.Esta patologiase dá devido a uma falha na sinalização entre o hormônio, proteínas intracelularese o GLUT4 em células musculares e adiposas. Alguns fatores contribuem para aumentar esta falha, como o aumento da fosforilação em serinado IRS -1 e atividade pró -inflamatória. Uma maneira indireta de se avaliar o grau de inflamação é analisar a expressão do SOCS3,que estámais expressoquando há maior atividade inflamatória. Aprática de exercício físico aparece como uma importante ferramenta, pois melhora a sensibilidade insulínica e pode aumentara expressão do transportador de glicose até mesmo em animais obesos. Oobjetivo do trabalho foiavaliar o efeito dos exercícios aeróbio contínuo e com pesoscombinadossobre a via de sinalização da insulina e expressão de transportador... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Obesity is a condition that affects manypeople and is related with some kindof diseases, like insulin resistance.This pathology occurs because of animpairmenton activation of GLUT4 machinery (insulin signaling, activation of intracellular proteins) in skeletal muscle and adipose tissue. Some factors contributedirectly with this illness, like increase IRS -1 serinephosphorylationand pro -inflammatoryactivity. An indirect way to measure inflammation istoanalyze expression of SOCS3, because when it is overexpressed means thatthere are more pro -inflammatory activity.Literature shows that physical exercise can improve insulin sensitivity by increases of GLUT4 expression even in obese rats. So we aimed... (Complete abstract click electronic access below) / Mestre

Chemerin e seu receptor (ChemR23) têm sido amplamente associados à disfunção endotelial, inflamação e resistência à insulina. No entanto, é ainda desconhecido se chemerin influencia diretamente a sinalização da insulina na vasculatura. A hipótese deste estudo é de que chemerin diminui a sinalização vascular da insulina, e que o uso de antagonista de ChemR23 (CCX 832) em um modelo de diabetes do tipo 2 relacionado à obesidade melhora as respostas vasculares a insulina. Mecanismos moleculares e vasculares foram investigados em artérias mesentéricas e células de músculo liso vascular em cultura (CMLV) de camundongos C57BL/6J, db/m (controles, não obesos, não diabéticos) e db/db (diabéticos, obesos), assim como em células endoteliais (CE) de humanos em cultura. Nossos resultados mostraram que chemerin diminui a vasodilatação induzida por insulina em camundongos C57BL/6J, efeito mediado por ChemR23, PI3K/Akt e estresse oxidativo. Em CMLV, chemerin, através de mecanismos dependentes de estresse oxidativo e ChemR23, diminui a fosforilação de IRS-1, PI3K e Akt e a translocação de GLUT4 para a membrana, induzidas por insulina. Chemerin também diminui a captação de glicose induzida por insulina via estresse oxidativo e ativação de AMPK e PI3K/Akt. Em CE, chemerin diminui a sinalização de óxido nítrico (NO) ativada pela insulina, novamente via ChemR23, estresse oxidativo e PI3K/Akt. CCX 832 diminui a massa corporal (sem alterar a ingestão de ração), os níveis de insulina e glicose (sem alterar a tolerância à glicose) e estresse oxidativo em aorta e rim de camundongos db/db. CCX 832 restaura parcialmente a disfunção vascular observada em camundongos db/db, sem modificar parâmetros estruturais destas artérias. Adicionalmente, CCX 832 diminui marcadores pró-inflamatórios em tecido adiposo perivascular (PVAT) e melhora a sinalização da insulina em aorta de camundongos db/db. Nossos achados destacam o sistema chemerin/ChemR23 como um novo e promissor alvo terapêutico para limitar a resistência à insulina e as complicações vasculares associadas ao diabetes relacionado à obesidade. / Chemerin and its G protein-coupled receptor (ChemR23) have been associated with endothelial dysfunction, inflammation and insulin resistance. Whether chemerin directly influences insulin signaling in the vasculature is unknown. We hypothesized that chemerin impairs vascular insulin signaling in obesity-related type 2 diabetes, effect that would be improved by the novel ChemR23 antagonist (CCX 832). Molecular and vascular mechanisms were probed in mesenteric arteries and cultured vascular smooth muscle cells (VSMC) from C57BL/6J, non-diabetic lean db/m and diabetic obese db/db mice as well as in human microvascular endothelial cells (EC). Chemerin decreased insulin-induced vasodilatation in C57BL/6J mice, effect mediated by ChemR23, PI3K/Akt and oxidative stress. In VSMC, chemerin, via oxidative stress- and ChemR23-dependent mechanisms, decreased insulin-induced IRS-1, PI3K and Akt phosphorylation, GLUT4 translocation to the membrane. In addition, chemerin decreases insulin-induced glucose uptake via oxidative stress and AMPK and PI3K/Akt activation. In EC, chemerin decreased insulin-activated nitric oxide (NO) signaling via ChemR23, oxidative stress and PI3K/Akt signaling pathway. CCX 832 decreases body weight (without altering food intake), insulin and glucose levels (without altering glucose tolerance) and oxidative stress in aorta and kidney from db/db mice. CCX 832 partially restored vascular dysfunction in db/db mice without modifying structural parameters. Additionally, CCX 832 decreases proinflammatory markers in perivascular adipose tissue (PVAT) and improves insulin signaling in aorta from db/db mice. Our findings highlight chemerin/ChemR23 system as a promising new therapeutic target to limit insulin resistance and vascular complications associated with obesity-related diabetes.

Ames dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells – somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice also have high circulating levels of anti-inflammatory and antidiabetic adiponectin. Plasma levels of this adipokine usually decrease with an increase in accumulation of visceral fat (VF). We thus believe that VF in df/df mice, developed in the absence of GH signaling, may be functionally different from the same fat depots in normal (N) mice and may be beneficial, rather than detrimental, to the overall health of the animal. We performed surgeries involving removal of VF depots (epididymal and perirenal fat) in both groups of mice and hypothesize that the beneficial effects of visceral fat removal (VFR) will be present exclusively in N mice as VF in df/df mice contributes to enhanced insulin sensitivity by producing decreased levels of pro-inflammatory adipokines like TNF and IL-6. We found that VFR improved insulin sensitivity only in N mice but not in the df/df mice. This intervention led to an upregulation of certain players of the insulin signaling pathway in the skeletal muscle of N mice only, with no alteration in df/df mice. The subcutaneous fat of df/df mice showed a downregulation of these insulin signaling genes upon VFR. Compared to N mice, epididymal fat of df/df mice (sham-operated) had increased gene expression of some of the players involved in insulin signaling and a decrease in transcript levels of TNFa. Ames dwarf mice had decreased levels of IL-6 protein in EF and in circulation. High circulating levels of adiponectin and iv decreased levels of IL-6 in circulation could contribute to the high insulin sensitivity observed in the Ames dwarf mice. Understanding the mechanisms responsible for VF having positive effects on insulin signaling in df/df mice would be important for future treatment of obese diabetic patients.

No description available.

The causes of Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease that results in skeletal muscle paralysis, remain unclear. However, a nuclear DNA and RNA binding protein called TAR DNA binding protein 43 (TDP-43) has emerged as a critical marker of ALS pathology. A previous drug screen conducted in the Zarnescu laboratory showed that anti-diabetic drugs can rescue lethality in a fruit fly model of ALS based on TDP-43. These results suggested that the insulin signaling pathway might be altered in motor neurons in a TDP-43 dependent manner. Therefore, we hypothesized that the insulin pathway is interacting with TDP-43 in vivo and may be contributing to TDP-43neurotoxicity. Using genetic interaction approaches in flies we found that TDP-43dependent locomotor defects are sensitive to the levels of insulin receptor activity. In addition, genetic interaction data suggest that Akt is hyperactivated in motor neurons expressing TDP-43, possibly as a compensatory mechanism to enable survival. Finally, upregulating protein synthesis through S6K and 4EBP appears to have beneficial effects. These findings support our hypothesis and provide insights into potential therapeutic strategies to help treat this devastating disease.