Chromium has been reported to increase glucose clearance in insulin resistant and diabetic populations. Skeletal muscle is the tissue primarily responsible for glucose clearance. We therefore tested the effect of chromium chloride (CrCl3) on skeletal muscle glucose uptake both in the absence and presence of a submaximal level of insulin via the rat hindlimb perfusion technique. 0.096 μM CrCl3 was used with and without 200 μU/ml insulin. Our testing showed that insulin significantly increased [H3]-2 deoxyglucose (2-DG) uptake in both the gastrocnemius and quadriceps muscles. Additionally, the combination of CrCl3 and insulin (Cr-sIns) led to greater amounts of 2-DG uptake than insulin alone (sIns) in both the gastrocnemius (Cr-sIns 6.49±0.75 μmol/g/h, sIns 4.83±0.42 μmol/g/h) and quadriceps (Cr-sIns 6.74±0.62 μmol/g/h, sIns 4.54±0.43 μmol/g/h). However, CrCl3 without insulin (Cr) had no affect on 2-DG uptake above basal (Bas) in both the gastrocnemius (Cr 1.45±0.14 μmol/g/h, Bas 1.61±30 μmol/g/h) and the quadriceps (Cr 1.35±0.15 μmol/g/h, Bas 1.27±0.13 μmol/g/h). It has been speculated that chromium works to increase glucose uptake by increasing insulin signaling. To examine this, we used western blotting analysis to test both Akt and AS160 phosphorylation in the mixed gastrocnemius. We found that insulin increased Akt and AS160 phosphorylation, but chromium had no affect on Akt (Cr-sIns 25%±2%, sIns 22%±4%) or AS160 (Cr-sIns 35%±5%, sIns 36%±4%) phosphorylation in the absence or presence of insulin. Our results suggest that supplementation with CrCl3 can lead to an increase in glucose uptake in skeletal muscle, but only in the presence of insulin. However, this effect of CrCl3 does not appear to be a result of enhanced insulin signaling. / text

This project focused on determining the effect of lithium on glucose uptake, glycogen synthesis, and insulin signaling proteins, protein kinase B (Akt1) and GSK-3, in isolated soleus muscle from female lean Zucker rats. We also investigated the role of the stress-activated p38 MAPK in the action of lithium to activate skeletal muscle glucose transport. In the absence of insulin, lithium (10 mM LiCl) increased basal glucose transport by 62% (p<0.05) and glycogen synthesis by 112%. Lithium did not alter phosphorylation of Akt ser473, but enhanced GSK-3β ser9 phosphorylation by 41%. Lithium further enhanced the effect of insulin on glucose transport (42%), glycogen synthesis (44%), and GSK-3ß phosphorylation (13%). Lithium increased phosphorylated p38 MAPK 31% without and 19% with insulin. Moreover, a selective p38 MAPK inhibitor, A304000, completely prevented the lithium-induced enhancement of glucose transport revealing the critical involvement of p38 MAPK phosphorylation in lithium-induced glucose transport in isolated skeletal muscle.

Made available in DSpace on 2014-06-11T19:22:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-01-27Bitstream added on 2014-06-13T20:27:33Z : No. of bitstreams: 1 pintojunior_dac_me_prud_prot.pdf: 1186583 bytes, checksum: e83d66af1d0fd4767ad1c9b69a60014b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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... / 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)

IGF1 and its receptor IGF1R have been correlated with the proliferation of granulosa cells as well as steroid synthesis. Studies have shown that conditional ablation of Igf1r in granulosa cells leads to follicular arrest at a secondary stage, absence of ovulation and infertility. With a high homology between IGF1R and INSR, the full effects of insulin signaling could be masked by just a single receptor knockout. Therefore, utilizing Esr2-iCre we generated a granulosa specific double knockout mouse model. These mice have severely disrupted follicular development, with a block at a primary stage. Granulosa cells do not proliferate, while the oocytes appear activated resulting in reduction of ovarian size, absence of estrous cyclicity and infertility. Since an early granulosa cell knockout leads to block in follicular development, it masks the receptor function during ovulation, and CL formation. With the use of Pgr-Cre, the follicular development goes undisturbed until the periovulatory stages. Pgr-Cre knockout of Insr and Igf1r results in reduced ovulation, and progesterone synthesis. Few oocytes, that do escape, get fertilized but fail to thrive, and do not implant. Pgr-Cre is also active in the uterine endometrium. Ablation of Insr and Igf1r in the uterus results in reduced endometrial proliferation during the preimplantation period, complete absence of implantation and decidualization. Collectively, these results indicate the importance of INSR and IGF1R during follicular development, and ovulation, as well as in uterine proliferation, implantation, and decidualization.

The endocannabinoid (EC) system has a role in metabolic homeostasis. The purpose of this study was to determine the effect of ECs and the fatty acids they are derived from on lipid metabolism and mitochondrial function in adipocytes. 3T3-L1 adipocytes on day 8 of differentiation were treated with ECs and fatty acids for 48 hours in the absence or presence of insulin and various inhibitors. Lysates were analyzed via Western immunoblotting, a lipolysis assay and Seahorse XF Analyzer for changes in protein levels, phosphorylation state, lipolysis, and oxygen consumption rate. Results showed that ECs (2-arachidonoyl glycerol) stimulated lipolysis via a novel AMPK-dependent pathway, while fatty acids had varying effects on insulin signaling and mitochondrial function . These data suggest adipose tissue EC receptors may be a suitable target for anti-obesity therapy. Further research is needed to understand how the dietary fatty acid profile may influence synthesis of ECs. / May 2016

In Drosophila, the insulin signaling pathway is at the interface between dietary conditions and control of growth and development, reproduction, stress responses and life span. Eight insulin like peptides (Dilp1-8), an insulin tyrosine kinase receptor (dInR) and its downstream components, as well as a relaxin-like receptor type (Lgr3) form the core of this signaling. Here we showed that the dInR mediates post-mitotic cell growth specifically in about 300 peptidergic neurons expressing the basic helix loop helix (bHLH) transcription factor Dimmed (Paper I).  Overexpression of dInR in Dimm positive neurons leads to increased size of cell body, Golgi apparatus and nucleus, whereas dInR knockdown causes an opposite effect. Manipulation of downstream components of insulin signaling induces similar changes in Dimm positive neurons. This mechanism is nutrient dependent. In Paper II, we further investigate the relation between Dimmed and dInR for regulation of cell growth. Coexpressing Dimm and dInR in a range of Dimm negative neurons results in increased cell size in both larval and adult stages. We provide further evidence that dInR regulates cell growth in a Dimm dependent manner and that DILP6 from glia cells is involved in this regulation. In addition, we find that Dimm alone is capable of triggering cell growth in certain neuron types at different developmental stages. Furthermore, ectopic Dimm alone can block apoptosis.  Dimm is a known master regulator of peptidergic cell fate. In paper III we find that ectopic expression of Dimm in Dimm negative motor neurons results in transformation the neurons towards a neuroendocrine phenotype. They acquire enlarged axon terminations and boutons, lose both pre- and postsynaptic markers, and display diminished levels of wingless and its receptor dFrizzled. Furthermore they show increased expression of several Dimm targets. Finally, combined ectopic Dimm and dInR expression gives rise to stronger phenotypes. In paper IV we studied another DILP possibly involved in growth regulation, the under-investigated DILP1. We generated Dilp1-Gal4 lines and anti DILP1 antibodies and found that DILP1 is transiently expressed in brain insulin producing cells (IPCs) from pupal stages to newly hatched adult flies. Diapausing virgin female flies display a high expression level of dilp1/DILP1 over at least 9 weeks of adult life. DILP1 expression is also correlated with the persistence of larval/pupal fat body and its expression is regulated by other DILPs and short neuropeptide F (sNPF). Flies mutant in dilp1 display increased food intake, but decreased stress resistance and life span. We found no obvious role of DILP1 in growth regulation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p>

Axonal regeneration within the mammalian central nervous system following traumatic damage is limited and interventions to enable regrowth is a crucial goal in regenerative medicine. The nematode Caenorhabditis elegans is an excellent model to identify the intrinsic genetic programs that govern axonal regrowth. Here we demonstrate that alterations in O-linked N- beta-acetylglucosamine (O-GlcNAc) post-translational modifications of proteins can increase the regenerative potential of individual neurons. O-GlcNAc are single monosaccharide protein modifications that occur on serines/threonines in nucleocytoplasmic compartments. Changes in O-GlcNAc levels serve as a sensor of cellular nutrients and acts in part through the insulin-signaling pathway. Loss of O-GlcNAc via mutation of the O-GlcNAc Transferase (OGT), the enzyme that adds O-GlcNAc onto target proteins, enhances regeneration by 70%. Remarkably, hyper-O-GlcNAcyation via mutation of the O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from target proteins, also enhances regeneration by 40%. Our results shed light on this apparent contradiction by demonstrating that O-GlcNAc enzyme mutants differentially modulate the insulin-signaling pathway. OGT mutants act through AKT1 to modulate glycolysis. In contrast, OGA mutants act through the FOXO/DAF-16 transcription factor to improve the mitochondrial stress response. These findings reveal for the first time the importance of O-GlcNAc post-translational modifications in axon regeneration and provide evidence that regulation of metabolic programs can dictate the regenerative capacity of a neuron. / 2021-02-20T00:00:00Z

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.

The formation of adipocytes is a complicated process in which insulin and IGF-1 signaling pathways and numerous transcription factors control the conversion of precursor cells to mature fat cells. The Rab5 protein acts as a rate-limiting protein during receptor-mediated endocytosis by switching between a GDP-bound inactive form and a GTP-bound active form. The inactivation and activation of Rab5 are regulated by several Rab5 GTPase activating proteins (GAPs) and Rab5 guanine nucleotide exchange factors (GEFs), respectively. This dissertation demonstrated that the activity of the small GTPase Rab5 and its regulators are essential for the differentiation of 3T3-L1 pre-adipocytes. Specifically, it showed that Rab5 activation is detrimental to the differentiation process. The overexpression of a dominant-negative Rab5:S34N mutant, but not an active counterpart (Rab5:Q79L), stimulated the differentiation of 3T3-L1 pre-adipocytes. Consequently, the expression of Rab5:S34N increased the expression of two adipogenic-specific transcriptional factors, PPARγ and C/EBPα. siRNA-mediated depletion of Rab5 inhibited the differentiation of 3T3-L1 pre-adipocytes, providing further evidence for the requirement of Rab5 in the process of adipogenesis. A dramatic decrease of the Rab5-GTP level is also observed during the differentiation of 3T3-L1 pre-adipocytes. Consistent with these observations, I found that the expression of Rab5 GEFs (i.e., RIN1, Rabex-5, and RAP6), which increased the GTP-bound form of Rab5, blocked the differentiation process. In contrast, the expression of Rab5 GAPs (i.e., RN-tre and RabGAP-5), which decreased the GTP-bound form of Rab5, stimulated differentiation of 3T3-L1 pre-adipocytes. I also found a novel interaction between the VPS9 domain of the Rab5 GEFs and the activated insulin receptor. This interaction is specific since the VPS9 domain did not interact with the catalytic inactive mutant of the insulin receptor and the Rab5 GAPs (no VPS9 domain) did not bind to the activated insulin receptor. The data point out that a reduction on the GTP-bound form of Rab5 is required for the rapid differentiation of 3T3-L1 pre-adipocytes, identifying Rab5 inactivation as an important contributor of adipogenesis. Also, these observations suggest a novel cellular mechanism of Rab5 activity in the adipogenesis process in connection with the insulin receptor, the Rab5 GAPs, and the Rab5 GEFs.

<p>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.</p><p>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.</p><p>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.</p>