Periapical lesions decrease insulin signal and cause insulin resistance

Introduction: Inflammatory cytokines are associated with decreased insulin signal transduction. Moreover, local oral inflammation, such as that accompanying periodontal disease, is associated with insulin resistance and type 2 diabetes mellitus. The aim of this study was to evaluate the effect of periapical lesions (PLs) on insulin signaling and insulin sensitivity in rats. We hypothesized that PLs alter systemic insulin signaling and insulin sensitivity via elevated plasmatic tumor necrosis factor α (TNF-α). Methods: Wistar rats were divided into control (CN) and PL groups. PLs were induced by exposing pulpal tissue to the oral environment. After 30 days, insulin sensitivity was measured using the insulin tolerance test. After euthanization, maxillae were processed for histopathology. Plasmatic concentrations of tumor necrosis factor α (TNF-α) were determined via the enzyme-linked immunosorbent assay. Insulin signal transduction was evaluated using insulin receptor substrate tyrosine phosphorylation status and serine phosphorylation status in periepididymal white adipose tissue via Western blotting. For insulin signaling and insulin tolerance tests, the analyses performed were analysis of variance followed by the Tukey post hoc test. For TNF-α analysis, the Student's t test was used. In all tests, P <.05 was considered significant. Results: The rats with PLs showed higher plasmatic TNF-α, lower constant rate for glucose disappearance values, and reduced pp185 tyrosine phosphorylation status but no change in serine phosphorylation status in white adipose tissue after insulin stimulation. Conclusions: PLs can cause alterations to both insulin signaling and insulin sensitivity, probably because of elevation of plasmatic TNF-α. The results from this study emphasize the importance of the prevention of local inflammatory diseases, such as PLs, with regard to the prevention of insulin resistance. Copyright © 2013 American Association of Endodontists.

Resveratrol reduces chronic inflammation and improves insulin action in the myocardium of high-fat diet-induced obeserats

OBJETIVO: Avaliar o efeito do resveratrol sobre a via de sinalização da insulina e melhora do quadro inflamatório no miocárdio de ratos Wistar obesos induzidos por dieta.MÉTODOS: Ratos Wistar foram divididos em grupos: controle (dieta padrão para roedores), obeso (dieta hiperlipídica) e obeso suplementado com resveratrol (20 mg/kg/dia), por 8 semanas (n=10). Ao final do período experimental, realizou-se o teste de tolerância à insulina, nos tempos 0 (sem insulina), 5, 10, 15, 20, 25 e 30 minutos após injeção intraperitoneal de insulina (2 U/kg). O peso corporal e o tecido adiposo epididimal foram mensurados. Fragmentos do miocárdio foram extraídos para análises da via da insulina e moléculas pró-inflamatórias através de Western blot.RESULTADOS: Os resultados indicam que a intervenção com resveratrol aumenta a constante de decaimento da glicose, fosforilação do receptor de insulina, substrato do receptor de insulina e da proteína quinase B. A suplementação de resveratrol também reduziu os níveis proteicos do fator de necrose tumoral alfa e de moléculas envolvidas com a transdução do sinal pró-inflamatório (quinase indutora do kappa B e fator nuclear kappa B). Os resultados ainda sugerem que a melhora na sensibilidade à insulina e a redução das moléculas pró-inflamatórias ocorreram independentemente da perda de peso corporal e da redução do tecido adiposo epididimal.CONCLUSÃO: A suplementação de resveratrol aumenta a sensibilidade à insulina, o que está relacionado à redução de fatores inflamatórios no miocárdio.

Downhill running excessive training inhibits hypertrophy in mice skeletal muscles with different fiber type composition

The aim of this study was to verify the effects of running overtraining protocols performed in downhill, uphill, and without inclination on the proteins related to hypertrophy signaling pathway in extensor digitorum longus (EDL) and soleus of C57BL/6 mice. We also performed histological and stereological analyses. Rodents were divided into control (CT; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up), and overtrained by running without inclination (OTR). The incremental load, exhaustive, and grip force tests were used as performance evaluation parameters. 36 h after the grip force test, EDL and soleus were removed and immediately used for immunoblotting analysis or stored at -80°C for histological and stereological analyses. For EDL, OTR/down decreased the protein kinase B (Akt) and tuberous sclerosis protein 2 (TSC2) phosphorylation (p), and increased myostatin, receptor-activated Smads (pSMAD2-3), and insulin receptor substrate-1 (pIRS-1; Ser307/636). OTR/down also presented low and high relative proportions of cytoplasm and connective tissue, respectively. OTR/up increased the mammalian target of rapamycin (pmTOR), 70-kDa ribosomal protein S6 kinase 1 (pS6K1) and pSMAD2-3, and decreased pTSC2. OTR decreased pTSC2 and increased pIRS-1 (Ser636). For soleus, OTR/down increased S6 ribosomal protein (pS6RP) and pSMAD2-3, and decreased pIRS-1 (Ser639). OTR/up decreased pS6K1, pS6RP and pIRS-1 (Ser639), and increased pTSC2 (Ser939), and pSMAD2-3. OTR increased pS6RP, 4E-binding protein-1 (p4E-BP1), pTSC2 (Ser939), and pSMAD2-3, and decreased pIRS-1 (Ser639). In summary, OTR/down inhibited the skeletal muscle hypertrophy with concomitant signs of atrophy in EDL. The effects of OTR/up and OTR depended on the analyzed skeletal muscle type. J. Cell. Physiol. 9999: 1-12, 2015. © 2015 Wiley Periodicals, Inc.

Double-Stranded RNA-Activated Protein Kinase Is a Key Modulator of Insulin Sensitivity in Physiological Conditions and in Obesity in Mice

The molecular integration of nutrient-and pathogen-sensing pathways has become of great interest in understanding the mechanisms of insulin resistance in obesity. The double-stranded RNA-dependent protein kinase (PKR) is one candidate molecule that may provide cross talk between inflammatory and metabolic signaling. The present study was performed to determine, first, the role of PKR in modulating insulin action and glucose metabolism in physiological situations, and second, the role of PKR in insulin resistance in obese mice. We used Pkr(-/-) and Pkr(+/+) mice to investigate the role of PKR in modulating insulin sensitivity, glucose metabolism, and insulin signaling in liver, muscle, and adipose tissue in response to a high-fat diet. Our data show that in lean Pkr(-/-) mice, there is an improvement in insulin sensitivity, and in glucose tolerance, and a reduction in fasting blood glucose, probably related to a decrease in protein phosphatase 2A activity and a parallel increase in insulin-induced thymoma viral oncogene-1 (Akt) phosphorylation. PKR is activated in tissues of obese mice and can induce insulin resistance by directly binding to and inducing insulin receptor substrate (IRS)-1 serine307 phosphorylation or indirectly through modulation of c-Jun N-terminal kinase and inhibitor of kappa B kinase beta. Pkr(-/-) mice were protected from high-fat diet-induced insulin resistance and glucose intolerance and showed improved insulin signaling associated with a reduction in c-Jun N-terminal kinase and inhibitor of kappa B kinase beta phosphorylation in insulin-sensitive tissues. PKR may have a role in insulin sensitivity under normal physiological conditions, probably by modulating protein phosphatase 2A activity and serine-threonine kinase phosphorylation, and certainly, this kinase may represent a central mechanism for the integration of pathogen response and innate immunity with insulin action and metabolic pathways that are critical in obesity. (Endocrinology 153:5261-5274, 2012)

Metabolic Disorders and Adipose Tissue Insulin Responsiveness in Neonatally STZ-Induced Diabetic Rats Are Improved by Long-Term Melatonin Treatment

Diabetes mellitus is a product of low insulin sensibility and pancreatic beta-cell insufficiency. Rats with streptozotocin-induced diabetes during the neonatal period by the fifth day of age develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, polyuria, and polydipsia aggravated by insulin resistance in adulthood. In this study, we investigated whether the effect of long-term treatment with melatonin can improve insulin resistance and other metabolic disorders in these animals. At the fourth week of age, diabetic animals started an 8-wk treatment with melatonin (1 mg/kg body weight) in the drinking water at night. Animals were then killing, and the sc, epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed, and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Blood samples were collected for biochemical assays. Melatonin treatment reduced hyperglycemia, polydipsia, and polyphagia as well as improved insulin resistance as demonstrated by constant glucose disappearance rate and homeostasis model of assessment-insulin resistance. However, melatonin treatment was unable to recover body weight deficiency, fat mass, and adipocyte size of diabetic animals. Adiponectin and fructosamine levels were completely recovered by melatonin, whereas neither plasma insulin level nor insulin secretion capacity was improved in diabetic animals. Furthermore, melatonin caused a marked delay in the sexual development, leaving genital structures smaller than those of nontreated diabetic animals. Melatonin treatment improved the responsiveness of adipocytes to insulin in diabetic animals measured by tests of glucose uptake (sc, EP, and RP), glucose oxidation, and incorporation of glucose into lipids (EP and RP), an effect that seems partially related to an increased expression of insulin receptor substrate 1, acetyl-coenzyme A carboxylase and fatty acid synthase. In conclusion, melatonin treatment was capable of ameliorating the metabolic abnormalities in this particular diabetes model, including insulin resistance and promoting a better long-term glycemic control. (Endocrinology 153: 2178-2188, 2012)

Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows

Gene expression of adipose factors, which may be part of the mechanisms that underlie insulin sensitivity, were studied in dairy cows around parturition. Subcutaneous fat biopsies and blood samples were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. In the adipose tissue samples, mRNA was quantified by real-time reverse transcription polymerase chain reaction for tumour necrosis factor alpha (TNFalpha), insulin-independent glucose transporter (GLUT1), insulin-responsive glucose transporter (GLUT4), insulin receptor, insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), regulatory subunit of phosphatidylinositol-3 kinase (p85) and catalytic subunit of phosphatidylinositol-3 kinase. Blood plasma was assayed for concentrations of glucose, beta-hydroxybutyric acid, non-esterified fatty acids (NEFA) and insulin. Plasma parameters followed a pattern typically observed in dairy cows. Gene expression changes were observed, but there were no changes in TNFalpha concentrations, which may indicate its local involvement in catabolic adaptation of adipose tissue. Changes in GLUT4 and GLUT1 mRNA abundance may reflect their involvement in reduced insulin sensitivity and in sparing glucose for milk synthesis in early lactation. Unchanged gene expression of IRS1, IRS2 and p85 over time may imply a lack of their involvement in terms of insulin sensitivity dynamics. Alternatively, it may indicate that post-transcriptional modifications of these factors came into play and may have concealed an involvement.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Insulin signaling and action in cultured skeletal muscle cells from lean healthy humans with high and low insulin sensitivity

The aim of these studies was to investigate whether insulin resistance is primary to skeletal muscle. Myoblasts were isolated from muscle biopsies of 8 lean insulin-resistant and 8 carefully matched insulin-sensitive subjects (metabolic clearance rates as determined by euglycemic-hyperinsulinemic clamp: 5.8 +/- 0.5 vs. 12.3 +/- 1.7 ml x kg(-1) x min(-1), respectively; P < or = 0.05) and differentiated to myotubes. In these cells, insulin stimulation of glucose uptake, glycogen synthesis, insulin receptor (IR) kinase activity, and insulin receptor substrate 1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity were measured. Furthermore, insulin activation of protein kinase B (PKB) was compared with immunoblotting of serine residues at position 473. Basal glucose uptake (1.05 +/- 0.07 vs. 0.95 +/- 0.07 relative units, respectively; P = 0.49) and basal glycogen synthesis (1.02 +/- 0.11 vs. 0.98 +/- 0.11 relative units, respectively; P = 0.89) were not different in myotubes from insulin-resistant and insulin-sensitive subjects. Maximal insulin responsiveness of glucose uptake (1.35 +/- 0.03-fold vs. 1.41 +/- 0.05-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.43) and glycogen synthesis (2.00 +/- 0.13-fold vs. 2.10 +/- 0.16-fold over basal for insulin-resistant and insulin-sensitive subjects, respectively; P = 0.66) were also not different. Insulin stimulation (1 nmol/l) of IR kinase and PI 3-kinase were maximal within 5 min (approximately 8- and 5-fold over basal, respectively), and insulin activation of PKB was maximal within 15 min (approximately 3.5-fold over basal). These time kinetics were not significantly different between groups. In summary, our data show that insulin action and signaling in cultured skeletal muscle cells from normoglycemic lean insulin-resistant subjects is not different from that in cells from insulin-sensitive subjects. This suggests an important role of environmental factors in the development of insulin resistance in skeletal muscle.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

Molecular mechanisms for the insulin sensitizing activity of rexinoids in skeletal muscle of diabetic (Db/Db) mice

Rexinoids are synthetic agonists for the retinoid X receptors (RXRs), a member of the nuclear receptor family of ligand-activated transcription factors. Rexinoids have been shown to lower serum glucose and insulin levels in animal models of type 2 diabetes. However the mechanisms that are responsible for the insulin-sensitizing action of rexinoids are largely unknown. Skeletal muscle accounts for the majority of insulin-regulated whole-body glucose disposal and impaired insulin action in muscle is an important contributor to the pathophysiology of type 2 diabetes. Glucose transport is a rate-limiting step in glucose utilization. The goal of these studies is to examine the mechanisms of the anti-diabetic activity of rexinoids in skeletal muscle of diabetic db/db mice. The results we have obtained showed that treatment of db/db mice with rexinoids for two weeks resulted in a significant increase in insulin-stimulated glucose transport activity in skeletal muscle. Insulin stimulates glucose transport in muscle via the regulation of both the insulin receptor substrate-1 (IRS-1)/Akt pathway and the Cbl-associated protein (CAP)/Cbl pathway. Rexinoids increased the insulin-stimulated IRS-1 tyrosine phosphorylation and Akt phosphorylation without effects on the activity of the CAP/Cbl pathway. The effects of rexinoids on the IRS-1/Akt pathway were associated with a decrease in the level of IRS-1 Serine 307 phosphorylation as well as qualitative and quantitative alterations in the fatty acyl-CoAs present within the muscle cells. In addition, rexinoids increased the expression of uncoupling protein 3 (UCP3) and activation of AMPK in diabetic muscle. This effect may also enhance the IRS-1/Akt signaling. We believe that it is the concerted activation of the IRS-1/Akt and AMPK signaling systems, a pharmacological mechanism that as far as we know, is unique to rexinoids, that results in the anti-diabetic effects of these drugs. Our results also suggest that the glucose-lowering mechanism of rexinoids is distinct from that of the thiazolidinediones (TZDs), peroxisome proliferator-activated receptor γ (PPARγ) agonists with well-characterized anti-diabetic activity. Rexinoids appear to represent a novel class of insulin sensitizers, with potential applications for the treatment of type 2 diabetes. ^

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Interaction of Class I Human Leukocyte Antigen (HLA-I) Molecules with Insulin Receptors and Its Effect on the Insulin-Signaling Cascade

Insulin receptor (IR) and class I major histocompatibility complex molecules associate with one another in cell membranes, but the functional consequences of this association are not defined. We found that IR and human class I molecules (HLA-I) associate in liposome membranes and that the affinity of IR for insulin and its tyrosine kinase activity increase as the HLA:IR ratio increases over the range 1:1 to 20:1. The same relationship between HLA:IR and IR function was found in a series of B-LCL cell lines. The association of HLA-I and IR depends upon the presence of free HLA heavy chains. All of the effects noted were reduced or abrogated if liposomes or cells were incubated with excess HLA-I light chain, β2-microglobulin. Increasing HLA:IR also enhanced phosphorylation of insulin receptor substrate-1 and the activation of phosphoinositide 3-kinase. HLA-I molecules themselves were phosphorylated on tyrosine and associated with phosphoinositide 3-kinase when B-LCL were stimulated with insulin.

Thyroid hormone receptor-associated proteins and general positive cofactors mediate thyroid hormone receptor function in the absence of the TATA box-binding protein-associated factors of TFIID

Coactivators previously implicated in ligand-dependent activation functions by thyroid hormone receptor (TR) include p300 and CREB-binding protein (CBP), the steroid receptor coactivator-1 (SRC-1)-related family of proteins, and the multicomponent TR-associated protein (TRAP) complex. Here we show that two positive cofactors (PC2 and PC4) derived from the upstream stimulatory activity (USA) cofactor fraction act synergistically to mediate thyroid hormone (T3)-dependent activation either by TR or by a TR-TRAP complex in an in vitro system reconstituted with purified factors and DNA templates. Significantly, the TRAP-mediated enhancement of activation by TR does not require the TATA box-binding protein-associated factors of TFIID. Furthermore, neither the pleiotropic coactivators CBP and p300 nor members of the SRC-1 family were detected in either the TR-TRAP complex or the other components of the in vitro assay system. These results show that activation by TR at the level of naked DNA templates is enhanced by cooperative functions of the TRAP coactivators and the general coactivators PC2 and PC4, and they further indicate a potential functional redundancy between TRAPs and TATA box-binding protein-associated factors in TFIID. In conjunction with earlier studies on other nuclear receptor-interacting cofactors, the present study also suggests a multistep pathway, involving distinct sets of cofactors, for activation of hormone responsive genes.

ClpA mediates directional translocation of substrate proteins into the ClpP protease

The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease. Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH2 or COOH terminus with fluorescent probes. Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP. We observed for both substrates that energy transfer occurs 2–4 s sooner with the COOH-terminally labeled molecules than with the NH2-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.

Evidence for a circulating islet cell growth factor in insulin-resistant states

Insulin resistance is a feature of many common disorders including obesity and type 2 diabetes mellitus. In these disorders, the β-cells compensate for the insulin resistance for long periods of time with an increase in secretory capacity, an increase in β-cell mass, or both. To determine whether the β-cell response might relate to a circulating growth factor, we have transplanted normal islets under the kidney capsule of normoglycemic insulin-resistant mice with two different models of insulin resistance: lean mice that have a double heterozygous deletion of the insulin receptor and insulin receptor substrate-1 (DH) or the obese, hyperglycemic ob/ob mice. In the grafts transplanted into both hosts, there was a marked increase in β-cell mitotic activity and islet mass that was comparable with that observed in the endogenous pancreas. By contrast, islets of the DH mouse transplanted into normal mice showed reduced mitotic index. These data suggest the insulin resistance is associated with a circulating islet cell growth factor that is independent of glucose and obesity.