Effects of troglitazone on cellular differentiation, insulin signaling, and glucose metabolism in cultured human skeletal muscle cells

To determine the immediate effect of thiazolidinediones on human skeletal muscle, differentiated human myotubes were acutely (1 day) and myoblasts chronically (during the differentiation process) treated with troglitazone (TGZ). Chronic TGZ treatment resulted in loss of the typical multinucleated phenotype. The increase of muscle markers typically observed during differentiation was suppressed, while adipocyte markers increased markedly. Chronic TGZ treatment increased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and membranous protein kinase B/Akt (PKB/Akt) Ser-473 phosphorylation more than 4-fold. Phosphorylation of p42/44 mitogen-activated protein kinase (42/44 MAPK/ERK) was unaltered. Basal glucose uptake as well as both basal and insulin-stimulated glycogen synthesis increased approximately 1.6- and approximately 2.5-fold after chronic TGZ treatment, respectively. A 2-fold stimulation of PI 3-kinase but no other significant TGZ effect was found after acute TGZ treatment. In conclusion, chronic TGZ treatment inhibited myogenic differentiation of that human muscle while inducing adipocyte-specific gene expression. The effects of chronic TGZ treatment on basal glucose transport may in part be secondary to this transdifferentiation. The enhancing effect on PI 3-kinase and PKB/Akt involved in both differentiation and glycogen synthesis appears to be pivotal in the cellular action of TGZ.

Brain Neuropeptides That Control Prolactin Secretion in Cattle

The objective was to test the hypothesis that dopamine regulates prolactin (PRL) secretion by determining acute changes in catecholamine concentrations in hypophyseal portal blood of cattle and their relation to peripheral blood concentration of PRL in hypophyseal stalk-transected (HST) and sham-operated control (SOC). Holstein heifers were subjected to neurosurgery to collect hypophyseal portal blood with a stainless steel cannula designed with a cuff placed under the pituitary stalk and peripheral blood via a jugular vein catheter. PRL plasma concentration was measured by radioimmunoassay, and dopamine and norepinephrine in portal plasma by radioenzymatic assay. During anesthesia before HST or SOC, PRL plasma concentration ranged from 20–40 ng/ml throughout 255 minutes. PRL abruptly increased and remained above 90 ng/ml after HST, compared with a steady decrease to <20 ng/ml in SOC heifers throughout 440 minutes. Within 5 minutes after severing of the hypophyseal stalk, dopamine in portal blood (>8 ng/ml) was significantly increased (P<0.05) compared with peripheral blood (<2 ng/ml). Norepinephrine concentration in portal blood was significantly greater (P<0.05) than in peripheral blood during the first 60 minutes. The sustained high PRL level in peripheral plasma after severing the hypophyseal stalk stimulated hypothalamic dopamine secretion from hypophyseal portal vessels during the prolonged period of blood collection. Norepinephrine concentration in these cattle was greater in hypophyseal portal blood than in peripheral blood, implicating both an important hypothalamic source of the catecholamine as well as an adrenal gland contribution during anesthesia.

Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion.

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

Metabolic inflexibility and insulin resistance in obese adolescents with non-alcoholic fatty liver disease.

BACKGROUND Non-alcoholic fatty liver disease (NAFLD) is a comorbidity of childhood obesity. OBJECTIVE We examined whole-body substrate metabolism and metabolic characteristics in obese adolescents with vs. without NAFLD. SUBJECTS Twelve obese (BMI ≥ 95th percentile) adolescents with and without NAFLD [intrahepatic triglyceride (IHTG) ≥5.0% vs. <5.0%] were pair-matched for race, gender, age and % body fat. METHODS Insulin sensitivity (IS) was assessed by a 3-h hyperinsulinemic-euglycemic clamp and whole-body substrate oxidation by indirect calorimetry during fasting and insulin-stimulated conditions. RESULTS Adolescents with NAFLD had increased (p < 0.05) abdominal fat, lipids, and liver enzymes compared with those without NAFLD. Fasting glucose concentration was not different between groups, but fasting insulin concentration was higher (p < 0.05) in the NAFLD group compared with those without. Fasting hepatic glucose production and hepatic IS did not differ (p > 0.1) between groups. Adolescents with NAFLD had higher (p < 0.05) fasting glucose oxidation and a tendency for lower fat oxidation. Adolescents with NAFLD had lower (p < 0.05) insulin-stimulated glucose disposal and lower peripheral IS compared with those without NAFLD. Although respiratory quotient (RQ) increased significantly from fasting to insulin-stimulated conditions in both groups (main effect, p < 0.001), the increase in RQ was lower in adolescents with NAFLD vs. those without (interaction, p = 0.037). CONCLUSION NAFLD in obese adolescents is associated with adverse cardiometabolic profile, peripheral insulin resistance and metabolic inflexibility.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.

Anti-Müllerian hormone and progesterone levels produced by granulosa cells are higher when derived from natural cycle IVF than from conventional gonadotropin-stimulated IVF.

BACKGROUND The study was designed to compare the effect of in vitro FSH stimulation on the hormone production and gene expression profile of granulosa cells (GCs) isolated from single naturally matured follicles obtained from natural cycle in vitro fertilization (NC-IVF) with granulosa cells obtained from conventional gonadotropin-stimulated IVF (c-IVF). METHODS Lutein granulosa cells from the dominant follicle were isolated and cultured in absence or presence of recombinant FSH. The cultures were run for 48 h and six days. Messenger RNA (mRNA) expressions of anti-Müllerian hormone (AMH) and FSH receptor were measured by quantitative polymerase chain reaction (qPCR). AMH protein and progesterone concentration (P4) in cultured supernatant were measured by ELISA and RIA. RESULTS Our results showed that the mRNA expression of AMH was significantly higher in GCs from NC- than from c-IVF on day 6 after treatment with FSH (1 IU/mL). The FSH stimulation increased the concentration of AMH in the culture supernatant of GCs from NC-IVF compared with cells from c-IVF. In the culture medium, the AMH level was correlated significantly and positively to progesterone concentration. CONCLUSIONS Differences in the levels of AMH and progesterone released into the medium by cultured GC as well as in AMH gene expression were observed between GCs obtained under natural and stimulated IVF protocols. The results suggest that artificial gonadotropin stimulation may have an effect on the intra-follicular metabolism. A significant positive correlation between AMH and progesterone may suggest progesterone as a factor influencing AMH secretion.

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. ^

Estradiol and insulin cross -talk in breast cancer

Approximately 33% of clinical breast carcinomas require estrogens to proliferate. Epidemiological data show that insulin resistance and diabetes mellitus is 2–3 times more prevalent in women with breast cancer than those with benign breast lesions, suggesting a clinical link between insulin and estradiol. Insulin and estradiol have a synergistic effect on the growth of MCF7 breast cancer cells, and long-term estradiol treatment upregulates the expression of the key insulin signaling protein IRS-1. The goal of this study was to further define the mechanism(s) of cross-talk between insulin and estradiol in regulating the growth of breast cancer. Using MCF7 cells, acute treatment with insulin or estradiol alone was found to stimulate two activities associated with growth: Erk MAP kinase and PI 3-kinase. However, combined acute treatment had an antagonistic effect on both activities. Acute estradiol treatment inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1 while increasing its serine phosphorylation; the serine phosphorylation was attenuated by the PI 3-kinase inhibitor wortmannin. The acute antagonism observed with combined estradiol and insulin are not consistent with the long-term synergistic effect on growth. In contrast, chronic estradiol treatment enhanced the insulin-sensitivity of breast cancer cells as measured by increases in total cellular insulin-stimulated tyrosine phosphorylation of IRS-1 and activation of PI 3-kinase. Estradiol stimulation of gene transcription was found to require PI 3-kinase activity but not MAP kinase activity. Insulin alone had no effect on ER transcriptional activity, but chronic treatment in combination with estradiol resulted in synergism of ER transcription. The synergistic effect of insulin and estradiol on MCF7 cell growth was also found to require PI 3-kinase but not MAP kinase activity. Therefore, chronic estradiol treatment increases insulin stimulation of PI 3-kinase, and PI 3-kinase is required for estradiol stimulation of gene transcription alone and in combined synergy with insulin. These data demonstrate that PI 3-kinase is the locus for the cross-talk between insulin and estradiol which results in enhanced breast cancer growth with long-term exposure to both hormones. This may have important clinical implications for women with high risk for breast cancer and/or diabetes mellitus. ^

Stimulation of renin secretion by nitric oxide is mediated by phosphodiesterase 3

This study aimed to characterize the cellular pathways along which nitric oxide (NO) stimulates renin secretion from the kidney. Using the isolated perfused rat kidney model we found that renin secretion stimulated 4- to 8-fold by low perfusion pressure (40 mmHg), by macula densa inhibition (100 μmol/liter of bumetanide), and by adenylate cyclase activation (3 nmol/liter of isoproterenol) was markedly attenuated by the NO synthase inhibitor nitro-l-arginine methyl ester (l-Name) (1 mM) and that the inhibition by l-Name was compensated by the NO-donor sodium nitroprusside (SNP) (10 μmol/liter). Similarly, inhibition of cAMP degradation by blockade of phosphodiesterase 1 (PDE-1) (20 μmol/liter of 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine) or of PDE-4 (20 μmol/liter of rolipram) caused a 3- to 4-fold stimulation of renin secretion that was attenuated by l-Name and that was even overcompensated by sodium nitroprusside. Inhibition of PDE-3 by 20 μmol/liter of milrinone or by 200 nmol/liter of trequinsin caused a 5- to 6-fold stimulation of renin secretion that was slightly enhanced by NO synthase inhibition and moderately attenuated by NO donation. Because PDE-3 is a cGMP-inhibited cAMP-PDE the role of endogenous cGMP for the effects of NO was examined by the use of the specific guanylate cyclase inhibitor 1-H-(1,2,4)oxodiazolo(4,3a)quinoxalin-1-one (20 μmol). In the presence of 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one the effect of NO on renin secretion was abolished, whereas PDE-3 inhibitors exerted their normal effects. These findings suggest that PDE-3 plays a major role for the cAMP control of renin secretion. Our findings are compatible with the idea that the stimulatory effects of endogenous and exogenous NO on renin secretion are mediated by a cGMP-induced inhibition of cAMP degradation.

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

Thiazolidinediones and insulin resistance: Peroxisome proliferatoractivated receptor γ activation stimulates expression of the CAP gene

c-Cbl-associated protein (CAP) is a signaling protein that interacts with both c-Cbl and the insulin receptor that may be involved in the specific insulin-stimulated tyrosine phosphorylation of c-Cbl. The restricted expression of CAP in cells metabolically sensitive to insulin suggests an important potential role in insulin action. The expression of CAP mRNA and proteins are increased in 3T3-L1 adipocytes by the insulin sensitizing thiazolidinedione drugs, which are activators of the peroxisome proliferator-activated receptor γ (PPARγ). The stimulation of CAP expression by PPARγ activators results from increased transcription. This increased expression of CAP was accompanied by a potentiation of insulin-stimulated c-Cbl tyrosine phosphorylation. Administration of the thiazolidinedione troglitazone to Zucker (fa/fa) rats markedly increased the expression of the major CAP isoform in adipose tissue. This effect was sustained for up to 12 weeks of treatment and accompanied the ability of troglitazone to prevent the onset of diabetes and its complications. Thus, CAP is the first PPARγ-sensitive gene identified that participates in insulin signaling and may play a role in thiazolidinedione-induced insulin sensitization.

Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1–3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

The Formation of an Insulin-responsive Vesicular Cargo Compartment Is an Early Event in 3T3-L1 Adipocyte Differentiation

Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.