Diminished GH secretion is a well known association of obesity. As in obese humans, Zucker fatty rats develop a progressive GH deficiency, present at 6 weeks of age and maximal at 10 to 12 weeks. The aim of this study was to investigate the GH dependence of IGF-I gene expression in liver and extrahepatic tissues of the obese Zucker rat as a model of progressive GH reduction during adult life. Six- and 11-week-old obese Zucker rats and their lean littermates were used to compare body weight, glycemia, insulinemia, serum GH and IGF-I levels and IGF-I mRNA expression in liver, heart, aorta, kidney and skeletal muscle. In comparison with lean controls, obese Zucker rats showed at both ages comparable glycemia, severe hyperinsulinemia (mU/ml, mean+/-s.e.m.; 6 weeks 138+/-10 vs 45+/-6 P<0.001; 11 weeks 147+/-14 vs 46+/-3, P<0.001) and lower GH (ng/ml; 6 weeks 1.7+/-0.9 vs 2.7+/-1.1; 11 weeks 1.5+/-0.9 vs 4.2+/-1.2) in the presence of similar circulating IGF-I levels (ng/ml; 6 weeks 774+/-26 vs 694+/-28; 11 weeks 1439+/-182 vs 1516+/-121). Hepatic IGF-I mRNA expression was already reduced at 6 weeks of age due to a significant decrease in the IGF-Ib transcript compared with lean controls (relative units; IGF-Ia: 99+/-2% vs 100+/-5%; IGF-Ib: 69+/-10% vs 100+/-2%, P<0.05) and this reduction was more marked in 11-week-old animals when both IGF-I transcripts were significantly diminished (relative units; IGF-Ia: 80+/-6% vs 100+/-1%, P<0.05; IGF-Ib: 65+/-5% vs 100+/-2%, P<0.01). Extrahepatic tissues expressed almost exclusively the IGF-Ia transcript, the amount of which relative to controls was: (1) similar at 6 weeks and decreased at 11 weeks in kidney and skeletal muscle extracts (relative units; kidney: 6 weeks 88+/-10% vs 100+/-2%; 11 weeks 76+/-3% vs 100+/-4%, P<0.05; vastus lateralis: 6 weeks 95+/-7% vs 100+/-10%; 11 weeks 59+/-4% vs 100+/-2%, P<0.001); (2) similar at both ages in thoracic aorta (relative units; 6 weeks 121+/-6% vs 105+/-5%; 11 weeks: 91+/-14% vs 100+/-4%); and (3) increased at both ages in left ventricle extracts (relative units; 6 weeks 114+/-2% vs 99+/-9%, P<0. 05; 11 weeks 119+/-7% vs 95+/-3%, P<0.05). -specific dependence of IGF-I mRNA on GH levels during adulthood, reflected by the different behavior of IGF-I expression for each tissue in conditions of progressive decrease of GH levels.
E Melian, B Gonzalez, R Ajo, N Gonzalez and F Sanchez Franco
F González, N S Rote, J Minium and J P Kirwan
Women with polycystic ovary syndrome (PCOS) are often insulin resistant and have chronic low-level inflammation. The purpose of this study was to determine the effects of hyperglycemia in vitro on tumor necrosis factor (TNF)-α release from mononuclear cells (MNC) in PCOS. Twelve reproductive-age women with PCOS (six lean, six obese) and 12 age-matched controls (six lean, six obese) were studied. Insulin sensitivity (ISHOMA) was estimated from fasting levels of glucose and insulin and percent truncal fat was determined by dual energy absorptiometry (DEXA). TNFα release was measured from MNC cultured under euglycemic and hyperglycemic conditions. ISHOMA was higher in obese women with PCOS than in lean women with PCOS (student’s t-test; 73.7 ± 14.8 vs 43.1 ± 8.6, P < 0.05), but similar to that of obese controls. ISHOMA was positively correlated with percent truncal fat (r=0.57, P < 0.04). Obese women with PCOS exhibited an increase in the percent change in TNFα release from MNC in response to hyperglycemia compared with obese controls (10 mM, 649 ± 208% vs 133 ± 30%, P < 0.003; 15 mM, 799 ± 347% vs 183 ± 59%, P < 0.04). The TNFα response directly correlated with percent truncal fat (r=0.45, P < 0.03) and ISHOMA (r=0.40, P < 0.05) for the combined groups, and with plasma testosterone (r=0.60, P < 0.05) for women with PCOS. MNC of obese women with PCOS exhibit an increased TNFα response to in vitro physiologic hyperglycemia. MNC-derived TNFα release may contribute to insulin resistance and hyperandrogenism, particularly when the combination of PCOS and increased adiposity is present.
A Acitores, N Gonzalez, V Sancho, I Valverde and ML Villanueva-Penacarrillo
Glucagon-like peptide-1 (GLP-1), an incretin with glucose-dependent insulinotropic and insulin-independent antidiabetic properties, has insulin-like effects on glucose metabolism in extrapancreatic tissues participating in overall glucose homeostasis. These effects are exerted through specific receptors not associated with cAMP, an inositol phosphoglycan being a possible second messenger. In rat hepatocytes, activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB), protein kinase C (PKC) and protein phosphatase 1 (PP-1) has been shown to be involved in the GLP-1-induced stimulation of glycogen synthase. We have investigated the role of enzymes known or suggested to mediate the actions of insulin in the GLP-1-induced increase in glycogen synthase a activity in rat skeletal muscle strips. We first explored the effect of GLP-1, compared with that of insulin, on the activation of PI3K, PKB, p70s6 kinase (p70s6k) and p44/42 mitogen-activated protein kinases (MAPKs) and the action of specific inhibitors of these kinases on the insulin- and GLP-1-induced increment in glycogen synthase a activity. The study showed that GLP-1, like insulin, activated PI3K/PKB, p70s6k and p44/42. Wortmannin (a PI3K inhibitor) reduced the stimulatory action of insulin on glycogen synthase a activity and blocked that of GLP-1, rapamycin (a 70s6k inhibitor) did not affect the action of GLP-1 but abolished that of insulin, PD98059 (MAPK inhibitor) was ineffective on insulin but blocked the action of GLP-1, okadaic acid (a PP-2A inhibitor) and tumour necrosis factor-alpha (a PP-1 inhibitor) were both ineffective on GLP-1 but abolished the action of insulin, and Ro 31-8220 (an inhibitor of some PKC isoforms) reduced the effect of GLP-1 while completely preventing that of insulin. It was concluded that activation of PI3K/PKB and MAPKs is required for the GLP-1-induced increment in glycogen synthase a activity, while PKC, although apparently participating, does not seem to play an essential role; unlike in insulin signaling, p70s6k, PP-1 and PP-2A do not seem to be needed in the action of GLP-1 upon glycogen synthase a activity in rat muscle.
C Gonzalez, A Alonso, N Alvarez, F Diaz, M Martinez, S Fernandez and AM Patterson
The mechanism for the development of insulin resistance in normal pregnancy is complex and is associated with serum levels of both progesterone and 17beta-estradiol. However, it remains unclear whether estrogens alone or progestins alone can cause insulin resistance, or whether it is a combination of both which produces this effect. We attempted to determine the role played by progesterone and/or 17beta-estradiol on the phenomena of sensitivity to insulin action that take place during pregnancy in the rat. Ovariectomized rats were treated with different doses of progesterone and/or 17beta-estradiol in order to simulate the plasma levels in normal pregnant rats. A euglycemic/hyperinsulinemic clamp was used to measure insulin sensitivity. At days 6 and 11, vehicle (V)- and progesterone (P)-treated groups were more insulin resistant than 17beta-estradiol (E)- and 17beta-estradiol+progesterone (EP)-treated groups. Nevertheless, at day 16, the V, EP and E groups were more resistant to insulin action than the P group. On the other hand, the V, EP and E groups were more insulin resistant at day 16 than at day 6, whereas the P group was more insulin resistant at day 6 than at day 16. Our results seem to suggest that the absence of female steroid hormones gives rise to a decreased insulin sensitivity. The rise in insulin sensitivity during early pregnancy, when the plasma concentrations of 17beta-estradiol and progesterone are low, could be due to 17beta-estradiol. However, during late pregnancy when the plasma concentrations of 17beta-estradiol and progesterone are high, the role of 17beta-estradiol could be to antagonize the effect of progesterone, diminishing insulin sensitivity.
Jorge N Artaza, Rajan Singh, Monica G Ferrini, Melissa Braga, James Tsao and Nestor F Gonzalez-Cadavid
Tissue fibrosis, the excessive deposition of collagen/extracellular matrix combined with the reduction of the cell compartment, defines fibroproliferative diseases, a major cause of death and a public health burden. Key cellular processes in fibrosis include the generation of myofibroblasts from progenitor cells, and the activation or switch of already differentiated cells to a fibrotic synthetic phenotype. Myostatin, a negative regulator of skeletal muscle mass, is postulated to be involved in muscle fibrosis. We have examined whether myostatin affects the differentiation of a multipotent mesenchymal mouse cell line into myofibroblasts, and/or modulates the fibrotic phenotype and Smad expression of the cell population. In addition, we investigated the role of follistatin in this process. Incubation of cells with recombinant myostatin protein did not affect the proportion of myofibroblasts in the culture, but significantly upregulated the expression of fibrotic markers such as collagen and the key profibrotic factors transforming growth factor-β1 (TGF-β1) and plasminogen activator inhibitor (PAI-1), as well as Smad3 and 4, and the pSmad2/3. An antifibrotic process evidenced by the upregulation of follistatin, Smad7, and matrix metalloproteinase 8 accompanied these changes. Follistatin inhibited TGF-β1 induction by myostatin. Transfection with a cDNA expressing myostatin upregulated PAI-1, whereas an shRNA against myostatin blocked this effect. In conclusion, myostatin induced a fibrotic phenotype without significantly affecting differentiation into myofibroblasts. The concurrent endogenous antifibrotic reaction confirms the view that phenotypic switches in multipotent and differentiated cells may affect the progress or reversion of fibrosis, and that myostatin pharmacological inactivation may be a novel therapeutic target against fibrosis.
MA Luque, N Gonzalez, L Marquez, A Acitores, A Redondo, M Morales, I Valverde and ML Villanueva-Penacarrillo
Glucagon-like peptide-1 (GLP-1) has been shown to have insulin-like effects upon the metabolism of glucose in rat liver, muscle and fat, and on that of lipids in rat and human adipocytes. These actions seem to be exerted through specific receptors which, unlike that of the pancreas, are not - at least in liver and muscle - cAMP-associated. Here we have investigated the effect, its characteristics, and possible second messengers of GLP-1 on the glucose metabolism of human skeletal muscle, in tissue strips and primary cultured myocytes. In muscle strips, GLP-1, like insulin, stimulated glycogen synthesis, glycogen synthase a activity, and glucose oxidation and utilization, and inhibited glycogen phosphorylase a activity, all of this at physiological concentrations of the peptide. In cultured myotubes, GLP-1 exerted, from 10(-13) mol/l, a dose-related increase of the D-[U-(14)C]glucose incorporation into glycogen, with the same potency as insulin, together with an activation of glycogen synthase a; the effect of 10(-11) mol/l GLP-1 on both parameters was additive to that induced by the equimolar amount of insulin. Synthase a was still activated in cells after 2 days of exposure to GLP-1, as compared with myotubes maintained in the absence of peptide. In human muscle cells, exendin-4 and its truncated form 9-39 amide (Ex-9) are both agonists of the GLP-1 effect on glycogen synthesis and synthase a activity; but while neither GLP-1 nor exendin-4 affected the cellular cAMP content after 5-min incubation in the absence of 3-isobutyl-1-methylxantine (IBMX), an increase was detected with Ex-9. GLP-1, exendin-4, Ex-9 and insulin all induced the prompt hydrolysis of glycosylphosphatidylinositols (GPIs). This work shows a potent stimulatory effect of GLP-1 on the glucose metabolism of human skeletal muscle, and supports the long-term therapeutic value of the peptide. Further evidence for a GLP-1 receptor in this tissue, different from that of the pancreas, is also illustrated, suggesting a role for an inositolphosphoglycan (IPG) as at least one of the possible second messengers of the GLP-1 action in human muscle.
Jorge N Artaza, Suzanne Reisz-Porszasz, Joan S Dow, Robert A Kloner, James Tsao, Shalender Bhasin and Nestor F Gonzalez-Cadavid
Myostatin (Mst) is a negative regulator of skeletal muscle in humans and animals. It is moderately expressed in the heart of sheep and cattle, increasing considerably after infarction. Genetic blockade of Mst expression increases cardiomyocyte growth. We determined whether Mst overexpression in the heart of transgenic mice reduces left ventricular size and function, and inhibits in vitro cardiomyocyte proliferation. Young transgenic mice overexpressing Mst in the heart (Mst transgenic mice (TG) under a muscle creatine kinase (MCK) promoter active in cardiac and skeletal muscle, and Mst knockout (Mst (−/−)) mice were used. Xiscan angiography revealed that the left ventricular ejection fraction did not differ between the Mst TG and the Mst (−/−) mice, when compared with their respective wild-type strains, despite the decrease in whole heart and left ventricular size in Mst TG mice, and their increase in Mst (−/−) animals. The expected changes in cardiac Mst were measured by RT-PCR and western blot. Mst and its receptor (ActRIIb) were detected by RT-PCR in rat H9c2 cardiomyocytes. Transfection of H9c2 with plasmids expressing Mst under muscle-specific creatine kinase promoter, or cytomegalovirus promoter, enhanced p21 and reduced cdk2 expression, when assessed by western blot. A decrease in cell number occurred by incubation with recombinant Mst (formazan assay), without affecting apoptosis or cardiomyocyte size. Anti-Mst antibody increased cardiomyocyte replication, whereas transfection with the Mst-expressing plasmids inhibited it. In conclusion, Mst does not affect cardiac systolic function in mice overexpressing or lacking the active protein, but it reduces cardiac mass and cardiomyocyte proliferation.
Johanna G Miquet, Lorena González, Marina N Matos, Christina E Hansen, Audreen Louis, Andrzej Bartke, Daniel Turyn and Ana I Sotelo
Chronically elevated levels of GH in GH-transgenic mice result in accelerated growth and increased adult body weight. We have previously described that the GH-induced JAK2/STAT5-signaling pathway is desensitized in the liver of transgenic mice overexpressing GH. However, these animals present increased circulating IGF-I levels, increased hepatic GHR expression, and liver organomegaly due to hypertrophy and hyperplasia, which frequently progress to hepatomas as the animals age, indicating that action of GH on the liver is not prevented. In the present study, we have evaluated other GH-signaling pathways that could be activated in the liver of GH-transgenic mice. Upon GH administration, normal mice showed an important increment in STAT3 phosphorylation level, but transgenic mice did not respond to acute GH stimulation. However, STAT3 was constitutively phosphorylated in transgenic mice, whereas its protein content was not increased. GH-transgenic mice showed overexpression of c-Src, accompanied by an elevation of its activity. Other signaling mediators including focal adhesion kinase, epidermal growth factor receptor, Erk, Akt, and mammalian target of rapamycin displayed elevated protein and basal phosphorylation levels in these animals. Thus, GH-overexpressing transgenic mice exhibit hepatic upregulation of signaling mediators related to cell proliferation, survival, and migration. The upregulation of these proteins may represent GH-signaling pathways that are constitutively activated in the presence of dramatically elevated GH levels throughout life. These molecular alterations could be implicated in the pathological alterations observed in the liver of GH-transgenic mice.
N Zmora, D Gonzalez-Martinez, JA Munoz-Cueto, T Madigou, E Mananos-Sanchez, SZ Doste, Y Zohar, O Kah and A Elizur
The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.
Shiao Y Chan, Laura A Hancox, Azucena Martín-Santos, Laurence S Loubière, Merlin N M Walter, Ana-Maria González, Phillip M Cox, Ann Logan, Christopher J McCabe, Jayne A Franklyn and Mark D Kilby
The importance of the thyroid hormone (TH) transporter, monocarboxylate transporter 8 (MCT8), to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with MCT8 (SLC16A2) mutations. Intrauterine growth restriction (IUGR), usually due to uteroplacental failure, is associated with milder neurodevelopmental deficits, which have been partly attributed to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and decreased cerebral thyroid hormone receptor expression. We postulate that altered MCT8 expression is implicated in this pathophysiology; therefore, in this study, we sought to quantify changes in cortical MCT8 expression with IUGR. First, MCT8 immunohistochemistry was performed on occipital and parietal cerebral cortex sections obtained from appropriately grown for gestational age (AGA) human fetuses between 19 weeks of gestation and term. Secondly, MCT8 immunostaining in the occipital cortex of stillborn IUGR human fetuses at 24–28 weeks of gestation was objectively compared with that in the occipital cortex of gestationally matched AGA fetuses. Fetuses demonstrated widespread MCT8 expression in neurons within the cortical plate and subplate, in the ventricular and subventricular zones, in the epithelium of the choroid plexus and ependyma, and in microvessel wall. When complicated by IUGR, fetuses showed a significant fivefold reduction in the percentage area of cortical plate immunostained for MCT8 compared with AGA fetuses (P<0.05), but there was no significant difference in the proportion of subplate microvessels immunostained. Cortical MCT8 expression was negatively correlated with the severity of IUGR indicated by the brain:liver weight ratios (r 2=0.28; P<0.05) at post-mortem. Our results support the hypothesis that a reduction in MCT8 expression in the IUGR fetal brain could further compromise TH-dependent brain development.