Daniel Turyn

Alterations in the early steps of the insulin-signaling system in skeletal muscle of GH-transgenic mice

Growth hormone (GH) excess is associated with insulin resistance, but the molecular mechanisms of this association are poorly understood. In the current work, we have examined the consequences of exposure to high GH levels on the early steps of the insulin-signaling system in the muscle of bovine (b) GH-transgenic mice. The protein content and the tyrosine phosphorylation state of the insulin receptor (IR), the IR substrate-1 (IRS-1), the association between IRS-1 and the p85 subunit of phosphatidylinositol (PI) 3-kinase, and the phosphotyrosine-derived PI 3-kinase activity in this tissue were studied. We found that in skeletal muscle of bGH-transgenic mice, exposure to high circulating GH levels results in 1) reduced IR abundance, 2) reduced IR tyrosine phosphorylation, 3) reduced efficiency of IRS-1 tyrosine phosphorylation, and 4) defective activation of PI 3-kinase by insulin. These alterations may be related to the insulin resistance exhibited by these animals.Growth hormone (GH) excess is associated with insulin resistance, but the molecular mechanisms of this association are poorly understood. In the current work, we have examined the consequences of exposure to high GH levels on the early steps of the insulin-signaling system in the muscle of bovine (b) GH-transgenic mice. The protein content and the tyrosine phosphorylation state of the insulin receptor (IR), the IR substrate-1 (IRS-1), the association between IRS-1 and the p85 subunit of phosphatidylinositol (PI) 3-kinase, and the phosphotyrosine-derived PI 3-kinase activity in this tissue were studied. We found that in skeletal muscle of bGH-transgenic mice, exposure to high circulating GH levels results in 1) reduced IR abundance, 2) reduced IR tyrosine phosphorylation, 3) reduced efficiency of IRS-1 tyrosine phosphorylation, and 4) defective activation of PI 3-kinase by insulin. These alterations may be related to the insulin resistance exhibited by these animals.

Angiotensin-(1-7) improves cardiac remodeling and inhibits growth-promoting pathways in the heart of fructose-fed rats.

The present study examined whether chronic treatment with angiotensin (ANG)-(1-7) reduces cardiac remodeling and inhibits growth-promoting signaling pathways in the heart of fructose-fed rats (FFR), an animal model of insulin resistance. Sprague-Dawley rats were fed either normal rat chow (control) or the same diet plus 10% fructose in drinking water. For the last 2 wk of a 6-wk period of the corresponding diet, control and FFR were implanted with osmotic pumps that delivered ANG-(1-7) (100 ng.kg(-1).min(-1)). A subgroup of each group of animals (control or FFR) underwent a sham surgery. We determined heart weight, myocyte diameter, interstitial fibrosis, and perivascular collagen type III deposition as well as the phosphorylation degree of ERK1/2, JNK1/2, and p38MAPK. FFR showed a mild hypertension that was significantly reduced after ANG-(1-7) treatment. Also, FFR displayed higher ANG II circulating and local levels in the heart that remained unaltered after chronic ANG-(1-7) infusion. An increased heart-to-body weight ratio, myocyte diameter, as well as left ventricular fibrosis and perivascular collagen type III deposition were detected in the heart of FFR. Interestingly, significant improvements in these cardiac alterations were obtained after ANG-(1-7) treatment. Finally, FFR that received ANG-(1-7) chronically displayed significantly lower phosphorylation levels of ERK1/2, JNK1/2, and p38MAPK. The beneficial effects obtained by ANG-(1-7) were associated with normal values of Src-homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) activity in the heart. In conclusion, chronic ANG-(1-7) treatment ameliorated cardiac hypertrophy and fibrosis and attenuated the growth-promoting pathways in the heart. These findings show an important protective role of ANG-(1-7) in the heart of insulin-resistant rats.

Angiotensin-(1–7) through AT2 receptors mediates tyrosine hydroxylase degradation via the ubiquitin–proteasome pathway

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1–7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang‐(1–7) down‐regulates tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang‐(1–7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from 3H‐l‐tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre‐incubated with Ang‐(1–7) showed a significant decrease in TH specific activity. Ang‐(1–7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT2 receptor antagonist and not by an AT1 or Mas receptor antagonist, suggesting the involvement of AT2 receptors. The proteasome inhibitor MG132 blocked the Ang‐(1–7)‐mediated TH reduction. In addition, Ang‐(1–7) increased the amount of TH–ubiquitin complexes, indicating that the Ang‐(1–7)‐mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang‐(1–7) down‐regulates TH activity and expression centrally leading to a decrease in the central NE system activity.

Angiotensin-(1-7) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stimulating STAT3 and STAT5a/b phosphorylation and inhibiting angiotensin II-stimulated ERK1/2 and Rho kinase activity.

Angiotensin (ANG) II contributes to cardiac remodelling by inducing the activation of several signalling molecules, including ERK1/2, Rho kinase and members of the STAT family of proteins. Angiotensin‐(1–7) is produced in the heart and inhibits the proliferative actions of ANG II, although the mechanisms of this inhibition are poorly understood. Accordingly, in the present study we examined whether ANG‐(1–7) affects the ANG II‐mediated activation of ERK1/2 and Rho kinase, STAT3 and STAT5a/b in rat heart in vivo. We hypothesized that ANG‐(1–7) inhibits these growth‐promoting pathways, counterbalancing the trophic action of ANG II. Solutions of normal saline (0.9% NaCl) containing ANG II (8 pmol kg−1) plus ANG‐(1–7) in increasing doses (from 0.08 to 800 pmol kg−1) were administered via the inferior vena cava to anaesthetized male Sprague–Dawley rats. After 5 min, hearts were removed and ERK1/2, Rho kinase, STAT3 and STAT5a/b phosphorylation was determined by Western blotting using phosphospecific antibodies. Angiotensin II stimulated ERK1/2 and Rho kinase phosphorylation (2.3 ± 0.2‐ and 2.1 ± 0.2‐fold increase over basal values, respectively), while ANG‐(1–7) was without effect. The ANG II‐mediated phosphorylation of ERK1/2 and Rho kinase was prevented in a dose‐dependent manner by ANG‐(1–7) and disappeared in the presence of the Mas receptor antagonist d‐Ala7‐ANG‐(1–7). Both ANG II and ANG‐(1–7) increased STAT3 and STAT5a/b phosphorylation to a similar extent (130–140% increase). The ANG‐(1–7)‐stimulated STAT phosphorylation was blocked by the AT1 receptor antagonist losartan and not by d‐Ala7‐ANG‐(1–7). Our results show a dual action of ANG‐(1–7), that is, a stimulatory effect on STAT3 and 5a/b phosphorylation through AT1 receptors and a blocking action on ANG II‐stimulated ERK1/2 and Rho kinase phosphorylation through Mas receptor activation. The latter effect could be representative of a mechanism for a protective role of ANG‐(1–7) in the heart by counteracting the effects of locally generated ANG II.

Cytokine-Inducible SH2 Protein Up-Regulation Is Associated with Desensitization of GH Signaling in GHRH-Transgenic Mice

The effects of continuous high GH levels on GH signal transduction through the GH receptor (GHR)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway as well as the desensitization of this pathway by suppressors of cytokine signaling (SOCS) were studied in transgenic mice overexpressing GHRH. In transgenic mice, hepatic GHR levels were 4.5-fold higher than in normal animals, whereas the protein contents of JAK2, STAT5a, and STAT5b did not vary. This same pattern was found for basal tyrosine phosphorylation (PY-): PY-GHR was 4.5-fold increased in transgenic mice, whereas there were no differences in PY-JAK2 and PY-STATs between normal and transgenic animals. After GH administration, tyrosine phosphorylation of GHR, JAK2, and STAT5s increased 3- to 7-fold in normal mice, but no significant changes were found in transgenic mice, indicating a decreased GH sensitivity in these animals. The content of cytokine-inducible SH2 protein, a member of the SOCS family, was 18-fold higher in GHRH-transgenic than in normal mice. Conversely, SOCS-3, present in normal mice, was hardly seen in transgenic animals, whereas SOCS-2 levels did not vary. These findings suggest that cytokine-inducible SH2 protein, significantly induced by continuously elevated GH levels, may be the SOCS protein responsible for the GH signaling desensitization in transgenic animals.

Upregulation of the angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas receptor axis in the heart and the kidney of growth hormone receptor knock-out mice

OBJECTIVEnGrowth hormone (GH) resistance leads to enhanced insulin sensitivity, decreased systolic blood pressure and increased lifespan. The aim of this study was to determine if there is a shift in the balance of the renin-angiotensin system (RAS) towards the ACE2/Ang-(1-7)/Mas receptor axis in the heart and the kidney of a model of GH resistance and retarded aging, the GH receptor knockout (GHR-/-) mouse.nnnDESIGNnRAS components were evaluated in the heart and the kidney of GHR-/- and control mice by immunohistochemistry and Western blotting (n=12 for both groups).nnnRESULTSnThe immunostaining of Ang-(1-7) was increased in both the heart and the kidney of GHR-/- mice. These changes were concomitant with an increased immunostaining of the Mas receptor and ACE2 in both tissues. The immunostaining of AT1 receptor was reduced in heart and kidney of GHR-/- mice while that of AT2 receptor was increased in the heart and unaltered in the kidney. Ang II, ACE and angiotensinogen levels remained unaltered in the heart and the kidney of GH resistant mice. These results were confirmed by Western blotting and correlated with a significant increase in the abundance of the endothelial nitric oxide synthase in both tissues.nnnCONCLUSIONSnThe shift within the RAS towards an exacerbation of the ACE2/Ang-(1-7)/Mas receptor axis observed in GHR-/- mice could be related to a protective role in cardiac and renal function; and thus, possibly contribute to the decreased incidence of cardiovascular diseases displayed by this animal model of longevity.

Ames dwarf (Prop1df/Prop1df) mice display increased sensitivity of the major GH-signaling pathways in liver and skeletal muscle

CONTEXTnGrowth hormone (GH) is an anabolic hormone that regulates growth and metabolism. Ames dwarf mice are natural mutants for Prop1, with impaired development of anterior pituitary and undetectable levels of circulating GH, prolactin and TSH. They constitute an endocrine model of life-long GH-deficiency. The main signaling cascades activated by GH binding to its receptor are the JAK2/STATs, PI-3K/Akt and the MAPK Erk1/2 pathways.nnnOBJECTIVESnWe have previously reported that GH-induced STAT5 activation was higher in Ames dwarf mice liver compared to non-dwarf controls. The aim of this study was to evaluate the principal components of the main GH-signaling pathways under GH-deficiency in liver and skeletal muscle, another GH-target tissue.nnnMETHODSnAmes dwarf mice and their non-dwarf siblings were assessed. Animals were injected i.p. with GH or saline 15min before tissue removal. Protein content and phosphorylation of signaling mediators were determined by immunoblotting of tissue solubilizates.nnnRESULTSnGH was able to induce STAT5 and STAT3 tyrosine phosphorylation in both liver and muscle, but the response was higher for Ames dwarf mice than for non-dwarf controls. When Erk1/2 activation was assessed in liver, only dwarf mice showed GH-induced phosphorylation, while in muscle no response to the hormone was found in either genotype. GH-induced Akt phosphorylation at Ser473 in liver was only detected in dwarf mice. In skeletal muscle, both normal and dwarf mice responded to a GH stimulus, although dwarf mice presented higher GH activation levels. The phosphorylation of GSK-3, a substrate of Akt, increased upon hormone stimulation only in dwarf mice in both tissues. In contrast, no differences in the phosphorylation of mTOR, another substrate of Akt, were observed after GH stimulus, either in normal or dwarf mice in liver, while we were unable to determine mTOR in muscle. Protein content of GH-receptor and of the signaling mediators studied did not vary between normal and dwarf animals in the assessed tissues.nnnCONCLUSIONnThese results show that several components of the main GH-signaling pathways exhibit enhanced sensitivity to the hormone in liver and muscle of Ames dwarf mice.

In vivo specific uptake of labeled insulin by liver, adipose tissue, pituitary, and adrenals in the turtle Chrysemys dorbigni

Insulin labeled with 125I was injected into turtles (Chrysemys dorbigni) to study its specific uptake by tissues. The maximum specific uptake of radioactivity by turtle tissues was obtained 1 hr after administration of [125I]iodoinsulin. Besides liver and adipose tissue, specific uptake of labeled insulin was detected in some endocrine glands, such as pituitary and adrenals. Both glands were as active in concentrating labeled insulin as liver and adipose tissue. A significant reduction of the uptake was observed when unlabeled insulin was injected together with the labeled hormone. This reduction was dose dependent, and the concentration of unlabeled insulin that prevented 50% of the tissue uptake of [125I]iodoinsulin was of 1 to 10 μg/kg body weight. These doses were able to induce blood glucose decrease in the turtle. Prolactin, growth hormone, or glucagon were unable to displace labeled insulin uptake. The major proportion of the radioactive material extracted from liver and pituitary 1 hr after [125I]iodoinsulin injection into turtle coeluted with [125I]iodoinsulin in Sephadex G-50 column. The presence of radioactive degradation products are consistent with the intracellular receptor mediated degradation hypothesis. These findings suggest the presence of specific insulin binding sites in liver, adipose tissue, pituitary, and adrenal glands from turtles.

Distribution and specific binding “in vivo” of iodinated growth hormones in the turtle Chrysemys dorbigni

Abstract Bovine (bGH) and human growth hormone (hGH) labeled with 125 I were injected into turtle Chrysemys dorbigni , in order to study their distributions in tissues. The radioactivity was basically concentrated by the liver and kidney, reaching a maximum 4 hr after the labeled hormone injection. Only the liver showed a significant reduction of radioactivity uptake, when labeled growth hormone was injected together with an excess of unlabeled hormone. This reduction was dose dependent. Injection of [ 125 I]iodo-hGH produced higher uptake of radioactivity by the liver than [ 125 I]iodo-bGH. The study performed suggests: (1) specific uptake of hGH or bGH by the liver; and (2) the presence in the liver of both somatogenic and lactogenic binding sites.

Prolactin (PRL) induction of cyclooxygenase 2 (COX2) expression and prostaglandin (PG) production in hamster Leydig cells

Serum prolactin (PRL) variations play a crucial role in the photoperiodic-induced testicular regression-recrudescence transition in hamsters. We have previously shown that cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins (PGs), is expressed mostly in Leydig cells of reproductively active hamsters with considerable circulating and pituitary levels of PRL. In this study, we describe a stimulatory effect of PRL on COX2/PGs in hamster Leydig cells, which is mediated by IL-1β and prevented by P38-MAPK and JAK2 inhibitors. Furthermore, by preparative isoelectric focusing (IEF), we isolated PRL charge analogues from pituitaries of active [isoelectric points (pI): 5.16, 4.61, and 4.34] and regressed (pI: 5.44) hamsters. More acidic PRL charge analogues strongly induced COX2 expression, while less acidic ones had no effect. Our studies suggest that PRL induces COX2/PGs in hamster Leydig cells through IL-1β and activation of P38-MAPK and JAK2. PRL microheterogeneity detected in active/inactive hamsters may be responsible for the photoperiodic variations of COX2 expression in Leydig cells.