Extracellular superoxide dismutase protects lung development in hyperoxia-exposed newborn mice

We tested the hypothesis that targeted transgenic overexpression of human extracellular superoxide dismutase (EC-SOD) would preserve alveolar development in hyperoxia-exposed newborn mice. We exposed newborn transgenic and wild-type mice to 95% oxygen (O2) or air x 7 days and measured bronchoalveolar lavage cell counts, and lung homogenate EC-SOD, oxidized and reduced glutathione, and myeloperoxidase. We found that total EC-SOD activity in transgenic newborn mice was approximately 2.5x the wild-type activity. Hyperoxia-exposed transgenic mice had less pulmonary neutrophil influx and oxidized glutathione than wild-type littermates at 7 days. We measured alveolar surface and volume density in animals exposed to 14 days more of air or 60% O2. Hyperoxia-exposed transgenic EC-SOD mice had significant preservation of alveolar surface and volume density compared with wild-type littermates. After 7 days 95% O2 + 14 days 60% O2, lung inflammation measured as myeloperoxidase activity was reduced to control levels in all treatment groups.     

Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression

To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27Kip1, p53, p16INK4a, and p19ARF, were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy.     

Myocardial hypertrophy in transgenic mice overexpressing human interleukin 1[alpha ]

BACKGROUND: Interleukin (IL)-1 has profound effects on nonimmune cells and organs, including the heart. The effects of IL-1 on transgenic hearts have not yet been described. METHODS AND RESULTS: We generated transgenic mice overexpressing the human IL-1 gene under control of the cytomegalovirus enhancer/chicken beta-actin promoter. Heart weight-body weight ratio increased 1.4- to 2.2-fold in transgenic mice compared with wild-type mice. Lung weight-body weight ratio also increased in transgenic mice, all of which died within 14 days of birth. Light microscopy revealed concentric hypertrophy with cardiomyocyte hypertrophy in all transgenic mice and pulmonary edema in some of them. Electron microscopy showed myofilament loss and an increased number of giant mitochondria, but no sarcomere disarray. Northern blotting showed that gene expression had been reprogrammed in the left ventricle of transgenic mice. Expression of fetal-type genes such as prepro-atrial natriuretic factor and beta-myosin heavy chain were increased, but voltage-dependent calcium channel messenger RNA expression was decreased in the left ventricle of transgenic mice compared with that of wild-type mice. CONCLUSIONS: IL-1 may cause structural and functional alterations in cardiac myocytes.     

Inhibition of protein phosphatase 1 by inhibitor-2 exacerbates progression of cardiac failure in a model with pressure overload

AIMS: The progression of human heart failure is associated with increased protein phosphatase 1 (PP1) activity, which leads to a higher dephosphorylation of cardiac regulatory proteins such as phospholamban. In this study, we tested the hypothesis whether the inhibitor-2 (I-2) of PP1 can mediate cardiac protection by inhibition of PP1 activity. METHODS AND RESULTS: We induced pressure overload by transverse aortic constriction (TAC) for 28 days in transgenic (TG) mice with heart-directed overexpression of a constitutively active form of I-2 (TG(TAC)) and wild-type littermates (WT(TAC)). Both groups were compared with sham-operated mice. TAC treatment resulted in comparable ventricular hypertrophy in both groups. However, TG(TAC) exhibited a higher atrial mass and an enhanced ventricular mRNA expression of beta-myosin heavy chain. The increased afterload was associated with the development of focal fibrosis in TG. Consistent with signs of overt heart failure, fractional shortening and diastolic function were impaired in TG(TAC) as revealed by Doppler echocardiography. The contractility was reduced in catheterized banded TG mice, which is in line with a depressed shortening of isolated myocytes. This is due to profoundly abnormal cytosolic Ca(2+) transients and a reduced stimulation of phosphorylation of phospholamban (PLB)(Ser16) after TAC in TG mice. Moreover, administration of isoproterenol was followed by a blunted contractile response in isolated myocytes of TG(TAC) mice. CONCLUSION: These results suggest that cardiac-specific overexpression of a constitutively active form of I-2 is deleterious for cardiac function under conditions of pressure overload. Thus, the long-term inhibition of PP1 by I-2 is not a therapeutic option in the treatment of heart failure.     

Insulin-like growth factor-1 attenuates the detrimental impact of nonocclusive coronary artery constriction on the heart.

Coronary artery narrowing (CAN) induces tissue injury, which may involve myocyte necrosis and apoptosis. Insulin-like growth factor (IGF)-1 may counteract cell death, modifying the detrimental effects of myocardial ischemia. On this basis, CAN was produced in female FVB.Igf+/- mice and nontransgenic littermates, and the animals were euthanized 7 days later. CAN consisted of an 82% reduction in the vessel luminal cross-sectional area in both groups of mice. Severe left ventricular dysfunction was present in CAN nontransgenic and transgenic mice, but heart and left ventricular weights increased more in littermates than in FVB.Igf+/- mice. Similarly, the changes in chamber volume and diastolic wall stress were greater in nontransgenic mice. Subacute tissue injury, represented by foci of replacement fibrosis, was 2.6-fold higher in CAN littermates than in FVB.Igf+/- mice. Ongoing myocyte necrosis was 5-fold greater in nontransgenic mice, whereas apoptosis was low and did not differ in the 2 groups of mice. In CAN nontransgenic mice, myocyte necrosis was 12-fold more frequent than apoptosis but, in CAN transgenic mice, these 2 types of cell death were comparable. alpha-Myosin and beta-myosin isoform mRNAs were affected by CAN, but alpha-myosin mRNA was reduced more in nontransgenic mice. In conclusion, myocyte necrosis and replacement fibrosis are the prevailing forms of myocardial damage induced by CAN. Constitutive overexpression of IGF-1 attenuates myocyte necrosis and tissue injury, having no effect on cell apoptosis. These factors limit ventricular dilation, myocardial loading, cardiac hypertrophy, and alterations in alpha- and beta-myosin isoform expression.     

The green fluorescent protein is an efficient biological marker for cardiac myocytes

There is a need for a non-toxic marker for cardiac myocytes in studies of cardiac development and in experimentally induced pathophysiologic states in adult animals. We investigated the possibility of using the enhanced green fluorescent protein (EGFP) gene as such a biological marker for cardiac myocytes in both whole animal and cell culture systems. Several lines of transgenic mice were constructed harboring an EGFP gene directed by a 2.38-kb promoter fragment of the hamster beta -myosin heavy chain gene. The transgene was preferentially expressed in the cardiac progenitor cells of embryos at E7.5, a developmental stage that precedes the formation of the cardiomyotube. It was specifically expressed in the cardiomyotube and myotomes along the somites of embryos at E8.5. The EGFP transgene expression continued in the heart throughout gestation and became very intense at birth. When neonatal cardiac cells were fractionated into myocytes and non-myocytes by a differential plating procedure, only myocytes from the transgenic mice showed specific green fluorescence of the transgene product that can be used as a marker for flow cytometry analysis. Although the expression levels were heterogeneous, EGFP expression persisted in the hearts of postnatal animals. In addition to the heart, some skeletal and smooth muscles from transgenic animals also expressed the transgene. The transgenic mice were healthy and had a normal life span, identical to their non-transgenic littermates. These results demonstrate that EGFP is an efficient non-toxic biological marker for cardiac myocytes.     

Inhibition of Na(+)-H(+) exchange prevents hypertrophy, fibrosis, and heart failure in beta(1)-adrenergic receptor transgenic mice

Chronic stimulation of the beta(1)-adrenergic receptor leads to hypertrophy and heart failure in beta(1)-adrenergic receptor transgenic mice and contributes to disease progression in heart failure patients. The cellular mechanisms underlying these detrimental effects are largely unknown. In this study, we have identified the cardiac Na(+)-H(+) exchanger (NHE1) as a novel mediator of adrenergically induced heart failure. beta(1)-Adrenergic receptor transgenic mice showed upregulation of both NHE1 mRNA (+140+/-6%) and protein (+42+/-19%). In order to test whether increased NHE1 is causally related to beta(1)-adrenergic-induced hypertrophy, fibrosis, and heart failure, beta(1)-adrenergic receptor transgenic (TG) and wild-type (WT) littermates were treated with a diet containing 6000 ppm of the NHE1 inhibitor cariporide or control chow for 8 months. There was significant hypertrophy of cardiac myocytes in beta(1)-adrenergic receptor transgenic mice (2.3-fold increase in myocyte cross-sectional area), which was virtually absent in cariporide-fed animals. Interstitial fibrosis was prominent throughout the left ventricular wall in nontreated beta(1)-adrenergic receptor transgenic mice (4.8-fold increase in collagen volume fraction); cariporide treatment completely prevented this development of fibrosis. Left ventricular catheterization showed that cariporide also prevented the loss of contractile function in beta(1)-adrenergic receptor transgenic mice: whereas untreated transgenic mice showed a significant decrease in left ventricular contractility (5250+/-570 mm Hg/s TG versus 7360+/-540 mm Hg/s WT, dp/dt(max)), this decrease was completely prevented by cariporide (8150+/-520 mm Hg/s TG cariporide). Inhibition of NHE1 prevented the development of heart failure in beta(1)-receptor transgenic mice. We conclude that the cardiac Na(+)-H(+) exchanger 1 is essential for the detrimental cardiac effects of chronic beta(1)-receptor stimulation in the heart.     

Cardiac contractile function is enhanced in isolated ventricular myocytes from growth hormone transgenic mice

Growth hormone (GH) plays a key role in cardiac growth and function. However, excessive levels of GH often result in cardiac dysfunction, which is the major cause of death in acromegalic patients. Transgenic mice with GH over-expression serve as useful models for acromegaly and exhibit impaired cardiac functions using echocardiography, similar to those of human acromegaly. However, the mechanism underscoring the impaired ventricular function has not been well defined. This study was designed to evaluate the cardiac excitation-contraction coupling in GH over-expressing transgenic mice at the single ventricular myocyte level. Myocytes were isolated from GH and age-matched wild-type mouse hearts. Mechanical properties were evaluated using an IonOptix MyoCam system. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS) and time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ properties were evaluated by fura-2. GH transgenic mice exhibited significantly increased body weights and enlarged heart and myocyte size. Myocytes from GH transgenic mice displayed significantly enhanced PS and+/-dL/dt associated with similar TPS and TR(90) compared with the wild-type littermates. Myocytes from GH transgenic mice displayed a similar resting intracellular Ca2+ level and Ca2+ removal rate but exhibited an elevated peak intracellular Ca2+ level compared with the wild-type group. Myocytes from both groups were equally responsive to increases in extracellular Ca2+ concentration and stimulating frequency. These results suggest that GH over-expression is associated with enhanced contractile function in isolated myocytes and that the impaired cardiac function observed in whole hearts may not be due to defects at the myocyte level.     

Expression of active alpha(1B)-adrenergic receptors in the heart does not alleviate ischemic reperfusion injury

Ischemic preconditioning reduces infarct size and improves cardiac function in various species, including mice. The mechanism for ischemic preconditioning protection is not entirely clear and activation of alpha(1B)-adrenergic receptors (AR) is believed to be involved. Transgenic mice expressing constitutively active mutant alpha(1B)-AR in the heart have enhanced alpha(1B)-AR activity and therefore can be used to test the role of alpha(1B)-AR in ischemic preconditioning. Wild-type and transgenic mice were subjected to 30- or 40-min periods of left coronary artery occlusion followed by 60-min reperfusion, or ischemic preconditioning prior to sustained ischemia-reperfusion. Risk and infarct zones were determined by staining with Evans blue and triphenyltetrazolium, respectively, and quantitated digitally. Infarct zone and infarct size were not different between wild-type and transgenic mice, nor was the extent of reduction in infarct size by preconditioning ischemia (wild-type mice: 45+/-3 to 18+/-3%, transgenic mice: 46+/-3 to 19+/-2% of the left ventricle, both P<0.01). Ventricular function was similar between wild-type and transgenic mice with or without ischemia-reperfusion injury. In conclusion, enhanced alpha(1B)-AR activity by cardiac-specific expression of constitutively active mutant alpha(1B)-AR in mice does not mimic ischemic preconditioning to protect against ischemia-reperfusion injury.     

Hepatitis B virus x gene and cyanobacterial toxins promote aflatoxin B1-induced hepatotumorigenesis in mice

AIM: To assess the combinative role of aflatoxin B1 (AFB1), cyanobacterial toxins (cyanotoxins), and hepatitis B virus (HBV) x gene in hepatotumorigenicity. METHODS: One-week-old animals carrying HBV x gene and their wild-type littermates were intraperitoneally (ip) injected with either single-dose AFB1 [6 mg/kg body weight (bw)], repeated-dose cyanotoxins (microcystin-LR or nodularin, 10μg/kg bw once a week for 15 wk), DMSO (vehicle control) alone, or AFB1 followed by cyanotoxins a week later, and were sacrificed at 24 and 52 wk post-treatment. RESULTS: AFB1 induced liver tumors in 13 of 29 (44.8%) transgenic mice at 52 wk post-treatment, significantly more frequent than in wild-type mice (13.3%). This significant difference was not shown in the 24-wk study. Compared with AFB1 exposure alone, MC-LR and nodularin yielded approximately 3-fold and 6-fold increases in the incidence of AFB1-induced liver tumors in wild-type animals at 24 wk, respectively. HBV x gene did not further elevate the risk associated with co-exposure to AFB1 and cyanotoxins. With the exception of an MC-LR-dosed wild-type mouse, no liver tumor was observed in mice treated with cyanotoxins alone at 24 wk. Neither DMSO-treated transgenic mice nor their wild-type littermates had pathologic alterations relevant to hepatotumorigenesis in even up to 52 wk. CONCLUSION: HBV x gene and nodularin promote the development of AFB1-induced liver tumors. Co-exposure to AFB1 and MC-LR tends to elevate the risk of liver tumors at 24 wk relative to exposure to one of them. The combinative effect of AFB1, cyanotoxins and HBVx on hepatotumorigenesis is weak at 24 wk.     

Reperfusion-activated Akt kinase prevents apoptosis in transgenic mouse hearts overexpressing insulin-like growth factor-1

Abstract -Hearts of wild-type and insulin-like growth factor-1 overexpressing (Igf-1(+/-)) transgenic mice were subjected to Langendorff perfusions and progressive periods of ischemia followed by reperfusion. Apoptosis was measured by DNA nucleosomal cleavage and a hairpin probe labeling assay to detect single-base overhang. Transgenic hearts subjected to 20 minutes of ischemia and 4 hours of reperfusion (I/R) sustained a rate of apoptosis of 1.8+/-0.3% compared with 4.6+/-1.1% for wild-type controls (n=4; P<0.03). Phosphorylation of the protein kinase Akt/protein kinase B was elevated 6.2-fold in transgenic hearts at baseline and increased another 4.4-fold within 10 minutes of reperfusion, remaining elevated for up to 2 hours. I/R activated Akt in wild-type hearts but to a lesser extent (1.6+/-0.3-fold). Pretreatment of transgenic hearts with wortmannin immediately before and during ischemia eliminated reperfusion-mediated activation of Akt and neutralized the resistance to apoptosis. The stress-activated kinase p38 was also activated during ischemia and reperfusion in both wild-type and transgenic hearts. Perfusion with the p38 inhibitor SB203580 (10 micromol/L) blocked both p38 activation and phosphorylation of Akt and differentially modulated apoptosis in wild-type and transgenic hearts. Pretreatment with SB203580 reduced apoptosis in wild-type hearts but increased apoptosis in transgenic hearts. These results demonstrate that Akt phosphorylation during I/R is modulated by IGF-1 and prevents apoptosis in hearts that overexpress the IGF-1 transgene.     

Proximal tubule overexpression of a locally acting IGF isoform, Igf-1Ea, increases inflammation after ischemic injury

ObjectiveIGF-1 is an important regulator of postnatal growth in mammals. In mice, a non-circulating, locally acting isoform of IGF-1, IGF-1Ea, has been documented as a central regulator of muscle regeneration and has been shown to improve repair in the heart and skin. In this study, we examine whether local production of IGF1-Ea protein improves tubular repair after renal ischemia reperfusion injury.DesignTransgenic mice in which the proximal-tubule specific promoter Sglt2 was driving the expression of an Igf-1Ea transgene. These animals were treated with an ischemic-reperfusion injury and the response at 24 h and 5 days compared with wildtype littermates.ResultsTransgenic mice demonstrated rapid and enhanced renal injury in comparison to wild type mice. Five days after injury the wild type and low expressing Igf-1Ea transgenic mice showed significant tubular recovery, while high expressing Igf-1Ea transgenic mice displayed significant tubular damage. This marked injury was accompanied by a two-fold increase in the number of F4/80 positive macrophages and a three-fold increase in the number of Gr1-positive neutrophils in the kidney. At the molecular level, Igf-1Ea expression resulted in significant up-regulation of proinflammatory cytokines such as TNF-α and Ccl2. Expression of Nfatc1 was also delayed, suggesting reduced tubular proliferation after kidney injury.ConclusionsThese data indicate that, unlike the muscle, heart and skin, elevated levels of IGF-1Ea in the proximal tubules exacerbates ischemia reperfusion injury resulting in increased recruitment of macrophages and neutrophils and delays repair in a renal setting.     

bcl-2 overexpression promotes myocyte proliferation

To determine the influence of Bcl-2 on the developmental biology of myocytes, we analyzed the population dynamics of this cell type in the heart of transgenic (TG) mice overexpressing Bcl-2 under the control of the alpha-myosin heavy chain promoter. TG mice and non-TG (wild type, WT) mice were studied at 24 days, 2 months, and 4 months after birth. Bcl-2 overexpression produced a significant increase in the percentage of cycling myocytes and their mitotic index. These effects were strictly connected to the expression of the transgene, as demonstrated in isolated myocytes. The formation of mitotic spindle and contractile ring was identified in replicating cells. These typical aspects of mitosis were complemented with the demonstration of karyokinesis and cytokinesis to provide structural evidence of cell division. Apoptosis was low at all ages and was not affected by Bcl-2. The higher cell replication rate in TG was conditioned by a decrease in the expression of the cell-cycle inhibitors, p21(WAF1) and p16(INK4a), and by an increase in Mdm2-p53 complexes. In comparison with WT, TG had 0.4 x 10(6), 0.74 x 10(6), and 1.2 x 10(6) more myocytes in the left ventricle at 24 days, 2 months, and 4 months, respectively. Binucleated myocytes were 12% and 25% larger in WT than in TG mice at 2 and 4 months of age. Taken together, these observations reveal a previously uncharacterized replication-enhancing function of Bcl-2 in myocytes in vivo in the absence of stressful conditions.     

Attenuated cardiac contractile responsiveness to insulin-like growth factor I in ventricular myocytes from biobreeding spontaneous diabetic rats

OBJECTIVE: Insulin-like growth factor I (IGF-1) stimulates cardiac growth and contraction, but resistance to its action has been reported in diabetes. This study was to determine if IGF-1-induced cardiac contractile action is altered in rats genetically predisposed to diabetes. METHOD: Ventricular myocytes were isolated from spontaneously biobreeding diabetes-prone (BB/DP) rats and their diabetes-resistant littermates (BB/DR). Mechanical properties were evaluated in cardiomyocytes using a video-based edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR(90)). Intracellular Ca(2+) transients were measured as changes in fura-2 fluorescence intensity (DeltaFFI). RESULTS: Myocytes from BB/DP rats displayed increased PS, prolonged TPS and TR(90,) as well as reduced resting FFI compared to the BB/DR group. IGF-1 (10(-10)-10(-6) M) caused a dose-dependent increase in PS in myocytes from BB/DR but not BB/DP rats. The increase of PS was blunted by IGF-1 antagonist H-1356, phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, but not tyrosine kinase inhibitor genistein. None of these agents affected responses to IGF-1 in BB/DP myocytes. Interestingly, IGF-1 elicited a comparable dose-dependent increase in Ca(2+) transients in myocytes from both BB/DR and BB/DP rats. CONCLUSION: These results suggest that the attenuation of IGF-1-induced cardiac contractile response in chemically-induced diabetes also exists in diabetes of genetic origin, possibly due to mechanisms involving PI-3 kinase and intracellular Ca(2+) sensitivity.     

Impaired expression of cardiac adiponectin in leptin-deficient mice with viral myocarditis

A mouse model of encephalomyocarditis (EMC) virus-induced myocarditis was used to investigate the expression of adiponectin in damaged cardiomyocytes. We intraperitoneally injected EMC virus into leptin-deficient ob/ob (OB) mice and wild-type (WT) mice. OB mice were divided into two subgroups consisting of mice with no intervention and mice receiving leptin replacement starting simultaneously with viral inoculation. We determined differences in heart weight, cardiac histological score, numbers of infiltrating and apoptotic cells in the myocardium, expression levels of adiponectin and TNF-alpha mRNA in the heart, adiponectin immunoreactivity in myocytes, adiponectin and TNF-alpha concentrations in the heart, and immunoreactivity of adiponectin receptors in myocytes between OB mice and WT mice. There was significantly decreased adiponectin mRNA expression, immunoreactivity, and protein level in the heart, and reduced immunoreactivity of adiponectin receptor 1 in myocytes from OB mice on days 4 and 8 after viral inoculation as compared with those in WT mice, together with increased cardiac weight, severe inflammatory myocardial damage, and increased levels of cardiac TNF-alpha mRNA and protein. Replacement of leptin in OB mice inhibited the development of severe myocarditis through augmentation of adiponectin mRNA, immunoreactivity, and protein level, increased adiponectin receptor 1 immunoreactivity in myocytes, and suppressed levels of TNF-alpha mRNA and protein. These results suggest that impaired expression of cardiac adiponectin may contribute to the progression of viral myocarditis through enhanced expression of TNF-alpha under a leptin-deficient condition.     

Alcohol suppresses insulin-like growth factor-1 gene expression in prepubertal transgenic female mice overexpressing the bovine growth hormone gene

BACKGROUND: Alcohol (ALC) delays puberty in female rats and alters the development of a normal menstrual pattern in rhesus monkeys. These actions are associated with depressed serum levels of growth hormone (GH), luteinizing hormone and insulin-like growth factor-1 (IGF-1). The mechanism of this ALC-induced depression in IGF-1 is not known, however, could be due to depressed GH and, possibly, to an alteration in the hepatic GH receptor. To assess whether ALC has a direct action at the liver, we used a transgenic mouse model that overexpresses GH, allowing assessment of potential direct actions of ALC on the level of either the GH receptor or the IGF-1-synthesizing machinery within the hepatocyte. METHODS: One group of transgenic mice was fed a liquid diet containing ALC. The second group was pair-fed the companion isocaloric control liquid diet. The third group of transgenic mice was fed Lab Chow and water. The fourth group consisted of normal (nontransgenic) littermates fed Lab Chow and water. Animals received their respective diets for 5 days. Mice were killed during their late juvenile stage of development, and tissues and blood collected and frozen. RESULTS: The ALC-fed transgenic mice showed a decrease (p < 0.01) in hepatic IGF-1a and IGF-1b messenger RNA levels compared with transgenic controls, and this paralleled a decrease (p < 0.01) in serum IGF-1. ALC did not alter the circulating levels of bovine GH held constant by the promotor and did not alter mouse GH receptor protein levels as analyzed by Western blotting. CONCLUSIONS: Using this transgenic animal model that maintains circulating GH in the presence of ALC, we found that the ability of ALC to suppress prepubertal Igf1 gene expression can also occur independently of any alterations in the level of circulating GH. This direct effect on the hepatocyte is a postreceptor event because the GH receptor protein levels were not altered by ALC exposure.