Effects of IGF-I on cardiac growth and expression of mRNAs coding for cardiac proteins after induction of heart hypertrophy in the rat.

Adult rat cardiomyocytes in long-term culture reexpress several fetal cardiac proteins which also reappear during overload heart hypertrophy in vivo. IGF-I decreases reexpression of some of these proteins and stimulates myofibrillogenesis. IGF-I might therefore contribute to enhancing readaptation of the heart to overload. In order to test this hypothesis, hypertension was induced in male Wistar Kyoto rats by constriction of the left renal artery, and an infusion of 500 microg/day of recombinant human IGF-I (rhIGF-I) or vehicle was started after the operation via intraabdominally implanted osmotic minipumps. In the vehicle-treated hypertensive animals body weight gain was reduced after 3, 7 and 14 days, whereas rhIGF-I-treated hypertensive animals continued to gain weight like sham-operated animals. Left ventricular weight and the left, but not the right ventricle/body weight ratio increased more in rhIGF-I- than in vehicle-infused rats. Left ventricular IGF-I mRNA levels remained unchanged after renal clipping in both vehicle- and rhIGF-I-treated rats. However, beta-myosin heavy chain (MHC) mRNA in the left ventricle was 6- to 10-fold increased in clipped controls during the whole postoperative period, and rhIGF-I reduced this increase by more than 50% on days 7 and 14. On the first postoperative day, rhIGF-I prevented the decrease (50%) of alpha-MHC mRNA and the increase (2.5-fold of atrial natriuretic factor mRNA in the left ventricle. Renal clipping did not alter cardiac alpha-actin, but enhanced skeletal alpha-actin mRNA expression in the left ventricle up to 2.5-fold. However, both mRNAs were unaffected by rhIGF-I treatment. Restoration of body weight gain and stimulation of left ventricular cardiac weight by rhIGF-I as well as partial reversion of hypertension-induced changes in cardiac protein expression may reflect beneficial effects contributing to enhance readaptation of the heart to overload.

Insulin-like growth factor-I stimulates myofibrillar genes and modulates atrial natriuretic factor mRNA in rat heart.

We have investigated the effect of a 6-day infusion of recombinant human (rh) IGF-I (0.3-1.0 mg/day) or rhGH (200 mU/day) into normal and hypophysectomized rats on the ventricular expression of myofibrillar genes (alpha- and beta-myosin heavy chain (MHC), skeletal and cardiac alpha-actin) and of atrial natriuretic factor (ANF). In normal rats, beta-MHC was not detectable either before or after IGF-I or GH, but alpha-MHC mRNA increased significantly (twofold) with GH (not statistically significant for IGF-I). In contrast to normal rats, hypophysectomized rats did not express alpha-MHC either before or after IGF-I or GH, but beta-MHC was strongly expressed and significantly stimulated (1.8-fold) by IGF-I (not statistically significantly with GH). Skeletal alpha-actin expression remained unchanged during IGF-I or GH treatment of normal rats, but was enhanced by both IGF-I and GH (2.5- and 2.8-fold respectively) in hypophysectomized rats. Expression of cardiac alpha-actin in normal and hypophysectomized rats was not altered by either treatment. IGF-I and GH decreased ventricular expression of ANF in normal rats by 63% and 45% respectively, but did not influence ANF expression in hypophysectomized rats. Our results show that IGF-I and GH (possibly via IGF-I) stimulate expression of myofibrillar genes and modulate ANF mRNA concentrations in rat heart ventricles in vivo, depending on the hormonal status of the animals. However, neither IGF-I nor GH caused a shift from the beta- to the alpha-MHC isoform in hypophysectomized rats.

Effects and fate of human IGF-binding protein-5 in rat osteoblast cultures.

Osteoblasts prepared from calvaria of newborn rats produce insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP), IGFBP-5 was discovered in bone extracts. However, we could not detect IGFBP-5 in the medium of newborn rat osteoblasts, although we found mRNA expression. To find an explanation for this discrepancy and to learn more about the physiological role of IGFBP-5 in these cells, we studied the biological activity and the fate of recombinant human (rh) IGFBP-5 in comparison to rhIGFBP-3. IGFBP-5 but not IGFBP-3 stimulated thymidine incorporation into DNA both in the absence and presence of IGF-I. However, IGFBP-5 did not enhance uridine incorporation into RNA and glucose incorporation into glycogen. 125I-rhIGFBP-5 but not 125I-rhIGFBP-3 rapidly disappeared from the culture medium consistent with the observation that endogenous (rat) IGFBP-3 but not IGFBP-5 accumulated in the medium. However, intact 125I-labeled or unlabeled rhIGFBP-5 was associated with the cell-layer matrix, whereas IGFBP-5 fragments appeared in the medium. Trapping of IGFBP-5 in the cell layer matrix may enhance local availability of IGF.

Triiodothyronine induces over-expression of alpha-smooth muscle actin, restricts myofibrillar expansion and is permissive for the action of basic fibroblast growth factor and insulin-like growth factor I in adult rat cardiomyocytes.

Effects of triiodothyronine (T3) on the expression of cytoskeletal and myofibrillar proteins in adult rat cardiomyocytes (ARC) were followed during two weeks of culture in the presence of 20% T3-depleted (stripped) FCS. Control cultures expressed mainly beta-myosin heavy chain (MHC) mRNA. T3 caused a switch to alpha-MHC expression and a dose-dependent increase of alpha-smooth muscle (alpha-sm) actin mRNA and protein. In parallel, the number of alpha-sm actin immunoreactive cells increased from 1% in controls to 29 and 62% in ARC treated with 5 and 100 nM T3. In the presence of T3, cells exhibited a higher beating rate than controls. The distribution of myofibrils in T3-treated cells was restricted to the perinuclear area with a sharp boundary. Only 5% of the control cells but 30 and 62% of the T3-treated (5 and 100 nM) ARC showed this restricted myofibrillar phenotype. Basic fibroblast growth factor (bFGF) which restricts myofibrillar growth and upregulates alpha-sm actin in ARC cultured with normal FCS had no effect on alpha-sm actin in ARC cultured in stripped FCS, but potentiated the effect of T3. In contrast, insulin-like growth factor I (IGF I), which suppresses alpha-sm actin and stimulates myofibrillogenesis in the presence of normal FCS suppressed T3-induced alpha-sm actin expression in stripped FCS. Thus, T3 appears to be permissive for the action of bFGF and IGF I on alpha-sm actin expression.

Reduced ob mRNA in hypophysectomised rats is not restored by growth hormone (GH), but further suppressed by exogenously administered insulin-like growth factor (IGF) I.

To examine whether GH and IGF-I participate in the regulation of obese (ob) mRNA expression we determined ob mRNA levels in epididydimal fat pads of hypophysectomised (hypox) rats, hypox rats treated with recombinant human (rh) GH or rhIGF-I and normal, weight-matched controls. We found that 1. ob mRNA was markedly suppressed after hypophysectomy (37 +/- 25% of controls), 2. GH infusion had no effect on ob mRNA, but stimulated IGF-I mRNA in fat pads, 3. IGF-I treatment further suppressed ob mRNA (3.5% +/- 0.6% of controls) and 4. serum insulin levels were decreased in all hypox groups (11.2 to 15.9% of controls). In conclusion, exogenous and GH-induced IGF-I differ in their effects on ob mRNA expression and GH is unable to restore ob mRNA towards normal at low insulin levels.

1 alpha,25-dihydroxyvitamin D3 increases IGF binding protein-5 expression in cultured osteoblasts.

Rat osteoblasts produce insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs). Expression of IGFBP-5, an IGFBP which stimulates DNA synthesis of osteoblasts, was studied in vitro under the influence of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH). These two calcium-regulating hormones stimulated the expression of IGFBP-5 mRNA in cultured rat osteoblasts at low concentrations (10 pM) and in a dose- and time-dependent manner. Intact IGFBP-5 was not detected in the culture medium, but was found attached to the cell layer. IGFBP-5 may thus direct IGFs to the sites of bone remodeling.

Triiodothyronine increases glucose transporter isotype 4 mRNA expression, glucose transport, and glycogen synthesis in adult rat cardiomyocytes in long-term culture.

Effects of T3 treatment (day 2-day 12) on the expression of the insulin-regulated GLUT4, on 2-deoxyglucose uptake, and on glycogen synthesis were studied in ARC after 12 days of culture in T3-depleted 20% fetal calf serum. GLUT4 mRNA expression was low in controls, but increased in a dose-dependent manner by T3 treatment up to 2.8-fold at 100 nM. In parallel, 100 nM T3 increased basal 2-deoxyglucose uptake 1.95-fold and insulin-stimulated uptake 1.75-fold. In addition, T3 enhanced basal and insulin-stimulated [14C] glucose incorporation into glycogen 1.86- and 1.5-fold, respectively. Hence, ARC may meet part of their increased energy requirements in response to T3 by enhancing GLUT4 expression.

Expression, effects, and fate of IGFBP-5 are different in normal and malignant osteoblastic cells.

Normal osteoblasts from newborn rat calvaria and human osteosarcoma (Saos-2) cells express IGFBP-5 mRNA. IGF I increases IGFBP-5 mRNA levels in both cell types, whereas retinoic acid stimulates IGFBP-5 mRNA expression in calvaria but suppresses it in Saos-2 cells. IGFBP-5 mRNA expression is stimulated in normal bone cells by parathyroid hormone. 30 nM IGFBP-5 stimulates 3H-thymidine incorporation in calvaria (which produce IGF I contributing to basal proliferation in serum-free medium) but not in Saos-2 cells which produce little IGF I and IGF II. Among the 5 rhIGFBPs tested (IGFBP-2 to -6), only IGFBP-5 stimulates DNA synthesis in calvaria cells, and only IGFBP-6 in Saos-2 cells. RhIGFBP-5 displays a short half-life (approximately 30 min) in serum-free medium of calvaria cells and a long half-life (approximately 15 h) in the medium of Saos-2 cells. Fragments of 20 and 14 kDa accumulate in the media of both cell types. Intact (31 kDa) IGFBP-5 is associated and remains with the extracellular matrix of mature calvaria osteoblasts but not of Saos-2 cells. Among the IGFBPs produced IGFBP-5 is unique with regard to its marked affinity to matrix of normal bone cells, its short half-life when released, and its stimulatory effects on DNA synthesis.

Expression and regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein messenger ribonucleic acid levels in tissues of hypophysectomized rats infused with IGF-I and growth hormone.

The expression and regulation of insulin-like growth factor-I (IGF-I) and IGF-binding protein-2 (IGFBP-2), -3, -4, and -5 messages were studied in liver, kidney, spleen, thymus, heart, brain, skeletal muscle, testes, and epididymal (white) adipose tissue (WAT) from hypophysectomized rats infused with saline, recombinant human (rh) IGF-I, or rhGH and compared with tissue messenger RNA (mRNA) levels in age-matched normal rats. The IGF-I message was present in all of these tissues. It was most abundant in liver and WAT, but was barely detectable in kidney, brain, and thymus. GH dependence was most pronounced in liver, skeletal muscle, and WAT and less so in heart, testes, kidney, spleen, and thymus. The IGF-I message in brain was not influenced by hypophysectomy. IGF-I infusion induced a small increase in its own mRNA in skeletal muscle and WAT, whereas it decreased its own message in liver. IGFBPs were expressed in a tissue-specific manner; IGFBP-2 mRNA was most abundant in testes and hypophysectomized liver, IGFBP-3 mRNA was most abundant in spleen, kidney, WAT, and liver, IGFBP-4 mRNA was most abundant in liver, and IGFBP-5 mRNA was most abundant in kidney, WAT, and skeletal muscle. After hypophysectomy, significant decreases in IGFBP expression were observed in liver (except IGFBP-2), skeletal muscle, brain, WAT (except IGFBP-4), and testes (except IGFBP-2), in contrast to heart, kidney, spleen, and thymus. GH infusion did not affect IGFBP-2 mRNA levels in liver (in contrast to IGF-I infusion) or brain. Like GH, IGF-I normalized IGFBP-3 mRNA levels in liver, but, in contrast to GH, had no effect on IGFBP-5 mRNA in WAT. It was considerably less effective than GH in raising IGFBP-5 mRNA levels in skeletal muscle. Thus, GH infusion can exert different effects on IGF-I and IGFBP expression than infused rhIGF-I. Differences may be due to direct actions of GH at the tissue level, including auto/paracrine effects of locally produced IGF-I.