Immunoneutralization of endogenous glucagon-like peptide-2 reduces adaptive intestinal growth in diabetic rats

Supraphysiological doses of glucagon-like peptide-2 (GLP-2) have been shown to induce intestinal growth by increasing villus height and crypt depth and by decreasing apoptosis, but a physiological effect of GLP-2 has not yet been demonstrated. Earlier, we found elevated levels of endogenous GLP-2 in untreated streptozotocin diabetic rats associated with marked intestinal growth. In the present study, we investigated the role of endogenous GLP-2 for this adaptive response. We included four groups of six rats: (1) diabetic rats treated with saline, (2) diabetic rats treated with non-specific antibodies, (3) diabetic rats treated with polyclonal GLP-2 antibodies and (4) non-diabetic control rats treated with saline. All animals were treated with once daily intraperitoneal injections for 13 days and killed on day 14. Diabetic rats treated with saline or non-specific antibodies had a significantly (P<0.01) increased area of mucosa (13.00+/-0.64 and 13.37+/-0.60 mm(2), respectively) in the proximal part of the small intestine compared with non-diabetic controls (7.97+/-0.70 mm(2)). In contrast, diabetic rats treated with GLP-2 antibodies had a significantly (P<0.01) smaller increase in area of mucosa in the proximal part of the small intestine (10.84+/-0.44 mm(2)). Antibody treatment had no effect on body weight, blood glucose concentrations and food intake. Thus, blocking of endogenous GLP-2 in a model of adaptive intestinal growth reduces the growth response, providing strong evidence for a physiological growth factor function of GLP-2.     

Enteral nutrients potentiate the intestinotrophic action of glucagon-like peptide-2 in association with increased insulin-like growth factor-I responses in rats

Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, intestinotrophic hormone derived from posttranslational processing of proglucagon in the distal bowel. GLP-2 is thought to act through indirect mediators, such as IGF-I. We investigated whether intestinal expression of GLP-2 and IGF-I system components are increased with the mucosal growth induced by enteral nutrient (EN) and/or a low dose of GLP-2 in parenterally fed rats. Rats were randomized to four treatment groups using a 2 x 2 design and maintained with parenteral nutrition (PN) for 7 days: PN alone, EN, GLP-2, and EN+GLP-2; n = 7-9. The two main treatment effects are +/-GLP-2 (100 microg.kg body wt(-1).day(-1)) and +/-EN (43% of energy needs, days 4-6). Combination treatment with EN+GLP-2 induced synergistic intestinal growth in ileum, resulting in greater mucosal cellularity, sucrase segmental activity, and gain of body weight (ENxGLP-2, P < 0.04). In addition, EN+GLP-2 induced a significant 28% increase in plasma concentration of bioactive GLP-2, a significant 102% increase in ileal proglucagon mRNA with no change in ileal dipeptidyl peptidase-IV (DPP-IV) specific activity, and significantly reduced plasma DPP-IV activity compared with GLP-2. This indicates that EN potentiates the intestinotrophic action of GLP-2. Proliferation of enterocytes due to GLP-2 infusion was associated with greater expression of ileal proglucagon, GLP-2 receptor, IGF-I, IGF binding protein-3 mRNAs, and greater IGF-I peptide concentration in ileum (P < 0.032). Ileal IGF-I mRNA was positively correlated with expression of proglucagon, GLP-2R, and IGFBP-5 mRNAs (R2 = 0.43-0.56, P < 0.0001). Our findings support the hypothesis that IGF-I is one of the downstream mediators of GLP-2 action in a physiological model of intestinal growth.     

Insulin-like growth factor I mediates high-fat diet-induced adipogenesis in Osborne-Mendel rats

To determine if high-fat (HF) diet-induced changes in adipose tissue cellularity are associated with the presence of paracrine growth factor(s) that alter preadipocyte proliferation, Osborne-Mendel rats were fed either a HF (76% energy) or a low-fat (LF, 12% energy) diet for 85 days. HF-fed rats had greater (P < 0.05) fat pad size, total fat cell number, number of small (30-70 microm) and large (80-140 microm) adipocytes, and percentage of 100- to 140-microm adipocytes compared with LF-fed rats. Preadipocytes in primary cell culture treated with inguinal adipose tissue conditioned medium (ATCM) prepared from HF-fed rats had greater (P < 0.05) proliferation compared with cultures treated with ATCM from LF-fed rats. Proliferative capacity of ATCM prepared from HF-fed rats was attenuated after the stripping of the medium of insulin-like growth factor I using an immunomagnetic bead separation system. These data are consistent with the concept that insulin-like growth factor I is involved in the paracrine regulation of adipogenesis.     

Hormonal responses to fasting and refeeding in chronic renal failure patients

To study anorexia in chronic renal failure (CRF) patients, we measured appetite-related hormones in seven CRF patients and four controls. Plasma concentrations and fractional changes from baseline (values from day 1, 0800) are listed as control vs. CRF (means +/- SE). Leptin, although higher in CRF (5.6 +/- 1.7 and 34 +/- 17 ng/ml), was suppressed after fasting; decrements were -51 +/- 9 and -55 +/- 8%. Nocturnal surge present during feeding was abolished upon fasting in both groups. Neuropeptide Y (NPY) was elevated in CRF (72 +/- 12 vs. 304 +/- 28 pg/ml, P = 0.0002). NPY rhythm, reciprocal to that of leptin, was muted in CRF. Basal cortisol was similar in both groups (17 +/- 3 and 17 +/- 2 microg/dl). In the controls, cortisol peaked in the morning and declined in the evening. CRF showed blunted cortisol suppression. Decrements were -61 +/- 3 and -20 +/- 9% at 1800 on day 1 (P = 0.008) and -61 +/- 8 and -26 +/- 8% at 2000 on day 2 (P = 0.02). Basal ACTH (25 +/- 5 and 54 +/- 16 pg/ml) as well as diurnal pattern was not statistically different between the groups. Baseline insulin was 6 +/- 1 and 20 +/- 9 microU/ml. During fasting, insulin was suppressed to -64 +/- 10 and -51 +/- 9%, respectively. Upon refeeding, increments were 277 +/- 96 and 397 +/- 75%. Thus, in our CRF patients, anorexia was not due to excess leptin or deficient NPY. Impaired cortisol suppression should favor eating. Insulin suppression during fasting and secretion after feeding should enhance both eating and anabolism. The constant high NPY suggests increased tonic hypersecretion.     

Direct and correlated responses to selection for post-weaning weight gain on ad libitum or restricted feeding in mice

Summary Selection for post-weaning weight gain in mice from 21 to 42 days, on either a full or restricted feeding level during this period was carried out for seven generations. Control lines were maintained for each feeding level. The rate of selection response was higher on full feeding due to a higher heritability and a larger phenotypic variance. Realised heritabilities of 0.29±0.05 and 0.19±0.04 for selection on full and restricted feeding respectively, were in close agreement with base population estimates.Selection on full feeding led to positive correlated responses in 21 day weight, 42 day weight, food intake and efficiency between 21 and 42 days, and 42 day tail length, but with little change in reproductive performance.Correlated responses to selection on restricted feeding were reduced 21 day weight, but an increase in 42 day weight and increased efficiency from 21 to 42 days. However, overall reproductive performance fell.     

Enzymatic- and renal-dependent catabolism of the intestinotropic hormone glucagon-like peptide-2 in rats

The intestinotropic hormone glucagon-like peptide (GLP)-2-(1-33) is cleaved in vitro to GLP-2-(3-33) by dipeptidyl peptidase IV (DP IV). To determine the importance of DP IV versus renal clearance in the regulation of circulating GLP-2-(1-33) levels in vivo, GLP-2-(1-33) or the DP IV-resistant analog [Gly(2)]GLP-2 was injected in normal or DP IV-negative rats and assayed by HPLC and RIA. Normal rats showed a steady degradation of GLP-2-(1-33) to GLP-2-(3-33) over time, whereas little or no conversion was detected for GLP-2-(1-33) in DP IV-negative rats and for [Gly(2)]GLP-2 in normal rats. To determine the role of the kidney in clearance of GLP-2-(1-33) from the circulation, normal rats were bilaterally nephrectomized, and plasma immunoreactive GLP-2 levels were measured. The slope of the disappearance curves for both GLP-2-(1-33) and [Gly(2)]GLP-2 were significantly reduced in nephrectomized compared with non-nephrectomized rats (P < 0.01). In contrast to both GLP-2-(1-33) and [Gly(2)]GLP-2, GLP-2-(3-33) did not stimulate intestinal growth in a murine assay in vivo. Thus the intestinotropic actions of GLP-2-(1-33) are determined both by the actions of DP IV and by the kidney in vivo in the rat.     

Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.     

Effect of short-term fasting/refeeding on epidermal growth factor content in the gastrointestinal tract of suckling rats.

Epidermal growth factor (EGF) is trophic for varying regions of the developing gastrointestinal tract (GIT) of suckling rats. The presence of large amounts of EGF in milk from various species, combined with low production of EGF by suckling animals, led to speculation that milk is a major source of EGF for suckling rats. We report that short-term fasting (8 hr) of 12-day-old suckling rats resulted in a significant decrease in the levels of immunoreactive EGF (irEGF) in the GIT. Pups refed by lactating mothers for 1 to 4 hr exhibited an increase in irEGF to original levels, whereas pups fed a rat milk substitute by gastric gavage did not have an increase in irEGF content. The irEGF levels in the GIT of pups that were manually fed normal rat milk, or rat milk substitute supplemented with EGF, returned to the prefasted levels. Fasted suckling rats refed 2 ml of rat milk in 2 h exhibited significantly higher level of irEGF in the GIT than did those refed with 0.5 ml in 45 min. Since rat milk irEGF exists in three distinct forms (A, B, and C; C is equal to authentic submandibular gland EGF, the irEGF forms in the GIT were characterized by native polyacrylamide gel electrophoresis. In the stomach luminal contents of the fed suckling rats, only the larger form, Peak B, was observed. Both the luminal content and the mucosa scrapings of all other segments of all groups contained only Form D (comigrating with desarginyl EGF), a metabolic derivative of EGF. All forms were immunoreactive, exhibited receptor binding, and stimulated DNA synthesis in growth-arrested fibroblasts. The rapid changes in EGF within the GIT of suckling rats suggest the EGF can acutely modify some GIT functions of suckling rats.     

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.     

Insulin-like growth factor I retards apoptotic signaling induced by ethanol in cardiomyocytes

Apoptosis of cardiac muscle cells may contribute to the development of cardiomyopathy and heart failure. Alcohol (ethanol) abuse is a major cause of cardiomyopathy, but its underlying mechanism remains unknown. To determine whether ethanol causes apoptosis in cardiac muscle and whether insulin-like growth factor I (IGF 1) improves cardiac muscle survival upon ethanol exposure, we have defined the effects of ethanol and IGF I in primary cardiomyocytes. Ethanol decreased cell viability in dose-response manner from 0.2% to 1%. In contrast, ethanol (0.2-1%) did not alter viability of cardiac fibroblasts. To assess the occurrence of apoptosis, DNA fragmentation was determined with quantitation of nucleosomes. Nucleosomes were increased in ethanol-treated cells, thus confirming the apoptotic effects of ethanol. The pro-apoptotic Bax protein and Caspase 3 are important proteins of apoptotic signaling. The content of Bax and the activities of Caspase 3 were increased upon ethanol exposure. IGF I partially suppressed Bax induction, Caspase 3 activation, DNA fragmentation, and increased cardiomyocyte survival. The effects of IGF I on ethanol-induced apoptosis can be inhibited with a chemical inhibitor of PI 3 Kinase (LY-294002), suggesting that anti-apoptotic actions of IGF I involves PI 3 Kinase. These results may have important implications on further understanding the pathogenesis of alcoholic heart disease and the development of new strategies to treat alcoholic cardiomyopathy.     

Neuronal Fos-like immunoreactivity associated with dexamethasone-induced hypertension in rats and effects of glucagon-like peptide-2

Aims

Dexamethasone-induced hypertension models have been used to study the mechanisms of glucocorticoid-induced hypertension, but the role of glucocorticoids in central cardiovascular regulation is not clearly understood. In the present study, we investigated the sites associated with dexamethasone-induced hypertension in the central nervous system in rats. We further investigated whether glucagon-like peptide-2 (GLP-2) was effective for dexamethasone-induced hypertension.

Main methods

Male Sprague–Dawley rats were treated with saline or dexamethasone (0.03 mg/kg/day, s.c) for 10 days. GLP-2 (60 μg/kg, i.v.) was given to rats after dexamethasone treatment. We measured systolic blood pressure by a tail-cuff method in conscious rats, and arterial blood pressure in anesthetized rats. Immunohistochemical techniques were used to detection of the c-fos protein (Fos).

Key findings

Fos-immunoreactivity (Fos-IR) in the dorsomedial hypothalamic nucleus (DMH) was higher in dexamethasone-treated rats than in saline-treated rats. However, Fos-IR in the infralimbic cortex, amygdala, and hippocampus was similar in saline-treated and dexamethasone-treated rats. Peripheral administration of GLP-2 reduced mean arterial blood pressure by 26%. After the peripheral administration of GLP-2, Fos-IR in the caudal ventrolateral medulla (CVLM) increased in dexamethasone-treated rats.

Significance

Chronic dexamethasone treatment induced Fos-IR in the DMH. Peripheral administration of GLP-2 suppressed dexamethasone-induced hypertension in rats by enhancing inhibitory neuronal activity.     

Insulin-like growth factor I binding and receptor kinase in red and white muscle

IGF-I receptors were partially purified from red and white skeletal muscle by lectin-affinity chromatography and the resultant fraction was depleted of insulin receptors by insulin affinity chromatography. Equilibrium binding of 125I-IGF-I to receptor preparations from red and white muscle yielded identical Scatchard plots. The integrity of the IGF-I receptor preparation in the two fiber types was identical as determined by affinity cross-linking. The tyrosine kinase activity of the receptor from red muscle was 2-3-fold more active towards exogenous substrates in both the basal and ligand-activated states as compared to white muscle. These data show that there is IGF-I-dependent kinase activity intrinsic to IGF-I receptors from skeletal muscle, and suggest that identical cellular factors may regulate the kinase activity of insulin and IGF-I receptors in a parallel manner in vivo.     

Insulin-like growth factor I and epidermal growth factor regulate the expression of transferrin receptors at the cell surface by distinct mechanisms

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.     

Insulin-like growth factor I in the developing and mature rat testis: immunohistochemical aspects

The distribution of insulin-like growth factor I (IGF-I; somatomedin C) was mapped in testes of different aged rats by using immunohistochemical techniques. The antiserum used, K 624, has been demonstrated to be specific for human IGF-I, as defined by several criteria. Antibodies to the M1 subunit of ribonucleotide reductase, a key enzyme in DNA synthesis, were used to visualize meiotic and mitotic cells. Cytoplasmic IGF-I-like immunoreactivity as demonstrable during the first two postnatal weeks in spermatogenic cells, in Sertoli cells, and in Leydig cells. The IGF-I-like immunoreactivity decreased in the Sertoli and Leydig cells during the third and fourth postnatal weeks, and in adult rats, only spermatogenic cells showed IGF-I-like immunoreactivity. In mature rat testes, the spermatocytes were strongly immunoreactive. During puberty and adulthood, the spermatogonia expressed subunit M1 ribonucleotide reductase immunoreactivity, whereas no IGF-I-like immunoreactivity could be detected. No extracellular immunoreactivity was observed. We propose that IGF-I and/or IGF-I-like substances, possibly formed by primary spermatocytes, are likely to be involved in differentiation processes, but not in the initiation of cell proliferation in adult testes. The autocrine and/or paracrine action of IGF-I and/or IGF-I-like substances may thus have different action in developing testes than in adult testes. Our results do, however, not allow firm statements about whether IGF-I and related substances exert their actions on Sertoli cells or spermatogenic cells.     

Insulin-like growth factor I prevents the development of sensitivity to kainate neurotoxicity in cerebellar granule cells

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.