Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the highest activity of systemic lupus erythematosus. Our results show that native circulating IgG complexes from active systemic lupus erythematosus patients expose fucosyl residues and their glycan core is accessible to soluble lectins. Two putative mechanisms may contribute to the increased exposure of these glycans: (1) the canonical N-glycosylation site of the IgG-CH2 domain; (2) an IgG binding non-IgG molecule, like complement or C-reactive protein. In both cases the complexed IgG may be alternatively targeted to lectin receptors of effector cells, e.g. dendritic cells.

Tumor necrosis factor alpha is involved in mouse growth and lymphoid tissue development.

Tumor necrosis factor alpha (TNF-alpha), a major mediator of inflammation, also possesses a wide pleiotropism of actions, suggesting its involvement in physiological conditions. TNF-alpha mRNA is present in mouse embryonic tissues and also in fetal thymus and spleen. Repeated injections of a monospecific polyclonal rabbit anti-mouse TNF-alpha antibody in mice, starting either during pregnancy or at birth, led to a severe but transient growth retardation, already present at birth, reaching a 35% decrease in body weight at 3 wk, with complete recovery at 8 wk. The insulin growth factor I (IGF-I) blood levels were decreased to about 50%; growth hormone release and other endocrine functions were unaltered. A marked atrophy of the thymus, spleen, and lymph nodes was also observed, with lymphopenia and impaired development of T and B cell peripheral lymphoid structures. The pathways involving TNF-alpha in IGF-I release and early body growth are probably distinct from those by which TNF-alpha participates in early development of lymphoid tissues, where its low physiological release may contribute to enhance lymphoid cell expansion.

Role of muscle insulin-like growth factors in nerve sprouting: suppression of terminal sprouting in paralyzed muscle by IGF-binding protein 4.

The protracted absence of muscle activation initiates complex cellular and molecular reactions aimed at restoring functional neuromuscular transmission and preventing degenerative processes. A central aspect of these reactions is the sprouting of intramuscular nerves in the vicinity of inactivated muscle fibers. Sprouts emerging from terminal nerve branches and nodes of Ranvier can reestablish functional contacts with inactive muscle fibers, and this is an essential restorative process in pathological conditions of the neuromuscular system. Due to their rapid upregulation in inactive skeletal muscle fibers and their ability to induce nerve sprouting in adult muscle, insulin-like growth factors (IGFs) are candidate signaling molecules to promote restorative reactions in the neuromuscular system. In this study we have exploited the high affinity and specificity of IGF-binding protein 4 (IGF-BP4) and IGF-BP5 for IGF1 and IGF2 to determine whether these growth factors are involved in the nerve sprouting reaction in paralyzed skeletal muscle. In tissue culture experiments with sensory- and motoneurons we demonstrate that the neurite promoting activity of IGF1 is blocked by IGF-BP4, and that a similar IGF-BP-sensitive activity is detected in muscle extracts from paralyzed, but not from control muscle. In in vivo experiments, we show that local delivery of IGF-BP4 to Botulinum toxin A-paralyzed skeletal muscle effectively prevents nerve sprouting in that muscle. Our findings indicate that muscle IGFs play an essential role in intramuscular nerve sprouting. In addition, these findings suggest that IGFs are major signaling factors from inactivated muscle to promote local restorative reactions, including interstitial cell proliferation and nerve sprouting.

Differential effects of insulin-like growth factor I and growth hormone on developmental stages of rat growth plate chondrocytes in vivo.

Skeletal growth depends upon enchondral ossification in growth plate cartilage, within which chondrocytes undergo well defined stages of maturation. We infused IGF-I or growth hormone (GH), two key regulators of skeletal growth, into hypophysectomized rats and compared their effects on growth plate chondrocyte differentiation using qualitative and quantitative autoradiography, stereology, and incident light fluorescence microscopy. Stem cell cycle time was shortened from 50 to 15 and 8 d after treatment with IGF-I and GH, respectively. Proliferating cell cycle time decreased from 11 to 4.5 and 3 d, and duration of the hypertrophic phase decreased from 6 to 4 and 2.8 d. Average matrix volume per cell at each differentiation stage was similar for normal, hormone-treated, and untreated hypophysectomized groups. Mean cell volume and cell height were significantly reduced by hypophysectomy at the proliferative and hypertrophic stages, but were restored to physiological values by IGF-I and GH. In contrast, cell productivity, i.e., increases in cell volume, height, and matrix production per unit of time, did not reach normal values with either IGF-I or GH, and this parameter was inversely proportional to cell cycle time or phase duration. IGF-I and GH are thus capable of stimulating growth plate chondrocytes at all stages of differentiation, albeit to variable degrees with respect to individual cell activities. Although it is generally accepted that GH acts at both the stem and proliferating phases of chondrocyte differentiation, our data represent the first evidence in vivo that IGF-I is also capable of stimulating stem cells.

Radioimmunological determination of insulinlike growth factors I and II in normal subjects and in patients with growth disorders and extrapancreatic tumor hypoglycemia.

Serum levels of immunoreactive insulinlike growth factors (IGF) I and II were determined by a modified IGF I and a new IGF II radioimmunoassay in normal children and adults, and in patients with acromegaly, isolated growth hormone deficiency, and extrapancreatic tumor hypoglycemia. Serum samples were gel filtered by a simple routine procedure at acidic pH to dissociate and separate IGF from the IGF carrier protein. Mean immunoreactive IGF I levels (+/- SD; corrected for crossreactivity of IGF II) were 193 +/- 58 ng/ml in normal adult subjects, 712 +/- 245 ng/ml in acromegalic patients and 24 +/- 14 ng/ml in patients with isolated growth hormone deficiency. The lack of growth hormone alone, irrespective of an otherwise normal hormonal status, appears to be responsible for the drastic decrease of IGF I levels. Oversecretion of growth hormone does not increase the levels of immunoreactive IGF II: mean levels (+/- SD; corrected for crossreactivity of IGF I) in normal and acromegalic subjects are virtually identical (647 +/- 126 and 641 +/- 189 ng/ml, respectively). Apparently, normal growth hormone levels stimulate IGF II production already maximally. However in growth hormone deficiency immunoreactive IGF II is significantly decreased (252 +/- 99 ng/ml). Thus, IGF II, like IGF I, is growth hormone dependent. But in contrast to IGF I, the growth hormone dependence of IGF II seems to become apparent only at subnormal growth hormone levels. In normal children IGF I is age dependent: it is low in newborn cord sera (51 +/- 20 ng/ml) and gradually rises into the adult range with increasing age. At the onset of and during puberty mean IGF I levels lie above prepubertal values. In contrast, IGF II levels in normal children are independent of age and pubertal stage beyond the first year of life, whereas newborns have significantly lower IGF II values. Hypoglycemia resulting from extrapancreatic tumors is not associated with increased immunoreactive IGF I or II levels. IGF I is decreased in most of the sera (mean level +/- SD:56 +/- 39 ng/ml) whereas IGF II lies in the normal range (556 +/- 195 ng/ml).

Posttranslational regulation of insulin-like growth factor binding protein-4 in normal and transformed human fibroblasts. Insulin-like growth factor dependence and biological studies.

Insulin-like growth factor binding protein-4 (IGFBP-4) is a 24-26-kD protein expressed by a variety of cell types in vivo and in vitro. Treatment of normal adult human fibroblasts with 10 nM insulin-like growth factor II (IGF-II) for 24 h resulted in an 85% decrease in endogenous IGFBP-4, as assessed by Western ligand blot analysis of the conditioned medium. Incubation of human fibroblast-conditioned medium (HFCM) with IGF-II under cell-free conditions led to a similar loss of IGFBP-4. This posttranslationally regulated decrease in IGFBP-4 appeared to be due to a protease in HFCM: (a) It could be prevented with specific protease inhibitors or incubation at 4 degrees C; (b) proteolysis of recombinant human (rh) IGFBP-4 required HFCM; (c) immunoblotting and radiolabeling confirmed cleavage of IGFBP-4 into 18- and 14-kD IGFBP-4 fragments. The protease was specific for IGFBP-4, and was strictly dependent on IGFs for activation. IGF-II was the most effective of the natural and mutant IGFs tested, inducing complete hydrolysis of rhIGFBP-4 at a molar ratio of 0.25:1 (IGF/IGFBP-4). Simian virus 40-transformed adult human fibroblasts also expressed IGFBP-4 and IGFBP-4 protease, as well as an inhibitor of IGFBP-4 proteolysis. In biological studies, intact rhIGFBP-4 potently inhibited IGF-I-stimulated [3H]aminoisobutyric acid uptake, whereas proteolyzed rhIGFBP-4 had no inhibitory effect. In conclusion, these data provide evidence for a novel IGF-dependent IGFBP-4-specific protease that modifies IGFBP-4 structure and function, and indicate a preferential role for IGF-II in its activation. Posttranslational regulation of IGFBP-4 may provide a means for cooperative control of local cell growth by IGF-I and IGF-II.

Acute metabolic effects and half-lives of intravenously administered insulinlike growth factors I and II in normal and hypophysectomized rats.

Insulinlike growth factors (IGF) act qualitatively like insulin on insulin target tissues in vitro. In the circulation in vivo they are bound to specific carrier proteins. In this form or when continuously infused into hypophysectomized (hypox) rats they do not exert acute insulinlike effects on glucose homeostasis. This study definitively shows that intravenous bolus injections of pure IGF I or II act acutely on glucose homeostasis: they lower the blood sugar, enhance the disappearance of U-[14C]glucose from serum and increase its incorporation into diaphragm glycogen in normal and hypox rats in the presence of antiinsulin serum. The same effects were obtained with recombinant human IGF I injected intravenously either with or without antiinsulin serum into normal rats. Free fatty acid levels decreased transiently only in normal animals. Lipid synthesis from glucose in adipose tissue was not stimulated in hypox and barely stimulated in normal rats. The half-life of injected IGF I or II in normal rats (approximately 4 h) is strikingly different from that in hypophysectomized rats (20-30 min) and appears to depend on the growth hormone-induced 150,000-200,000-mol wt IGF carrier protein that is lacking in hypophysectomized rats. 15 min after the bolus serum IGF I and II concentrations were similar to steady state levels during long-term infusion in hypox rats. Free IGF was barely detectable, however, in the infused animals, whereas 40-100% was found free 15 min after the bolus. These observations for the first time confirm the hypothesis that only free IGF, but not the IGF carrier protein complex, is bioavailable to insulin target tissues.

Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia?

The pathogenesis of extrapancreatic tumor hypoglycemia has been related to the secretion of big insulin-like growth factor (IGF) II by the tumor. In 25 of 28 patients with this type of hypoglycemia we found 1.5-8-fold elevated serum levels of immunoreactive big (15-25 kD), but decreased levels of normal IGF II. After removal of the tumor, big IGF II disappeared and normal IGF II increased. Tumors contained elevated levels of IGF II, 65-80% in the big form. The insulin-like bioactivity of big IGF II and its affinity towards IGF-binding proteins (IGFBP)-2 and -3 are similar to those of normal IGF II, but two- to threefold higher on a molar basis. Big IGF II is mainly bound to the 50-kD IGFBP complex. The latter contains approximately 10 times more of this peptide than in normal serum and displays three- to fourfold increased insulin-like bioactivity. The formation of the 150-kD IGFBP complex with 125I-recombinant human IGFBP-3 is impaired in tumor serum. This results in sequestration of IGFBP-3 and predominant association of big IGF II with IGFBP-2 and -3 in the 50-kD complex. Increased bioavailability of big IGF II in this complex due to unrestricted capillary passage and enhanced insulin bioactivity of this big IGF II pool provide a continuous increased insulin-like potential available to insulin and type 1 IGF receptors of insulin-sensitive tissues and thus may lead to sustained hypoglycemia.

Inhibition of the action of nonsuppressible insulin-like activity on isolated rat fat cells by binding to its carrier protein.

Nonsuppressible insulin-like activity extracted and purified from human serum (NSILA-S) mimics all insulin-like effects in vitro and, after injection, in vivo in the presence of excess insulin antibodies. However, there is no evidence that it exerts acute insulin-like effects in its native form in the circulation, where it is almost completely bound to a specific large molecular weight carrier protein. In this paper we show that partially purified NSILA-S-carrier protein, devoid of endogenous insulin-like activity, inhibits the stimulatory effect of NSILA-S, but not of insulin, on 3-0-methylglucose transport and on lipogenesis from [U-(14)C]glucose in isolated rat fat cells. Concomitantly, it prevents binding of (125)I-labeled NSILA-S to the insulin receptor and to the NSILA-S-binding site. The following explanation is, therefore, offered for the absence of acute insulin-like effects of native NSILA-S in vivo: In native serum NSILA-S occurs almost exclusively as NSILA-S-carrier complex. According to recent findings the passage of this complex through blood capillaries is restricted. The present results indicate that, in addition, it is metabolically inactive, or, at least, possesses reduced metabolic activity. The well-known phenomenon that whole serum, nevertheless, exerts pronounced nonsuppressible insulin-like effects on adipose tissue in vitro seems, therefore, to be mainly caused by the presence of a large molecular weight insulin-like protein not identical to the NSILA-S-carrier complex.

Activity in fetal bovine serum that stimulates erythroid colony formation in fetal mouse livers is insulinlike growth factor I.

In the present study, the erythropoietic activity of fetal serum was characterized. Using fetal bovine serum (FBS) as a source of the erythropoietic activity and serum-free cultures of fetal mouse livers (FMLC assay) as a detection system, we found that FBS stimulated colony formation from late erythroid progenitor cells (CFU-E) in a dose-dependent fashion. The slope of the dose-response curve, however, was significantly different from that for erythropoietin (Ep), the best-characterized erythropoietic activity so far. The erythropoietic activity of FBS was found in the 120-160- and 40-70-kD range at neutral pH. In the presence of 1 M acetic acid, however, the erythropoietic activity had an apparent molecular mass between 3 and 13 kD. From ion exchange experiments with DEAE-cellulose, the isoionic point of the activity was estimated to about pH 5. Furthermore, the erythropoietic activity of FBS was found to be co-eluted on Sephadex G-150 with the binding proteins of insulinlike growth factors (IGF). The IGF I concentration determined by radioimmunoassay was 70 ng IGF I/ml. The Ep activity of FBS was less than 5 mU/ml when determined with the posthypoxic polycythemic mouse assay for Ep. These results suggest that the erythropoietic activity of FBS is related to IGF and not to Ep. The erythropoietic activity of FBS was abolished by an antiserum against IGF I. Furthermore, IGF I was a factor of approximately 40 more potent than IGF II in stimulating erythroid colony formation. All of these findings suggest that the erythropoietic activity of FBS is IGF I.

Insulin-like growth factor I stimulates lipid oxidation, reduces protein oxidation, and enhances insulin sensitivity in humans.

To elucidate the effects of insulin-like growth factor I (IGF-I) on fuel oxidation and insulin sensitivity, eight healthy subjects were treated with saline and recombinant human (IGF-I (10 micrograms/kg.h) during 5 d in a crossover, randomized fashion, while receiving an isocaloric diet (30 kcal/kg.d) throughout the study period. On the third and fourth treatment days, respectively, an L-arginine stimulation test and an intravenous glucose tolerance test were performed. A euglycemic, hyperinsulinemic clamp combined with indirect calorimetry and a glucose tracer infusion were performed on the fifth treatment day. IGF-I treatment led to reduced fasting and stimulated (glucose and/or L-arginine) insulin and growth hormone secretion. Basal and stimulated glucagon secretion remained unchanged. Intravenous glucose tolerance was unaltered despite reduced insulin secretion. Resting energy expenditure and lipid oxidation were both elevated, while protein oxidation was reduced, and glucose turnover rates were unaltered on the fifth treatment day with IGF-I as compared to the control period. Enhanced lipolysis was reflected by elevated circulating free fatty acids. Moreover, insulin-stimulated oxidative and nonoxidative glucose disposal (i.e., insulin sensitivity) were enhanced during IGF-I treatment. Thus, IGF-I treatment leads to marked changes in lipid and protein oxidation, whereas, at the dose used, carbohydrate metabolism remains unaltered in the face of reduced insulin levels and enhanced insulin sensitivity.

Human fetal and adult chondrocytes. Effect of insulinlike growth factors I and II, insulin, and growth hormone on clonal growth.

Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life.

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.

Comparison of the effects of growth hormone and insulin-like growth factor I on substrate oxidation and on insulin sensitivity in growth hormone-deficient humans.

Insulin-like growth factor-I (IGF-I) is considered to be the mediator of the growth-promoting effects of growth hormone (GH). The metabolic effects of these two hormones, however, are different. Whereas GH treatment leads to elevated insulin and glucose levels, reduced insulin sensitivity, and impaired glucose tolerance, IGF-I treatment leads to reduced insulin and GH levels and enhanced insulin sensitivity. IGF-I may, therefore, not only be the mediator of the growth-promoting effects of GH but also a modulator of the effects of GH on insulin action and glucose metabolism. To study the influence of GH and IGF-I on substrate metabolism and insulin sensitivity (assessed by euglycemic, hyperinsulinemic clamping combined with indirect calorimetry and glucose tracer infusion), we have treated eight GH-deficient adults with GH (2 IU/m2 daily subcutaneously [s.c.]), IGF-I (10 micrograms/kg.h s.c.), or both hormones together for 7 d, respectively, and compared the effects of these treatment regimens with a control phase. Our findings suggest that (a) both GH and IGF-I promote lipolysis and lipid oxidation, albeit by different mechanisms; (b) treatment with either hormone is followed by enhanced energy expenditure and reduced protein oxidation; and (c) IGF-I reverses the insulin resistance induced by GH.

Intravenously injected insulin-like growth factor (IGF) I/IGF binding protein-3 complex exerts insulin-like effects in hypophysectomized, but not in normal rats.

Insulin-like growth factor (IGF) circulates in blood in two large molecular mass forms of 150 and 40 kD. Under normal conditions, most of the IGF is bound to the 150-kD complex by which it is retained in the circulation and therefore unable to exert acute insulin-like actions. The aim of this study was to answer the question whether or not IGF in the 40-kD complex is bioavailable to insulin target tissues and thus can cause acute insulin-like effects in vivo. Intravenously injected 1:1 molar recombinant human (rh) IGF I/rhIGF binding protein (BP)-3 complex lowered blood glucose and stimulated glycogen synthesis in diaphragm of hypophysectomized, but not of normal rats. The serum half-lives of the two components of the complex were similar to each other, but considerably shorter in hypox than in normal rats. On neutral gel filtration of serum both components of the injected complex appeared predominantly in the 150-kD region in normal rats. In hypox rats which lack the 150-kD complex they were found in the 40-kD region and disappeared rapidly from the circulation. We conclude that in the absence of the 150-kD complex, IGF associated with the 40-kD complex can rapidly leave the vascular compartment, reach insulin or type 1 IGF receptors and exert acute insulin-like effects.

Regulation of binding proteins for insulin-like growth factors (IGF) in humans. Increased expression of IGF binding protein 2 during IGF I treatment of healthy adults and in patients with extrapancreatic tumor hypoglycemia.

UNLABELLED: Insulin-like growth factors (IGFs) in blood form two complexes with specific binding proteins (BPs): a large, growth hormone (GH)-dependent complex with restricted capillary permeability, and a smaller complex, inversely related to GH, with high turnover of its IGF pool and free capillary permeability. The distribution of BPs and of IGFs I and II between these complexes was studied in sera from healthy adults treated with IGF I or/and GH and from patients with extrapancreatic tumor hypoglycemia. Like GH, IGF I administration raises IGF I and two glycosylation variants of IGFBP-3 in the large complex, but unlike GH drastically reduces IGF II. During IGF I infusion, IGFBP-3 appears in the small complex whose IGFBP-2 and IGF I increase three- to fivefold and fivefold, respectively. GH treatment, associated with elevated insulin levels, suppresses IGFBP-2 and inhibits its increase owing to infused IGF I. The small complex of tumor sera contains increased amounts of IGFBP-2 and -3, and two- to threefold elevated IGF II. CONCLUSIONS: low GH and/or insulin during IGF I infusion and in extrapancreatic tumor hypoglycemia enhance expression of IGFBP-2 and favor partition of IGFBP-3 into the small complex. Free capillary passage and high turnover of its increased IGF I or II pools may contribute to compensate for suppressed insulin secretion during IGF I infusion or to development of tumor hypoglycemia.

Direct effects of leptin on brown and white adipose tissue.

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

Evaluation of IGF-1 levels in cats with transient and permanent diabetes mellitus.

It was investigated if IGF-1 levels in cats which experience diabetic remission (i.e. transient diabetes mellitus) differ from those in cats with permanent disease. Thirteen of 32 diabetic cats showed remission within 16 weeks after initiating insulin therapy, 19 cats continued to need insulin therapy. IGF-1 concentrations were measured before (t(0)), 1-3 (t(1)) and 4-8 (t(2)) weeks after initiating insulin therapy. No difference in IGF-1 levels was found between cats with transient and permanent diabetes at any point in time. In both groups of cats IGF-1 concentrations were significantly lower compared to those of controls before insulin administration. After starting insulin therapy IGF-1 increased significantly in both groups. In cats with transient diabetes IGF-1 levels were not different from controls already at t(1), whereas in cats with permanent diabetes it took until t(2). Although IGF-1 levels seem to normalize faster in cats with transient diabetes mellitus, measurement is not helpful to predict the course of the disease.

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.

BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Crystal structure of a phosphatase-resistant mutant of sporulation response regulator Spo0F from Bacillus subtilis.

BACKGROUND: Spo0F, a phosphotransferase containing an aspartyl pocket, is involved in the signaling pathway (phosphorelay) controlling sporulation in Bacillus subtilis. It belongs to the superfamily of bacterial response regulatory proteins, which are activated upon phosphorylation of an invariant aspartate residue. This phosphorylation is carried out in a divalent cation dependent reaction catalyzed by cognate histidine kinases. Knowledge of the Spo0F structure would provide valuable information that would enable the elucidation of its function as a secondary messenger in a system in which a phosphate is donated from Spo0F to Spo0B, the third of four main proteins that constitute the phosphorelay. RESULTS: We have determined the crystal structure of a Rap phosphatase resistant mutant, Spo0F Tyr13-->Ser, at 1.9 A resolution. The structure was solved by single isomorphous replacement and anomalous scattering techniques. The overall structural fold is (beta/alpha)5 and contains a central beta sheet. The active site of the molecule is formed by three aspartate residues and a lysine residue which come together at the C terminus of the beta sheet. The active site accommodates a calcium ion. CONCLUSIONS: The structural analysis reveals that the overall topology and metal-binding coordination at the active site are similar to those of the bacterial chemotaxis response regulator CheY. Structural differences between Spo0F and CheY in the vicinity of the active site provide an insight into how similar molecular scaffolds can be adapted to perform different biological roles by the alteration of only a few amino acid residues. These differences may contribute to the observed stability of the phosphorylated species of Spo0F, a feature demanded by its role as a secondary messenger within the phosphorelay system which controls sporulation.