Elsevier

Journal of Hepatology

Volume 35, Issue 5, November 2001, Pages 558-567
Journal of Hepatology

Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6

https://doi.org/10.1016/S0168-8278(01)00170-2Get rights and content

Abstract

Background/Aims: Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1β and tumor necrosis factor α (TNFα) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells.

Methods: mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [125I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate.

Results: In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1β or TNFα were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6.

Conclusions: The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.

Introduction

The insulin-like growth factors (IGF-I, -II) are structurally related polypeptides involved in various metabolic, proliferative, and differentiation processes mediated by endocrine, autocrine and paracrine mechanisms [1]. IGFs are bound with high affinity to a family of six structurally related binding proteins (IGFBP-1 to -6) [2], [3], [4], [5], performing a variety of functions including carrier proteins in the circulation, storage of IGFs in specific tissue compartments, inhibition of IGF action by preventing access to IGF receptors, or potentiation of the mitogenic response by providing a stable source of available growth factor [4]. In plasma, most IGFs circulate in a 150 kDa ternary complex (150 kDa complex) that consists of one molecule each of IGF-I (or IGF-II), IGFBP-3, and the acid-labile subunit (ALS) [6]. In adult rats, the liver has been recognized as a main source of circulating IGFs, individual IGFBPs and of the ALS [3]. Rat hepatocytes have been shown to secrete IGFBP-1, -2 and -4 and ALS [7], [8], [9], [10]. IGFBP-3 expression, however, was only found in Kupffer cells (KC), endothelial cells (EC) and hepatic stellate cells (HSC) [11], [12], [13], [14], [15], [16], [17]. IGF-I is predominantly released from hepatocytes [16], [18], [19], [20].

The metabolic response in critically ill patients after trauma, extensive surgery and thermal injury, during chronic infection and inflammation is associated with weight loss, negative nitrogen balance, and severe muscle wasting that cannot be solely explained by a reduced caloric intake [21]. Various lines of evidence support the role of acute phase cytokines, such as interleukin (IL)-6, IL-1 and tumor necrosis factor α (TNFα), as putative mediators for many of the metabolic manifestations of trauma and chronic illness [22]. These catabolic conditions are associated with alterations in multiple components of the IGF axis. The most consistent changes are a decrease in IGF-I and an elevation of IGFBP-1 in the plasma. In experimental models of these conditions (e.g. injection of lipopolysaccharides (LPS), thermal injury in rats), the reduction of plasma IGF-I and the increase of IGFBP-1 levels are closely correlated with an altered IGF-I and IGFBP-1 protein content and mRNA expression in the liver [23], [24], [25], [26], [27], [28]. In addition, comparable alterations of IGF-I and IGFBP-1 serum concentrations were observed after the administration of recombinant IL-1 or TNFα to control animals [29], [30], [31], while neutralizing TNFα antibodies and IL-1 receptor antagonists attenuated the sepsis-induced alterations of IGF-I and IGFBP-1 serum concentrations [29], [30]. So far, a direct effect of acute phase mediators on IGF-I and IGFBP biosynthesis has only been performed in HepG2 cells [25], [32], [33] and in primary cultures of rat hepatocytes [34], [35]. These studies demonstrated that the acute phase mediators inhibited the growth hormone (GH)-stimulated IGF-I synthesis and increased that of IGFBP-1. In the present paper, we demonstrate that the cytokines IL-6, IL-1β and TNFα increased the biosynthesis of IGFBP-1, -3 and -4 in primary cultures of hepatocytes, KC and in cocultures of both liver cells, while that of IGF-I was inhibited. These data support the concept of a direct effect of these cytokines on the hepatic production of the IGF axis components.

Section snippets

Materials

Sodium [125I]iodide (specific activity, 16.85 mCi/μg) was purchased from Amersham Pharmacia Biotech (Freiburg, Germany), and [32P]dCTP (specific activity, 3000 Ci/mmol) from Hartmann Analytic (Braunschweig, Germany). Recombinant human IGF-I (Dr Märki, Ciba Geigy, Basel, Switzerland) was iodinated by the chloramine-T method to a specific activity of about 60–80 μCi/μg. Recombinant human non-glycosylated IGFBP-3 (rhIGFBP-3; gift from Dr A. Sommer and Dr C. Maack, Celtrix, Santa Clara, CA) was

Regulation of IGF-I secretion by IL-6

IGF-I peptides were measured in media from liver cells conditioned under serum-free conditions for 24 h. KC released approximately 1.4±0.3 ng IGF-I/mg secreted protein within 24 h into the medium, whereas hepatocytes secreted approximately 9.8±1.3 ng IGF-I/mg protein per 24 h under identical conditions (Table 1). Addition of 100 ng/ml of IL-6 decreased IGF-I secretion by KC to 0.5±0.1 ng/mg protein per 24 h (Table 1). Maintenance of hepatocytes in the presence of 100 ng/ml of IL-6 decreased

Discussion

Several lines of evidence are implicating acute phase cytokines as mediators of multiple alterations of the IGF axis during catabolic conditions in humans, as well as in animal models. The most consistent changes comprise a decrease of IGF-I and an elevation of IGFBP-1 in serum being dependent on an altered hepatic gene expression of these components [23], [24], [25], [26], [27], [28]. In the present study, cocultures of rat hepatocytes with KC were used as a model to study the regulation of

Acknowledgements

The technical assistance of Sieglinde Zachmann and Susanne Hupe is gratefully acknowledged. This work was supported by grants from the Deutsche Forschungsgemeinschaft, SFB 402 ‘Molekulare und zelluläre Hepatogastroenterologie‘ (projects A5), GRK 335/2 ‘Klinische, zelluläre und molekulare Biologie Innerer Organe’ and project Scha 700/1-2.

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