ABSTRACT

The effects of insulin and insulin-like growth factor-I (IGF-I) on glucose transport were compared in myotubes derived from chicken breast muscle satellite cells in vitro. Myotubes were incubated (for 0·5 or 4 h) with or without glucose in the presence or absence of insulin or IGF-I. Glucose uptake was subsequently measured by the incorporation of 2-[1,2-³H(N)] deoxy-d-glucose ([³H]2DG) in glucose-free medium (10 min at 20 °C). Glucose uptake was almost completely abolished by the addition of cytochalasin B or phloretin. It was increased by a decrease in glucose concentration in the incubation medium. Insulin (5 mg/l) stimulated [³H]2DG uptake to a maximum of 43 ± 10% above basal after 30-min incubation and 101 ± 15% after 4-h incubation. IGF-I and insulin at equimolar concentrations (25 μg/l and 20 μg/l respectively) were almost equipotent after 0·5 h but after 4-h incubation IGF-I was 17-fold more potent, suggesting that this 'late' effect was mediated through the IGF-I receptor. Incubation with cycloheximide suggested that the effect of IGF-I involved increased protein synthesis. The results suggest that chicken myotubes express a glucose transporter which is regulated by IGF-I and glucose concentration. However, they do not appear to express a typical insulin-responsive transport system.

Journal of Endocrinology (1993) 137, 465–472

Protein kinase D (PKD) is emerging as an important kinase regulating energy balance and glucose metabolism; however, whether hepatic PKD activity can be targeted to regulate these processes is currently unclear. In this study, hepatic PKD activity was reduced using adeno-associated virus vectors to express a dominant-negative (DN) version of PKD1, which impairs the action of all three PKD isoforms. In chow-fed mice, hepatic DN PKD expression increased whole-body glucose oxidation, but had only mild effects on glucose and insulin tolerance and no effects on glucose homeostasis following fasting and refeeding. However, circulating VLDL cholesterol was reduced under these conditions and was associated with hepatic fatty acid accumulation, but not lipids involved in lipoprotein synthesis. The limited effects on glucose homeostasis in DN PKD mice was despite reduced expression of gluconeogenic genes under both fasted and refed conditions, and enhanced pyruvate tolerance. The requirement for PKD for gluconeogenic capacity was supported by in vitro studies in cultured FAO hepatoma cells expressing DN PKD, which produced less glucose under basal conditions. Although these pathways are increased in obesity, the expression of DN PKD in the liver of mice fed a high-fat diet had no impact on glucose tolerance, insulin action, pyruvate tolerance or plasma VLDL. Together, these data suggest that PKD signalling in the liver regulates metabolic pathways involved in substrate redistribution under conditions of normal nutrient availability, but not under conditions of overnutrition such as in obesity.

The genetic events involved in thyroid carcinogenesis are still incompletely understood. Several rearrangements and mutations of oncogenes have been implicated in the development of thyroid papillary carcinomas, follicular adenomas and carcinomas. However, none of these molecular alterations is suitable either as a general marker for the diagnosis of thyroid carcinomas or to differentiate between thyroid follicular adenomas and carcinomas. In order to identify new genes with altered expression which could serve as such markers, we analyzed RNA from thyroid tumor and normal tissue using a novel technique called restriction-mediated differential display. Several differentially expressed genes were identified, including the gene for IgG Fc binding protein (FcgammaBP). Differential expression of FcgammaBP was confirmed by quantitative real-time RT-PCR. Our experiments showed that IgG Fc binding protein (FcgammaBP) is differentially expressed in normal thyroid tissue, thyroid adenomas and thyroid carcinomas. While the FcgammaBP gene is constitutively expressed in normal thyroid tissue, its expression is significantly increased in follicular thyroid adenomas and significantly decreased in papillary and follicular thyroid carcinomas. Thus, measurement of the expression levels of FcgammaBP in thyroid biopsies might help to make the otherwise difficult distinction between a thyroid follicular adenoma and a follicular carcinoma.

Adiponectin and, especially, its oligomeric complex composition have been suggested to be critical in determining insulin sensitivity. Pro-inflammatory cytokines play an important role in the development of insulin resistance in obesity and associated diseases. Therefore, we investigated the effect of long-term exposure of tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and interferon (IFN)-γ on total insulin-sensitizing adiponectin secretion and adiponectin complex formation from human adipocytes. In parallel, adipocyte delipidation and leptin production levels were monitored. The present study demonstrates that TNF-α, IL-1β, and IFN-γ dose and time dependently suppressed total adiponectin secretion within 7 days (60, 70, and 35% reduction respectively). IL-6 was also able to reduce (50%) adiponectin production, although only in combination with exogenous soluble IL-6 receptors (sIL-6R). However, the oligomeric distribution (high, middle, and low molecular weight (HMW) complexes) of secreted adiponectin was not altered by any of these cytokines. All studied pro-inflammatory cytokines resulted in delipidation and reduction of lipid-laden adipocyte numbers. Despite this reduction of lipid-laden adipocytes, TNF-α, IL-6/sIL-6R, and IL-1β stimulated leptin release. Our data indicate that (i) long-term pro-inflammatory cytokine exposure downregulates total adiponectin secretion from delipidizing adipocytes and (ii) pro-inflammatory cytokines are not important regulators of adipocyte-derived adiponectin oligomerization. Hence, their individual contribution to low expression of HMW adiponectin found in insulin-resistant conditions seems unlikely. Furthermore, delipidizing adipocytes and preadipocytes are active leptin producers when stimulated by TNF-α, IL-6/sIL-6R, and IL-1β.

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.