Abstract In the current investigation, hypothalamic–pituitary–adrenal (HPA) axis function was examined in young and aged male Long‐Evans rats that were initially assessed on a version of the Morris water maze sensitive to cognitive impairment during ageing. In behaviourally characterized rats, a 1‐h restraint stress paradigm revealed that plasma corticosterone concentrations in aged cognitively impaired rats took significantly longer to return to baseline following the stressor than did those in young or aged cognitively unimpaired rats. No differences in basal or peak plasma corticosterone concentrations, however, were observed between young or aged rats, irrespective of cognitive status. Using ribonuclease protection assays and in situ hybridization, we evaluated mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) mRNA abundance in young and aged rats characterized on the spatial task. Abundance of MR mRNA was decreased as a function of age in stratum granulosum but not hippocampus proper, and the decrease in MR mRNA was largely unrelated to cognitive status. However, GR mRNA was significantly reduced in several hippocampal subfields (i.e. stratum granulosum and temporal hippocampus proper) and other related cortical structures (medial prefrontal and olfactory regions) of aged cognitively impaired rats compared to either young or aged cognitively unimpaired cohorts, and was significantly correlated with spatial learning ability among the aged rats in each of these brain regions. In agreement with previous stereological data from this ageing model, no changes were detected in neuron density in the hippocampus of the rats used in the in situ hybridization analysis. These data are the first to describe a coordinated decrease in GR mRNA in a functional brain system including hippocampus and related cortical areas that occurs in tandem with impairments of the HPA response to stress and cognitive decline in ageing.
Insulin-like growth factor I (IGF-I) and transforming growth factor-β1 (TGF-β1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-β1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-β1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of α-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-β1 inhibited basal DNA synthesis. TGF-β1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-β1 but not IGF-I potently stimulated expression of α-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-β1 attenuated stress fiber formation without reducing α-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-β1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-β1 pretreatment. IGF-I potently stimulated proliferation of TGF-β1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-β1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-β1 and IGF-I, but they may act in concert to increase collagen deposition.
Normal luminal bacteria and bacterial cell wall polymers are implicated in the pathogenesis of chronic intestinal inflammation. To determine the direct involvement of bacteria and their products on intestinal fibrogenesis, the effects of purified bacterial cell wall polymers on collagen and cytokine synthesis were evaluated in intestinal myofibroblast cultures established from normal fetal and chronically inflamed cecal tissues. In this study, the intestines of Lewis rats were intramurally injected with peptidoglycan-polysaccharide polymers. Collagen and transforming growth factor (TGF)-β1 mRNA levels were measured and correlated with mesenchymal cell accumulation by immunohistochemistry. The direct effects of cell wall polymers on fibrogenic cytokine and collagen α1 (type I) expression were evaluated in intestinal myofibroblast cultures. We found that intramural injections of bacterial cell wall polymers induced chronic granulomatous enterocolitis with markedly increased collagen synthesis and concomitant increased TGF-β1 and interleukin (IL)-6 expression. Intestinal myofibroblast cultures were established, which both phenotypically and functionally resemble the mesenchymal cells that are involved in fibrosis in vivo. Bacterial cell wall polymers directly stimulated collagen α1 (I), TGF-β1, IL-1β, and IL-6 mRNA expression in the intestinal myofibroblasts derived from both normal and inflamed cecum. Neutralization of endogenous TGF-β1 inhibited in vitro collagen gene expression. From our results, we conclude that increased exposure to luminal bacterial products can directly activate intestinal mesenchymal cells, which accumulate in areas of chronic intestinal inflammation, thus stimulating intestinal fibrosis in genetically susceptible hosts.
The studies comparing the actions of dried thyroid gland (Thyroideum-Polfa) with L-thyroxine sodium (L-T4) were carried out in 20 female patients with hypothyroidism, including 19 patients with the primary hypothyroidism and 1 patient with hypothyroidism secondary to pituitary deficiency. Administration of the dried thyroid gland did not normalize blood serum T4 an TSH in any patient. Normal serum T4 or even slightly increased was achieved in all patients treated with L-T4. Serum TSH was normalized in 17 patients with the primary hypothyroidism. The following conclusions have been drawn: 1. Dried thyroid gland (Thyroideum-Polfa) is ineffective in the treatment of hypothyroidism. 2. Serum TSH remains elevated despite normal serum T3 in cases of the primary hypothyroidism with decreased serum T4 levels. 3. Sodium salt of L-thyroxine should be used for the treatment of hypothyroidism. 1-Triiodothyronine sodium may be used as an adjuvant therapy.
To investigate the influence of the insulin-like growth factor binding-proteins (IGFBPs) on the nutritional regulation of IGF-I's actions, we compared the gene expression of IGF-I and the six IGFBPs in liver and kidney of protein-restricted (P5) and normally fed (P15) young rats. Using poly(A)+ Northern blot analysis, we observed a decrease in IGF-I messenger RNA (mRNA) at steady state in liver (-50%) and kidney (-60%). The increases in IGFBP-1 mRNA were parallel in these two tissues (liver, 5.7-fold; kidney, 4-fold). In contrast, the expression of the other IGFBP genes exhibited organ-specific regulation during protein restriction; although IGFBP-2 mRNA increased in liver in the P5 group (3-fold), it decreased slightly in kidney (-15%). IGFBP-3 mRNA declined by 30% in liver and was unchanged in kidney. IGFBP-4 mRNA increased by 50-88% in liver and was not modified in kidney. IGFBP-5 mRNA was not detected in liver and was identical in kidney of P15 and P5 rats. IGFBP-6 mRNA was not changed in either liver or kidney during protein restriction. To determine whether the changes in IGFBP mRNAs induced by protein restriction were associated with changes in the respective peptides, IGFBPs in supernatants of liver homogenates and in serum of the same rats were measured by ligand blot analyses. IGFBP-1 and IGFBP-2 Western immunoblot analyses were also performed in serum. By ligand blot, a 45,000 mol wt (M(r)) band (IGFBP-3) decreased in liver and serum of P5 rats, paralleling the changes in liver IGFBP-3 mRNA. A 30,000 M(r) band, consistent with IGFBP-1 and/or IGFBP-2, increased in liver. By immunoblot in serum, IGFBP-1 was only detectable in P5 rats, whereas IGFBP-2 decreased in the P5 group. By ligand blot, a 24,000 M(r) band (IGFBP-4) declined slightly in serum (not detected in liver). Our study shows that protein restriction regulates the expression of four of six IGFBPs in rats, and this regulation is organ specific. The nutritional regulation of IGFBP peptides in biological fluids, in particular serum, seems to involve additional mechanisms.
The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5′ ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.
This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen α1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, α-smooth muscle actin (A), vimentin (V), desmin (D), and c- kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and “normal” ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen α1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V + cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V + /A − /D − ), myofibroblasts (V + /A + /D + ), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V − /A + /D + ). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.
Dietary protein restriction in young rats stunts growth and decreases serum insulin-like growth factor I (IGF-I) concentrations. To investigate the possibility of a diet-induced accelerated clearance of IGF-I, the clearance of 125I-labeled IGF-I was determined by measuring the radioactivity in serum obtained at different intervals after bolus injection in 5-wk-old rats that had received either a normal 15% protein diet (P15; n = 6) or a 5% protein isocaloric diet (P5; n = 7) for 1 wk. The clearance and volume of distribution of 125I-IGF-I were increased in P5 rats by 58 and 75%, respectively, whereas the terminal elimination half-life was not changed. The calculated IGF-I production rate was decreased by 40% in P5 rats. The intermediate distribution phase (alpha) was twofold faster in P5 than in P15 animals, and 125I-IGF-I was preferentially bound to IGF binding proteins in the 40-kDa complex in P5 rats. These observations might explain the increased clearance and degradation of IGF-I in the P5 rats. Dietary protein restriction appears to lower serum IGF-I concentrations by both decreasing its production rate and increasing its clearance.
