We have recently identified in serum an acid protease which is capable of generating des(1-3)IGF-I from intact IGF-I. Here we have utilized a synthetic substrate with the sequence, biotin-G-P-E-T-L-C-BSA which contains the N-terminal sequence of IGF-I, to investigate the levels of this protease activity in streptozotocin-diabetic rats. Protease activity, quantified in terms of the amount of the biotin label lost, was determined in serum and hepatic extracts from normal control rats, diabetic rats and insulin-treated diabetic rats. Both the serum protease activity and protease activity in hepatic extracts were significantly increased in diabetic rats compared with control rats (P<0.02 and P<0.005). Following acute administration of insulin, a rapid and marked reduction in serum protease activity was observed; with an approximately 50% reduction apparent at 30 min (P<0.001). Chronic insulin treatment of diabetic rats also significantly reduced the serum and hepatic protease activity to the levels seen in control rats. A positive correlation between protease activity and serum glucose level was observed (r=0.58, P<0.005). The abundance of Spi 2.1 mRNA, a serine protease inhibitor, capable of inhibiting the IGF-I protease activity in vitro, was significantly decreased in the liver of diabetic rats and insulin treatment of diabetic rats did not normalize Spi 2.1 mRNA levels. These data suggest that the conversion of IGF-I to the more active des(1-3)IGF-I variant may be enhanced in diabetic animals. Since serum IGF-I levels are reduced in diabetic rats, increased des(1-3)IGF-I-generating protease activity would enhance the functional activity of the circulating IGF-I.
OBJECTIVE: IGF-I, IGF-I receptor and IGF-binding proteins (IGFBPs) are expressed in thyroid tissue and are associated with the function and growth of the thyroid. This study investigated the in vivo and in vitro effects of increased IGFBP-1 levels on the function and growth of the thyroid gland. DESIGN: Transgenic mice which constitutively overexpress IGFBP-1 were used. These mice have a phenotype consistent with partial inhibition of IGF-I action. METHODS: Thyroid growth, morphology and hormonogenesis were determined in transgenic mice treated with goitrogens, sodium perchlorate and methimazole. In vitro cell proliferation in thyroid follicles was assessed in response to IGF-I and TSH. RESULTS: Thyroid weight was increased in transgenic mice, relative to their body mass, whereas serum tri-iodothyronine (T(3)), thyroxine and T(3)-binding capacity were reduced, compared with wild-type. While an inverse relationship between T(3) and TSH was observed in both groups of goitrogen-treated mice, the slope of the line of best fit was less steep in transgenic mice compared with wild-type mice. Thyroid growth was less marked in transgenic than wild-type mice in response to goitrogens, although TSH levels were higher in goitrogen-treated transgenics. In vitro proliferative response of isolated thyroid follicles to IGF-I, but not to TSH, was reduced in transgenic, compared with wild-type mice. CONCLUSIONS: The results of this study suggest that, while overexpression of IGFBP-1 attenuates IGF-I action in vitro, it enhances thyroid growth in vivo, presumably as a result of perturbations in thyroid function at multiple levels.
The effect of thyroxine replacement dosage on serum total T4, fT4, T3, basal TSH and TSH-response to TRH stimulation were investigated in 33 patients receiving 50~200μg of thyroxine per day for at least 3 months together with 60 normal subjects as control. As compared with controls, the T4-treated patients had much higher serum total T4 and fT4, and yet much lower basal TSH levels. Those who received thyroxine more than 150μg/day frequently had elevated serum fT4 and suppressed TSH-response to TRH stimulation. Serum total T4 and fT4 levels were positively correlated with thyroxine dosage, while TSH-response to TRH stimulation was negatively correlated to it and serum T4 and fT4 levels as well.
These findings suggested that serum. T4 and fT4 assays might be used as sensitive indices in monitoring T4 dosage during thyroxine replacement therapy.
The insulin-like growth factor-binding proteins (IGFBPs) represent a family of conserved proteins that share the ability to bind the insulin-like growth factors, IGF-I and IGF-II, with affinities comparable to their respective receptors. Unlike the transmembrane IGF receptors, the IGFBPs are secreted proteins. Although many of the IGFBPs are able to interact with cell surface membranes, there is no convincing evidence as yet that these binding sites function as signal transducers. The IGFBPs are present in the serum, virtually all biological fluids, tissue extracts and conditioned medium from a variety of cell lines. They appear to be present and conserved in lower species, suggesting a functionally important role throughout evolution. Although the presence of IGFBPs was first suspected in the early days of somatomedin research in the 1960s, the elucidation of their nature and complexity had to await the development of molecular techniques. Six high-affinity members of this family of proteins have been identified and the cognate cDNAs for these binding proteins have been cloned from a variety of species. An additional four potential members of the IGFBP family have been identified through computer analysis of GenBank sequence data, on the basis of the presence of the IGFBP motif, GCGCCXXC (Rosenfeld 1998). These have considerably reduced binding affinity for the IGFs and, at the recent Fourth International IGF Symposium, the consensus opinion was that these proteins should be categorized as IGFBP-related proteins (IGFBPRPs-1 to -4). In the past decade, there has been a multitude of publications dealing with the IGFBPs. Whereas initial research focused on their role as modulators of IGF action, it is now appreciated that at least some of IGFBPs have additional IGF-independent functions. It is also important to remember that the vast majority of data concerning the biological functions of the IGFBPs have been obtained from in vitro cell culture conditions, in which concentrations of the IGFs and their binding proteins used may not match those encountered in vivo. Supraphysiological concentrations and nonphysiological conditions may well account for the confusing and often contradictory results obtained in vitro: for example, both IGFBPs-1 and -3 have been shown to have stimulatory and inhibitory effects on IGF-I-induced actions in cell culture. In vivo studies of IGFBP action have been limited until recently by the lack of large quantities of purified IGFBP. With development of transgenic and knockout mouse models, some insights into the physiological role of the IGFBPs are now starting to emerge. However, the limited phenotype of the IGFBP-3-overexpressing mice (Murphy et al. 1995) and the IGFBP-2 and IGFBP-4 knockout mice (Wood et al. 1993, Pintar et al. 1997) have led to the need to reconsider the potential physiological roles of these binding proteins. In the absence of a clear understanding of the physiological role of the individual IGFBPs, some investigators have speculated that there may be considerable functional redundancy in the IGFBP family. Here, I will review some of the similarities and differences between the IGFBPs and some recent insights into the biological functions of these proteins.
Transgenic mice that overexpressed IGFBP-1 are hyperinsulinemic in the first week of life and gradually develop fasting hyperglycemia. In adult transgenic mice, the hypoglycemic response to IGF-I but not insulin or des (1-3) IGF-I was attenuated (P < 0.05) compared with wild-type mice. Furthermore, in isolated adipocytes from transgenic mice, the stimulatory effect of IGF-I but not insulin on 2-deoxy-[3H]-glucose uptake was reduced (P < 0.02). In contrast, in isolated soleus muscle, the effects of both IGF-I and insulin on 2-deoxy-3H-glucose uptake and on [3H]-glucose incorporation into glycogen were significantly reduced compared to wild-type mice. The decline in specific activity of the 2-deoxy-3H-glucose, a measure of glucose appearance in the circulation, was more marked in transgenic animals (P < 0.05). In addition, tissue uptake of glucose was significantly higher in diaphragm, heart, intestine, liver, soleus muscle, and adipose tissue from fasting transgenic mice. Plasma concentrations of alanine, lysine, and methionine were also elevated in transgenic mice. These data suggest that overexpression of IGFBP-1 attenuates the hypoglycemic effect of endogenous IGF-I, which is initially compensated for by enhanced pancreatic insulin production. However, in adult mice pancreatic insulin content is reduced, insulin resistance is demonstrable in skeletal muscle and fasting hyperglycemia develops.
Differentiation of precursor cells into mature fat cells is accompanied by enhanced expression of insulin-like growth factor (IGF)-I and is stimulated by multiple hormones including growth hormone, glucocorticoids, IGF-I and insulin. We used transgenic mice that overexpress insulin-like growth factor binding protein-1 to investigate the role of IGF-I in the accumulation of fat tissue. In response to a sucrose-enriched diet, transgenic mice gained significantly less body weight and the epididymal fat mass was significantly reduced compared with wild-type mice. The increase in adipocyte size was also significantly reduced in transgenic mice compared with wild-type mice. Fewer colonies were generated from adipose tissue from transgenic mice and the mitogenic response of these cells to IGF-I was significantly reduced compared with those from wild-type mice. Induction of glycerol-3-phosphate dehydrogenase, a measure of adipocyte differentiation, by IGF-I but not insulin, was reduced in preadipocytes from transgenic mice. These data indicate that IGF-I has a critical role in the proliferation of adipocyte precursors, the differentiation of preadipocytes and the development of obesity in response to calorie excess.
OBJECTIVE: Adipose tIssue regulates insulin sensitivity via the circulating adipocytokines, leptin, resistin and adiponectin. The objective of this study was to compare the levels of resistin, adiponectin and leptin in lean and obese subjects and determine the relationship between circulating adipocytokines and insulin resistance. METHODS: We examined plasma levels of resistin, adiponectin and leptin in 17 lean subjects with a mean body mass index (BMI) of approximately 23 and 34 non-diabetic obese individuals with a mean BMI approximately 33. Insulin resistance was assessed using the homeostasis model assessment ratio (HOMA-R) formula derived from fasting insulin and glucose levels. RESULTS: Resistin levels were not significantly different between the two groups but were significantly higher in women compared with men, 35.4+/-6.5 (s.e.) vs 15.4+/-2.9 microg/L, P<0.01. Resistin did not correlate with BMI but did significantly correlate with HOMA-R, P<0.01, and this correlation remained significant after adjustment for gender and BMI. Adiponectin levels were significantly lower in obese compared with lean subjects, P<0.005, and higher in women, P<0.001, but showed no significant correlation with HOMA-R. Leptin levels were significantly higher in obese subjects and women and correlated with HOMA-R and resistin. DISCUSSION: In this small group of patients we demonstrated that insulin resistance correlated most strongly with leptin levels. A significant correlation between resistin levels and insulin resistance was also observed. Although a similar trend was apparent for adiponectin, the correlation with insulin resistance did not achieve statistical significance.
This paper reports on the growth and crystallography of organic pigment particles during solvent treatment. Transmission electron microscopy (TEM) and parallel electron energy loss spectroscopy (PEELS) have been used to study crystal growth during the pigmentation process. The action of the solvent has been studied as a function of solvent concentration and time. TEM images indicated that the particles were not of uniform thickness. This was then confirmed using PEELS on a VG HB5 STEM. The information gained gives evidence for growth by both coalescence and ripening
Transgenic mice which overexpress insulin-like growth factor binding protein-1 (IGFPB-1) demonstrate fasting hyperglycemia, hyperinsulinemia and glucose intolerance in adult life. Here we have examined the ontogeny of pancreatic endocrine dysfunction and investigated islet cell proliferation and apoptosis in this mouse model. In addition we have examined pancreatic insulin content in transgenic mice derived from blastocyst transfer into non-transgenic mice. Transgenic mice were normoglycemic at birth but had markedly elevated plasma insulin levels, 56.2 +/- 4.5 versus 25.4 +/- 1.5 pmol/l, p < 0.001, and pancreatic insulin concentration, 60.5 +/- 2.5 versus 49.0 +/- 2.6 ng/mg of tissue, P < 0.01, compared with wild-type mice. Transgenic mice derived from blastocyst transfer to wild-type foster mothers had an elevated pancreatic insulin content similar to that seen in pups from transgenic mice. There was an age-related decline in pancreatic insulin content and plasma insulin levels and an increase in fasting blood glucose concentrations, such that adult transgenic mice had significantly less pancreatic insulin than wild-type mice. Pancreatic islet number and the size of mature islets were increased in transgenic animals at birth compared with wild-type mice. Both islet cell proliferation, measured by 5-bromo-2'-deoxyuridine labeling, and apoptosis, assessed by the in situ terminal deoxynucleotidyl transferase and nick translation assay, were increased in islets of newborn transgenic mice compared with wild-type mice. In adult mice both islet cell proliferation and apoptosis were low and similar in transgenic and wild-type mice. Islets remained significantly larger and more numerous in adult transgenic mice despite a reduction in pancreatic insulin content. These data suggest that overexpression of IGFBP-1, either directly or indirectly via local or systemic mechanisms, has a positive trophic effect on islet development.
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