Increased plasma IGF-1 levels but lack of changes in adipocyte glucose transport in weanling rats with dorsomedial hypothalamic nucleus lesions 1 year after lesion production.

Experimental destruction of the dorsomedial hypothalamic nuclei (DMN) in weanling rats exerts an antiaging effect by preventing microalbuminuria and kidney lesions both 1 month and 1 year after lesion production. In the present study we report further on antiaging effects of DMN lesions (DMNL) by measuring glucose transport into adipocytes and plasma levels of insulin-like growth factors 1 and 2 (IGF-I, IGF-II). Male and female weanling Sprague-Dawley rats received bilateral electrolytic lesions in the DMN; sham-operated animals served as controls (SCON). The rats were maintained for 1 year and food intake was measured 3 weeks after surgery and 3 weeks prior to sacrifice. As expected, DMNL resulted in profound reductions of body weight and food intake, with male DMNL rats showing higher body weights and body weight gains than their female counterparts. The same was true of the respective SCON. In male DMNL rats, carcass fat in absolute terms was significantly reduced vs. SCON, but it was comparable among all groups when expressed in percent. Lean body mass (LBM), although significantly reduced in absolute terms in DMNL rats vs. SCON, was, however, significantly higher in male DMNL vs. SCON when expressed in percent, but not in females. LBM laid down per food energy taken in was higher in DMNL rats of both sexes than in their respective SCON. Efficiency of food utilization was normal in male DMNL vs. male SCON but was higher in female DMNL vs. SCON. Both male and female DMNL rats had significantly higher plasma IGF-1 concentrations than their respective SCON, and male DMNL rats had higher values than female DMNL rats. Plasma concentrations of IGF-II were significantly higher in DMNL vs. SCON, but only in females. Under both basal and insulin-stimulated conditions, DMNL rats had normal 3-0-methylglucose flux in adipocytes from epididymal fat pads vs. SCON. However, DMNL and SCON responded similarly to the stimulating effect of insulin. Although one-year-old rats may not be considered "aged", we do consider the observed lack of a drop in plasma IGF-I levels that occurs with aging as an "anti-aging" effect of DMN lesions.

Differential distribution of insulin-like growth factors and their binding proteins within bone: relationship to bone mineral density.

To evaluate the possibility that insulin-like growth factors (IGFs) and their binding proteins (BPs) in bone play a role in regulating cortical bone formation in growing animals, we compared changes in IGF and IGF BP levels with changes in bone mineral density (BMD) at three different regions (proximal, middle, and distal) along the rabbit femoral shaft. BMD measured by dual-energy x-ray absorptiometry decreased progressively from proximal to distal regions of the shaft, from 0.449 +/- 0.005 to 0.354 +/- 0.002 g/cm2 (mean +/- SEM; n = 9), respectively; total protein concentrations also decreased toward the distal region. We extracted the IGFs and their BPs from bone by demineralization in 10% EDTA and 4 M guanidine-HCl (pH 4.5). The IGFs were then separated from their BPs by size exclusion HPLC. The pH of the extraction buffer profoundly influenced the recoveries of the IGFs and, to a lesser extent, the total protein; at least 100% more IGFs were recovered at acid (4.5) pH than at neutral (7.5) or basic (10.5) pH. The levels of IGF-I decreased markedly from proximal to distal regions, from 273 +/- 27 to 100 +/- 38 ng human IGF-I equivalent/g bone (or 103 +/- 10 to 52 +/- 11 ng human IGF-I equivalent/mg protein), respectively. IGF-II was uniformly distributed (385 +/- 17 ng human IGF-II equivalent/g bone; mean of all three regions). Levels of the predominant 28-32 kD IGF BP doublet increased by about 100% from proximal to distal segments, regardless of whether the data were expressed per unit mass or protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The differential regulation of insulin-like growth factor (IGF) binding proteins by IGF-I during the life span of the rat.

To evaluate the therapeutic potential of insulin-like growth factor-I (IGF-I) as an anabolic agent during aging, we determined its effects on IGF binding proteins (BPs) in male rats of 2, 8, 16, and 24 months of age. In control animals, a striking increase (143%) in the predominant 39-45 kDa serum IGFBP (BP-3), with little change in serum IGF-I, accompanied the marked deceleration of growth which occurred between 2 and 8 months; the levels of IGF-I and its BPs declined by 15% and 34%, respectively, later in life. Infusion of IGF-I (1.2 mg/kg/day) for 2 weeks produced progressively larger increases in circulating IGF-I with age, from 24% to 95% between 2 and 24 months, consistent with an age-related decrease in exogenous IGF-I clearance. We attributed these results to the large increase in IGFBPs that occurred with maturation, as well as an induction of IGFBP-3 (34-68%) and a larger increase in the 30-34 kDa IGFBP (BP-2; 136-235%) following IGF-I treatment in the older (16-24 months) animals. Anabolic actions of IGF-I, which were seen only in the older rats, included modes increases in weight velocity (5.2 +/- 1.2 g/week), serum phosphorous (20%), and alkaline phosphatase (26%) compared to age-matched controls. In conclusion, differential changes in the relative levels of the different IGFBPs with IGF-I treatment in older animals appeared to profoundly influence both the half-life and tissue accessibility of exogenous IGF-I, thus modulating the potential benefits of IGF-I as an anabolic agent during aging.

Dissociation of bone mineral density from age-related decreases in insulin-like growth factor-I and its binding proteins in the male rat.

We evaluated the possibility that age-related decreases in circulating and/or bone-associated insulin-like growth factor-I (IGF-I) and its binding proteins (BPs) were associated with the development of osteopenia in 8-, 16-, and 24-month-old specific pathogen-free Brown Norway/Fischer 344 male rats. We measured bone mineral densities (BMD) of femurs by dual-energy x-ray absorptiometry. IGFs and IGFBPs were extracted from bone and separated by molecular exclusion HPLC before quantitation by specific radioligand assays. BMD did not change significantly between 8 and 24 months of age. IGF-I levels decreased by about 30% between 8 and 24 months in both serum and bone. Similarly, both circulating and bone-derived IGFBPs also declined (30% and 60%, respectively) with age. Thus, maintenance of femoral BMD throughout most of the adult rat life span was dissociated from the age-related decline in circulating and bone-associated IGF-I and IGFBPs.

Negative feedback regulation of insulin-like growth factor-II gene expression in differentiating myoblasts in vitro.

We have previously reported that autocrine secretion of insulin-like growth factor-II (IGF-II) plays a critical role in stimulating spontaneous myogenic differentiation in vitro. Myogenesis and IGF-II gene expression are both negatively controlled by high serum growth medium, and it is likely that serum inhibits terminal differentiation at least in part by blocking autocrine secretion of IGF-II. To investigate this possibility, we assessed the effects of various serum fractions and growth factors on endogenous IGF-II gene expression in rat L6A1 myoblasts. Unexpectedly, we found that IGF-I, IGF-II, and high concentrations of insulin were potent inhibitors of IGF-II gene expression. This is the first example we have seen in which IGFs regulate their own expression by a negative feedback mechanism. Feedback inhibition was not dependent on the stimulation of cell proliferation by IGFs, and differentiated L6A1 myotubes remained sensitive to this action of the IGFs. Results with IGF analogs suggested that the inhibition of IGF-II gene expression by IGFs was mediated by the type I IGF receptor and was strongly suppressed by L6A1-secreted IGF-binding proteins. Human primary myoblasts also exhibited feedback inhibition by the IGFs, whereas the rapidly fusing mouse Sol 8 cell line did not. We conclude that IGF-II gene expression in differentiating L6A1 myoblasts is regulated by a negative feedback mechanism (unusual for the IGFs) that acts primarily through the type I IGF receptor and appears to be inhibited by IGF-binding proteins secreted by L6A1 myoblasts in low serum differentiation medium.

Ontogenesis of insulin processing in fetal rat hepatocytes.

We studied insulin processing and hepatic glycogenesis in cultured hepatocytes isolated from rat fetuses of 17, 19, and 21 days of gestation. Steady-state insulin binding increased by 250% between days 17 and 19, from 145 +/- 8 to 361 +/- 52 fmol/mg protein, and by an additional 40% (405 +/- 69 fmol/mg protein) by 21 days of gestation. At 37 degrees C, 125I-insulin was rapidly (t 1/2 less than 5 min) internalized by hepatocytes at all three ages, reaching maximal levels (63-76% of the total cell-associated radioactivity) by 15 min. 125I-labelled degradation products appeared rapidly (t 1/2 less than 15 min) within the cells. Yet, the majority (68-77%) of the intracellular radioactivity consisted of intact 125I-insulin, even after 4 h at 37 degrees C. Hepatocytes pre-loaded with 125I-insulin and then acid-stripped of surface-bound radioactivity, rapidly released both intact 125I-insulin (retroendocytosis) and its radiolabelled degradation products. While intact insulin was initially released more rapidly (t 1/2 less than 6 min), and reached a plateau after 15-30 min, the degradation products continued to accumulate in the medium for at least 4 h. Methylamine inhibited intracellular 125I-insulin degradation at all three gestational ages and also blocked insulin-stimulated glycogenesis in 19- and 21-day hepatocytes, without altering basal glycogen synthesis. Insulin-stimulated glycogenesis was not induced in 17-day fetal rat hepatocytes in control or methylamine-treated cultures. We conclude that both degradative and retroendocytotic pathways for processing insulin are present in fetal rat hepatocytes by 17 days of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of age and IGF-I administration on elastin gene expression in rat aorta.

An age-related decrease in elasticity of arteries has been found in clinical and experimental studies done during the past two decades. We have investigated molecular and endocrine aspects of that decrease by examining the effects of age and insulin-like growth factor-I (IGF-I) on rat aorta elastogenesis. For comparison, pulmonary elastogenesis was examined in the same experimental animals. Different aged groups of male Fischer 344 rats (barrier protected) were implanted with minipumps for a two-week infusion of either 0.1 N acetic acid (vehicle solution) or IGF-I (1.2 mg/kg/day). The DNA content (micrograms DNA/g tissue) decreased with age in aorta but remained fairly constant in lung. Administration of IGF-I increased the aortic DNA content in all but the oldest rats. Conversely, the DNA content of pulmonary tissue was significantly increased in only the youngest animals. The steady-state levels of tropoelastin mRNA decreased dramatically in both aorta and lung with increased age. The decrease was greater in lung than aorta. Administration of IGF-I elevated aortic tropoelastin mRNA steady-state levels, whereas lung tropoelastin mRNA levels were unaffected by IGF-I administration. Aortic tissue synthesized decreased amounts of insoluble elastin with increased age. These results establish a direct relationship between aortic tropoelastin mRNA levels and the synthesis of insoluble elastin in aging. Administration of IGF-I increased aortic elastin synthesis throughout the life span of the rat, although the proportionate increase diminished with age.

Evidence for insulin-like growth factor-I regulation of chick aortic elastogenesis.

The potential role of insulin-like growth factor-I (IGF-I) as a modulator of chick aortic embryogenesis was examined. Studies were designed to investigate the in vivo relationships between embryonic IGF-I serum concentrations, liver and aortic IGF-I mRNA steady-state levels and the inception and perpetuation of aortic elastogenesis. In addition to aortic tissue, elastogenesis was measured in heart and lung tissues in order to compare the responses of functionally unique elastin-containing tissues to the developmental appearance of IGF-I in embryonic serum. Our results demonstrate that the induction of aortic tropoelastin mRNA steady-state levels coincides with a major increase in serum IGF-I concentration. This is not the case with either lung or heart elastogenic responses. All three tissues examined (aorta, lung, and heart) exhibited different developmental patterns of tropoelastin mRNA steady-state levels during the embryonic ages studied (8- through 10-day). Only aortic tropoelastin mRNA levels paralleled the rise and fall of IGF-I serum levels. Steady-state levels of liver IGF-I mRNA peaked one day (9-day) prior to detectable IGF-I serum levels but otherwise mirrored the gradual, but steady decrease in IGF-I serum levels through 16-day. Aortic tissue also expresses IGF-I mRNA beginning at 8-day and continuing throughout the embryonic ages examined (16-day). Although the relative levels of aortic IGF-I mRNA are very low in comparison to corresponding mRNA levels in liver, the fact that IGF-I mRNA is transcribed in the aorta points to the possibility that autocrine and/or paracrine mechanisms of IGF-I action may be operative in aortic elastogenesis.

Effect of growth hormone on growth and glucose tolerance of normal rats.

To determine the effect of growth hormone (GH) administration on growth rate, ultimate size, and glucose tolerance of intact rats, we administered ovine GH to male Sprague-Dawley weanling rats for six weeks. Growth hormone, in a dose sufficient to double the growth rate and increase sixfold the somatomedin C (SM-C) levels of hypophysectomized rats, failed to increase the rate of growth or size of visceral organs of intact rats. Their SM-C levels increased only 10%. Following glucose administration, the mean blood glucose levels were higher at every time point measured in those treated with GH compared with the intact control animals. We conclude that, although there was no difference in somatic or visceral growth, the dose of exogenous GH administered increased SM-C levels and decreased glucose tolerance in the intact rats. While a larger GH dose might increase the final body size of non-GH-deficient animals, impaired glucose tolerance could be a significant side effect.

The influence of epidermal growth factor on surface morphology of fetal rat hepatocytes in primary culture.

In an attempt to understand the hormonal regulation of somatomedin secretion in the fetus, we have confirmed that epidermal growth factor (EGF) stimulates fetal rat hepatocytes in primary culture to secrete somatomedin in a time and a dose-dependent fashion. Transmission electron microscopy (TEM) revealed that the cultured cells had ultrastructural features consistent with those of fetal hepatocytes. Scanning electron microscopy (SEM) showed that cells grown in either Medium 199 or EGF supplemented Medium 199 formed cellular aggregates within 6 h. The surface features of cells in control and experimental cultures were indistinguishable up until 24 h after exposure to EGF. At this point in time, morphological differences between treatment groups were first apparent with SEM. In the presence of EGF, cellular aggregates were thicker, cells were more rounded in contour, and the number of microvilli and cytoplasmic excrescences (blebs) was greater than in control cultures. These differences were further accentuated at 48 h after exposure to the growth factor. Since the appearance of microvilli and blebs coincides with increasing production of somatomedin, they may represent morphological evidence of secretory activity.

Hormonal regulation of somatomedin secretion by fetal rat hepatocytes in primary culture.

To determine which hormones might regulate somatomedin secretion in the fetus, we measured somatomedin levels in conditioned medium from primary cultures of fetal rat hepatocytes. We employed a bioassay [( 3H]thymidine incorporation into DNA of chick embryo fibroblasts), a displacement assay [competition for binding of radiolabeled multiplication-stimulating activity (rat insulin-like growth factor II) to the somatomedin-binding protein] for total somatomedin, and the RIA for somatomedin-C. Epidermal growth factor and dexamethasone were the most active hormones tested; total somatomedin levels were 2-3 times above control levels. Rat GH was much less stimulatory. Human placental lactogen, glucagon, and insulin had little or no effect. Stimulation of somatomedin secretion by both epidermal growth factor and dexamethasone was time and dose dependent. The maximal response occurred at 48 h at a concentration of about 1 X 10(-7) M of either hormone. In the bioassay, stimulation by epidermal growth factor, but not dexamethasone, was detected. The steroid enhanced the secretion of an inhibitor that completely masked the mitogenic activity of the increased somatomedin levels. The somatomedin secreted by fetal hepatocytes exhibited immunological cross-reactivity with human somatomedin-C, but the levels were 500-fold less than those measured by our displacement assay. This suggests that the predominant fetal rat somatomedin is not somatomedin-C. We conclude that epidermal growth factor and dexamethasone, but not GH or placental lactogen, stimulated the secretion by fetal hepatocytes of a somatomedin which resembled multiplication-stimulating activity.