Maternal obesity causes fetal hypothalamic insulin resistance and disrupts development of hypothalamic feeding pathways.

OBJECTIVE: Perinatal exposure to maternal obesity results in predisposition of offspring to develop obesity later in life. Increased weight gain in offspring exposed to maternal obesity is usually associated with hyperphagia, implicating altered central regulation of food intake as a cause. We aimed to define how maternal obesity impacts early development of the hypothalamus to program lasting dysfunction in feeding regulatory pathways. METHODS: Mice offspring of diet-induced obese mothers were compared to the offspring of lean control mothers. We analysed gene expression in the fetal hypothalamus, alongside neurosphere assays to investigate the effects of maternal obesity on neural progenitor cell proliferation in vitro. Western blotting was used to investigate the insulin signalling pathway in the fetal hypothalamus. Characterisation of cell type and neuropeptide profile in adulthood was linked with analyses of feeding behaviour. RESULTS: There was a reduction in the expression of proliferative genes in the fetal hypothalamus of offspring exposed to maternal obesity. This reduction in proliferation was maintained in vitro when hypothalamic neural progenitor cells were grown as neurospheres. Hypothalamic fetal gene expression and neurosphere growth correlated with maternal body weight and insulin levels. Foetuses of obese mothers showed hypothalamic insulin resistance, which may be causative of reduced proliferation. Furthermore, maternal obesity activated the Notch signalling pathway in neonatal offspring hypothalamus, resulting in decreased neurogenesis. Adult offspring of obese mothers displayed an altered ratio of anorexigenic and orexigenic signals in the arcuate nucleus, associated with an inability to maintain energy homeostasis when metabolically challenged. CONCLUSIONS: These findings show that maternal obesity alters the molecular signature in the developing hypothalamus, which is associated with disrupted growth and development of hypothalamic precursor cells and defective feeding regulation in adulthood. This is the first report of fetal hypothalamic insulin resistance in an obese pregnancy and suggests a mechanism by which maternal obesity causes permanent changes to hypothalamic structure and function.

The tolerability of dothiepin: a review of clinical studies between 1963 and 1990 in over 13,000 depressed patients.

1. A total of 13,834 depressed patients were exposed to dothiepin most frequently at a dose of 150 mg/day and over 6 weeks, in 116 clinical studies between 1963 and 1990. 2. Overall, 2,066 (15%) patients were withdrawn prematurely from dothiepin for a variety of reasons, the most commonly specified reason being due to drug-related events in 500 (4%) patients. 3. In the remaining 11,768 patients, there were 9,312 reports of unwanted events most typically associated with the pharmacological effects of a tricyclic antidepressant although no serious sequelae were reported. 4. This review indicates that the incidence of serious adverse events associated with dothiepin at therapeutic doses is very low.

Effects of dietary fatty acid saturation on serum and tissue cholesterol concentrations and fecal sterol excretion: a chronic metabolic study in the rat.

We studied the effect of the degree of fatty acid saturation on cholesterol metabolism in rats fed either a low cholesterol-low fat diet (control group), high cholesterol-low fat diet (Chol group), high cholesterol-high saturated fat diet (Chol-SF) or high cholesterol-high PUF diet (Chol-PUF). The highest serum cholesterol levels were found in the Chol-SF group. No significant difference in serum cholesterol was found between the Chol and Chol-PUF groups. The Chol-PUF group showed the greatest accumulation of cholesterol in the liver. There was no significant difference in fecal excretion of acidic sterols, cholesterol and coprostanol between the Chol-PUF and Chol-SF groups. We conclude that in chronic feeding experiments in rats: a) high dietary cholesterol intake results in hypercholesterolemia; b) the hypercholesterolemia is accentuated by high dietary SF but is unaffected by PUF; c) addition of PUF to high cholesterol diet leads to massive cholesterol accumulation in the liver which can partly account for the apparent hypocholesterolemic effect of PUF relative to SF; and d) differences in serum and tissue cholesterol levels between SF and PUF supplemented groups are unlikely to be due to their effects on fecal sterol excretion.

Alkaline phosphatase positive precursors of adipocytes in the human bone marrow.

Developing fat cells in the bone marrow of leukaemic patients treated with chemotherapy were found to be endowed with membrane-bound alkaline phosphatase. Since alkaline phosphatase is a cytochemical marker of 'reticular' cells, this observation provides cytochemical evidence that reticular cells may convert to adipocytes when marrow cellularity abruptly decreases.

The effect of glucocorticoids on plasma insulin-like growth factor I concentration in the rat fetus.

Radioimmuno- and radioreceptor-assayable insulin-like growth factor I (IGF-I) concentration was determined in plasma pools of rat fetuses on gestation day 21 following treatment of the pregnant rats with pharmacologic doses of betamethasone, dexamethasone, or cortisone on gestation days 12 and 13. Dose-related reduction of plasma concentration of IGF-I occurred after betamethasone or dexamethasone treatment. There was no change in IGF-I concentration after cortisone treatment. The changes in fetal IGF-I concentration after steroid treatment were parallel between the two assays. Comparison of the results with previous data showing the effects of identical treatment of pregnant rats on fetal body and organ growth suggests that the IGF-I changes correlate better with reduction of liver/body ratio than with reduction of body weight. The findings indicate that growth retardation after steroid treatment in the fetus is in part the result of factors other than IGF-I. This may include a direct effect of the glucocorticoids on skeletal tissue. Reduced IGF-I concentration may contribute to the growth deficit.

Expression of tartrate-resistant acid phosphatase in bone marrow macrophages.

Bone marrow macrophages were found to express tartrate-resistant acid phosphatase (TRAP) under pathological conditions. In chronic granulocytic leukemia and metastatic carcinoma in the bone marrow this phenomenon was striking, all or almost all of the marrow macrophages being reactive. In other conditions, such as hypertransfusion or chemotherapy-induced marrow aplasia, the phenomenon did occur but was clearly a minor one. These observations indicate that tissue macrophages may become TRAP positive under the effect of unknown stimuli operating in certain pathological conditions. The results further suggest that the synthesis of the isoenzyme of acid phosphatase resistant to tartrate inhibition is a marker of macrophage activation rather than of differentiation towards particular subsets of the mononuclear phagocyte system.

Dissociation of catch-up growth control and neural control of growth hormone secretion in the stunted head-irradiated rat.

Male Long-Evans rats were irradiated to the head only at 2 days of age; littermates of the same sex were sham-irradiated. At 40 days of age the irradiated rats were divided into two groups, one of which was fasted 48 h and the other fed a normal diet. The irradiated rats, fasted and nonfasted, were cannulated in the superior vena cava at 48 to 50 days of age. Between 54 and 58 days of age the cannulated undisturbed rats had blood samples withdrawn at 15-min intervals over an 18-h period (9 h light and 9 h dark). Body weight and tail length data showed characteristic stunting following irradiation. The superimposed fast caused transient growth retardation; on refeeding, the fasted rats showed a pattern of catch-up growth limited to the irradiated non-fasted body size. Plasma growth hormone (GH) concentration in the fasted-refed rats as compared with the nonfasted irradiated rats showed no change in the average period of the bursts of GH secretion, the numbers of values in ranges of GH concentration, or the area under the curve of the plasma GH concentration versus time. No difference in these parameters was present in light or dark, considered separately. We conclude 1) that the link between the catch-up growth control and neural mechanisms controlling GH secretion is impaired as a consequence of the neonatal head-irradiation and 2) that catch-up growth acceleration is not dependent on increased GH secretion.

Comparative cytotoxicity of naphthalene and its monomethyl- and mononitro-derivatives in the mouse lung.

Male Swiss-Webster mice were exposed to naphthalene, 1-methyl-, 2-methyl-, 1-nitro- and 2-nitronaphthalene by intraperitoneal injection of peanut oil solutions over a dose range of 0.5-3.0 mmol kg-1 body weight. Treated mice were killed at times from 6 hours to 14 days post-treatment. Tissues were analyzed for cytotoxic effects by optical and electron microscopy, and for cell proliferation by autoradiography following in vitro labeling of lung slices with 3H-thymidine. The naphthalene derivatives varied widely in their cytotoxic effects. The most toxic was 1-nitronaphthalene with no mice surviving doses greater than 1 mmol kg-1. Naphthalene and 2-methylnaphthalene were about equally toxic, followed by 2-nitro- and 1-methylnaphthalene, in decreasing order of toxicity. In all cases the first evidence of cytotoxic effects was seen in the Clara cells of the bronchiolar epithelium, and, at the highest doses, toxic effects were found in the adjacent ciliated cells. Changes could be detected at the ultrastructural level at all doses, and within 6 hours after treatment. Only slight effects were seen in other cell types. Increased cell proliferation following chemical treatment was seen only in the bronchiolar epithelium, among cells tentatively identified as Clara cells or their precursors. Cytotoxic effects of naphthalene and its 1- and 2-methyl derivatives were confined to the lung, with minimal evidence of toxicity in the liver and kidney. The mononitronaphthalenes both produce small areas of centrizonal necrosis in the liver, but no discernible effects in the kidney. The experiments demonstrate the effect of small structural differences on the cytotoxicity of this group of environmental pollutants and also illustrate the sensitivity of the Clara cell as a target for xenobiotics.

The effects of different steroids on costal and epiphyseal cartilage of fetal and adult rats.

The effects of different doses of various steroids on growth, and on costal and epiphyseal chondrocytes, have been studied in prenatal, immature, and adult Long-Evans rats using histochemical techniques, and both light and electron microscopy. Both prenatal and postnatal treatments have been employed. The steroids used were cortisone (CA), betamethasome (BM), and, in the prenatal group only, dexamethasone (DM). Body weight is reduced in all treated rats (except the low dose of CA) by day 17 of gestation, with greater weight reductions occurring in rats receiving the higher dose level of each steroid. In rats treated prenatally or neonatally, and sacrificed postnatally on days 39-43 or days 116-127, body weights, and tibial and tail lengths, are less than in correspondingly aged controls, thus showing a persistence of the effects of treatment. Costal and epiphyseal cartilages in prenatal rats show cellular, synthetic, and ultrastructural alterations induced by treatment with glucocorticoids but the responses are not necessarily comparable. Except for the low dose of DM, the higher doses of each steroid are more effective in inhibiting, or altering, growth and cellular differentiation in the developing fetuses. Surprisingly, a low dose of DM has a more devastating effect on the cells and extracellular matrix of both costal and epiphyseal cartilage, than do higher dose-levels of the various steroids. Low doses of CA and BM are also effective in inhibiting or altering growth and cellular differentiation, but their effectiveness is largely limited to 17 days of gestation. The order of effect of the various doses of the different steroids on fetal cartilage, listed in decreasing order of severity, is as follows: 0.12 DM, 0.24 DM, 0.42 BM, 50 CA, with 25 CA and 0.18 BM being approximately equal and only slightly different from control cartilages. The effect of prenatal or neonatal glucocorticoid treatment on chondrocytes is minimal in the 30-43 days, or 116-127 day, postnatal groups. In immature and adult rats, cortisone affects the chondrocytes more deleteriously than does betamethasone, and a 5.0 mg dose of CA seems to affect chondrocytes, body weight, and tibial and tail lengths more than 0.2 or 7.5 mg doses.

Growth hormone secretion in the stunted head-irradiated rat.

Pulsatile secretion profiles of pituitary growth hormone (GH) and size and number of cells of brain, heart ventricles, liver, kidney, and gastrocnemius muscle were determined in male Long-Evans rats which received 600 rad x-irradiation to the head only at 2 days of age. Controls consisted of sham-irradiated littermates. The irradiated rats showed significant stunting of body weight and tail length beginning prior to weaning and lasting throughout the period (64 days) of observation. In irradiated rats at 20-21 days of age, just prior to weaning, organ weight was significantly reduced in all organs studied. Brain showed a decrease in organ/body ratio (p less than 0.0005) and in total DNA content (p less than 0.0005), but these values were not significantly changed in the other organs. DNA/organ ratio was increased significantly in heart (p less than 0.025) and gastrocnemius muscle (p less than 0.025); brain, liver, and kidney had nonsignificant increases. Protein/DNA ratios were decreased significantly in brain (p less than 0.005), heart (p less than 0.01), and gastrocnemius muscle (p less than 0.05); liver and kidney had nonsignificant decreases. Blood samples were removed for GH determination from cannulated undisturbed irradiated and control rats at 15-min intervals for 18-h periods (9 h light and 9 h dark) at 47-64 days of age. Irradiated rats had normal periodicity of bursts of GH secretion. The area under the curve of GH concentration versus time of the irradiated rat was decreased in light (p less than 0.025) and in dark (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased secretion of growth hormone in rats undergoing catch-up growth after fasting.

The pattern of pulsatile secretion of growth hormone (GH) was determined in catheterized undisturbed male Long-Evans rats which had been fasted for 48 hours beginning at 39 days of age and then fed a stock diet ad lib. Plasma GH was determined at 15 minute intervals for 6 and 24 hour periods at 12 to 26 days into recovery during which time catch-up growth is known to occur in this fasted rat model. Secretion in fasted-refed rats occurred in characteristic periodic bursts of pulses resulting in plasma values that tended to be above 200 ng/ml; the intervening troughs had values under one ng/ml. The mean period of secretory bursts ranged from 2.4 to 3.1 hours in different groups and in light and dark periods; periods did not differ significantly by t test between fasted-refed and control rats. In fasted-refed rats sampled for 24 hours the area under the curve of GH concentration plotted against tie was significantly greater in light than in dark; this difference did not occur in controls. In rats sampled for 6 hours in light the area under the curve of GH concentration was significantly greater in the fasted-refed rats than in the controls. The findings show that the GH secretion in rats is increased in the light phase of the diurnal light/dark cycle during catch-up growth after fasting; the increased secretion occurs within the normal periodic pattern of GH secretion. We conclude that GH secretion is linked to the catch-up phenomenon but that the role of GH in catch-up growth remains unclear.

Effect of dimethylthiourea on paraquat toxicity in rats.

We evaluated the utility of dimethylthiourea ( DMTU ), a hydroxyl radical scavenger, as potential therapy for paraquat poisoning. Seventy-five male Sprague-Dawley rats were divided into four groups ( DMTU -paraquat, paraquat, DMTU , control) and studied for up to 10 days. Clinical observations (dyspnea, weakness), mortality, hepatic and renal function tests, body weights, and histologic studies were performed. There was no mortality in the DMTU and control groups although various histologic and biochemical studies suggested that DMTU may be both hepatotoxic and nephrotoxic. In contrast, 74% of DMTU -paraquat rats and 39% of paraquat rats died (p less than 0.01). In addition, toxicity was clearly earlier in the DMTU -paraquat group than in the paraquat group. Hence, rather than being protective, treatment with dimethylthiourea was associated with increased mortality in paraquat-poisoned rats.

Syncytioskeletons in choriocarcinoma in culture.

Indirect immunofluorescence microscopy using anti-actin serum has been used to investigate the distribution of actin-containing polymers in BeWo cells. This cell line, derived from a human choriocarcinoma, contains tissue that, like its tissue of origin, is partly syncytial. The syncytial nature has been inferred from study of Nomarski optical sections and from transmission electron microscopy. The multinucleated plaques of tissue possess a syncytioskeleton with a number of actin-containing features characteristic of cultured cells. These include stress fibres, cortical layers and ruffled membranes. Other actin-containing structures are more typical of the related non-pathological syncytiotrophoblast. These include a dense population of microvilli. The overall organization of the actin syncytioskeletons bears no obvious relationship to the number or position of nuclei in the syncytium. Indirect immunofluorescence microscopy has also been employed to localize the protein tubulin in BeWo cells. The microtubules do not appear to be spatially organized by a particular nucleus. Rather, there are numerous microtubule-organizing centres (MTOCs) that exist in the cytoplasm and do not have the expected numerical and positional relationship to nuclei. From these data it appears that polymeric cytoskeletal elements in these syncytia are organized in a manner not immediately subordinate to syncytial nuclei.

Effect of neonatal head X-irradiation on growth of costal and tibial cartilage in rats: a histochemical and electron microscopic study.

Long-Evans rats were exposed to a single dose of head X-irradiation (600 rads) at 2 days of age. Experimental and sham irradiated rats were sacrificed at 14, 20-21, 23, 41-45, and 70-71 days. Tibial epiphyseal width and the number of cells in the epiphyseal plate were determined. Histochemical and electron microscopic studies were carried out on both costal and epiphyseal cartilage. Histochemical techniques revealed a reduction in chondroitin sulfate at 14 days in both costal and epiphyseal cartilage of X-irradiated rats. Epiphyseal cartilage demonstrated recovery subsequently, and this was followed by a normal decrease of chondroitin sulfate with increasing age, but costal cartilage did not recover. Collagen synthesis was also reduced in both costal and epiphyseal cartilage, but not as dramatically as chondroitin sulfate. Except for some electron dense cells and reduced scalloping of the cell membrane, costal chondrocytes from irradiated rats did not show major ultrastructural alterations. In contrast, epiphyseal chondrocytes demonstrated radiation induced alterations in organelles, in enhanced glycogen deposition, and in retardation of chondrocyte maturation. Extracellularly in both costal and epiphyseal cartilage of irradiated rats, collagen density and matrix granules were reduced, while calcification of the matrix was enhanced. Beyond 45 days, the effects of irradiation were markedly reduced. Comparisons of the histochemical results with metabolic studies carried out previously in cartilage from the same animals indicated a more direct concordance of the histochemical results with the pattern of physical growth and supported the usefulness of morphologic and histochemical techniques in the analysis of the growth disorder in the head-irradiated rat.

Cartilage metabolism during growth retardation following irradiation of the head of the neonatal rat.

The heads of 2-day-old male and female rats were irradiated with a single dose of 600 rads X irradiation, a dose which is known to stunt body weight, tibial length, and tail length, in order to ascertain its effects on synthesis by cartilage of sulfated proteoglycans, DNA, chondroprotein, and collagen as determined by utilization of [35S]sulfate, [Me-3H]thymidine, [1-14C]leucine, and [3,4-3H]proline, respectively. Data have been collected at 20-21, 23, 41-45, and 70-71 days of age. In comparison to controls, growth in body weight, tibial length, and tail length was significantly retarded in irradiated rats of both sexes. Although slow catch-up growth was observed with respect to tail length in both sexes and tibial length in females, a significant deficit in body weight in irradiated rats in both sexes remained at 70-71 days. Cartilage metabolism as evidenced by incorporation of the labeled substances showed no significant disturbance just prior to weaning (20-21 days) or after completion of the principal growth surge (70-71 days). Reduced sulfate and thymidine incorporation attributable to a brief period of undernutrition associated with weaning occurred in head-irradiated rats immediately following weaning (23 days). Increased isotope incorporation occurred at 41-45 days of age in cartilage of irradiated rats incubated with labeled sulfate, leucine, and proline; it did not increase with labeled thymidine. We conclude that neonatal head irradiation slows the rate of growth through the age of most rapid postnatal growth in normal rats. The pattern of cartilage metabolism during this time can be the result either of stimulation by a factor other than somatomedin, or selective inhibition of cartilage thymidine incorporation acting in combination with somatomedin.

The effect of neonatal head-irradiation and subsequent fasting on the mechanisms of catch-up growth.

The heads of 2 day old male and female rats were X-irradiated with 600 rad. Non-irradiated littermates served as controls. At 40 days of age groups of irradiated and non-irradiated rats were subjected to a 48 hour fast. Non-fasted groups of irradiated and non-irradiated rats were fed ad lib. and were used for comparative studies. Growth of body weight and tail length was recorded at intervals through 70 days of age. At sacrifice, pituitary weight, tibial length, and tibial epiphyseal width were also determined. The results confirm earlier findings that whole head irradiation produces reduced growth of body weight and of tail length which remains uncompensated by catch-up growth. After fasting and then refeeding normal catch-up growth acceleration occurred in both male and female irradiated and nonirradiated animals. The fasted non-irradiated animals caught up to the non-irradiated control rat size for both body weight and tail length. Similarly, the fasted irradiated rats caught up to the irradiated, non-fasted rat size, but did not catch up to the size of the non-irradiated controls. Pituitary weight and tibial length were significantly reduced in irradiated males and females. At sacrifice, no significant difference existed between the fasted and non-fasted subgroups. The tibial epiphyseal growth plate was not narrowed in irradiated rats; fasted rats had increased epiphyseal width during recovery in only one group. We conclude that the catch-up growth control is intact in the head-irradiated stunted rat. The findings suggest that the mechanism which recognizes normal body size (set-point for body size) and which determines the limit of catch-up growth acceleration is reset for a smaller body size by the head-irradiation.

Calcifications of cardiac valve bioprostheses. Biochemical, histologic, and ultrastructural observations in a subcutaneous implantation model system.

To study the process of calcification in bioprosthesis, 108 glutaraldehyde-treated porcine aortic valve leaflets were implanted subcutaneously in rabbits and removed 1 day to 6 months later; morphologic findings were correlated with biochemically determined levels of calcium (Ca++) and gamma-carboxyglutamic acid (Gla), a vitamin K-dependent Ca++-binding amino acid known to be present in a variety of tissues with pathological calcification. Gla and Ca++ levels began to increase about 2 months after implantation and increased progressively with time. Ca/Gla molar ratios were comparable to those in leaflets of bioprostheses explanted from patients, 22 to 64 months after implantation. Morphologically evident calcification began at the same time that Gla and Ca++ increases were detected biochemically and also increased in severity with time. Electron microscopy showed that calcification primarily involved the surface of collagen fibrils and the interfibrillar spaces. The biochemical and morphological findings in this experimental system are similar to those described in calcified porcine bioprosthetic valve leaflets removed from patients, but occurred much more rapidly. As with pathological calcification of other tissues, progressive calcification was accompanied by increased Gla levels, suggesting that Gla plays a role in the calcification of the leaflets. This model allows rapid comparative evaluation of large numbers of valve leaflets maintained under similar host conditions.

Ultrastructural evidence of pulmonary capillary endothelial damage from paraquat.

Because of lack of agreement concerning the toxicity of paraquat to the pulmonary microvasculature, we have undertaken an electron microscopic study of lungs of paraquat-treated rats. Rats were injected with paraquat or sterile water (controls) intraperitoneally; the animals were then killed at 24-h intervals for 10 days post-injection. In the control animals, lung ultrastructure remained normal throughout the study. In treated animals, the initial evidence of alveolar epithelial injury occurred 24 h post-paraquat. By 48 h, severe fragmentation and desquamation of membranous pneumocytes occurred, and both alveolar and interstitial edema were present. Epithelial damage was maximal 72-96 h post-paraquat. Pulmonary capillary endothelial abnormalities were less extensive than the alveolar epithelial lesions. Endothelial damage was first observed 48 h post-paraquat. In endothelial cells on the septal (thick) side of the capillaries, the number of pinocytotic vesicles was significantly increased (P less than 0.05) from 48 to 96 h post-paraquat. In endothelium adjacent to damaged epithelium, abnormalities included hydration, fragmentation, discontinuity, and widened intercellular junctions; these were maximal 72-96 h post-paraquat. Although other mechanisms are probably important, damaged pulmonary capillary endothelium seems to be a factor favoring paraquat-induced pulmonary edema.