Stachybotrys chartarum alters surfactant-related phospholipid synthesis and CTP:cholinephosphate cytidylyltransferase activity in isolated fetal rat type II cells.

Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.

Resistance to diet-induced obesity is associated with increased proopiomelanocortin mRNA and decreased neuropeptide Y mRNA in the hypothalamus.

Mechanisms mediating genetic susceptibility to diet-induced obesity have not been completely elucidated. Elevated hypothalamic neuropeptide Y (NPY) and decreased hypothalamic proopiomelanocortin (POMC) are thought to promote the development and maintenance of obesity. To assess the potential role of hypothalamic neuropeptide gene expression in diet-induced obesity, the present study examined effects of a high-fat diet on hypothalamic NPY and POMC mRNA in three strains of mice that differ in susceptibility to develop diet-induced obesity. C57BL/6J, CBA, and A/J mice were fed either normal rodent chow or a high-fat diet for 14 weeks after which hypothalamic gene expression was measured. On the high-fat diet, C57BL/6J mice gained the most weight, whereas A/J mice gained the least weight. On the high-fat diet, NPY mRNA significantly decreased as body weight increased in CBA and A/J mice, but not in C57BL/6J mice. In addition, POMC mRNA significantly increased as body weight increased in A/J mice, but not in CBA and C57BL/6J mice. Since decreased NPY mRNA and increased POMC mRNA would presumably attenuate weight gain, these results suggest that a high-fat diet produces compensatory changes in hypothalamic gene expression in mice resistant to diet-induced obesity but not in mice susceptible to diet-induced obesity.

Hyperphagia and weight gain after gold-thioglucose: relation to hypothalamic neuropeptide Y and proopiomelanocortin.

Genetic obesity is associated with increased neuropeptide Y (NPY) messenger RNA (mRNA) and decreased POMC mRNA in the hypothalamus of ob/ob and db/db mice, or impaired sensitivity to alphaMSH (derived from POMC) in the yellow agouti mouse. Acquired obesity can be produced by chemically lesioning the hypothalamus with either monosodium glutamate (MSG) in neonates or gold thioglucose (GTG) in adult mice. The present study examined whether elevated NPY mRNA and/or decreased POMC mRNA in the hypothalamus are associated with obesity due to hypothalamic lesions. GTG injection into adult mice produced a profound obese phenotype, including hyperphagia, increased body weight, and increased leptin mRNA and peptide, in association with reduced hypothalamic NPY mRNA and POMC mRNA. MSG treatment produced virtual elimination of NPY mRNA in the arcuate nucleus and a reduction of hypothalamic POMC mRNA, and led to elevated leptin. MSG pretreatment did not attenuate GTG-induced hyperphagia and obese phenotype. These results do not support a role for NPY-synthesizing neurons in the arcuate nucleus in mediating hypothalamic acquired obesity, but are consistent with the hypothesis that decreased activity of hypothalamic neurons synthesizing POMC play a role in mediating hypothalamic obesity.

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin.

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.

Injection with gold thioglucose impairs sensitivity to glucose: evidence that glucose-responsive neurons are important for long-term regulation of body weight.

It has been proposed, but never demonstrated, that glucose-responsive neurons are essential for the long-term regulation of body weight, and that mice injected with gold-thio-glucose (GTG) become obese due to destruction of glucose-responsive neurons. To assess these hypotheses, mice were injected with either saline (control) or a dose of GTG that produces obesity, and the effects on feeding of peripheral injection of saline, glucose, 2-deoxyglucose (2-DG), or cholecystokinin (CCK) were measured. In control mice, 2-DG increased, whereas glucose and CCK decreased, food intake significantly. In contrast, in GTG-treated mice, 2-DG and glucose did not have a significant effect on food intake. The GTG-treated mice remained sensitive to the inhibitory effect of CCK on food intake. These data indicate that i.p. injection of GTG, which produces obesity, also destroys glucose-responsive neurons, consistent with the hypothesis that glucose-responsive neurons contribute to the long-term regulation of body weight.

Ventromedial hypothalamic lesions produced by gold thioglucose do not impair induction of NPY mRNA in the arcuate nucleus by fasting.

There is increasing evidence that neuropeptide Y (NPY) plays an important role in the regulation of food intake. Neuropeptide Y mRNA in the arcuate nucleus increases after fasting and it has been proposed that this increase in NPY activity occurs as a result of the decreased circulating levels of both insulin and glucose associated with a fast. Glucose-responsive neurons in the ventromedial nucleus (VMN) of the hypothalamus alter their activity in response to changes in circulating glucose levels and these neurons have been proposed to be involved in the regulation of feeding behavior and metabolism. However, it is not known if these glucose-responsive neurons are involved in the response of NPY mRNA in the arcuate nucleus to fasting. To address this relationship, mice were injected with either saline or gold thioglucose (GTG), which appears to act on glucose-responsive neurons, and killed 6 weeks later after a 72 h fast or under ad lib fed conditions. In situ hybridization histochemistry for NPY mRNA was performed on hypothalamic sections containing the arcuate nucleus. The number of labelled cells was counted and the density of autoradiographic silver grains overlying the cells was also quantified (i.e. pixels per cell). Fasting resulted in increased levels of total NPY mRNA (number of labelled cells multiplied by the pixels per cell) in the arcuate nucleus of both control and GTG-treated mice. In addition, the relative fasting-induced increase (i.e. the fasted to fed ratio) in number of cells detected, number of pixels per cell, and total NPY mRNA was similar in both the control and GTG-treated mice. These data suggest that GTG-sensitive VMN neurons play little role in the induction of NPY mRNA by fasting in the arcuate nucleus.

In situ hybridization for creatine kinase-B messenger RNA in rat uterus and brain.

Creatine kinase-B (CKB) is present in both uterus and brain, and in uterus its synthesis (protein and mRNA) is regulated by estrogen. In the present study we have used in situ hybridization to detect CKB mRNA in uterus and brain, and to determine whether there is cell type specific induction of CKB by estrogen in these tissues. Tissue was taken from ovariectomized (ovx) rats that had been injected with either estrogen (17 beta-estradiol-3-benzoate and/or 17 beta-estradiol) or vehicle alone, 2, 8, 24 and 72 h previously. The brains and uteri were removed, frozen, cryostat-sectioned, and processed for in situ hybridization histochemistry. The uterine and brain sections were incubated with a tritiated DNA probe complementary to a 3' fragment of CKB mRNA, or a control sense probe to the same 3' fragment. In uterine smooth muscle cells, a 2.5- and 3.5-fold induction of CKB mRNA was observed 2 and 24 h after estrogen administration, respectively, and levels approached ovx controls at 72 h. A smaller induction (1.9-fold, 2 h) was observed in uterine epithelium, with little induction of CKB mRNA in stroma. In the brain CKB mRNA was detected in neurons, but not in clearly identified glia, and only occasionally in ependymal cells. In brain regions containing estrogen receptors there was no evidence of a significant estrogen effect on CKB mRNA levels. Some brain regions had higher neuronal expression than others (e.g. medial septum vs. preoptic area), but expression was widespread and not limited to neuroendocrine sites.

Differential regulation of luteinizing hormone-releasing hormone and galanin messenger ribonucleic Acid levels by alpha(1) adrenergic agents in the ovariectomized rat.

α(1) -Adrenergic control of luteinizing hormone-releasing hormone (LHRH) and galanin mRNA levels was examined in ovariectomized rats. Each rat was ovariectomized and a permanent bilateral cannula was implanted 1 mm dorsal to the preoptic area. Eleven to 14 days later, each rat received one of three treatments: prazosin (α(1) antagonist, n = 8), methoxamine (α(1) agonist, n = 5) or control (no drug, n = 7). Each drug was suspended in a polyacrylamide gel matrix and delivered to the preoptic area via the bilateral cannula. After 24 h of continuous exposure to the adrenergic agents (or control), rats were anesthetized, decapitated and brains were stored in liquid nitrogen until sectioned (7 µm) on a cryostat. In situ hybridization was performed using a [(32) P]-end-labelled 59mer complementary to LHRH mRNA. Reduced silver grains, proportional to LHRH mRNA content, were quantified for number of LHRH cells per section, number of grains per labelled cell and total number of grains in labelled cells. Compared to the controls, prazosin caused a 32% decrease (P<0.05) in the number of cells expressing the LHRH gene. The LHRH cells from untreated animals had a median of 53 grains/cell with a smooth distribution above and below the median. Treatment with prazosin reduced the median number of grains/cell to 36 (P<0.05). When the number of grains in labelled cells were totalled, prazosin decreased (P<0.01) the number of grains by 47%. Surprisingly, methoxamine caused no quantitative changes in any of the parameters examined. This might be explained if LHRH transcription in control animals was proceeding at near-maximal rates supported, in part, by an endogenous α(1) ligand. Alternatively, continuous exposure to this agonist may have resulted in desensitization to its stimulatory effects. When anatomically matched brain tissue sections from these animals were examined for galanin mRNA, no differences among experimental groups were detected. In conclusion, administration of an α(1) -adrenergic antagonist into the preoptic area suppressed levels of LHRH mRNA but not galanin mRNA. Therefore, the data suggest that an endogenous α(1) ligand, such as norepinephrine (or epinephrine), is required to maintain a high level of LHRH gene expression in the ovariectomized rat.

Effects of gamma-aminobutyric acid receptor agonists and antagonist on LHRH-synthesizing neurons as detected by immunocytochemistry and in situ hybridization.

Gamma-aminobutyric acid (GABA) is thought to play an important role in the regulation of luteinizing hormone-releasing hormone (LHRH) release but its role in the regulation of LHRH gene expression and LHRH synthesis is not known. We hypothesized that since GABA appears to have primarily inhibitory effects on LHRH cells (at the level of the cell body), GABA may act to decrease LHRH gene expression and peptide synthesis. This hypothesis was tested by examining the effect of GABA receptor activation and GABA receptor blockade on LHRH mRNA and peptide levels employing in situ hybridization and immunocytochemistry. Cells in the preoptic area (POA) of ovariectomized (ovx) rats were selectively exposed in vivo to specific GABA-ergic receptor agonists or an antagonist for up to 24 h. THIP, a specific GABAA receptor agonist, did not have a significant effect on either the intensity of LHRH immunoreactivity, or the number of LHRH-ir cells, observed as compared to controls. Baclofen, a GABAB receptor agonist appeared to decrease the number of cells with the greatest intensity of LHRH immunoreactivity, compared to controls. In situ hybridization, with either a tritiated RNA probe or a 32P-labelled oligonucleotide, complementary to LHRH mRNA, revealed that THIP either had no effect on the labelling intensity (32P-labelled oligonucleotide) or (contrary to our hypothesis) a slight excitatory effect on the level of LHRH mRNA detected per cell (tritiated RNA probe). Bicuculline (a specific GABAA receptor antagonist) decreased both the number of labelled cells observed per section through the POA, and the intensity of labelling observed in sections hybridized with the 32P-labelled oligonucleotide. These results suggest that in the POA GABAA receptors do not exert an inhibitory effect on LHRH gene expression, but rather could affect LH perhaps by electrically inhibiting LHRH neurons. In contrast, baclofen appeared to exert an inhibitory effect on LHRH gene expression, since the number of grains per labelled cell in the POA of baclofen-treated rats was lower than the grains per labelled cell of control rats. Also, similar to the results obtained with immunocytochemistry, in situ hybridization following baclofen treatment suggested that activation of GABAB receptors is able to reduce the number of neurons with the highest levels of LHRH mRNA. Thus, in the POA, GABA acting through GABAB receptors could be effective through changes in mRNA or peptide synthesis.