Transcriptional Basis for Rhythmic Control of Hunger and Metabolism within the AgRP Neuron.

The alignment of fasting and feeding with the sleep/wake cycle is coordinated by hypothalamic neurons, though the underlying molecular programs remain incompletely understood. Here, we demonstrate that the clock transcription pathway maximizes eating during wakefulness and glucose production during sleep through autonomous circadian regulation of NPY/AgRP neurons. Tandem profiling of whole-cell and ribosome-bound mRNAs in morning and evening under dynamic fasting and fed conditions identified temporal control of activity-dependent gene repertoires in AgRP neurons central to synaptogenesis, bioenergetics, and neurotransmitter and peptidergic signaling. Synaptic and circadian pathways were specific to whole-cell RNA analyses, while bioenergetic pathways were selectively enriched in the ribosome-bound transcriptome. Finally, we demonstrate that the AgRP clock mediates the transcriptional response to leptin. Our results reveal that time-of-day restriction in transcriptional control of energy-sensing neurons underlies the alignment of hunger and food acquisition with the sleep/wake state.

Sewage sludge ash characteristics and potential for use in bricks, tiles and glass ceramics.

The characteristics of sewage sludge ash (SSA) and its use in ceramic applications pertaining to bricks, tiles and glass ceramics have been assessed using the globally published literature in the English medium. It is shown that SSA possesses similar chemical characteristics to established ceramic materials and under heat treatment achieves the targeted densification, strength increases and absorption reductions. In brick and tile applications, technical requirements relating to strength, absorption and durability are achievable, with merely manageable performance reductions with SSA as a partial clay replacement. Fluxing properties of SSA facilitate lower firing temperatures during ceramics production, although reductions in mix plasticity leads to higher forming water requirements. SSA glass ceramics attained strengths in excess of natural materials such as granite and marble and displayed strong durability properties. The thermal treatment and nature of ceramic products also effectively restricted heavy metal leaching to low levels. Case studies, predominantly in bricks applications, reinforce confidence in the material with suitable technical performances achieved in practical conditions.

Use of recycled aggregates from construction and demolition waste in geotechnical applications: A literature review.

The use of recycled aggregates (RA) in construction constitutes a significant step towards a more sustainable society and also creates a new market opportunity to be exploited. In recent years, several case-studies have emerged in which RA were used in Geotechnical applications, such as filling materials and in unbound pavement layers. This paper presents a review of the most important physical properties of different types of RA and their comparison with natural aggregates (NA), and how these properties affect their hydraulic and mechanical behaviour when compacted. Specifically, the effects of compaction on grading size distribution curves and density are analysed, as well as the consequences of particle crushing on the resilient modulus, CBR and permeability. The paper also contains an analysis of the influence of incorporating different RA types on the performance of unbound road pavement layers as compared with those built with NA by means of the International Roughness Index and deflection values. The results collected from the literature indicate that the performance of most RA is comparable to that of NA and can be used in unbound pavement layers or in other applications requiring compaction.

Effects of perilipin 2 antisense oligonucleotide treatment on hepatic lipid metabolism and gene expression.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. We previously showed that Perilipin 2 (Plin2), a member of lipid droplet protein family, is markedly increased in fatty liver, and its reduction in the liver of diet-induced obese mice by antisense oligonucleotide (ASO) decreased steatosis and enhanced insulin sensitivity. Plin2-ASO treatment markedly suppressed lipogenic gene expression. To gain a better understanding of the biological role of Plin2 in liver, we performed microarray analysis to determine genes differentially regulated by Plin2-ASO compared with a control (scrambled) oligonucleotide (Cont). Male C57BL/6J mice on a high-fat diet were treated with Plin2- or Cont-ASO for 4 wk. Plin2-ASO decreased hepatic triglycerides, and this was associated with changes in expression of 1,363 genes. We analyzed the data for functional clustering and validated the expression of representative genes using real-time PCR. On the high-fat diet, Plin2-ASO decreased the expression of enzymes involved in fatty acid metabolism (acsl1, lipe) and steroid metabolism (hmgcr, hsd3b5, hsd17b2), suggesting that Plin2 affects hepatic lipid metabolism at the transcriptional level. Plin2-ASO also increased the expression of genes involved in regulation of hepatocyte proliferation (afp, H19), mitosis (ccna2, incenp, sgol1), and extracellular matrix (col1a1, col3a1, mmp8). Plin2-ASO had similar effects on gene expression in chow-fed mice. Together, these results indicate that Plin2 has diverse metabolic and structural roles in the liver, and its downregulation promotes hepatic fibrosis and proliferation.

Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration.

Fatty liver disease is associated with obesity and type 2 diabetes, and hepatic lipid accumulation may contribute to insulin resistance. Histone deacetylase 3 (Hdac3) controls the circadian rhythm of hepatic lipogenesis. Here we show that, despite severe hepatosteatosis, mice with liver-specific depletion of Hdac3 have higher insulin sensitivity without any changes in insulin signaling or body weight compared to wild-type mice. Hdac3 depletion reroutes metabolic precursors towards lipid synthesis and storage within lipid droplets and away from hepatic glucose production. Perilipin 2, which coats lipid droplets, is markedly induced upon Hdac3 depletion and contributes to the development of both steatosis and improved tolerance to glucose. These findings suggest that the sequestration of hepatic lipids in perilipin 2­coated droplets ameliorates insulin resistance and establish Hdac3 as a pivotal epigenomic modifier that integrates signals from the circadian clock in the regulation of hepatic intermediary metabolism.

Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice.

Lipid droplets in the liver are coated with the perilipin family of proteins, notably adipocyte differentiation-related protein (ADRP) and tail-interacting protein of 47 kDa (TIP47). ADRP is increased in hepatic steatosis and is associated with hyperlipidemia, insulin resistance, and glucose intolerance. We have shown that reducing ADRP in the liver via antisense oligonucleotide (ASO) treatment attenuates steatosis and improves insulin sensitivity and glucose tolerance. We hypothesized that TIP47 has similar effects on hepatic lipid and glucose metabolism. We found that TIP47 mRNA and protein levels were increased in response to a high-fat diet (HFD) in C57BL/6J mice. TIP47 ASO treatment decreased liver TIP47 mRNA and protein levels without altering ADRP levels. Low-dose TIP47 ASO (15 mg/kg) and high-dose TIP47 ASO (50 mg/kg) decreased triglyceride content in the liver by 35% and 52%, respectively. Liver histology showed a drastic reduction in hepatic steatosis following TIP47 ASO treatment. The high dose of TIP47 ASO significantly blunted hepatic triglyceride secretion, improved glucose tolerance, and increased insulin sensitivity in liver, adipose tissue, and muscle. These findings show that TIP47 affects hepatic lipid and glucose metabolism and may be a target for the treatment of nonalcoholic fatty liver and related metabolic disorders.

Metabolic dysfunction associated with adiponectin deficiency enhances kainic acid-induced seizure severity.

Metabolic syndrome has deleterious effects on the CNS, and recent evidence suggests that obesity rates are higher at presentation in children who develop epilepsy. Adiponectin is secreted by adipose tissue and acts in the brain and peripheral organs to regulate glucose and lipid metabolism. Adiponectin deficiency predisposes toward metabolic syndrome, characterized by obesity, insulin resistance, impaired glucose tolerance, hyperlipidemia, and cardiovascular morbidity. To investigate the relationship between metabolic syndrome and seizures, wild-type C57BL/6J and adiponectin knock-out mice were fed a high-fat diet, followed by treatment with low doses of kainic acid to induce seizures. Adiponectin deficiency in mice fed a high-fat diet resulted in greater fat accumulation, impaired glucose tolerance, hyperlipidemia, increased seizure severity, and increased hippocampal pathology. In contrast, there were no adverse effects of adiponectin deficiency on metabolic phenotype or seizure activity in mice fed a normal (low-fat) chow diet. These findings demonstrate that metabolic syndrome modulates the outcome of seizures and brain injury.

Estrogen sulfotransferase regulates body fat and glucose homeostasis in female mice.

Estrogen regulates fat mass and distribution and glucose metabolism. We have previously found that estrogen sulfotransferase (EST), which inactivates estrogen through sulfoconjugation, was highly expressed in adipose tissue of male mice and induced by testosterone in female mice. To determine whether inhibition of estrogen in female adipose tissue affects adipose mass and metabolism, we generated transgenic mice expressing EST via the aP2 promoter. As expected, EST expression was increased in adipose tissue as well as macrophages. Parametrial and subcutaneous inguinal adipose mass and adipocyte size were significantly reduced in EST transgenic mice, but there was no change in retroperitoneal or brown adipose tissue. EST overexpression decreased the differentiation of primary adipocytes, and this was associated with reductions in the expression of peroxisome proliferator-activated receptor-γ, fatty acid synthase, hormone-sensitive lipase, lipoprotein lipase, and leptin. Serum leptin levels were significantly lower in EST transgenic mice, whereas total and high-molecular-weight adiponectin levels were not different in transgenic and wild-type mice. Glucose uptake was blunted in parametrial adipose tissue during hyperinsulinemic-euglycemic clamp in EST transgenic mice. In contrast, hepatic insulin sensitivity was improved but muscle insulin sensitivity did not change in EST transgenic mice. These results reveal novel effects of EST on adipose tissue and glucose homeostasis in female mice.

CRTC2 (TORC2) contributes to the transcriptional response to fasting in the liver but is not required for the maintenance of glucose homeostasis.

The liver contributes to glucose homeostasis by promoting either storage or production of glucose, depending on the physiological state. The cAMP response element-binding protein (CREB) is a principal regulator of genes involved in coordinating the hepatic response to fasting, but its mechanism of gene activation remains controversial. We derived CRTC2 (CREB-regulated transcription coactivator 2, previously TORC2)-deficient mice to assess the contribution of this cofactor to hepatic glucose metabolism in vivo. CRTC2 mutant hepatocytes showed reduced glucose production in response to glucagon, which correlated with decreased CREB binding to several gluconeogenic genes. However, despite attenuated expression of CREB target genes, including PEPCK, G6Pase, and PGC-1alpha, no hypoglycemia was observed in mutant mice. Collectively, these results provide genetic evidence supporting a role for CRTC2 in the transcriptional response to fasting, but indicate only a limited contribution of this cofactor to the maintenance of glucose homeostasis.

Inhibition of ADRP prevents diet-induced insulin resistance.

Diets with high fat content induce steatosis, insulin resistance, and type 2 diabetes. The lipid droplet protein adipose differentiation-related protein (ADRP) mediates hepatic steatosis, but whether this affects insulin action in the liver or peripheral organs in diet-induced obesity is uncertain. We fed C57BL/6J mice a high-fat diet and simultaneously treated them with an antisense oligonucleotide (ASO) against ADRP for 4 wk. Glucose homeostasis was assessed with clamp and tracer techniques. ADRP ASO decreased the levels of triglycerides and diacylglycerol in the liver, but fatty acids, long-chain fatty acyl CoAs, ceramides, and cholesterol were unchanged. Insulin action in the liver was enhanced after ADRP ASO treatment, whereas muscle and adipose tissue were not affected. ADRP ASO increased the phosphorylation of insulin receptor substrate (IRS)1, IRS2, and Akt, and decreased gluconeogenic enzymes and PKCepsilon, consistent with its insulin-sensitizing action. These results demonstrate an important role for ADRP in the pathogenesis of diet-induced insulin resistance.

Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone.

CYP2C11, the most commonly expressed isoform of cytochrome P450 in male rat liver, was measured in spleen, thymus and bone marrow by quantitative real-time PCR and enhanced Western blotting. CYP2C11 concentrations in the lymphoid tissues were a fraction of that observed in liver, but like the liver, were sexually dimorphic (M>F) with mRNA and protein levels in agreement. Although the response to hypophysectomy varied according to tissue and sex, expression levels of CYP2C11 in all measured tissues remained greater in males. Further differences in CYP2C11 expression between liver and lymphoid tissue were observed following restoration of the circulating masculine growth hormone profile in hypophysectomized rats. In contrast to the liver where the renaturalized growth hormone profile elevated CYP2C11 expression in both sexes, the response was opposite in spleen and thymus with isoform concentrations declining in both sexes. Lastly, the divergent response of CYP2C11 between the liver and immune system was examined in cultured splenocytes exposed to different mitogens. In contrast to the dramatic depletion of CYP2C11 reported in proliferating hepatocytes, mitogen-stimulation resulted in a significant elevation in splenocyte CYP2C11 expression. In summary, we report for the first time that thymus, spleen and bone marrow express, albeit nominal, sex-dependent levels of CYP2C11 (M>F) whose regulation appears to be under some hormonal control, but very different from that of the hepatic isoform.

Attenuated expression of episodic growth hormone-induced CYP2C11 in female rats associated with suboptimal activation of the Jak2/Stat5B and other modulating signaling pathways.

Inherent sex differences in various parameters of growth, musculoskeletal function, metabolism, and cytochrome P450 (P450)-dependent drug metabolism have been reported in rats and humans administered typical intermittent/episodic growth hormone (GH) replacement therapy. Having infused and monitored the identical physiologic masculine (episodic) growth hormone profile to both hypophysectomized male and female rats, we observed that induction levels of hepatic CYP2C11 were 35 to 40% lower in females. Associated with the reduced expression of the P450 isoform in the episodic GH-treated females were dramatically lower activation levels of Janus kinase (Jak2), signal transducers and activators of transcription (Stat5A and 5B) as well as 50% less binding of Stat5B to the CYP2C11 promoter. Because the Jak2/Stat5B signaling pathway mediates the effects of the masculine GH profile on its target cells, we conclude that the lower induction level of CYP2C11 in females exposed to the masculine GH profile is probably due, at least in part, to the suboptimum activation of the Jak2/Stat5B pathway. In addition to the reduced activation of the Jak2/Stat5B pathway, we observed lower activational levels of mitogen-activated protein kinase (p44/p42) and, indirectly, nuclear factor-kappaB in the episodic GH-treated females that may be involved in attenuating the activity of the Jak2/Stat5B pathway diminishing CYP2C11 expression levels.

Sexually dimorphic regulation of hepatic isoforms of human cytochrome p450 by growth hormone.

Sex differences in drug metabolism have been reported in numerous species, including humans. In rats and mice, sex-dependent differences in circulating growth hormone profiles are responsible for the differential expression of multiple sex-dependent isoforms of cytochrome P450, which is the basis for the sexual dimorphism in drug metabolism. In contrast, very little is known about sex differences in human isoforms of cytochrome P450 and their regulation by growth hormone. In this study, we have examined the effects of physiologic-like exposure doses to dexamethasone and/or pulsatile (masculine) or constant (feminine) human growth hormone on expression levels of CYP3A4, 1A2, 2D6, and 2E1 and the glucocorticoid and growth hormone receptors in hepatocyte cultures obtained from men and women donors. We report that growth hormone can regulate expression of CYP3A4, 1A2, and 2D6. The masculine-like pulsatile growth hormone profile suppresses dexamethasone-induced CYP3A4, 1A2, and 2D6, whereas the feminine-like constant profile is permissive allowing isoform expression to be equal to or greater than glucocorticoid induction alone. There are intrinsic sexual differences in hepatocytes of men and women resulting in different levels of responsiveness of CYP3A4, 1A2, and hormone receptor expression to the same sexually dimorphic growth hormone profiles. Last, although real, the sexually dimorphic effects of growth hormone on human cytochrome P450 expression are not as dramatic as those observed in rats and could easily be overlooked by the heterogeneous backgrounds of human populations.

Inadequacy of the Janus kinase 2/signal transducer and activator of transcription signal transduction pathway to mediate episodic growth hormone-dependent regulation of hepatic CYP2C11.

CYP2C11, the most commonly expressed hepatic cytochrome P450 isoform in male rats, is induced by the masculine "episodic" secretory growth hormone profile. A considerable number of reports have indicated that episodic growth hormone effects are mediated by the activation of the Janus kinase 2 (Jak2)/signal transducer and activator of transcription (Stat)5B signal transduction pathway. We observed that restoration of the normal masculine plasma growth hormone pulse in hypophysectomized male rats did indeed rapidly activate (phosphorylate) Jak2, shortly followed by activation and nuclear translocation of Stat5B. Infusion of a growth hormone pulse with an amplitude that was 10% of the normal height induced a dramatic overexpression of CYP2C11, had little effect activating Jak2, but induced a more rapid and greater accumulation of activated nuclear Stat5B. Restoration of a growth hormone pulse with an amplitude of only 1% of normal had little effect phosphorylating Jak2, activated and translocated to the hepatic nucleus approximately 70% of the normally induced levels of Stat5B, but had no inductive effect on CYP2C11. Last, the hypophysectomized male rat receiving no growth hormone replacement expressed 25 to 35% of normal concentrations of CYP2C11 despite no measurable activation of either Jak2 or Stat5B. These results raise concerns regarding the requisite role of the Jak2/Stat5B pathway in mediating episodic growth hormone regulation of CYP2C11. However, accumulation of activated extracellular signal-regulated kinase (ERK)1 and ERK2 were the only transducers measured in the study not affected by the 1% replacement pulse of growth hormone and were elevated 2- to 3-fold above normal when the pulse was renaturalized to 10% of physiological amplitude, suggesting the possible involvement of mitogen-activated protein kinase in episodic growth hormone regulation of CYP2C11.

Studies on LHRBI of sheep corpora lutea--a preliminary report on in vivo administration to rhesus monkeys.

An LH-receptor binding inhibitor (LHRBI), previously isolated from sheep corpora lutea in a partially purified form was subjected to wheat germ lectin chromatography. The unadsorbed fraction (UM-2R-III A) thus obtained had maximum LHRBI activity. This preparation was utilized to develop polyclonal antibodies. The purified fraction could be radioiodinated, suggesting its peptide nature. Intravenous injections of UM-2R-IIIA at 200 microg protein per dose in two doses per day at 9.30 h and 16.30 h on days 1 and 2 of the menstrual cycle to regularly cycling monkeys resulted in a shortening of the length of the cycle by 2 to 3 days. In addition, serum levels of progesterone increased prior to ovulation and remained high through the cycle of all three treated monkeys. It is possible that LHRBI induced enhanced synthesis and/or secretion of progesterone by the ovarian follicles thus suggesting a role for LHRBI in ovarian function.

Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats.

Humans as well as other mammals experience an aging-related decline in drug metabolism as well as a diminution in growth hormone secretion. In the case of rats, these events are more pronounced in senescent males, whose expression of male-specific isoforms of cytochrome P450, the major drug-metabolizing enzymes and constituting approximately 60-70% of the total cytochrome P450 in male rat liver, is completely suppressed, whereas female-dependent isoforms are remarkably induced to female-like levels. Overlooked in these independently reported studies is the fact that "signals" inherent in the masculine episodic and female continuous growth hormone profiles regulate expression and/or suppression of the dozen or so sex-dependent cytochrome P450 isoforms in rat liver. Whereas previous studies identified profound reductions in the pulse amplitudes of the masculine growth hormone profile as the cause for the diminished hormone secretion during aging, pulse heights are not recognized by the cytochromes as regulatory signals. Instead, we have shown that just a nominal secretion of growth hormone during the usual growth hormone-devoid interpulse period in the masculine episodic profile can explain the complete repression of male-specific CYP2C11, CYP3A2, and CYP2A2 and induction of female-dependent CYP2C12, CYP2C6, and CYP2A1 observed in senescent male rats.

Comparative studies with penicillinase, horseradish peroxidase, and alkaline phosphatase as enzyme labels in developing enzyme immunoassay of cortisol.

Relative merit of different enzyme labels for measuring cortisol directly in serum by competitive enzyme immunoassay (ELISA) was examined. Cortisol-21-hemisuccinate was labeled separately with penicillinase, horseradish peroxidase (HRP), and alkaline phosphatase (ALP) under identical reaction conditions. Antibody developed in rabbits against cortisol-3-0-(carboxymethyl)-oxime-bovine serum albumin was used to coat polystyrene tubes that were precoated with anti-rabbit gamma globulin (ARGG). Cortisol standards were prepared in steroid-free human serum in buffer (1:4) contaning 8-anilino-1-naphthalene sulfonic acid (8-ANS). Assay buffer also consisted 8-ANS. The assay involved adding standard cortisol or serum sample to antibody-coated tubes, followed by addition of enzyme label and buffer, and incubation for 2 h at 37 degrees C. The whole procedure took 3 h for completion. All three labels proved to be sensitive, with a slope around -2.0. Although penicillinase as an enzyme label was highly sensitive and stable compared with others, the assays were not always accurate and precise, especially at low concentrations of cortisol. This was mainly due to the color reagent used for measuring penicillinase activity. Serum samples that underwent 2-3 freeze-thaw cycles gave high values with HRP label compared with ALP. Therefore, utilizing ALP as an enzyme label, an ELISA was developed and its performance was comparable with some of the commercial kits already in the market.

Prevention of latently expressed CYP2C11, CYP3A2, and growth hormone defects in neonatally monosodium glutamate-treated male rats by the N-methyl-D-aspartate receptor antagonist dizocilpine maleate.

Neonatal administration of monosodium glutamate (MSG) can produce latently expressed defects in drug metabolism and growth hormone secretion as well as stunted growth and obesity. Instead of secreting growth hormone in the masculine episodic profile, plasma hormone levels are generally undetectable in affected adult male rats. Moreover, male-specific isoforms of cytochrome P450 (P450; e.g., CYP2C11 and CYP3A2), whose combined levels comprise the bulk of the total hepatic P450 in adult male rats, are similarly undetectable in these animals. Since "signaling elements" in the masculine episodic growth hormone profile are solely responsible for the elevated characteristic male-like expression levels of CYP2C11 and CYP3A2, suppression of the isoforms in the MSG-treated rats appeared to be caused by the simple absence of the hormone from the circulation. However, the reported failures of restored physiologic masculine growth hormone profiles to correct the P450 defects suggested the occurrence of direct MSG-induced liver damage independent of the well known hypothalamic lesions produced by the amino acid. Concurrent administration of dizocilpine maleate (MK-801), a selective and highly potent noncompetitive N-methyl-D-aspartate receptor antagonist of glutamate, completely prevented the adverse effects of neonatal MSG treatment on P450 expression, growth hormone secretion, and growth parameters, indicating that the amino acid-induced defects are solely a result of neuronal (i.e., hypothalamic) damage produced at the time of MSG exposure. The irreversibility of the P450 damage is described as resulting from secondary defects initially induced by the neuronal lesions.

Middle-age alterations in the sexually dimorphic plasma growth hormone profiles: involvement of growth hormone-releasing factor and effects on cytochrome p450 expression.

Rat liver, as well as other species, contains numerous sex-dependent isoforms of cytochrome P450 (P450) that are regulated by the sexually dimorphic profiles of circulating growth hormone. During puberty, young adulthood, and senescence, changes in the hormonal profiles appear to be responsible for alterations in age-associated expression levels of selective P450 isoforms. In contrast, little is known about the growth hormone secretory profiles and their P450-dependent expression levels during middle age. In the present study, we observed subtle changes in the hormonal concentrations, and frequencies of peaks and interpulse periods in the sexually dimorphic growth hormone profiles of 1-year-old male and female rats correlated to suppression of male-specific isoforms CYP2C11 and CYP2C13 and female-predominant CYP2C7. To identify possible causes for the age-associated changes in the circulating growth hormone profiles, the responsiveness of the hypothalamic-pituitary axis to growth hormone secretagogues clonidine and growth hormone-releasing factor (GRF) were examined in middle-aged male and female rats. In spite of the same sexually dimorphic response in young adult and middle-aged rats to both secretogogues (males > females), the pituitary somatotrophs in the older animals exhibited a dramatic decrease in sensitivity to clonidine, characterized by subnormal growth hormone release levels and an inordinate delay in pituitary response to clonidine stimulation. Results from similar studies conducted on middle-aged arcuate nucleus-lesioned rats suggest that a decline in GRF secretion is a possible contributor to the age-associated alterations in plasma growth hormone profiles during middle age. These changes in GRF-induced, sexually dimorphic secretory growth hormone profiles and the accompanying decline in P450 expression levels may anticipate similar, but more profound, changes to occur during senescence.