Increase of PTEN gene expression in insulin resistance.

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a regulator of insulin sensitivity in the insulin target tissue. However, whether PTEN gene expression is changed in insulin resistance remains unclear. We observed that both the mRNA and protein level of PTEN in soleus muscle isolated from the obese Zucker rats (Fa/Fa) were increased compared to the age-matched lean group. Similarly, both the mRNA and protein level of PTEN in soleus muscle of the fructose-fed lean Zucker rats (Fa/Fa) showing the higher glucose-insulin index were higher than that of the regular chow fed group. These results suggest that increase of PTEN gene expression seems to be related to the development of insulin resistance.

Signals in the activation of opioid mu-receptors by loperamide to enhance glucose uptake into cultured C2C12 cells.

In an attempt to understand the signal pathways of opioid mu-receptors for glucose metabolism, we used loperamide to investigate the glucose uptake into the myoblast C2C12 cells. Loperamide enhanced the uptake of radioactive deoxyglucose into C2C12 cells in a concentration-dependent manner that was abolished in cells pre-incubated with naloxone or naloxonazine at concentrations sufficient to block opioid mu-receptors. Pharmacological inhibition of phospholipase C (PLC) by U73122 resulted in a concentration-dependent decrease in loperamide-stimulated uptake of radioactive deoxyglucose into C2C12 cells. This inhibition of glucose uptake by U73122 was specific since the inactive congener, U73343, failed to modify loperamide-stimulated glucose uptake. Moreover, both chelerythrine and GF 109203X diminished the action of loperamide at concentrations sufficient to inhibit protein kinase C (PKC). The obtained data suggest that an activation of opioid mu-receptors in C2C12 cells by loperamide may increase glucose uptake via the PLC-PKC pathway.

Synthesis, antiplatelet and vasorelaxing effects of monooxygenated flavones and flavonoxypropanolamines.

A series of flavones and flavonoxypropanolamines were synthesized and tested in-vitro for their ability to inhibit aggregation of washed rabbit platelets and human platelet-rich plasma (PRP), and for vasoconstriction of rat thoracic aorta. The various substituted positions of the hydroxyl group in flavone ring B and the various oxypropanolamine side chains substituted at position C-2' of flavone modified the antiplatelet effects. All the compounds tested in human PRP showed significant inhibition of secondary aggregation induced by adrenaline (epinephrine), suggesting that the antiplatelet effect of these compounds is mainly due to an inhibitory effect on thromboxane formation. Compounds 11 and 12 also had potent vasorelaxant effects in rat thoracic aorta. Phenylephrine- and high-K+-induced 45Ca2+ influx in aorta were both inhibited by the selected compound 11. This result indicates that the inhibitory effect of 11 on the contractile response caused by high-K+ medium and noradrenaline (norepinephrine) in rat thoracic aorta is mainly due to inhibition of Ca2+ influx through both voltage-dependent and receptor-operated Ca2+ channels.

Synthesis and anticancer evaluation of certain gamma-aryloxymethyl-alpha-methylene-gamma-phenyl-gamma-butyrolactones.

Certain gamma-aryloxymethyl-alpha-methylene-gamma-phenyl- gamma-butyrolactones were synthesized and evaluated for their anticancer activity. These compounds demonstrated a strong growth inhibitory activity against leukemia cell lines but are relatively inactive against non-small cell lung cancers and CNS cancers. The anticancer potency for aryl portion is in an order of quinoline > 8-hydroxyquinoline > 2-methylquinoline >> naphthalene >> benzene.

Synthesis and antiplatelet evaluation of alpha-methylene-gamma-butyrolactone bearing 2-methylquinoline and 8-hydroxyquinoline moieties.

In a search for inhibitors of platelet aggregation, some alpha-methylene-gamma-butyrolactones bearing 2-methylquinoline and 8-hydroxyquinoline moieties were synthesized and evaluated for antiplatelet activities against thrombin (Thr)-, arachidonic acid (AA)-, collagen (Col)-, and platelet-activating factor (PAF)-induced aggregation in washed rabbit platelets. With the exception of 2-[[2,3,4,5-tetrahydro-4-methylene-5-oxo-2-(4-phenylphenyl)-2 -furanyl]methoxy]-8-hydroxyquinoline (8f), these alpha-methylene-gamma-butyrolactones completely inhibited the platelet aggregation induced by AA and Col. The 2-methylquinoline derivatives were also active against Thr- and PAF-induced aggregation, while their 8-hydroxyquinoline counterparts were relatively inactive.

Roles of leukotrienes in bovine corpus luteum regression: an in vivo microdialysis study.

A microdialysis system (MDS) was surgically implanted into the corpora lutea (CL) of 12 normally cycling Holstein heifers. Heifers were either allowed to undergo spontaneous luteolysis (Spontaneous, n = 6) or received an intramuscular injection of prostaglandin F2 alpha (PGF2 alpha) on Day 12 of the estrous cycle (Induced, n = 6). The MDS was implanted on Day 11 in the induced heifers and on Day 17 in Spontaneous heifers. CL were perfused with Ringer's solution at a flow rate of 3 ml/hr beginning immediately after surgery. Dialysate samples were collected hourly for 3-4 days. Samples were assayed for progesterone (P4), oxytocin (OT), PGF, and leukotrienes B (LTB) and C (LTC). Dialysate OT was undetected in all but one Spontaneous and one induced heifer. Lipoxygenase products of arachidonic acid (AA) metabolism (LTB and LTC) in the dialysate were found to be closely associated with luteal regression. In Spontaneous heifers, the mean interval from the first hormone peak to the onset of P4 decline was similar for PGF, LTB, and LTC, with the first peak occurring at 12.8 +/- 8.1, 22.0 +/- 6.1, and 11.0 +/- 8.9 hr before the onset of P4 decline, respectively. The peak LTC value was greater (P < 0.05) than peak LTB or PGF. The 12-hr sampling interval with the highest LTC peak frequency was highly correlated (r = 1.0; P < 0.01) with the onset of P4 decline, but the highest LTB and PGF peak frequencies were not associated with the onset of P4 decline. Indeed, the mean numbers of PGF and LTB hormone peaks were higher (P < 0.05) after the onset of P4 decline than before. Administration of PGF2 alpha on Day 12 of the estrous cycle stimulated a decline in P4 secretion and an increase in the secretion of PGF, LTB, and LTC from the CL. In induced animals, the peak level of PGF was greater (P < 0.05) than peak LTB. These results suggest that the AA metabolites LTB and, especially, LTC play important roles during normal regression of the bovine CL.

Intracytoplasmic glutathione concentration and the role of beta-mercaptoethanol in preimplantation development of bovine embryos.

In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mg/ml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (P < 0.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 pM/embryo) and reaching the highest (P < 0.03) level at the hatched-blastocyst stage (36.7 pM/embryo). A significantly (P < 0.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 pM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of beta-mercaptoethanol (2-ME) to promote glutathione synthesis, higher (P < 0.05) percentages of embryos developed to the 9- to 16-cell, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 microM than after no supplementation with 2-ME. However, when 16-cell embryos were cultured in BECM supplemented with 6.25 and 12.5 microM of 2-ME, blastocyst formation was not significantly (P > 0.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2 x 2 factorial design, there was a significant (P < 0.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (P < 0.001) affected development after the fourth cleavage (morula compaction and blastocyst formation), but there was no significant (P > 0.11) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation.

Synthesis and antiplatelet effects of omega-aminoalkoxylxanthones.

A series of omega-aminoalkoxylxanthones were synthesized and tested in-vitro for their ability to inhibit aggregation of rabbit washed platelets and human platelet-rich plasma (PRP) induced by various inducers. Nine of these compounds showed more potent antiplatelet effects than natural norathyriol tetraacetate on collagen-induced aggregation. The various omega-aminoalkoxyl side chains of the synthesized compounds modified the antiplatelet effects. All the compounds tested in human PRP showed significant inhibition of secondary aggregation induced by adrenaline, suggesting that the antiplatelet effects of these compounds is mainly due to an inhibitory effect on thromboxane formation. These compounds at high concentration also cause vasorelaxing action in rat thoracic aorta.

gamma-Pyrone compounds. 5. Synthesis and antiplatelet effects of xanthonoxypropanolamines and related compounds.

A series of simple xanthonoxypropanolamines and related compounds were synthesized. 3-[3-(Cyclopropylamino)propoxy]-xanthone showed same potent antiplatelet effects as norathyriol tetraacetate on arachidonate-induced aggregation. 3-[3-(Cyclohexylamino)-2-hydroxypropoxy]xanthone showed more potent antiplatelet effects than norathyriol tetraacetate on collagen-induced aggregation. The various amino groups of the oxypropanolamine or oxypropylamine side chains of the synthesized compounds regulated the antiplatelet effects.

Xanthonolol: a calcium channel and beta-adrenoceptor blocker with vasodilating properties.

1. Xanthonolol (0.1-5.0 mg/kg, i.v.) reduced the blood pressure, heart rate, and L-isoproterenol (0.05 microgram/kg, i.v.)-induced tachycardia in rats. 2. In the isolated guinea-pig right atrium, xanthonolol (10(-6)-3 x 10(-4) M) produced long-lasting negative, inhibited L-isoproterenol-induced positive chronotropic effects, prevented the rate-increasing effects of increased extracellular Ca2+ (3.0-9.0 mM), and inhibited Ca2+ (3.0-9.0 mM)-induced heart rate-increase. 3. In the isolated guinea-pig thoracic aorta, the contractions induced by CaCl2 (0.1-5.0 mM) were inhibited by xanthonolol (10(-6)-10(-4) M). 4. Xanthonolol is suggested to have a calcium channel and beta adrenergic blocking effect with vasodilating properties.

Gamma-pyrone compounds as potential anti-cancer drugs.

The gamma-pyrones, artomunoxanthotrione epoxide, cyclocommunol, cyclomulberrin, and cyclocommunin exhibited potent inhibition of human PLC/PRF/5 and KB cells in-vitro. Dihydroisocycloartomunin showed significant and potent inhibition of human PLC/PRF/5 and KB cells in-vitro, respectively. Cyclomorusin, dihydrocycloartomunin and artomunoxanthone showed significant inhibition of KB cells in-vitro. Based on the above finding and the reported antileukaemic activity of xanthone psorospermin, a series of natural gamma-pyrones was prepared and the inhibition of human PLC/PRF/5 and KB cells in-vitro was measured. Structure-activity analysis indicated the epoxide group substituted at 3-hydroxyl and 2,6-; 3,6-; and 3,5-dihydroxyl xanthone enhanced the anti-tumour activity. The epoxide group substituted at the 6-hydroxyl group of 1,6-dihydroxyxanthone did not show anti-tumour activity.

gamma-Pyrone compounds. IV: Synthesis and antiplatelet effects of mono- and dioxygenated xanthones and xanthonoxypropanolamine.

Xanthodilol, mono- and dioxygenated xanthones, and 1,3-, 2,3-, 3,4-, 3,5-, 1,6-, 2,6- and 3,6-dioxygenated xanthones were synthesized from benzophenone precursors by Friedel-Crafts acylation and subsequent base-catalyzed cyclization to eliminate methanol. 3-Hydroxy-xanthone, xanthodilol, 2,3-dihydroxyxanthone diacetate, and 3,4-dihydroxyxanthone and its diacetate showed potent antiplatelet effects on arachidonate- and collagen-induced aggregation. 3,5-Dihydroxyxanthone and its diacetate, 1,6-dimethoxyxanthone, and 3,6-dihydroxyxanthone and its diacetate showed potent antiplatelet effects on arachidonate-induced aggregation.

Gamma-pyrone compounds. II: Synthesis and antiplatelet effects of tetraoxygenated xanthones.

Norathyriol and its analogues, 1,3,5,6-, 3,4,5,6-, 3,4,6,7- and 2,3,6,7-tetrahydroxyxanthone, were synthesized from benzophenone precursors by Friedel-Crafts acylation and subsequent base-catalyzed cyclization to eliminate methanol. Both 3,4,6,7- and 2,3,6,7-tetrahydroxyxanthone tetraacetate showed potent anti-platelet aggregation effects on arachidonic acid-induced platelet aggregation. 3,4,6,7-Tetrahydroxyxanthone tetraacetate and 1,3,5,6-tetrahydroxyxanthone showed potent and significant anti-platelet aggregation effects on collagen-induced platelet aggregation.

Abnormal growth hormone receptor gene expression in the sex-linked dwarf chicken.

Sex-linked dwarfism is a recessive mutation that causes a reduction in body weight gain and long bone growth of chickens. We examined the effect of the dwarfing gene on body weight, hepatic GH-binding activity, and the structure and expression of the growth hormone receptor (GHR) gene in two different lines of sex-linked dwarf (SLD) broiler chickens. Liver samples from one line of dwarf chicken were obtained from Arbor Acres Farm, Inc. (Glastonbury, CT) and fertile eggs from the second line of SLD were obtained from the University of Georgia. In the GA line, the average body weight of homozygous (dwdw) males at 11 weeks of age was 43% lower than that of normal (DwDw) males, while heterozygous (Dwdw) males were only 9% below normal. In the CT line, hepatic GH-binding activity of 35-week-old chickens was high (20% specific binding) in normal (DwDw) males and undetectable in liver membranes prepared from dwdw males. At 11 weeks of age, hepatic GH-binding activity of Dwdw males (3.9% specific binding) in the GA line was reduced by 44% and that of dwdw males was almost undetectable (0.34% specific binding) when compared to the average of normal GA males (7.1% specific binding). Southern and Northern blot analyses revealed different abnormalities in the GHR gene from the two separate lines of SLD. A restriction fragment length polymorphism in DNA and an aberrantly sized transcript (mRNA) were detected in the CT line of SLD chickens.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of the chicken growth hormone receptor complementary deoxyribonucleic acid: mutation of the gene in sex-linked dwarf chickens.

A novel complementary DNA (cDNA) encoding the chicken GH receptor was isolated from a chicken liver cDNA library, using polymerase chain reaction with primers derived from highly conserved sequences of the mammalian GH receptor. The nucleotide sequence predicts a mature protein of 592 amino acids and a 16 amino acid signal peptide that are partially homologous to the sequence reported for the rabbit (53%), rat (58%), and human (50%) GH receptors. Despite this low level of homology, a number of structural features of the GH receptor are conserved, including 7 cysteine residues in the extracellular domain and 5 in the intracellular region. Three transcripts of approximately 4.7, 4.0, and 1.0 kilobases are present on Northern blots of total RNA prepared from the livers of 35-week-old male chickens. Expression of the GH receptor was also detected in a wide range of tissues. The chicken GH receptor cDNA was then used as a probe in Southern and Northern blot analyses of DNA and RNA prepared from livers of sex-linked dwarf chickens, which have undetectable levels of hepatic GH-binding activity, in addition to other endocrine abnormalities. A restriction fragment length polymorphism was found in DNA, and an aberrantly-sized transcript was found in hepatic RNA of the dwarf chicken. These results indicate that a mutation in the GH receptor gene is responsible for the phenotype of the sex-linked dwarf chicken. This type of dwarfism resembles Laron-type dwarfism in humans, where a defect in the GH receptor gene has recently been identified. These receptor-deficient chickens should serve as a unique model system for studying the role of the GH receptor in growth and development.

Vasorelaxation of rat thoracic aorta caused by norathyriol isolated from Gentianaceae.

The pharmacological effects of norathyriol on isolated rat thoracic aorta were examined. In the high-K+ (60 mM) medium, Ca2+ (0.03 to 3 mM)-induced vasocontraction was inhibited concentration dependently by norathyriol. Given as pretreatment norathyriol (20 to 200 microM) also inhibited the norepinephrine (NE, 3 microM)-induced tonic contraction. However, the phasic contraction was inhibited only by high concentrations of norathyriol (200 and 400 microM). The tonic contraction elicited by NE was also relaxed by the addition of norathyriol. This relaxing effect of norathyriol was not antagonized by methylene blue (50 microM) or indomethacin (20 microM) and was still seen in denuded rat aorta. Although the cAMP level was not changed by norathyriol, the cGMP level was increased by a high concentration of norathyriol (400 microM). [3H]Inositol monophosphate formation caused by NE was not affected by norathyriol at concentration of either 100 or 400 microM. The 45Ca2+ influx caused by either NE or high K+ was inhibited by norathyriol in a concentration-dependent manner. It is concluded that norathyriol relaxed the rat thoracic aorta mainly by suppressing the Ca2+ influx through both voltage-dependent and receptor-operated calcium channels.

Dietary thyrotropin-releasing hormone stimulates growth rate and increases the insulin: glucagon molar ratio of broiler chickens.

The effects of thyroid manipulation on growth, feed efficiency, and plasma hormone levels were determined in rapidly growing chickens. Beginning at 3 weeks of age, eight broiler cockerels were provided with control feed (CF) or feed containing either 1 ppm of triiodothyronine (T3), 1 ppm of thyroxine (T4), 0.3% propylthiouracil (PTU), or 5 ppm of thyrotropin-releasing hormone (TRH) for 3 weeks. Blood samples were taken at 4, 5, and 6 weeks for determination of plasma levels of growth hormone, insulin-like growth factor, T3, T4, insulin, glucagon, glucose, and nonesterified fatty acids. Dietary TRH increased (P less than 0.05) the growth rate of chickens by 14% when compared with the CF group. Plasma growth hormone levels were reduced (P less than 0.05) 65% by dietary T3 and 33% by treatment with either T4 or TRH when compared with the CF group. Plasma insulin-like growth factor levels were 16% lower (P less than 0.05) in PTU-fed birds than the other treatment groups. Plasma T3 levels were elevated (P less than 0.05) 3-fold by dietary T3 and 38% by TRH whereas plasma T3 in the PTU group was 38% below the average of CF birds. Plasma T4 levels were increased (P less than 0.05) by 12-fold in T4-fed birds, decreased 48% in TRH-fed birds, and nondetectable in birds treated with either T3 or PTU. Compared with the other treatments, dietary PTU increased (P less than 0.01) plasma insulin levels 4.3-fold whereas TRH provided a 2.7-fold increase in plasma insulin. Plasma glucagon levels were 26% higher (P less than 0.05) in T3-fed birds than those fed either T4 or PTU. These observations indicate that thyroid activity plays an important role in regulating secretion of GH and the pancreatic hormones. Furthermore, our study demonstrates the potential use of TRH as an orally active growth promoter for poultry.

Growth, metabolic and endocrine responses of broiler cockerels given a daily subcutaneous injection of natural or biosynthetic chicken growth hormone.

The effects of daily injection of natural chicken growth hormone (ncGH) or recombinant-derived chicken growth hormone (rcGH) on growth, heat production rate, plasma hormone levels and body composition were determined in rapidly growing broiler chickens. Beginning at 3 wk of age, eight broiler chickens were given a daily injection of either bicarbonate buffer (control), 100 or 200 micrograms ncGH/kg body wt, or 200 micrograms rcGH/kg body wt for 14 d. Blood samples were taken preinjection and 4 h postinjection on d 7 and 14 of chicken growth hormone (GH) treatment. Compared to preinjection levels, plasma GH levels at 4 h postinjection were significantly (P less than 0.05) elevated by daily injection (per kg body wt) of 100 micrograms ncGH (2.3-fold), 200 micrograms ncGH (5.5-fold) or 200 micrograms rcGH (6.4-fold). Although exogenous chicken GH treatment failed to increase body weight gain, ncGH injections did increase (P less than 0.05) body fat content to 117% that of the control group. Daily injection of chicken GH did not alter plasma levels of immunoreactive insulin-like growth factor-I (IGF-I), thyroid hormones, insulin, glucagon or glucose. Feed efficiency, heat production rate and respiratory quotient were also not affected by chicken GH treatment. Plasma levels of nonesterified fatty acids were elevated (P less than 0.05) by treatment with 200 micrograms ncGH/kg body wt. In contrast to domestic mammals, it is apparent that exogenous chicken GH can not be used to increase lean body mass or improve productive efficiency in chickens. Our results indicate that exogenous chicken GH exerts a strong lipogenic, rather than lipolytic, action in rapidly growing broiler cockerels.

Photoperiodic regulation of plasma melatonin levels in the laying chicken (Gallus domesticus)1.

Plasma melatonin profiles were determined in laying chickens maintained on either a 16L:8D or a 20L:4D light-dark cycle (LDC). The range of plasma melatonin concentrations was low during the light period (40-100 pg/ml) and higher during the dark period (150-390 pg/ml). Compared to the light period, plasma concentrations of melatonin were 4- to 5-fold higher (P less than 0.05) during the dark period. The amplitude of melatonin concentrations was greater when hens were held under a short dark period (4.6-fold, 20L:4D) than when hens were exposed to a 16L:8D LDC (3.8-fold). In the laying chicken, a broad peak in concentration of plasma melatonin was found in the dark period. It was different from that previously reported in the chick. Since the time of oviposition in the hen occurs in a limited period after the onset of darkness in a LDC, a modified physiological function of melatonin for birds of different ages may account for the difference in the nocturnal pattern of plasma melatonin. The elevated level of plasma melatonin in the dark period of a LDC is considered to be essential for regulating the time of oviposition in the laying chicken under a particular LDC.