Akt substrate of 160 kDa dephosphorylation rate is reduced in insulin-stimulated rat skeletal muscle after acute exercise.

Because greater Akt substrate of 160 kDa (AS160) phosphorylation has been reported in insulin-stimulated skeletal muscles without improved Akt activation several hours post-exercise, we hypothesized that prior exercise would result in attenuated AS160 dephosphorylation in insulin-stimulated rat skeletal muscle. Epitrochlearis muscles were isolated from rats that were sedentary (SED) or exercised 3 h earlier (3 h post-exercise; 3hPEX). Paired muscles were incubated with [(3)H]-2-deoxyglucose (2-DG) without insulin or with insulin. Lysates from other insulin-stimulated muscles from SED or 3hPEX rats were evaluated using AS160(Thr642) and AS160(Ser588) dephosphorylation assays. Prior exercise led to greater 2-DG uptake concomitant with greater AS160(Thr642) phosphorylation and a non-significant trend (P=0.087) for greater AS160(Ser588). Prior exercise also reduced AS160(Thr642) and AS160(Ser588) dephosphorylation rates. These results support the idea that attenuated AS160 dephosphorylation may favor greater AS160 phosphorylation post-exercise.

The B2 receptor of bradykinin is not essential for the post-exercise increase in glucose uptake by insulin-stimulated mouse skeletal muscle.

Bradykinin can enhance skeletal muscle glucose uptake (GU), and exercise increases both bradykinin production and muscle insulin sensitivity, but bradykinin's relationship with post-exercise insulin action is uncertain. Our primary aim was to determine if the B2 receptor of bradykinin (B2R) is essential for the post-exercise increase in GU by insulin-stimulated mouse soleus muscles. Wildtype (WT) and B2R knockout (B2RKO) mice were sedentary or performed 60 minutes of treadmill exercise. Isolated soleus muscles were incubated with [³H]-2-deoxyglucose +/-insulin (60 or 100 microU/ml). GU tended to be greater for WT vs. B2RKO soleus with 60 microU/ml insulin (P=0.166) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). Both genotypes had significant exercise-induced reductions (P<0.05) in glycemia and insulinemia, and the decrements for glucose (approximately 14 %) and insulin (approximately 55 %) were similar between genotypes. GU tended to be greater for exercised vs. sedentary soleus with 60 microU/ml insulin (P=0.063) and was significantly greater for muscles with 100 microU/ml insulin (P<0.05). There were no significant interactions between genotype and exercise for blood glucose, plasma insulin or GU. These results indicate that the B2R is not essential for the exercise-induced decrements in blood glucose or plasma insulin or for the post-exercise increase in GU by insulin-stimulated mouse soleus muscle.

Rapid reversal of insulin-stimulated AS160 phosphorylation in rat skeletal muscle after insulin exposure.

Increased phosphorylation of Akt substrate of 160 kDa (AS160) is essential to trigger the full increase in insulin-stimulated glucose transport in skeletal muscle. The primary aim of this study was to characterize the time course for reversal of insulin-stimulated AS160 phosphorylation in rat skeletal muscle after insulin removal. The time courses for reversal of insulin effects both upstream (Akt phosphorylation) and downstream (glucose uptake) of AS160 were also determined. Epitrochlearis muscles were incubated in vitro using three protocols which differed with regard to insulin exposure: no insulin (never exposed to insulin), transient insulin (30 min with 1.8 nmol/l insulin, then incubation without insulin for 10, 20 or 40 min), or sustained insulin (continuously incubated with 1.8 nmol/l insulin). After removal of muscles from insulin, Akt and AS160 phosphorylation reversed rapidly, each with a half-time of <10 min and essentially full reversal by 20 min. Glucose uptake reversed more slowly (half time between 10 and 20 min with essentially full reversal by 40 min). Removal of muscles from insulin resulted in a rapid reversal of the increase in AS160 phosphorylation which preceded the reversal of the increase in glucose uptake, consistent with AS160 phosphorylation being essential for maintenance of insulin-stimulated glucose uptake.

Relationship between protein O-linked glycosylation and insulin-stimulated glucose transport in rat skeletal muscle following calorie restriction or exposure to O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate.

AIMS AND BACKGROUND: Protein O-linked glycosylation is regulated in vivo by the concentration of hexosamine substrates. Calorie restriction (60% of ad libitum intake) for 20 days causes decreased UDP-N-acetylhexosamine levels and increased insulin-mediated glucose transport in rat skeletal muscle. Conversely, prolonged incubation (19 h) of muscle with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenyl-carbamate (PUGNAc; an inhibitor of N-acetyl-beta-D-glucosaminidase) is characterized by increased O-linked glycosylation and insulin resistance. We aimed to determine the calorie restriction effect on O-linked glycosylation and characterize the temporal relationship between PUGNAc-induced O-linked glycosylation and insulin resistance. HYPOTHESIS: A calorie restriction protocol characterized by decreased muscle hexosamine levels will result in a global reduction in O-linked glycosylated proteins in muscle, and PUGNAc-induced insulin resistance will coincide with increased O-linked glycosylation. METHODS: Plantaris muscle and liver from rats (ad libitum or calorie restricted) were analysed for O-linked glycosylation using two antibodies against different O-linked N-acetylglucosamine epitopes. Also, rat epitrochlearis muscles were incubated for 8.5 h +/- 100 mum PUGNAc prior to measurement of [(3)H]-3-O-methylglucose transport and O-linked glycosylation. RESULTS: Calorie restriction did not alter protein O-linked glycosylated levels in muscle or liver. Incubation with PUGNAc for 8.5 h resulted in increased in O-linked glycosylation but unaltered basal or insulin-stimulated glucose transport. CONCLUSIONS: The delay between O-linked glycosylation and insulin resistance in muscle incubated with PUGNAc suggests an indirect, relatively slow mechanism for insulin resistance. The effect of calorie restriction on insulin action in muscle is unlikely to be the direct result of a global change in protein O-linked glycosylation.

Role of kallikrein-kininogen system in insulin-stimulated glucose transport after muscle contractions.

Serum proteins [molecular weight (MW) > 10,000] are essential for increased insulin-stimulated glucose transport after in vitro muscle contractions. We investigated the role of the kallikrein-kininogen system, including bradykinin, which is derived from kallikrein (MW > 10,000)-catalyzed degradation of serum protein kininogen (MW > 10,000), on this contraction effect. In vitro electrical stimulation of rat epitrochlearis muscles was performed in 1) rat serum +/- kallikrein inhibitors; 2) human plasma (normal or kallikrein-deficient); 3) rat serum +/- bradykinin receptor-2 inhibitors; or 4) serum-free buffer +/- bradykinin. 3-O-methylglucose transport (3-MGT) was measured 3.5 h later. Serum +/- kallikrein inhibitors tended (P = 0.08) to diminish postcontraction insulin-stimulated 3-MGT. Contractions in normal plasma enhanced insulin-stimulated 3-MGT vs. controls, but contractions in kallikrein-deficient plasma did not. Supplementing rat serum with bradykinin receptor antagonist HOE-140 during contraction did not alter insulin-stimulated 3-MGT. Muscles stimulated to contract in serum-free buffer plus bradykinin did not have enhanced insulin-stimulated 3-MGT. Bradykinin was insufficient for postcontraction-enhanced insulin sensitivity. However, results with kallikrein inhibitors and kallikrein-deficient plasma suggest kallikrein plays a role in this improved insulin action.

Absence of insulin receptor substrate-1 expression does not alter GLUT1 or GLUT4 abundance or contraction-stimulated glucose uptake by mouse skeletal muscle.

The purpose of this study was to determine the influence of insulin receptor substrate-1 (IRS-1) expression on GLUT1 and GLUT4 glucose transporter protein abundance, contraction-stimulated glucose uptake, and contraction-induced glycogen depletion by skeletal muscle. Mice (6 months old) from three genotypes were studied: wild-type (IRS-1(+/+)), heterozygous (IRS-1(+/-)) for the null allele, and IRS-1 knockouts (IRS-1(-/-)) lacking a functional IRS-1 gene. In situ muscle contraction was induced (electrical stimulation of sciatic nerve) in one hindlimb using contralateral muscles as controls. Soleus and extensor digitorum longus were dissected and 2-deoxyglucose uptake was measured in vitro. 2-Deoxyglucose uptake was higher in basal muscles (no contractions) from IRS-1(-/-) vs. both other genotypes. Contraction-stimulated 2-deoxyglucose uptake and glycogen depletion did not differ among genotypes. Muscle IRS-1 protein was undetectable for IRS-1(-/-) mice, and values were approximately 40 % lower in IRS-1(+/-) than in IRS-1(+/+) mice. No difference was found in IRS-1(+/+) compared to IRS-1(-/-) groups regarding muscle abundance of GLUT1 and GLUT4. Substantial reduction or elimination of IRS-1 did not alter the hallmark effects of contractions on muscle carbohydrate metabolism--activation of glucose uptake and glycogen depletion.

Exercise training eliminates age-related differences in skeletal muscle insulin receptor and IRS-1 abundance in rats.

Insulin resistance is common in old age, and exercise training can improve insulin sensitivity. The purpose of this study was to determine the influence of age (6 vs 26 months) and exercise training (10 weeks of treadmill running) on insulin signaling protein abundance in skeletal muscle from male Fisher 344 rats. Muscle levels of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and Akt1, a serine-threonine kinase, were determined. IRS-1 was reduced with aging, IR and PI3K abundance was greater in old rats, and Akt1 was unchanged. IRS-1 was increased by training in old but not young rats, and IR was increased by training in young but not old rats. PI3K tended to increase and Akt1 did not change with training, regardless of age. Aging does not uniformly affect insulin signaling protein abundance, and exercise differentially alters IR and IRS-1 in young and old rats, thereby eliminating age-related differences in these proteins.

Characteristics of an oviductal glycoprotein and its potential role in fertility control.

At the time of ovulation the lining epithelium of the mammalian oviduct consists of columnar ciliated and secretory cells. These mature cells are dependent on ovarian steroids in carnivores. Oestradiol induces differentiation of these cells and maintains their mature functional state, and progesterone induces dedifferentiation. The secretory cells synthesize and secrete an oestrogen-dependent high molecular weight glycoprotein. The cDNAs encoding oviductal glycoproteins from several species have been sequenced and show high similarity. The human cDNA hybridized with a single message on northern blots of total oviduct RNA obtained from oestradiol-treated cats (about 2.3 kb) and dogs (about 2.1 kb). This glycoprotein is the major nonserum protein present in the oviductal lumen at the time of ovulation, fertilization and early embryonic development. The glycoproteins associate with the zona pellucida of oviductal eggs in all species studied to date. Recent studies suggest that the bovine glycoprotein facilitates sperm capacitation and significantly increases the ability of bovine spermatozoa to fertilize bovine oocytes in vitro, that the hamster glycoprotein increases the sperm penetration rate of the zona pellucida by three times and that the human glycoprotein increases sperm binding to the zona pellucida by three times. All of the evidence for a biological function for this glycoprotein is derived from studies performed in several different species at reproductive stages before fertilization. The biological actions of this glycoprotein suggest a potential role for the glycoprotein in fertility control. Specifically, purified or recombinant glycoprotein may improve success in IVF procedures by enhancing binding of spermatozoa to the zona pellucida and improving fertilization rates. The glycoprotein may also be a potential immunocontraceptive target since antibodies generated against the oviductal glycoprotein may prevent fertilization by preventing binding of spermatozoa to the zona pellucida.

The baboon oviduct: characteristics of an oestradiol-dependent oviduct-specific glycoprotein.

The baboon oviductal epithelium differentiates into a tall columnar epithelium consisting of ciliated and secretory cells during the follicular phase of the menstrual cycle in response to rising oestradiol levels. The apical tips of these secretory cells are filled with membrane-bound secretory granules. During the luteal phase when progesterone levels are elevated, the epithelium regresses and deciliation occurs. Analysis of secretory proteins obtained from explant culture media by SDS-PAGE followed by fluorography or Western blots has revealed that the baboon oviduct synthesizes and secretes a high molecular weight glycoprotein during the follicular phase of the cycle. Immunocytochemistry demonstrated that this oviductal glycoprotein is localized to the secretory granules of epithelial secretory cells, is oviduct specific, and that following secretion the oviductal glycoprotein binds to the zona pellucida and perivitelline space of ovulated oocytes and embryos within the oviduct. Similar proteins have been characterized in other mammalian species. cDNA data show that the complete coding sequence is 2228 bp for a protein of 623 amino acids. A Genbank search showed that baboon oviductal glycoprotein has high homology to other oviductal glycoprotein sequences at both the nucleotide and amino acid levels. Studies conducted to date probing the biological function of oviductal glycoprotein indicate that this protein plays a role in prefertilization reproductive events (sperm capacitation; sperm-zona binding; zona penetration). Additional experiments are needed to reveal a specific function and mechanism for this molecule.

Regional distribution and hormonal control of estrogen-dependent oviduct-specific glycoprotein messenger ribonucleic acid in the baboon (Papio anubis).

Our objective in this study was to complete the sequence of the baboon oviductal glycoprotein, examine the hormonal regulation of the oviductal glycoprotein mRNA, and determine whether there was a regional variation within the oviduct in the level of oviductal glycoprotein mRNA expression. Finally, because of the structural similarity of the amino terminal end of the oviductal glycoprotein to chitinases, we sought to determine whether the oviductal glycoprotein functions as a glycosyl hydrolase. The total transcript length of the baboon oviductal glycoprotein was determined to be 2228 nucleotides in length plus a poly(A) tail. The largest open reading frame was 623 amino acids, which would produce a protein of 69.3 kDa. The first 420 amino acids were highly homologous to the amino acid sequence of other oviductal glycoproteins, but the remainder of the sequence differed considerably from that of all other species except the human. Although the N-terminal region exhibited sequence similarity to chitinases, the oviductal glycoprotein did not exhibit any activity towards typical chitinase substrates. The oviductal glycoprotein mRNA levels were elevated to approximately the same extent in the fimbria, ampulla, and isthmus of the oviduct after estradiol treatment and in the late follicular stage of the menstrual cycle. The oviductal glycoprotein mRNA levels were lower in the early follicular stage and early luteal stage and were not detectable in the late luteal stage or in progesterone-treated baboons. These results indicate that the oviductal glycoprotein mRNA is induced by estradiol and is present at the highest levels at the time of fertilization. Although there is structural homology with chitinases, no such glycosyl hydrolase activity could be detected. However, the common structure of the N-terminal region of the oviductal glycoproteins implies that it has the same, as yet unknown, function in all species.

Complementary deoxyribonucleic acid cloning and molecular characterization of an estrogen-dependent human oviductal glycoprotein.

A 120-kDa oviduct-specific glycoprotein is synthesized and secreted into the oviductal lumen during estrogen dominance in the human. The objective of this investigation was to clone, sequence, and characterize the cDNA to this core protein. Rapid amplification of cDNA ends was used to clone a contiguous 3' CDNA end and 5' cDNA end. The total length of the cDNA was determined to be 2.2 kb by sequence analysis and exhibited a 92% sequence identity with the comparable overlapping baboon cDNA (1.2 kb). A high degree of homology was found to the N-terminal sequence of hamster oviductin and the partial sequence of a homologous baboon and bovine oviduct glycoprotein. Northern blots revealed a single mRNA species of 2.4 kb. Using RNA from various tissues of an estrogen-treated baboon, we found that the mRNA for the oviductal glycoprotein was present only in the oviduct. Hybridization was detected to an mRNA of similar size from oviductal tissue of the baboon, hamster, and mouse and to an mRNA of slightly smaller size in the rabbit, cow, and cat but not to any mRNA species from rat oviductal RNA. Slot-blot analysis showed that the message was present in significantly greater (p < 0.05) concentrations in RNA from oviductal tissue from the late follicular stage than from the early follicular, early or late luteal, or postpartum stages. In conclusion, we have isolated the complete cDNA for a human oviductal glycoprotein. The presence of significantly greater amounts of the mRNA during the late follicular phase of the menstrual cycle is consistent with the proposed estrogen control. The mRNA for the oviductal glycoprotein is present only in the oviduct of an estrogen-treated baboon, and a cross-hybridizing RNA is found in oviductal RNA from various mammals.

El control de los sistemas de atencion medica conceptualizacion y mecanica operativa. / The control of the medical care systems. Conceptualization and operative mechanics

La creciente dificultad a la que se hallan sometidos los sistemas de atencion medica, lleva a meditar sobre la necesidad de implementacion de mecanismos que sirvan a la evaluacion y al consecuente proceso de toma de decision. El contar con una informacion sistematizada basicamente comparable coadyuva en favor de una mas eficiente administracion de recursos. La desigualdad cualicuantitativa de la atencion medica que se brinda sera dificil de corregir si no se cuenta con los mecanismos de regulacion adecuados. Este trabajo pretende dar pautas generales y una metodologia aplicable a las distintas modalidades del trabajo medico vigentes en el pais

El control de los sistemas de atencion medica conceptualizacion y mecanica operativa. / The control of the medical care systems. Conceptualization and operative mechanics

La creciente dificultad a la que se hallan sometidos los sistemas de atencion medica, lleva a meditar sobre la necesidad de implementacion de mecanismos que sirvan a la evaluacion y al consecuente proceso de toma de decision. El contar con una informacion sistematizada basicamente comparable coadyuva en favor de una mas eficiente administracion de recursos. La desigualdad cualicuantitativa de la atencion medica que se brinda sera dificil de corregir si no se cuenta con los mecanismos de regulacion adecuados. Este trabajo pretende dar pautas generales y una metodologia aplicable a las distintas modalidades del trabajo medico vigentes en el pais