Research Article
Abstract
Glucose tolerance is determined by both insulin action and insulin-independent effects, or "glucose effectiveness," which includes glucose-mediated stimulation of glucose uptake (Rd) and suppression of hepatic glucose output (HGO). Despite its importance to tolerance, controversy surrounds accurate assessment of glucose effectiveness. Furthermore, the relative contributions of glucose's actions on Rd and HGO under steady state and dynamic conditions are unclear. We performed hyperglycemic clamps and intravenous glucose tolerance tests in eight normal dogs, and assessed glucose effectiveness by two independent methods. During clamps, glucose was raised to three successive 90-min hyperglycemic plateaus by variable labeled glucose infusion rate; glucose effectiveness (GE) was quantified as the slope of the dose-response relationship between steady state glucose and glucose infusion rate (GE[CLAMP(total)]), Rd (GE[CLAMP(uptake)]) or HGO (GE[CLAMP(HGO)]). During intravenous glucose tolerance tests, tritiated glucose (1.2 microCi/kg) was injected with cold glucose (0.3 g/kg); glucose and tracer dynamics were analyzed using a two-compartment model of glucose kinetics to obtain Rd and HGO components of glucose effectiveness. All experiments were performed during somatostatin inhibition of islet secretion, and basal insulin and glucagon replacement. During clamps, Rd rose from basal (2.54+/-0.20) to 3.95+/-0.54, 6.76+/-1.21, and 9.48+/-1.27 mg/min per kg during stepwise hyperglycemia; conversely, HGO declined to 2.06+/-0.17, 1.17+/-0.19, and 0.52+/-0.33 mg/min per kg. Clamp-based glucose effectiveness was 0.0451+/-0.0061, 0.0337+/-0.0060, and 0.0102+/-0.0009 dl/min per kg for GE[CLAMP(total)], GE[CLAMP(uptake)], and GE[CLAMP(HGO)], respectively. Glucose's action on Rd dominated overall glucose effectiveness (72.2+/-3.3% of total), a result virtually identical to that obtained during intravenous glucose tolerance tests (71.6+/-6.1% of total). Both methods yielded similar estimates of glucose effectiveness. These results provide strong support that glucose effectiveness can be reliably estimated, and that glucose-stimulated Rd is the dominant component during both steady state and dynamic conditions.
Authors
M Ader, T C Ni, R N Bergman
Abstract
PEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, was recently identified as the candidate gene for X-linked hypophosphatemia. In the present study, we cloned mouse and human Pex/PEX cDNAs encoding part of the 5' untranslated region, the protein coding region, and the entire 3' untranslated region, determined the tissue distribution of Pex/PEX mRNA, and characterized the Pex mutation in the murine Hyp homologue of the human disease. Using the reverse transcriptase/polymerase chain reaction (RT/PCR) and ribonuclease protection assays, we found that Pex/PEX mRNA is expressed predominantly in human fetal and adult mouse calvaria and long bone. With RNA from Hyp mouse bone, an RT/PCR product was generated with 5' but not 3' Pex primer pairs and a protected Pex mRNA fragment was detected with 5' but not 3' Pex riboprobes by ribonuclease protection assay. Analysis of the RT/PCR product derived from Hyp bone RNA revealed an aberrant Pex transcript with retention of intron sequence downstream from nucleotide 1302 of the Pex cDNA. Pex mRNA was not detected on Northern blots of poly (A)+ RNA from Hyp bone, while a low-abundance Pex transcript of approximately 7 kb was apparent in normal bone. Southern analysis of genomic DNA from Hyp mice revealed the absence of hybridizing bands with cDNA probes from the 3' region of the Pex cDNA. We conclude that Pex/PEX is a low-abundance transcript that is expressed predominantly in bone of mice and humans and that a large deletion in the 3' region of the Pex gene is present in the murine Hyp homologue of X-linked hypophosphatemia.
Authors
L Beck, Y Soumounou, J Martel, G Krishnamurthy, C Gauthier, C G Goodyer, H S Tenenhouse
Abstract
Features characteristic to rheumatoid joint destruction, including synovial overgrowth and bone resorption, are experimentally produced by augmenting c-fos gene expression. We tested here if arthritic joint destruction was inhibited upon inactivation of the c-fos/AP-1 signal by administering short double-stranded AP-1 DNA oligonucleotides into mice with collagen-induced arthritis to compete for the binding of AP-1 in vivo at the promoter binding site. Arthritic joint destruction was inhibited in a sequence-specific and dose-dependent manner by oligonucleotides containing the AP-1 sequence. The oligonucleotides inhibited gene expression at the transcriptional level. Nucleotide sequences besides AP-1 also appeared to be important structurally for binding of AP-1 onto DNA and for the stability of oligonucleotides against nucleases. Immunohistochemical chase experiment administering biotinylated oligonucleotides into arthritic mice showed that AP-1 oligonucleotides reached the inflamed joint. Thus, activation of c-fos/AP-1 appears essentially important in arthritic joint destruction.
Authors
S Shiozawa, K Shimizu, K Tanaka, K Hino
Abstract
Heat shock proteins (HSP) are components of the steroid receptor complex and are released into the cell cytosol after hormone binding. We tested whether HSPs released from steroid receptors mediate an increase in calcineurin phosphatase activity by steroid hormones. Aldosterone increased calcineurin activity in microdissected rat cortical collecting ducts (CCD) and connecting tubules, but not in proximal tubules, medullary thick ascending limb, or outer medullary collecting ducts. In contrast, 5 microM dexamethasone increased calcineurin activity in both CCD and proximal tubules. Aldosterone increased CCD calcineurin activity after 30 min and this response was blocked by spironolactone, but not by actinomycin D. An antibody recognizing HSP-56 did not change basal calcineurin activity, but completely blocked the stimulation of calcineurin by aldosterone. Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid receptor complex, also blocked the aldosterone response, whereas HSP-90 or HSP-70 increased calcineurin activity in permeabilized CCD. In summary, (a) aldosterone increases calcineurin activity in CCD through a transcription-independent process; (b) maneuvers inactivating HSP-56 or slowing HSP disassociation from the receptor complex blocks stimulation of calcineurin by steroid hormones; (c) HSP-90 and HSP-70 increase CCD calcineurin activity in the absence of steroid hormone. We conclude that HSPs released from transformed steroid receptors can stimulate calcineurin activity through a transcription-independent pathway.
Authors
J A Tumlin, J P Lea, C E Swanson, C L Smith, S S Edge, J S Someren
Abstract
It is well documented that the activity of Na+,K+-ATPase can be inhibited by the arachidonic acid metabolite, 20-hydroxyeicosa-tetraenoic acid (20 HETE). Evidence is presented here that this effect is mediated by protein kinase C (PKC). PKC inhibitors abolished 20 HETE inhibition of rat Na+,K+-ATPase in renal tubular cells. 20 HETE caused translocation of PKC alpha from cytoplasm to membrane in COS cells. It also inhibited Na+,K+-ATPase activity in COS cells transfected with rat wild-type renal Na+,K+-ATPase alpha1 subunit, but not in cells transfected with Na+,K+-ATPase alpha1, where the PKC phosphorylation site, serine 23, had been mutated to alanine. PKC-induced phosphorylation of rat renal Na+,K+-ATPase, as well as of histone was strongly enhanced by 20 HETE at the physiologic calcium concentration of 1.3 microM, but not at the calcium concentration of 200 microM. The results indicate that phospholipase A2-arachidonic acid-20 HETE pathway can exert important biological effects via activation of PKC and that this effect may occur in the absence of a rise in intracellular calcium.
Authors
S Nowicki, S L Chen, O Aizman, X J Cheng, D Li, C Nowicki, A Nairn, P Greengard, A Aperia
Abstract
Cartilage specimens from osteoarthritis (OA)-affected patients spontaneously released PGE2 at 48 h in ex vivo culture at levels at least 50-fold higher than in normal cartilage and 18-fold higher than in normal cartilage + cytokines + endotoxin. The superinduction of PGE2 production coincides with the upregulation of cyclooxygenase-2 (COX-2) in OA-affected cartilage. Production of both nitric oxide (NO) and PGE2 by OA cartilage explants is regulated at the level of transcription and translation. Dexamethasone inhibited only the spontaneously released PGE2 production, and not NO, in OA-affected cartilage. The NO synthase inhibitor HN(G)-monomethyl-L-arginine monoacetate inhibited OA cartilage NO production by > 90%, but augmented significantly (twofold) the spontaneous production of PGE2 in the same explants. Similarly, addition of exogenous NO donors to OA cartilage significantly inhibited PGE2 production. Cytokine + endotoxin stimulation of OA explants increased PGE2 production above the spontaneous release. Addition of L-NMMA further augmented cytokine-induced PGE2 production by at least fourfold. Inhibition of PGE2 by COX-2 inhibitors (dexamethasone or indomethacin) or addition of exogenous PGE2 did not significantly affect the spontaneous NO production. These data indicate that human OA-affected cartilage in ex vivo conditions shows (a) superinduction of PGE2 due to upregulation of COX-2, and (b) spontaneous release of NO that acts as an autacoid to attenuate the production of the COX-2 products such as PGE2. These studies, together with others, also suggest that PGE2 may be differentially regulated in normal and OA-affected chondrocytes.
Authors
A R Amin, M Attur, R N Patel, G D Thakker, P J Marshall, J Rediske, S A Stuchin, I R Patel, S B Abramson
Abstract
Antibody and T cell-mediated immune responses to oligodendroglial autoantigens transaldolase (TAL) and myelin basic protein (MBP) were examined in patients with multiple sclerosis (MS). Immunohistochemical studies of postmortem brain sections revealed decreased staining by MBP- and TAL-specific antibodies in MS plaques, indicating a concurrent loss of these antigens from demyelination sites. By Western blot high titer antibodies to human recombinant TAL were found in 29/94 sera and 16/23 cerebrospinal fluid samples from MS patients. Antibodies to MBP were undetectable in sera or cerebrospinal fluid of these MS patients. Proliferative responses to human recombinant TAL (stimulation index [SI] = 2.47+/-0.3) were significantly increased in comparison to MBP in 25 patients with MS (SI = 1.37+/-0.1; P < 0.01). After a 7-d stimulation of PBL, utilization of any of 24 different T cell receptor Vbeta gene segments in response to MBP was increased less than twofold in the two control donors and six MS patients investigated. In response to TAL-H, while skewing of individual Vbeta genes was also less than twofold in healthy controls, usage of specific Vbeta gene segments was differentially increased ranging from 2.5 to 65.9-fold in patients with MS. The results suggest that TAL may be a more potent immunogen than MBP in MS.
Authors
E Colombo, K Banki, A H Tatum, J Daucher, P Ferrante, R S Murray, P E Phillips, A Perl
Abstract
Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pathway. Needle biopsies of vastus lateralis muscle were taken from nine subjects at rest and after 60 min of cycle ergometer exercise. In all subjects, exercise increased MAPK phosphorylation, and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2. Furthermore, exercise increased the activities of the upstream regulators of MAPK, MAP kinase kinase, and Raf-1. When two additional subjects were studied using a one-legged exercise protocol, MAPK phosphorylation and p90 ribosomal S6 kinase 2, MAP kinase kinase 1, and Raf-1 activities were increased only in the exercising leg. These studies demonstrate that exercise activates the MAPK cascade in human skeletal muscle and that this stimulation is primarily a local, tissue-specific phenomenon, rather than a systemic response to exercise. These findings suggest that the MAPK pathway may modulate cellular processes that occur in skeletal muscle in response to exercise.
Authors
D Aronson, M A Violan, S D Dufresne, D Zangen, R A Fielding, L J Goodyear
Abstract
The aim of this study was to determine whether elements of the human renin-angiotensin system (RAS) could functionally replace elements of the mouse RAS by complementing the reduced survival and renal abnormalities observed in mice carrying a gene-targeted deletion of the mouse angiotensinogen gene (mAgt). Double transgenic mice containing the human renin (HREN) and human angiotensinogen (HAGT) genes were bred to mice heterozygous for the mAgt deletion and the compound heterozygotes were identified and intercrossed. The resulting progeny (n = 139) were genotyped at each locus and the population was stratified into two groups: the first containing both human transgenes (RA+) and the second containing zero or one, but not both human transgenes (RA-). Despite appropriate Mendelian ratios of RA- mice that were wildtype (+/+), heterozygous (+/-), and homozygous (-/-) for the deletion of mAgt at birth, there was reduced survival of RA- mAgt-/- mice to adulthood (P < 0.001 by chi2). In contrast, we observed appropriate Mendelian ratios of RA+ mAgt+/+, RA+ mAgt+/-, and RA+ mAgt-/- mice at birth and in adults (P > 0.05 by chi2). These results demonstrate that the presence of both human transgenes rescues the postnatal lethality in mAgt-/- mice. The renal histopathology exhibited by RA- mAgt-/- mice, including thickened arterial walls, severe fibrosis, lymphocytic infiltration, and atrophied parenchyma, was also rescued in the RA+ mAgt-/- mice. Direct arterial blood pressure recordings in conscious freely moving mice revealed that BP (in mmHg) varied proportionally to mAgt gene copy number in RA+ mice (approximately 20 mmHg per mAgt gene copy, P < 0.001). BP in RA+ mAgt-/- mice (132+/-3, n = 14) was intermediate between wild-type (RA- mAgt+/+, 105+/-2, n = 9) and RA+ mAgt+/+ (174+/-3, n = 10) mice. These studies establish that the human renin and angiotensinogen genes can functionally replace the mouse angiotensinogen gene, and provides proof in principle that we can examine the regulation of elements of the human RAS and test the significance of human RAS gene variants by a combined transgenic and gene targeting approach.
Authors
R L Davisson, H S Kim, J H Krege, D J Lager, O Smithies, C D Sigmund
Abstract
Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. We report here the clinical, endocrinological, and molecular analyses of two unrelated Japanese kindreds of 46,XX subjects affected with lipoid CAH who manifested spontaneous puberty. Phenotypic female infants with 46,XX karyotypes were diagnosed with lipoid CAH as newborns based on a clinical history of failure to thrive, hyperpigmentation, hyponatremia, hyperkalemia, and low basal values of serum cortisol and urinary 17-hydroxycorticosteroid and 17-ketosteroid. These patients responded to treatment with glucocorticoid and 9alpha-fludrocortisone. Spontaneous thelarche occurred in association with increased serum estradiol levels at the age of 10 and 11 yr, respectively. Pubic hair developed at the age of 12 yr 11 mo in one subject and menarche was at the age of 12 yr in both cases. Both subjects reported periodic menstrual bleeding and subsequently developed polycystic ovaries. To investigate the molecular basis of the steroidogenic lesion in these patients, the StAR gene was characterized by PCR and direct DNA sequence analyses. DNA sequence analysis revealed that one patient is homozygous for the Gln 258 Stop mutation in exon 7 and that the other patient is a compound heterozygote with the Gln 258 Stop mutation and a single A deletion at codon 238 in the other allele causing a frame-shift, which renders the StAR protein nonfunctional. These findings demonstrate that ovarian steroidogenesis can be spared to some extent through puberty when the StAR gene product is inactive. This is in marked contrast to the early onset of severe defects in testicular and adrenocortical steroidogenesis which are characteristics of this disease.
Authors
K Fujieda, T Tajima, J Nakae, S Sageshima, K Tachibana, S Suwa, T Sugawara, J F Strauss 3rd
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