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PubMed1991

Secretion of the cell surface lipoprotein pullulanase in Escherichia coli. Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway.

The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion. When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles. In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis. Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes. A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled. We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.

Base Sequence↗
PubMed2001

Axonal secretion of Reelin by Cajal-Retzius cells: evidence from comparison of normal and Reln(Orl) mutant mice.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.

Acetylcholine↗
PubMed1988

The molecular basis of growth hormone deficiency.

A well-known law states that 'if a thing can go wrong it will go wrong'. This clearly applies to the hypothalamic-pituitary-somatic axis as to many other physiological and biochemical systems. Defects of this axis, giving rise to stunted growth, can occur at several different points, as has been discussed in detail in this review. Defects at the level of the brain can lead to inadequate production or secretion of the factors that control growth hormone secretion. Defects at the level of the pituitary can lead to failure to produce or secrete adequate quantities of growth hormone, or to production of inactive hormone. Defects at the level of target organs can lead to inability to respond to growth hormone or somatomedins. The axis involved in the production and effects of growth hormone is a complex one, and defects have been identified at most of the points that 'could go wrong', although in many cases the molecular details are far from fully understood. Increased understanding of the biochemistry and physiology of the hormonal control of growth, and of the impairments to which it is subject, should provide an improved basis for treatment of growth defects. Nevertheless, there remain many points at which our knowledge is very incomplete. The field is a rapidly moving one and further developments in both basic understanding and clinical treatment are to be expected during the next few years.

Animals↗
PubMed1990

ATP-dependent efflux of GSSG and GS-conjugate from isolated rat hepatocytes.

The driving force for efflux of dinitrophenyl-glutathione (GS-DNP) and oxidized glutathione (GSSG) from freshly isolated rat hepatocytes was studied. Incubation of hepatocytes in Krebs with increasing K+ concentrations (equivalently replaced for Na+) or in Krebs with 3 mM ouabain led to a partial or complete dissipation of the plasma membrane potential, as measured by the equilibrium distribution of 36Cl-. This had no effect on the initial efflux rate of GSSG and GS-DNP. On the other hand, partial depletion of the cellular ATP content via different independent mechanisms significantly reduced the initial efflux rate of these compounds. Titration of the cellular ATP content by incubation of the cells with different concentrations of atractyloside revealed a linear relation between the cellular ATP content and the initial efflux rate of GS-DNP. The efflux of GS-DNP was also studied in hepatocytes from mutant rats with hepatobiliary transport defect (TR- rats). These rats have a hereditary canalicular secretion defect for a number of organic anions including GS-DNP. As we have shown previously, the efflux of GS-DNP from TR- rat hepatocytes is significantly slower than from normal hepatocytes (J. Clin. Invest. 84: 476-483, 1989). Depletion of the cellular ATP content in these cells had no significant effect on the residual efflux of GS-DNP. From these studies, we conclude that an ATP-dependent transport system for oxidized glutathione and glutathione conjugates is involved in the biliary transport of these compounds. The possible relation of this transport system with that described in other cell types and tissues, like erythrocytes and heart sarcolemma, is discussed.

Adenosine Triphosphate↗
PubMed2000

A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides.

Nonphysiological truncations of apolipoprotein (apo) B-100 cause familial hypobetalipoproteinemia (FHBL) in humans and mice. An elucidation of the mechanisms underlying the FHBL phenotypes may provide valuable information on the metabolism of apo B-containing lipoproteins and the structure-function relationship of apo B. To generate a faithful mouse model of human FHBL, a subtle mutation was introduced into the mouse apo B gene by targeting embryonic stem cells using homologous recombination followed by removal of the selection marker gene by Cre-loxP-mediated site-specific recombination. The engineered mice bear a premature stop codon at residue 1767 and a 42-base pair loxP inserted into intron 24 of the apo B gene, thus closely resembling the apo B-38.9-producing mutation in humans. Apo B-38.9 was the sole apo B protein in homozygote (apob(38.9/38.9)) plasma. In heterozygotes (apob(+/)(38. 9)), apo B-100 and apo B-48 were reduced by 75 and 40%, respectively, and apo B-38.9 represented 20% of total circulating apo B. Hepatic apo B-38.9 mRNA levels were reduced by 40%. In cultured apob(+/)(38. 9) hepatocytes, apo B-100 was produced in trace quantities, and the synthesis rate of apo B-38.9 relative to apo B-48 was reduced by 40%. However, almost equimolar amounts of apo B-38.9 and apo B-48 were secreted into the media. Pulse-chase studies revealed that apo B-38. 9 was secreted at a faster rate and more efficiently than apoB-48. Nevertheless, both apob(+/)(38.9) and apob(38.9/38.9) mice had reduced hepatic triglyceride secretion rates and fatty livers. Thus, low mRNA levels or defective secretion of apo B-38.9 may not be responsible for the FHBL phenotypes caused by the apo B-38.9 mutation. Rather, a reduced capacity of apo B-38.9 for triglyceride transport may account for the fatty livers in these mice.

Animals↗
PubMed1985

Abnormalities of islet B-cell function, insulin action, and fat distribution in women with histories of gestational diabetes: relationship to obesity.

Although obese women with histories of gestational diabetes mellitus (former GDM) are highly predisposed to develop noninsulin-dependent diabetes mellitus (NIDDM), lean former GDM women are less predisposed. To explore reasons for this difference, we performed measures of islet B-cell function and insulin action in eight lean former GDM women [ideal body weight (IBW), 107 +/- 2% (mean +/- SEM)], 11 obese former GDM (IBW, 161 +/- 11%), and 19 normal women subjects who were individually pair-matched to former GDM for % IBW and age. The first phase (0-10 min) insulin secretory response to iv glucose was significantly lower in both lean and obese former GDM compared to that in normal women (3,480 +/- 548% vs. 8,234 +/- 1,337% basal . min and 3,444 +/- 682 vs. 10,251 +/- 2,465). The second phase (10-60 min) insulin response to glucose was also significantly lower in lean former GDM women and tended to be lower in obese former GDM women compared to that in their respective controls. Insulin action was assessed by the insulin sensitivity index (SI) using Bergman's minimal modeling technique. SI values in lean former GDM women were similar to those in their controls (4.42 +/- 1.3 X 10(-4) ml min-1 microU-1 vs. 5.19 +/- 1.2 X 10(-4). In contrast, SI values in obese former GDM women were significantly lower than those in their controls (0.77 +/- 0.28 X 10(-4) vs. 2.04 +/- 0.43 X 10(-4). To assess whether differences in fat distribution and fat cell size were associated with these differences in insulin sensitivity, the waist to thigh circumference ratio, the waist to hip ratio, and abdominal fat cell diameter were measured. All three were significantly greater in the obese former GDM women than in controls. Thus, an abnormal central distribution of adiposity appears to be associated with the insulin action defect in obese former GDM women. We conclude that both lean and obese former GDM women have insulin secretion defects. Although a modest insulin action defect in lean former GDM women may have been missed by this technique, only in the obese former GDM women, who have a higher risk for future NIDDM, was an insulin action defect demonstrable. Thus, impairments of both insulin secretion and insulin action may be necessary to cause a marked predisposition toward NIDDM.

Adipose Tissue↗
PubMed2004

Influence of lipoteichoic acid D-alanylation on protein secretion in Lactococcus lactis as revealed by random mutagenesis.

Lactococcus lactis, a food-grade nonpathogenic lactic acid bacterium, is a good candidate for the production of heterologous proteins of therapeutic interest. We examined host factors that affect secretion of heterologous proteins in L. lactis. Random insertional mutagenesis was performed with L. lactis strain MG1363 carrying a staphylococcal nuclease (Nuc) reporter cassette in its chromosome. This cassette encodes a fusion protein between the signal peptide of the Usp45 lactococcal protein and the mature moiety of a truncated form of Nuc (NucT). The Nuc secretion efficiency (secreted NucT versus total NucT) from this construct is low in L. lactis (approximately 40%). Twenty mutants affected in NucT production and/or in secretion capacity were selected and identified. In these mutants, several independent insertions mapped in the dltA gene (involved in D-alanine transfer in lipoteichoic acids) and resulted in a NucT secretion defect. Characterization of the dltA mutant phenotype with respect to NucT secretion revealed that it is involved in a late secretion stage by causing mature NucT entrapment at the cell surface.

Alanine↗
PubMed1993

Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa.

The process of extracellular secretion in Pseudomonas aeruginosa requires specialized machinery which is widely distributed among bacteria that actively secrete proteins to the extracellular medium. One of the components of this machinery is the product of the xcpR gene, which is homologous to pilB, a gene encoding a protein essential for the biogenesis of type IV pili. Both XcpR and PilB are characterized by the presence of a conserved ATP-binding motif (Walker sequence). The codons of highly conserved glycine residues within the Walker sequences of xcpR and pilB were altered to encode a serine, and the effects of these substitutions were examined. Bacteria expressing mutant XcpR or PilB were unable to secrete exotoxin A or assemble pili, respectively. In addition, high-level expression of mutant XcpR in wild-type P. aeruginosa led to a pleiotropic extracellular secretion defect, resulting in the periplasmic accumulation of enzymes that are normally secreted from the cell. These studies show that the putative ATP-binding sites of XcpR and PilB are essential for their functions in protein secretion and assembly of pili, respectively. Moreover, the observed dominant negative phenotype of mutant XcpR suggests that this protein functions as a multimer or, alternatively, interacts with another essential component of the extracellular protein secretion machinery.

Adenosine Triphosphate↗
PubMed2004

Openers of ATP-dependent K+-channels protect against a signal-transduction-linked and not freely reversible defect of insulin secretion in a rat islet transplantation model of Type 2 diabetes.

AIMS/HYPOTHESIS: We tested whether chronic overstimulation by levels of hyperglycaemia commonly found in Type 2 diabetes can irreversibly desensitise beta cells and, if so, whether desensitisation relates to the reduction of insulin content and/or the number of beta cells. METHODS: We transplanted islets from Wistar-Furth rats under the kidney capsule to neonatally streptozotocinised recipients. Recipients received daily vehicle, diazoxide (100 mg/kg) or the selective activator of beta cell type K(+)-ATP channels 6-chloro -3-(1-methylcyclopropyl) amino-4 H-thienol [3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NN414) (3 mg/kg) intragastrically for at least 9 weeks. Endpoint measurements were made exactly 7 days after cessation of treatment. RESULTS: Blood glucose did not differ between groups (mean of total: 13.2+/-1.4 mmol/l). C-peptide levels were significantly depressed in drug- versus vehicle-treated rats 3 to 4 hours after the last gastric tubing event, but not at endpoint. Insulin responses to 27 mmol/l glucose from perifused grafts were not significant after vehicle (median increment 18 x 10(-3) microU.islet(-1).min(-1)) but were significant per se and versus vehicle in the diazoxide and NN414 groups (median 107 and 83 x 10(-3) respectively). Rising second-phase secretion was seen only in the drug-treated groups. Stimulation by 25 mmol/l KCl, together with 0.5 mmol/l 3-isobutyl-1-methylxanthine and 3.3 mmol/l glucose, was enhanced in the drug-treated groups (p<0.05 versus vehicle). Graft insulin content did not differ between groups, nor did percentage of beta cells (between 67 and 68% of endocrine cells). CONCLUSIONS/INTERPRETATION: Chronic overstimulation by moderate hyperglycaemia damages signalling events including those required for glucose-induced insulin secretion. This signal transduction defect occurs in the absence of any effect on islet macro-morphometry or insulin stores.

Animals↗
PubMed1988

Apolipoprotein C-II deficiency: identification of a structural variant ApoC-II Padova.

Apolipoprotein(apo) C-II DNA, RNA and protein from a patient with a familial deficiency of apoC-II were evaluated and compared to normal individuals. No major defect of the apoC-II gene could be detected by Southern blot hybridization. Northern and slot blot analyses of total liver RNA documented normal levels of a normal sized apoC-II mRNA. Immunohistochemical studies of the liver of the apoC-II deficient patient revealed a normal to slightly elevated intracellular content of the C-II apolipoprotein. Plasma apoC-II was 3 to 5% of normal apoC-II levels and exhibited abnormal electrophoretic mobility on two dimensional gel electrophoresis and immunoblotting. We postulate that at the molecular level, the deficiency of apoC-II in the plasma of this patient results from a structural defect in the coding portion of the apoC-II gene leading to either defective secretion of cellular apoC-II or increased catabolism of a structurally defective apoC-II in plasma.

Apolipoprotein C-II↗
PubMed2000

Mutations in the S1 subunit of pertussis toxin that affect secretion.

Pertussis toxin is a member of the AB(5) family of toxins and is composed of five subunits (S1 to S5) present in a 1:1:1:2:1 ratio. Secretion is a complex process. Each subunit has a secretion signal that mediates transport to the periplasm, where processing and assembly occur. Secretion of the assembled 105-kDa toxin past the outer membrane is mediated by the nine proteins encoded in the ptl operon. Previous studies have shown that S1, the catalytically active A subunit of pertussis toxin, is necessary for efficient secretion, suggesting that a domain on S1 may be required for interaction with the secretion apparatus. Previously, recombinant S1 from four different mutants (serine 54 to glycine, serine 55 to glycine, serine 56 to glycine, and arginine 57 to lysine) was shown to retain catalytic activity. We introduced these mutations into Bordetella pertussis and monitored pertussis toxin production and secretion. No pertussis toxin was detected in the serine 54-to-glycine mutant. The other S1 mutants produced periplasmic pertussis toxin, but little pertussis toxin secretion was observed. The arginine 57-to-lysine mutant had the most dramatic secretion defect. It produced wild-type levels of periplasmic pertussis toxin but secreted only 8% as much toxin as the wild-type strain. This phenotype was similar to that observed for strains with mutations in the ptl genes, suggesting that this region may have a role in pertussis toxin secretion.

Animals↗
PubMed1988

Sympathetic denervation fails to produce beta adrenergic supersensitivity in adult rat parotid gland.

Parotid acinar cells, prepared from pharmacologically sympathectomized adult rats (reserpine, 0.1 mg/kg/day for 1 week), display decreased responsiveness to beta adrenergic stimulation in vitro compared to cells from control and surgically sympathectomized rats. Both methods of denervation increase amylase content (amylase activity per microgram of DNA). Percent release of amylase activity and percent release of CCl3COOH-precipitable [14C]leucine were used as indicators of protein secretion. Exposure of cells from pharmacologically sympathectomized rats to the beta adrenergic agonist, isoproterenol, resulted in a marked reduction in receptor-coupled secretion (67% and 75% relative to controls, respectively). 8-Bromo-cyclic AMP, like isoproterenol, was unable to surmount this reserpine-induced inhibition of stimulated secretion, suggesting that an alteration in receptor-adenylate cyclase coupling is not responsible for the observed secretion defect. Cells prepared from surgically sympathectomized rats displayed modest decreases in stimulated secretion when the same secretory markers were monitored (30% and 25% relative to controls, respectively). The number of beta adrenoreceptors [( 3H]dihydroalprenolol binding sites) increased (35%), with no change in binding affinity, in membrane preparations from reserpine-treated rats. Thus, the observed inhibition of beta adrenergic agonist-induced secretion is not likely the result of alterations in beta adrenergic receptor characteristics. Short-term (1 week) surgical denervation had no effect on the number of beta adrenergic receptor sites; however, an increase in ligand binding affinity was noted. The decrease in the apparent Kd (30%) was not the result of a shift in receptor subtype as determined by competition studies with specific beta-1 (atenolol) and beta-2 (ICI 118,551) receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

[Biochemical characteristics of new thermosensitive secretory mutants of yeasts].

New thermosensitive mutants of the yeast Saccharomyces cerevisiae which block the secretion of periplasmic enzymes at restriction temperature have been obtained. These mutants accumulate active low molecular weight and mature invertase species in the cell; the buoyant density of the cells in a Percoll gradient is higher than that in the wild strain cells. The mutant cells transferred to permissive temperature (25 degrees C) in the absence of protein synthesis can secrete some amount of accumulated invertase. It was found that the secretory defects of conditional mutants do not affect the activity of cytoplasmic enzymes (e.g., alcohol dehydrogenase) or the level of total protein synthesis and glycosylation and do not induce non-specific disturbances in energy metabolism and plasma membrane functions at restriction temperature. Some strains of new secretory mutants revealed uncoupled defective secretion of periplasmic enzymes and intrinsic membrane proteins (proline permease). The possibility of branching of the secretory pathway for periplasmic enzymes and cytoplasmic membrane proteins is discussed.

Acid Phosphatase↗
PubMed2006

Regulation of insulin secretion by uncoupling protein.

UCPs (uncoupling proteins) can regulate cellular ATP production by uncoupling oxidative phosphorylation. UCP2 is expressed in islet beta-cells and its induction reduces glucose-stimulated insulin secretion. Under physiological conditions, superoxide, formed as a by-product of respiration, activates UCP2. This leads to reduced ATP production, which impairs closure of the ATP-dependent K+ channels to prevent insulin secretion. It is suggested that the physiological role of UCP2 is to prevent excessive superoxide generation through a feedback loop. UCP2 induction may also alter fatty acid metabolism by altering NAD/NADH or by facilitating cycling of fatty acid anions. Recently, UCP2 has been proposed to keep insulin secretion low during starvation, a function under the control of the transcription co-repressor, surtuin-1, which has been shown to bind to the UCP2 promoter. Pathological UCP2 expression or activation may suppress glucose-stimulated insulin secretion to the extent that diabetes onset is hastened. In ob/ob mice, induction of UCP2 at age 5 weeks precedes development of insulin secretion defects and hyperglycaemia. Activating protein kinase A-dependent pathways can normalize insulin secretion in UCP2-overexpressing islets. Conversely, lowering UCP2 expression may promote increased insulin secretion. UCP2 knockout mice were protected from the diabetogenic effects of a high-fat diet and their islets exhibited increased sensitivity to glucose and elevated ATP/ADP. These results support a role for UCP2 as a gene contributing to the pathogenesis of Type 2 diabetes.

Diabetes Mellitus↗
PubMed1996

XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer.

XpsD is an outer membrane lipoprotein, required for the secretion of extracellular enzymes by Xanthomonas campestris pv. campestris. Our previous studies indicated that when the xpsD gene was interrupted by transposon Tn5, extracellular enzymes were accumulated in the periplasm (Hu, N.-T., Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C. Huang, H.-Y. and Wu, C.-Y. (1992) J. Bacteriol. 174, 2679-2687). In this study, we constructed a series of substitutions and deletion mutant xpsD genes to investigate the roles of NH2- and COOH-terminal halves of XpsD in protein secretory function. Among these secretion defective xpsD mutations, one group (encoded by pCD105, pYLA, pKdA6, and pKD2) caused secretion interference when co-expressed with wild type xpsD, but the other (encoded by pMH7, pKdPs, and pKDT) did not. Cross-linking studies and gel filtration chromatography analysis indicated that the wild type XpsD protein forms a multimer in its native state. Similar gel filtration analysis of xpsD mutants revealed positive correlations between multimer formation and secretion interfering properties exerted by the mutant XpsD proteins in the parental strain XC1701. Those mutant XpsD proteins (encoded by pCD105, pYL4, pKdA6, and pKD2) that caused secretion interference formed multimers that are similar to the wild type XpsD multimers and those (encoded by pMH7, pKdPs, and pKDT) that did not formed smaller ones. Furthermore, gel filtration and anion exchange chromatography analyses indicated that the wild type XpsD protein co-fractionated with XpsD (delta 29-428) or XpsD (delta 448-650) protein but not with XpsD (delta 74-303) or XpsD (delta 553-759) protein. We propose that the mutant XpsD (delta 29-428) protein caused secretion interference primarily by forming mixed nonfunctional multimers with the wild type XpsD protein in XC1701 (pCD105), whereas the mutant XpsD (delta 74-303) did so by competing for unknown factor(s) in XC1701(pYL4).

Bacterial Outer Membrane Proteins↗
PubMed1992

An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca.

The last gene (pulO) of the pulC-O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC-O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post-translationally. The products of two pulG derivatives with internal inframe deletions were also processed by PulO, but the three PulG-PhoA hybrids, two PulJ-PhoA hybrids and the single PulH-PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction experiments showed that both precursor and mature forms of PulG appear to be associated with low-density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.

Amino Acid Sequence↗
PubMed1999

Pathophysiological and genetic characterization of the major diabetes locus in GK rats.

Genetic studies of the type 2 diabetes-like GK rat have revealed several susceptibility loci for the compound diabetes phenotype. Congenic strains were established for Niddm1, the major quantitative trait locus (QTL) for postprandial glucose levels, by transfer of GK alleles onto the genome of the normoglycemic F344 rat. Despite the polygenic nature of diabetes in GK, the locus-specific diabetes phenotype was retained in the congenic strain Niddmla, containing a GK-derived genomic fragment of 52 cM from the Niddm1 locus. Furthermore, Niddm1 was divided into two non-overlapping loci, physically separated in the two congenic strains Niddmlb and Niddm1i with distinct metabolic phenotypes. Both strains displayed postprandial hyperglycemia and reduced insulin action in isolated adipose cells. Furthermore, Niddm1i already exhibits a pronounced in vivo insulin secretion defect at 65 days, while Niddm1b develops a relative insulin secretory defect at 95 days. This suggests that Niddm1i impairs mechanisms common to insulin secretion in pancreatic B-cells and insulin action in adipocytes. Niddm1b rats show signs of increasing insulin resistance with age associated with obesity, hyperinsulinemia, and dyslipidemia. Moreover, the data indicated nonallelic interaction (epistasis) between Niddm1b and Niddm1i on the postprandial glucose levels. These data emphasize the pathophysiological complexity of diabetes, even within an apparently single QTL, and demonstrate the potential of the GK model in transforming the multifactorial diabetes phenotype into single traits, suitable for positional cloning.

Adipose Tissue↗
PubMed2006

Dexamethasone suppresses phospholipase C activation and insulin secretion from isolated rat islets.

Dexamethasone inhibits insulin secretion from isolated islets. In the present experiments, possible underlying biochemical mechanisms responsible for defective secretion were explored. Dexamethasone (1 micromol/L) had no immediate deleterious effect on 15 mmol/L glucose-induced insulin release from perifused rat islets. However, a 3-hour preincubation period with 1 micromol/L dexamethasone resulted in parallel reductions in both the first (64%) and second phases (74%) of 15 mmol/L glucose-induced insulin secretion monitored during a dynamic perifusion. When measured after the perifusion, there were no differences in insulin content or in the capacity of control or dexamethasone-treated islets to use glucose. Dexamethasone (1 micromol/L) preexposure also reduced phorbol ester- and potassium-induced secretion. In additional experiments, islets were labeled for 3 hours with 3H-inositol in the presence or absence of 1 micromol/L dexamethasone. The steroid did not affect total 3H-inositol incorporation during the labeling period. However, the capacity of 15 mmol/L glucose, 30 mmol/L KCl, and 100 micromol/L carbachol to activate phospholipase C (PLC), monitored by the accumulation of labeled inositol phosphates, was significantly reduced in dexamethasone-pretreated islets. Inclusion of the nuclear glucocorticoid receptor antagonist RU486 (mifepristone, 10 micromol/L) abolished the adverse effects of dexamethasone on both glucose-induced inositol phosphate accumulation and insulin secretion. Quantitative Western blot analyses revealed that the islet contents of PLCdelta1, PLCbeta1, beta2, beta3, and protein kinase C alpha were unaffected by dexamethasone pretreatment. These findings demonstrate that dexamethasone pretreatment impairs insulin secretion via a genomic action and that impaired activation of the PLC/protein kinase C signaling system is involved in the evolution of its inhibitory effect on secretion.

Animals↗