Lytic transglycosylase contributes to the survival of lipooligosaccharide-deficient, colistin-dependent Acinetobacter baumannii.

OBJECTIVES: The phenomenon of colistin dependence in Acinetobacter baumannii has been described in a situation in which colistin is now considered as the last resort for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. In this study, we aimed to reveal a gene associated with colistin dependence in A. baumannii. METHODS: The colistin-dependent A. baumannii H08-391D strain was isolated from a patient, and target gene-inactivation mutants were constructed. We investigated the effects of target gene on colistin dependence with quantitative real-time PCR and endotoxin assay. Also, we observed the change of cell morphology by electron microscopy. RESULTS: The expression of ACICU_02898, encoding a soluble lytic transglycosylase associated with cell-wall degradation and recycling, was increased by eight-to 42-fold in colistin-dependent mutants, and deletion of ACICU_02898 in a colistin-dependent strain led to colistin susceptibility (MIC = 8 mg/L). Endotoxin activity was significantly low in a colistin-dependent derivative ACICU_02898-inactivated mutant and a complemented mutant. In addition, the ACICU_02898-inactivated mutant showed a highly reduced growth rate. The colistin-dependent derivative and ACICU_02898-inactivated mutant showed clearly distinguished absorption profiles in the red/green fluorescence dot blot with regard to their membrane potential. Electron microscopy revealed that the deletion mutant cells were elongated compared to the colistin-susceptible wild-type strain and colistin-dependent strain. CONCLUSIONS: A colistin-dependent A. baumannii strain exhibited a deficiency in its outer membrane integrity and high expression of lytic transglycosylase was required for survival. This study reveals why the colistin-dependent mutant can tolerate high antibiotic concentrations.

Influence of dietary avilamycin on ileal and cecal microbiota in broiler chickens.

The mechanisms by which antibiotic growth promoters (AGP) enhance growth rates, feed efficiencies, and disease resistance in poultry need to be understood for designing safer and alternative strategies to replace AGP. Avilamycin has been widely used as an AGP in poultry, but its impact on the structure and function of the gut microbiome of broiler chickens has not been fully elucidated. In this study, we investigated the bacterial communities of the ileum and cecum in broiler chickens fed with an avilamycin-supplemented diet, by high-throughput sequencing of bacterial 16S rRNA genes. Alpha diversity metrics indicated that the ileal bacterial diversity was higher in avilamycin-fed chickens than in the control group, whereas the opposite was true for the cecum. Multivariate analyses revealed that the ileal microbiota of the avilamycin-fed group were clearly distinguished from those of the control group, whereas the cecal bacterial communities were apparently not influenced by feeding diets containing avilamycin. In the ilea, 2 operational taxonomic units (OTU) that matched Lactobacillus reuteri and Clostridium were enriched (P = 0.016 and P = 0.007, respectively) in the avilamycin-fed group, and an OTU belonging to Lactobacillus crispatus was decreased (P = 0.016). In the cecal microbiota showing much higher diversity with 1,286 non-singleton OTU, 12 OTU were decreased, and 3 were increased in response to avilamycin treatment (P = 0.005-0.047). Functional profiling of bacterial communities based on PICRUSt analysis revealed that 10 functional categories were enriched by avilamycin treatments, and 4 functional categories were decreased. In conclusion, our results demonstrated that the influence of avilamycin supplementation on the diversity, taxonomic composition, and functional profiles of the microbiota was evidently different in the ileum and cecum. These results further our understanding of the impact of AGP on the composition and activity of commensal bacteria in the chicken gastrointestinal tract to develop novel feeding strategies for improving animal health and performance.

Spatial heterogeneity and stability of bacterial community in the gastrointestinal tracts of broiler chickens.

Bacterial communities in the different regions of gastrointestinal tract (GIT) of broiler chickens were analyzed by pyrosequencing approach to understand microbial composition and diversity. The DNA samples extracted from 7 different regions along the GIT were subjected to bacterial-community analysis by pyrosequencing of the V1-V3 region of 16S rRNA gene. Major bacterial phyla in the chicken-gut microbiota included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria, but Firmicutes were mostly dominant (67.3 ± 16.1% of the total sequence reads identified). Among Firmicutes, Lactobacillales, including the genera Lactobacillus and Enterococcus, were the most dominant (51.8 ± 34.5% of the total sequence reads identified) from the crop to ileum. In contrast, in the cecum and large intestine, those genera were rarely detected, and Clostridiales were dominant (55.9 ± 31.4%). Fast UniFrac analysis showed that microbial communities from the crop to jejunum of the same individual chicken were grouped together, and those from ileum, cecum, and large intestine were clustered in a more GIT-specific manner. The numbers of shared operational taxonomic units between the neighboring segments of GIT were low, ranging from 2.9 to 20.3%. However, the abundance of shared operational taxonomic units in each segment was relatively high, ranging from 61.7 to 85.0%, suggesting that substantial proportions of microbial communities were shared between each segment and its neighboring segments, comprising a core microbiota. Our results suggested that the microbial communities of 7 main segments in the chicken GIT were distinctive according to both individuals and the different segments of GIT, but their stability was maintained along the GIT.

Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites.

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans.

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.

Biological production of optically active muconolactones by Rhodococcus rhodochrous.

Optically active (-)-3-methylmuconolactone was biologically produced using a mutant strain of Rhodococcus rhodochrous N75 that is capable of metabolizing 4-methylcatechol via a modified ortho-cleavage pathway. The mutant strain (CJ30) was prepared by mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine and found to be blocked in the degradation of 3-methyl-muconolactone. Cells of the mutant CJ30, which had been previously grown on yeast extract and induced with p-toluate, transformed p-toluate (11.5 mM) to optically active (-)-3-methylmuconolactone with a yield of 53%. The structure of 3-methylmuconolactone was confirmed by NMR spectroscopy and mass spectrometry. Cell-free extracts of R. rhodochrous N75 also transformed a range of 4-alkylcatechols, such as 4-ethylcatechol, 4-iso-propylcatechol, and 4-tert-butylcatechol, to the corresponding 4-alkyl-substituted muconolactones.

Biotransformation of malachite green by the fungus Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.

Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome 1q25.

We have previously identified megakaryocyte stimulating factor (MSF) gene expression by synovial fibroblasts as the origin of lubricin in the synovial cavity. Lubricin is a mucinous glycoprotein responsible for the boundary lubrication of articular cartilage. MSF has a significant homology to vitronectin and is composed of 12 exons. RNA was purified from human synovial fibroblasts and articular chondrocytes grown in vitro from tissue explants obtained from subjects without degenerative joint disease. RT-PCR was used with multiple complimentary primer pairs spanning the central mucin expressing exon 6 of the MSF gene and individual exons on both the N- and C-terminal sides of exon 6. Exons 2, 4 and 5 appear to be variably expressed by synovial fibroblasts and articular chondrocytes. Lubricating mucin, in the form of MSF, is expressed by both chondrocytes and synovial fibroblasts in vitro. Both lubricin and superficial zone protein (SZP), a related proteoglycan, share a similar primary structure but could differ in post-translational modifications with O-linked oligosaccharides which are predominant in lubricin and with limited amounts chondroitin and keratan sulfate found in SZP. Since most of the MSF exons are involved in the expression of lubricating mucin, a strong homology to vitronectin persists. It is therefore appropriate to consider that both SZP and lubricin occupy a new class of biomolecules termed tribonectins. Screening of a human genome bacterial artificial chromsome (BAC) library with a cDNA primer pair complimentary for exon 6 identified two clones. Both clones were complimentary for chromosome 1q25 by in situ hybridization. This same locus was previously implicated in camptodactyl-arthropathy-pericarditis syndrome (CAP) by genetic mapping. It is hypothesized that CAP, a large joint arthropathy, may be associated with ineffective boundary lubrication provided by synovial fluid.

Boundary lubrication by lubricin is mediated by O-linked beta(1-3)Gal-GalNAc oligosaccharides.

Lubrication of mammalian joints is mediated by lubricin, a product of megakaryocyte stimulating factor gene (MSF; GenBank accession #U70136) expression. Lubricin (M(r) approximately 240 kDa) is a mucinous glycoprotein which is 50% (w/w) post-translationally modified with beta(1-3)Gal-GalNAc incompletely capped with NeuAc, and lubricates apposed cartilaginous surfaces in the boundary mode through an unknown mechanism. Both bovine and human lubricin were purified from synovial fluid and digested with recombinant glycosidases. Released oligosaccharides were identified and quantified by fluorophore assisted carbohydrate electrophoresis (FACE). Corresponding digests of human lubricin were also assayed in a friction apparatus oscillating latex rubber against polished glass at a pressure of 0.35 x 10(6) N/m(2) and the coefficient of friction (mu) was measured. Digestion with alpha2,3-neuraminidase decreased lubricating ability by 19.3%. Partial removal of beta(1-3)Gal-GalNAc moieties by endo-alpha-N-acetyl-D-galactosaminidase reduced lubricating ability by 77.2%. Human lubricin digested with combined alpha2,3-neuraminidase and beta1-3,6-galactosidase continued to lubricate at 52.2% of its nominal value. Both bovine and human lubricin released 48.6% and 54.4% of total beta(1-3)Gal-GalNAc sidechains following digestion with endo-alpha-N-acetyl-D-galactosaminidase. Biological boundary lubrication by synovial fluid in vitro is provided primarily by extensive O-linked beta(1-3)Gal-GalNAc.

Lubricin is a product of megakaryocyte stimulating factor gene expression by human synovial fibroblasts.

OBJECTIVE: The boundary lubricating ability of human synovial fluid has been attributed to lubricin, a mucinous glycoprotein. We investigated the primary structure of lubricin and its cellular origin. METHODS: Lubricin was purified from pooled synovial fluid aliquots with normal lubricating activity obtained from patients with osteoarthritis. Lubricating ability of lubricin was assayed in a friction apparatus that oscillates natural latex against a ring of polished glass. Native and lubricin deglycosylated with O-glycosidase DS and NANase III were trypsinized and sequenced by liquid chromatography mass spectrometry. Sequence results were compared to known structures in GenBank. Sequence data from strong matches were used in creating cDNA primers for reverse transcription-polymerase chain reaction (RT-PCR) with RNA from human synovial fibroblasts obtained intraoperatively. RESULTS: Purified lubricin possesses an apparent molecular weight of 280 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation decreased the apparent molecular weight on SDS-PAGE to 120 kDa. Sequences specific for megakaryocyte stimulating factor precursor (MSF) were identified in GenBank. A 100% match was observed for exons 6 though 9 of MSF. Lubricin/MSF reduced the coefficient of friction (m) in the latex:glass bearing from 0.131 to 0.047. MSF is 1404 amino acids in size with multiple functional domains similar to vitronectin. The reported structure of MSF contains a centrally located mucin (exon 6) with 76 repeats of the degenerate motif of KEPAPTT, the presumed site of extensive O-linked glycosylation. RT-PCR with primers complementary for Pro214- Ala307 in exon 6 and RNA from human synovial fibroblasts produced the predicted product size of 280 bp. CONCLUSION: Lubricin is secreted by synovial fibroblasts via expression of the MSF gene. Lubricin is constructed of MSF exons 6 through 9 but the presence of other exons cannot be excluded. Lubricin/MSF is the only lubricating component in the final lubricating fraction of human synovial fluid.

The effect of phospholipase digestion upon the boundary lubricating ability of synovial fluid.

OBJECTIVE: To identify the boundary lubricant in synovial fluid (SF). Is synovial lubrication mediated by surface active phospholipid as opposed to mucinous glycoprotein? METHODS: A sonicated preparation of phosphatidylcholine and bovine SF were tested in vitro in a bearing of latex oscillating against polished glass under a load of 0.35 x 10(6) N/m2. The friction apparatus isolates conditions of boundary lubrication and has been validated against a cartilaginous bearing. Coefficient of friction (mu) was measured and compared against mu from physiologic saline, which served as a control. Separate digestions were carried out upon the SF with trypsin, phospholipase C, and phospholipase A2 in the presence and absence of proteolytic inhibitors. RESULTS: Digestions of bovine SF by phospholipase C in the presence of protease inhibitors did not remove boundary lubricating ability compared to an undigested control (p = 0.89). Digestion of bovine SF with trypsin removed all lubricating ability and raised friction (p = 0.004). Commercial purified phospholipase C contained trypsin-like activity when activity was tested with N alpha-benzoyl-L-arginine ethyl ester as substrate. Similar results were observed for phospholipase A2, which possesses a lower amount of trypsin activity. CONCLUSION: The results indicate that phospholipid does not play a prominent role in synovial fluid's ability to lubricate an artificial bearing. Rather, the boundary lubricating ability of SF is attributable to lubricin, a mucinous glycoprotein.

The modified beta-ketoadipate pathway in Rhodococcus rhodochrous N75: enzymology of 3-methylmuconolactone metabolism.

Rhodococcus rhodochrous N75 is able to metabolize 4-methylcatechol via a modified beta-ketoadipate pathway. This organism has been shown to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) prior to hydrolysis of the butenolide ring. A lactone-CoA synthetase is induced by growth of R. rhodochrous N75 on p-toluate as a sole source of carbon. The enzyme has been purified 221-fold by ammonium sulfate fractionation, hydrophobic chromatography, gel filtration, and anion-exchange chromatography. The enzyme, termed 3-methylmuconolactone-CoA synthetase, has a pH optimum of 8.0, a native Mr of 128,000, and a subunit Mr of 62,000, suggesting that the enzyme is homodimeric. The enzyme is very specific for its 3-methylmuconolactone substrate and displays little or no activity with other monoene and diene lactone analogues. Equimolar amounts of these lactone analogues brought about less than 30% (most brought about less than 15%) inhibition of the CoA synthetase reaction with its natural substrate.

Cerebral metabolic response to hypoglycemia in severe intrauterine growth-retarded rat pups.

This study was to examine the effects of hypoglycemia on the brain metabolism of severe intrauterine growth retardation (IUGR) rat pups. IUGR pups were produced by ligating one of the uterine arteries of the dams. The pups on the opposite uterine horn were used as the control. They were delivered by cesarean section on day 21 and received regular insulin 5 units/kg or equivalent volume of normal saline at 40 min of age. The plasma glucose, lactate, blood gas values and brain glucose, lactate, ATP contents were determined at 5 and 100 min of age. The IUGR pups had higher plasma and brain lactate concentrations than the control throughout the study period. They had lower plasma glucose, oxygen and pH values, brain glucose and ATP contents than control at 5 min of age. Despite insulin-induced hypoglycemia, brain ATP contents of the IUGR recovered to normal levels at 100 min of age when the oxygenation and pH improved. These data indicate that the brain energy metabolism of IUGR rat pups was suppressed by asphyxia and hypoglycemia. However, even in the continuing presence of hypoglycemia, brain energy metabolism returned to normal. The recovery is probably related to better oxygenation and utilization of alternative energy fuels, such as lactate.

Cerebral metabolic response to hypoglycemia in severe intrauterine growth-retarded rat pups.

This study was to examine the effects of hypoglycemia on the brain metabolism of severe intrauterine growth retardation (IUGR) rat pups. IUGR pups were produced by ligating one of the uterine arteries of the dams. The pups on the opposite uterine horn were used as the control. They were delivered by cesarean section on day 21 and received regular insulin 5 units/kg or equivalent volume of normal saline at 40 min of age. The plasma glucose, lactate, blood gas values and brain glucose, lactate, and ATP contents were determined at 5 and 100 min of age. The IUGR pups had higher plasma and brain lactate concentrations than the control throughout the study period. They had lower plasma glucose, oxygen and pH values, brain glucose and ATP contents than control at 5 min of age. Despite insulin-induced hypoglycemia, brain ATP contents of the IUGR recovered to normal levels at 100 min of age when the oxygenation and pH improved. These data indicate that the brain energy metabolism of IUGR rat pups was suppressed by asphyxia and hypoglycemia. However, even in the continuing presence of hypoglycemia, brain energy metabolism returned to normal. The recovery is probably related to better oxygenation and utilization of alternative energy fuels, such as lactate.

Comparison of the boundary-lubricating ability of bovine synovial fluid, lubricin, and Healon.

Purified human umbilical hyaluronate and a commercial preparation of rooster comb hyaluronate (Healon) intended for intra-articular viscosupplementation did not demonstrate the same degree of boundary-lubricating ability as bovine synovial fluid or its purified lubricating mucin, lubricin (p < 0.01). Boundary lubrication was measured in vitro in an arthrotripsometer oscillating natural latex against polished glass under a load of 0.35 MPa with an entraining velocity of 0.37 mm/s. The two hyaluronate solutions possessed the same hyaluronate concentration as synovial fluid, but Healon was 4.5 times more viscous. Present practice of viscosupplementation therapy for degenerative joint disease is limited and fails to implicate the important role of synovial mucin. Boundary lubrication provided by synovial mucin, independent of its viscosity, is not replicated by hyaluronate hydrogels.

Isolation, propagation, and characterization of rat liver serosal mesothelial cells.

Although rat liver epithelial cell (RLEC) lines have been developed by a number of laboratories, the identity of the clonogenic nonparenchymal progenitors is unknown. To provide insight into the derivation of RLEC, we immunoisolated serosal liver mesothelial cells (LMC) and bile duct epithelial cells and attempted to propagate each epithelial cell population using culture conditions routinely employed to establish RLEC lines. Briefly, the selective reactivity of LMC with two bile duct cell surface markers, OC.2 and BD.2, was exploited to develop an immunocytochemical technique to isolate LMC. Livers were collagenase dissociated, the mesothelial capsule was "peeled" and digested with pronase to destroy contaminating hepatocytes, and rare biliary ductal epithelial cells were immunodepleted using OC.2. LMC were subsequently isolated by selective binding to magnetic beads adsorbed with BD.2 and cultured in supplemented Waymouths 752/1 media containing 10% fetal calf serum. Proliferating BD.2+ LMC rapidly formed epithelial-like monolayers that could be continuously subcultured after trypsinization. In contrast, attempts to establish cell lines from purified OC.2+ bile duct epithelial cells were unsuccessful. Results from reverse transcriptase polymerase chain reaction analysis confirmed that LMC expressed Wilms' tumor transcripts, a lineage marker for mesodermally-derived cells. In summary, our findings clearly demonstrate that LMC can be continuously propagated using culture conditions routinely employed to establish RLEC lines, an observation that supports the contention that some RLEC lines may be derived from LMC.

Evaluation of insulin sensitivity in obese offspring of diabetic rats by hyperinsulinemic-euglycemic clamp technique.

Young adult macrosomic offspring of streptozotocin-induced mildly hyperglycemic rats exhibit accelerated growth through the first 10 wk of age. At 10 wk, oral glucose loading resulted in elevated plasma insulin and glucose concentrations compared to controls. To assess the mechanism of the abnormal glucose tolerance in vivo, hyperinsulinemic-euglycemic clamp studies were performed. Ten-wk-old rats were fasted overnight, and porcine insulin was infused (2.4 mU.kg-1.min-1). Glucose was infused concurrently at varying rates to maintain euglycemia for 40-60 min. Insulin levels were raised from a baseline value of 163 +/- 57 pmol/L (23 +/- 8 microU/mL) (SD) to 476 +/- 57 pmol/L (67 +/- 8 microU/mL) at steady state for males and from 178 +/- 43 pmol/L (25 +/- 6 microU/mL) to 454 +/- 43 pmol/L (64 +/- 6 microU/mL) for females. The results showed that the macrosomic male and female animals were significantly less sensitive to the effects of insulin than were their respective controls; this was evident by a lower increment in glucose disposal rate per unit increase in insulin (0.04 +/- 0.01 versus 0.11 +/- 0.03 for males and 0.05 +/- 0.03 versus 0.18 +/- 0.07 mg.kg-1 per microU/mL for females). The endogenous glucose production by the liver in the basal (fasted) state in the macrosomic group compared to controls was higher, suggesting possible hepatic insulin resistance. However, endogenous glucose production was suppressed to the same degree between the experimental and control groups during the hyperinsulinemic period, suggesting that the hepatic insulin resistance can be overcome by high insulin levels.(ABSTRACT TRUNCATED AT 250 WORDS)

The cross-generation effect of neonatal macrosomia in rat pups of streptozotocin-induced diabetes.

To study the cross-generation effect of enhanced growth in macrosomic newborn rats, we induced mild hyperglycemia in 15 pregnant Sprague Dawley rats by intraperitoneal injection of streptozotocin, 35 mg/kg body weight, on the 5th d of gestation. As reported previously, we produced hyperinsulinemia and accelerated growth in the fetuses of hyperglycemic dams. We also showed that the macrosomic female pups (second generation) continued to have a higher growth rate through the first 12 wk of life. In this study, the second-generation female rats were mated with macrosomic second-generation males; they demonstrated glucose intolerance during late pregnancy and delivered pups (third generation) with higher birth weight and plasma insulin levels than the pups from control second-generation rats. When the macrosomic third-generation pups were raised under identical nutritional and environmental conditions as controls, the macrosomic rats showed accelerated growth and higher fat tissue weight during the first 12 wk of life. Furthermore, the macrosomia was associated with glucose intolerance and higher insulin to glucose ratios compared to controls. We also mated the offspring of second-generation streptozotocin-injected nonmacrosomic as well as the offspring of macrosomic pups of buffer-injected dams; none of the pups from these matings were significantly macrosomic. Therefore, we conclude that the perpetuation of obesity and possibly glucose intolerance across generations in this rat model is predominantly a result of abnormal intrauterine metabolic environment rather than genetic factor driven.

Glucose metabolism in adipocytes of obese offspring of mild hyperglycemic rats.

Six pregnant rats were made mildly hyperglycemic by intraperitoneal injection of streptozotocin on d 5 of gestation. Four control rats were injected with citrate buffer. Thirty pups born to experimental dams who had increased birth weight (birth weight greater than 1.7 SD of mean birth weight of pups from control dams) maintained accelerated growth through 10 wk of age. At 10 wk, oral glucose tolerance tests showed higher glucose and insulin levels than the controls (n = 37). In addition to the higher body weight, the experimental rats also had higher fat weight to body weight ratios. Adipocytes of epididymal fat from obese males and periovarian fat from obese females had higher lipid content with significantly larger cell size than the adipocytes of the controls. The adipocytes of macrosomic rats showed attenuated response to insulin-stimulated glucose conversion to total lipid and fatty acid when compared with the responses seen in the adipocytes of the control rats. Interestingly, although the insulin-stimulated glucose conversion to CO2 was similar in macrosomic and control males, the response in the macrosomic female was blunted when compared with that of the control females. Insulin receptor binding capacities of the macrosomic rats were lower than those of the controls, which is consistent with a phenomenon of down-regulation. However, the receptor affinities were higher in the experimental animals than in controls. Therefore, a postreceptor defect may account for the abnormality in glucose metabolism in the obese rats. In conclusion, the abnormal response to oral glucose loading in these experimental obese, hyperinsulinemic rats is due to peripheral tissue insulin resistance that is probably postreceptor in nature.

Neonatal sympathoadrenal response to acute hypoxia: impairment after experimental intrauterine growth retardation.

Sympathoadrenal system function may be altered following intrauterine growth retardation (IUGR). We tested the hypothesis that the growth-retarded newborn rat pup has increased basal sympathoadrenal activity under normoxic conditions and a blunted sympathoadrenal response to acute hypoxia. IUGR was established by uterine artery ligation on d 18 of gestation in Sprague-Dawley rats. Growth-retarded pups were chosen as those whose birth wt was more than 2 x SD below the mean birth wt of control pups delivered to sham-operated dams. At 24 +/- 12 h of age cardiac sympathetic neuronal activity (CSNA) was determined by 3H-norepinephrine tracer and alpha-methyltyrosine techniques. Adrenal medullary catecholamine synthesis (CAT SYN) was measured by 14C-tyrosine precursor methods, and adrenal catecholamine release (CAT REL) was determined using alpha-methyl-tyrosine. IUGR and control pups were studied over a 120-min period of normoxia or hypoxia (FiO2 = 0.09). Under normoxic conditions, the IUGR pups had increased CSNA and increased adrenal CAT SYN and CAT REL compared to controls. Adrenal CAT REL in normoxic IUGR pups was selective for epinephrine. In response to acute hypoxia, control pups had increased CSNA and increased adrenal CAT SYN and CAT REL compared to normoxic controls, with the proportion of norepinephrine and epinephrine released mimicking the ratio of the two amines in the adrenal. In contrast, in hypoxic IUGR pups CSNA and adrenal CAT SYN did not increase, and norepinephrine alone was released from the adrenal medulla.(ABSTRACT TRUNCATED AT 250 WORDS)