Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.     

Postnatal recruitment of brown adipose tissue is induced by the cold stress experienced by the pups. An analysis of mRNA levels for thermogenin and lipoprotein lipase.

In order to investigate the postnatal recruitment process, gene expression in the brown adipose tissue of rat pups was followed during the first 20 h of life. In normal pups, the level of mRNA coding for the uncoupling protein thermogenin increased markedly but gradually within the first 24 h. Lipoprotein lipase and actin mRNA levels were relatively low and remained constant. In pups exposed to thermoneutral temperature (35 degrees C) for the first 12 h after birth, no increase in thermogenin mRNA or lipoprotein lipase mRNA was observed, whereas in pups exposed to 28 degrees C a clear increase in both thermogenin and lipoprotein lipase mRNA levels was found. Actin mRNA levels were not affected by the environmental temperature under these circumstances. It was concluded that the postnatal recruitment in brown adipose tissue is a consequence of the cold stress experienced by the newborn pups. Thus, postnatal recruitment is not ontogenically predetermined.     

Genetic and developmental regulation of the lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structural gene control enzyme expression

We report here a study of the developmental and genetic control of tissue-specific expression of lipoprotein lipase, the enzyme responsible for hydrolysis of triglycerides in chylomicrons and very low density lipoproteins. Lipoprotein lipase (LPL) mRNA is present in a wide variety of adult rat and mouse tissues examined, albeit at very different levels. A remarkable increase in the levels of LPL mRNA occurs in heart over a period of several weeks following birth, closely paralleling developmental changes in lipase activity and myocardial beta-oxidation capacity. Large increases in LPL mRNA also occur during differentiation of 3T3L1 cells to adipocytes. As previously reported, at least two separate genetic loci control the tissue-specific expression of LPL activity in mice. One of the loci, controlling LPL activity in heart, is associated with an alteration in LPL mRNA size, while the other, controlling LPL activity in adipose tissue, appears to affect the translation or post-translational expression of LPL. To examine whether these genetic variations are due to mutations of the LPL structural locus, we mapped the LPL gene to a region of mouse chromosome 8 using restriction fragment-length polymorphisms and analysis of hamster-mouse somatic cell hybrids. This region is homologous to the region of human chromosome 8 which contains the human LPL gene as judged by the conservation of linked genetic markers. Genetic variations affecting LPL expression in heart cosegregated with the LPL gene, while variations affecting LPL expression in adipose tissue did not. Furthermore, Southern blotting analysis indicates that LPL is encoded by a single gene and, thus, the genetic differences are not a consequence of independent regulation of two separate genes in the two tissues. These results suggest the existence of cis-acting elements for LPL gene expression that operate in heart but not adipose tissue. Our results also indicate that two genetic mutations resulting in deficiencies of LPL in mice, the W mutation on chromosome 5 and the cld mutation on mouse chromosome 17, do not involve the LPL structural gene locus. Finally, we show that the gene for hepatic lipase, a member of a gene family with LPL, is unlinked to the gene for LPL. This indicates that combined deficiencies of LPL and hepatic lipase, observed in humans as well as in certain mutant strains of mice, do not result from focal disruptions of a cluster of lipase genes.     

Localization and function of metabotropic glutamate receptor 8 in the enteric nervous system

The enteric nervous system (ENS) contains glutamatergic neurons, transporters, and functional ionotropic and groups I and II metabotropic glutamate receptors (mGluRs). The aim of this study was to determine whether the ENS contains functional group III mGluRs. RT-PCR demonstrated the expression of mGluR7 and mGluR8 mRNA in rat myenteric ganglia. Western blot analysis confirmed the presence of mGluR8 protein. Immunocytochemistry, in conjunction with confocal microscopy, demonstrated mGluR8 immunoreactivity in the ENS of several species, including humans. mGluR8 immunoreactivity was localized to the membrane of nerve cell bodies that received glutamatergic input. Significant receptor internalization of mGluR8 was observed on activation, and localization to membrane was observed on blocking with the mGluR III antagonist (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG). mGluR8-positive myenteric neurons contained glutamate or nitric oxide synthase (NOS), a marker of inhibitory motorneurons. Enteric group III mGluRs are functional because mGluR8 agonists inhibited forskolin-induced accumulation of cAMP in isolated myenteric ganglia, and CPPG reduced this effect. In addition, an accelerating effect on guinea pig colonic motility was observed after the application of mGluR8 agonists. Increase in motility was specific, because CPPG inhibited it. Moreover, in the presence of hexamethonium or Nomega-nitro-l-arginine methyl ester, an inhibitor of NOS, responses caused by mGluR8 agonists were abolished. mGluR8 agonists also increased longitudinal muscle contractions. These findings suggest that mGluR8 agonists increase motility by inhibiting nitrergic relaxation and possibly by facilitating cholinergic contractions.     

Complementation of the radiosensitive M059J cell line

M059J is a radiosensitive cell line established from a human glioblastoma tumor that fails to express the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs, now known as PRKDC). Another cell line, M059K, established from the same tumor is radioresistant. Neither M059J nor M059K cells have been fully characterized, beyond the lack of expression of PRKDC and low expression of ATM in M059J cells. To determine whether its radiosensitive phenotype is due to a defect in the gene that encodes PRKDC, we show here that M059J cells can be complemented with the PRKDC gene by introducing a fragment of human chromosome 8 containing a copy of the human PRKDC gene. Two hybrid cell lines that retain an extra copy of PRKDC display active kinase activity and are radioresistant, demonstrating that the primary defect in M059J cells is in PRKDC. In addition, these cell lines derived from M059J cells provide us with a closer genetic match to M059J than M059K cells in studies to elucidate the function of DNA-PK.     

Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry

A sensitive method for determination of metallothionein (MT) isoform levels in rat liver by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry was developed. Critical steps in sample preparation, like MT extraction, MT saturation with Cd and protein separation, were optimized. This method is capable of measuring levels of 2.0 μg/g liver for metallothionein-1 (MT-1) and 1.3 μg/g liver for metallothionein-2 (MT-2), respectively, with a high recovery of 103% on average. The method described, thus, proved suitable for analyzing metallothionein isoform concentrations even in untreated animals. The ratio of MT-1 to MT-2 was found to be 1:1 on average. MT decomposition during storage was very high in whole livers, but could be reduced by about 80% when extracted liver samples were used.     

Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system

BackgroundIn the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.ResultsWe investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.ConclusionsOur results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0643-z) contains supplementary material, which is available to authorized users.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.