High fat diet consumption differentially affects adipose tissue inflammation and adipocyte size in obesity-prone and obesity-resistant rats.

BACKGROUND/OBJECTIVES: Expanding visceral adiposity is associated with increased inflammation and increased risk for developing obesity-related comorbidities. The goal of this study was to examine high fat diet (HFD)-induced differences in adipocyte size and cytokine/chemokine expression in visceral and subcutaneous adipose depots in obesity-prone (OP) and obesity-resistant (OR) rats. METHODS: OP and OR rats were fed either a low fat diet (LFD, 10% kilocalories from fat) or HFD (60% kilocalories from fat) for 7 weeks. Adipocyte size and the presence of crown-like structures in epididymal and inguinal adipose tissue were determined. A multiplex cytokine/chemokine panel was used to assess the expression of inflammatory markers in epididymal and inguinal adipose tissues. RESULTS: A higher percentage of large adipocytes (>5000 µm2) was detected in the epididymal and inguinal adipose tissues of OP rats and a higher percentage of small adipocytes (<4000 µm2) was detected in the epididymal and inguinal adipose tissues of OR rats. More crown-like structures were identified in epididymal adipose tissue of OP rats fed a LFD, compared to OR rats. Consumption of a HFD increased the number of crown-like structures in OR, but not OP rats. Epididymal expression of pro-inflammatory cytokines (IL-1ß and TNF-α) was higher in OP rats, compared to OR rats fed LFD. HFD consumption increased epididymal expression of GM-CSF, IL-1α, IL-1ß, IL-6, MIP-2 and TNF-α in OP and OR rats. Inguinal expression of pro-inflammatory cytokines (IL-1α, IL-1ß and TNF-α) was higher in OP rats, compared to OR rats. CONCLUSIONS: Overall, these data suggest that a higher susceptibility to developing obesity is characterized by large adipocytes and increased visceral adipose inflammation. Interestingly, in OR rats, the detrimental effects of HFD consumption on visceral adipose inflammation are evident with only small increases in weight and adiposity, suggesting that HFD also increases the risk for obesity-related comorbidities in OR rats.

Hypothalamic QRFP: regulation of food intake and fat selection.

QRFP, a member of the RFamide-related peptide family, is a strongly conserved hypothalamic neuropeptide that has been characterized in various species. Prepro-QRFP mRNA expression is localized to select regions of the hypothalamus, which are involved in the regulation of feeding behavior. The localization of the peptide precursor has led to the assessment of QRFP on feeding behaviors and the orexigenic effects of QRFP have been detected in mice, rats, and birds. QRFP acts in a macronutrient specific manner in satiated rats to increase the intake of a high fat diet, but not the intake of a low fat diet, and increases the intake of chow in food-restricted rats. Studies suggest that QRFP's effects on food intake are mediated by the adiposity signal, leptin, and hypothalamic neuropeptides. Additionally, QRFP regulates the expression and release of hypothalamic Neuropeptide Y and proopiomelanocortin/α-Melanocyte-Stimulating Hormone. QRFP binds to receptors throughout the brain, including regions associated with food intake and reward. Taken together, these data suggest that QRFP is a mediator of motivated behaviors, particularly the drive to ingest high fat food. The present review discusses the role of QRFP in the regulation of feeding behavior, with emphasis on the intake of dietary fat.

Sensitivity to the satiating effects of exendin 4 is decreased in obesity-prone Osborne-Mendel rats compared to obesity-resistant S5B/Pl rats.

BACKGROUND: Osborne-Mendel (OM) rats are prone to obesity when fed a high-fat diet, whereas S5B/Pl (S5B) rats are resistant to diet-induced obesity when fed the same diet. OM rats have a decreased satiation response to fatty acids infused in the gastrointestinal tract, compared to S5B rats. One possible explanation is that OM rats are less sensitive to the satiating hormone, glucagon-like peptide 1 (GLP-1). GLP-1 is produced in the small intestine and is released in response to a meal. The current experiments examined the role of GLP-1 in OM and S5B rats. METHODS: Experiment 1 examined preproglucagon mRNA expression in the ileum of OM and S5B rats fed a high-fat (55% kcal) or low-fat (10% kcal) diet. Experiment 2 investigated the effects of a 2 h high-fat meal after a 24 h fast in OM and S5B rats on circulating GLP-1 (active) levels. Experiment 3 examined the effects of exendin-4 (GLP-1 receptor agonist) administration on the intake of a high-fat or a low-fat diet in OM and S5B rats. RESULTS: Preproglucagon mRNA levels were increased in the ileum of OM rats compared to S5B rats and were increased by high-fat diet in OM and S5B rats. OM and S5B rats exhibited a similar meal-initiated increase in circulating GLP-1 (active) levels. Exendin-4 dose dependently decreased food intake to a greater extent in S5B rats compared to OM rats. The intake of low-fat diet, compared to the intake of high-fat diet, was more sensitive to the effects of exendin-4 in these strains. CONCLUSIONS: These results suggest that though OM and S5B rats have similar preproglucagon mRNA expression in the ileum and circulating GLP-1 levels, OM rats are less sensitive to the satiating effects of GLP-1. Therefore, dysregulation of the GLP-1 system may be a mechanism through which OM rats overeat and gain weight.

Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy.

Removal of adrenal steroids by adrenalectomy (ADX) slows or reverses the development of many forms of obesity in rodents, including those that are leptin or leptin receptor deficient. Obesity is associated with hyperleptinemia and leptin resistance. We hypothesized that glucocorticoids impair leptin receptor signaling and that removal thereof would activate the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway. The inhibitory effect of leptin (2.5 microg icv) on food intake was enhanced in ADX rats. A combination of ribonuclease protection assays, RT-PCR, Western blots, and mobility shift assays was used to evaluate the leptin signaling pathway in whole hypothalami from sham-operated, ADX and corticosterone-replaced ADX (ADX-R) Sprague-Dawley rats that were treated acutely with either saline vehicle or leptin intracerebroventricularly. ADX increased the expression of leptin receptor mRNA, increased STAT-3 mRNA and protein levels, induced constitutive STAT-3 phosphorylation and DNA binding activity, and also reduced suppressor of cytokine signaling-3 (SOCS-3) mRNA and protein levels. ADX and leptin treatment increased STAT-3 phosphorylation, but with no concomitant increase in DNA binding activity. Leptin and ADX decreased NPY mRNA expression, but their combination did not further decrease NPY mRNA. Corticosterone supplementation of ADX rats partially reversed many of these effects. In conclusion, ADX through activation of STAT-3 and inhibition of SOCS-3 activates the JAK-STAT signaling pathway. These effects most probably explain the ability to prevent the development of obesity by removal of adrenal steroids.

The effects of a high fat diet on leptin mRNA, serum leptin and the response to leptin are not altered in a rat strain susceptible to high fat diet-induced obesity.

Osborne-Mendel (OM) and S5B/Pl rats differ in their sensitivity to develop obesity when fed a high fat (HF) diet; OM rats become obese, whereas S5B/Pl rats remain thin. We have investigated the possibilities that either an impaired leptin response or resistance to leptin action underlies the sensitivity to this form of obesity in OM rats. In Experiment 1, OM and S5B/Pl rats fed a nonpurified diet were killed at d 0 or were fed either a HF (56% fat energy) or a low fat (LF, 10% fat energy) diet for 2 or 7 d. The HF diet increased serum leptin significantly by d 2 to levels that were similar in both rat strains. At 7 d, leptin levels were lower than at d 2 but remained higher than levels in the d 0 control groups. The leptin mRNA:18S RNA ratio in epididymal adipose tissue increased to higher levels in HF-fed OM rats than in S5B/Pl rats fed that diet. However, although the LF diet had no effect in S5B/Pl rats, it increased leptin mRNA levels in epididymal adipose tissue of OM rats compared with the controls fed the nonpurified diet. In Experiment 2, OM and S5B/Pl rats were fed HF or LF diets for 5 wk. At that time, their feeding response to a range of leptin doses (0, 1, 5 or 10 microgram) given intracerebroventricularly was tested after overnight food deprivation. There was a similar dose-dependent reduction in energy intake in response to leptin in both OM and S5B/Pl rats. These responses were independent of the diet. The data suggest that the susceptibility of OM rats to HF diet-induced obesity is not related to either a loss of central sensitivity to leptin or a failure to enhance leptin production acutely, although the failure to maintain chronically increased levels of serum leptin could contribute to the obesity.

Differential expression of insulin receptor tyrosine kinase inhibitor (fetuin) gene in a model of diet-induced obesity.

The Differential Display technique has been used to identify differences in mRNA expression in adipose tissue after the introduction of a high fat diet to two strains of rat (OM and S5B/PI) that differ in their susceptibility to develop obesity on this diet. The insulin receptor tyrosine kinase inhibitor protein (fetuin) was shown to be differentially expressed in OM but not S5B/PI rats. This circulating protein may play a role in the development of peripheral insulin resistance associated with high fat diets.

Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12.

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.

Differences in binding of hepatic nuclear proteins from lean and obese rats to the 5'-upstream region of tyrosine aminotransferase.

The glucocorticoid effects on liver tyrosine aminotransferase mRNA levels have been studied in young, lean, and obese Zucker (fa/fa) rats and 5'-upstream regions of the tyrosine aminotransferase (TAT) gene have been used in gel retardation studies to investigate nuclear protein binding. Hepatic TAT mRNA levels were increased in obese fa/fa rats but were normalized seven days after adrenalectomy. Corticosterone replacement to adrenalectomized rats restored the increased levels of TAT mRNA in the obese animals. A 60-bp fragment of upstream TAT DNA (-2463 to -2403) was identified which showed higher levels of band shifting after incubation with hepatic nuclear proteins of obese rats compared with the proteins from lean animals. This differential level of gel retardation was substantially reduced by alkaline phosphatase treatment of nuclear proteins. Gel retardation was reduced when nuclear proteins were prepared from adrenalectomized obese rats, and increased with nuclear proteins from adrenalectomized rats replaced with corticosterone. DNA affinity chromatography and gel electrophoresis identified three proteins of approximately 58, 62, and 65 kDa in the DNA-protein complex. Increased amounts of these three proteins were purified from nuclei of obese rats. HNF3 alpha antibodies induced hypershift of the gel retardation pattern implicating HNF3 alpha as one of the proteins that binds to the 60 bp DNA fragment. The data support the hypothesis that decreased phosphorylation of nuclear proteins in obese rats is glucocorticoid-dependent and may contribute to the altered transcriptional activity of glucocorticoid-responsive genes.

Purification and N-terminal amino acid sequences of two polypeptides encoded by the mcrB gene from Escherichia coli K-12.

This report provides a purification method for the two proteins, 51 kDa and 33 kDa, both encoded by the same mcrB gene of the McrBC restriction system in Escherichia coli K-12. The two proteins were produced in large quantity using a T7 expression system and copurified to near homogeneity by DEAE-Sepharose and Affi-Gel blue column chromatography. The N-terminal amino acid sequences of these purified McrB proteins were the same as those predicted from the mcrB DNA sequence by Ross et al. [J. Bacteriol. 171 (1989b) 1974-1981]. The 33-kDa protein totally overlaps the C-terminal part of the 51-kDa protein.

DNA restriction polymorphism in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus.

Restriction endonuclease analysis was used to examine variation in DNA of 22 wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV). Eleven of the 15 isolated from Louisiana were distinguishable based on restriction fragment profiles from the enzymes BamHI, HindIII, and EcoRI. There was significant genetic variation in SfNPV isolates within single agricultural fields. Nucleotide sequence divergence values, based on restriction fragment profiles, indicated that genetic variation among isolates foreign to Louisiana (Ohio, Ecuador, Mexico, Georgia, Colombia, and Venezuela) was greater than that among the Louisiana isolates. However, certain foreign isolates were similar to or identical with Louisiana isolates. Genetic variation of the viral DNA was not influenced by the insect's host plan species.

Overproduction and purification of McrC protein from Escherichia coli K-12.

The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989).

Nomenclature relating to restriction of modified DNA in Escherichia coli.

At least three restriction systems that attack DNA containing naturally modified bases have been found in common Escherichia coli K-12 strains. These systems are McrA, McrBC, and Mrr. A brief summary of the genetic and phenotypic properties so far observed in laboratory strains is set forth, together with a proposed nomenclature for describing these properties.

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.

Serratia marcescens rpr gene sensitizes Escherichia coli wild-type, xth, and nfo strains to methyl methanesulphonate.

It is reported here that the rpr DNA repair gene of Serratia marcescens does not complement an Escherichia coli xth nfo AP endonuclease mutation for resistance to methyl methanesulphonate (MMS). Rather, rpr sensitized Escherichia coli wild-type, xth, and nfo strains to MMS. Also, it was found that rpr could not complement a triple tag alkA recA mutation in E. coli, indicating that there are limits to rpr complementing capabilities. It was determined that rpr gene dosage was not a factor in recA complementation. MMS sensitization of an E. coli wild-type strain, however, was directly related to rpr copy number. These data indicate that Rpr does not have an associated AP endonuclease activity, and that it is incapable of substituting for Tag I, Tag II, and RecA in a tag alkA recA background.

Evidence for unique DNA repair activity encoded by a cloned Serratia marcescens gene: suppression of Escherichia coli mutations that reduce repair of alkylated DNA.

A recombinant plasmid containing a Serratia marcescens DNA repair gene has been analyzed biochemically and genetically in Escherichia coli mutants deficient for repair of alkylated DNA. The cloned gene suppressed sensitivity to methyl methanesulfonate of an E. coli strain deficient in 3-methyladenine DNA glycosylases I and II (i.e., E. coli tag alkA) and two different E. coli recA mutants. Attempts to suppress the methyl methanesulfonate sensitivity of the E. coli recA mutant by using the cloned E. coli tag and alkA genes were not successful. Southern blot analysis did not reveal any homology between the S. marcescens gene and various known E. coli DNA repair genes. Biochemical analysis with the S. marcescens gene showed that the encoded DNA repair protein liberated 3-methyladenine from alkylated DNA, indicating that the DNA repair molecular is an S. marcescens 3-methyladenine DNA glycosylase. The ability to suppress both types of E. coli DNA repair mutations, however, suggests that the S. marcescens gene is a unique bacterial DNA repair gene.

Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12.

The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that it encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methyl-cytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.

Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.

Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair.

We report here the molecular isolation of a DNA fragment which encodes Tag-like activity from the Gram-negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag-like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3-methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA-damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII-SmaI fragment. Labelling of plasmid-encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42,000 and 16,000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3-methyladenine DNA glycosylase I of E. coli.

Phosphate starvation induces uptake of glyphosate by Pseudomonas sp. strain PG2982.

Pseudomonas sp. strain PG2982 has the ability to use the phosphonate herbicide, glyphosate, as a sole phosphorus source (J. K. Moore, H. D. Braymer, and A. D. Larson, Appl. Environ. Microbiol. 46:316-320, 1983). Glyphosate uptake is maximal in the late log phase of growth and is induced by phosphate starvation. Uptake is inhibited by phosphate and arsenate, but not by the amino acids glycine and sarcosine. The Km and Vmax for glyphosate uptake were calculated to be 23 microM and 0.97 nmol/mg (dry weight) per min, respectively. A phosphate transport system with a broad substrate specificity may be responsible for glyphosate uptake.

Organization and sequence of the hsd genes of Escherichia coli K-12.

The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12. The three hsd genes are transcribed in the same direction, but from two promoters. hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS. pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod. Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated. A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues. The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.