Human adenovirus Ad-36 induces adipogenesis via its E4 orf-1 gene.

OBJECTIVE: Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process. DESIGN AND RESULTS: Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-beta and peroxisome proliferator-activated receptor gamma2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted. CONCLUSION: Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

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.

Abnormal regulation of hepatic glucocorticoid receptor mRNA and receptor protein distribution in the obese Zucker rat.

This study examines the cellular distribution of glucocorticoid receptor (GR) protein and transcriptional activity of the GR gene in the liver of Zucker obese (fa/fa) rats. Immunoabsorption and Western blotting showed an increase in nuclear GR protein level but a decrease in cytosolic GR levels in the liver of 5-week old male obese rats (fa/fa) compared to their lean littermates (Fa/-). These changes were confirmed by receptor-ligand binding assays with [3H]-dexamethasone which showed a sixfold increase in average obese nuclear GR binding and a twofold reduction in cytosolic GR binding. HSP90, but not HSP70, levels were reduced in hepatic cytosol and increased in hepatic nuclei prepared from obese rats. Using Northern blot analysis of hepatic RNA, we demonstrated a twofold increase in hepatic mRNAs for GR, malic enzyme (ME), tyrosine aminotransferase (TAT), and glyceraldehyde 3-PO4-dehydrogenase in the obese rat. Increased transcription of GR and ME mRNAs in obese nuclei was indicated in nuclear run-on assays. These data suggest that there is increased nuclear localization of GR in the liver of obese rats and suggests that increased transcription of GR gene may contribute to this effect. The described changes may contribute to the abnormal regulation by glucocorticoids of some hepatic genes in the Zucker fa/fa rat.

Regulation of beta 3-adrenergic receptor mRNA by sympathetic nerves and glucocorticoids in BAT of Zucker obese rats.

Impaired brown adipose tissue (BAT) thermogenesis in the genetically obese Zucker fatty (fa/fa) rat is restored to normal by adrenalectomy. We investigated the role of the sympathetic nervous system in modulating the effects of adrenalectomy by studying beta3-adrenergic receptor (AR) and uncoupling protein (UCP) mRNA levels in unilaterally sympathectomized interscapular BAT of lean and obese rats. UCP mRNA levels were increased by adrenalectomy. Sympathetic denervation prevented this adrenalectomy-induced increase in lean rats but not in obese rats. beta 3-AR mRNA was decreased in BAT of obese rats. Adrenalectomy decreased and denervation increased beta 3-AR mRNA in lean rats but the opposite response was observed to both of these manipulations in obese rats. beta 3-AR mRNA and UCP mRNA were negatively correlated in lean rats but positively correlated in obese rats. Norepinephrine increased UCP mRNA levels in denervated BAT of both lean and obese rats and decreased beta 3-AR mRNA in lean rats but not obese rats. These data suggest that the regulation of the beta 3-AR gene in response to sympathetic stimuli and glucocorticoids is abnormal in the obese rat.

Adrenalectomy of the obese Zucker rat: effects on the feeding response to enterostatin and specific mRNA levels.

The effects of adrenalectomy on the feeding response to enterostatin and the mRNA levels of its parent protein, pancreatic colipase, have been investigated in lean (fa/?) and genetically obese (fa/fa) rats. Adrenalectomy reduced body weight gain and food intake of obese rats. Enterostatin inhibited the intake of high-fat diet in obese rats but not in lean rats. Adrenalectomy reduced food intake of all rats and abolished the response to enterostatin in the obese group. Obese rats had low levels of colipase mRNA, but these were normalized after adrenalectomy. The ability to respond to exogenous enterostatin is possibly linked to low levels of production of the peptide. The effects of adrenalectomy on brown adipose tissue uncoupling protein (UCP) mRNA and beta 3-adrenergic receptor (beta 3-AR) mRNA were also investigated. Northern blot analysis showed low levels of both UCP mRNA and beta 3-AR mRNA in obese rats that were restored to or toward the normal levels of lean rats by adrenalectomy. Adrenalectomy had no significant effects on mRNA levels in lean rats.

Structure and variability of recently inserted Alu family members.

The HS subfamily of Alu sequences is comprised of a group of nearly identical members. Individual subfamily members share 97.7% nucleotide identity with each other and 98.9% nucleotide identity with the HS consensus sequence. Individual subfamily members are on the average 2.8 million years old, and were probably derived from a single source 'master' gene sometime after the human/great ape divergence. The recent Alu family member insertions provide a better image of the structure of Alu retroposons before they have had the opportunity to change significantly. All of the HS subfamily members are flanked by perfect direct repeats as a result of insertion at staggered nicks. The 'master' gene from which the HS subfamily members were derived had an oligo-dA rich tail at least 40 bases long. The 'master' gene is very rich in CpG dinucleotides, but nucleotide substitutions within subfamily members accumulated in a random manner typical for Alu sequence with CpG substitutions occurring 9.2 fold faster than non-CpG substitutions.

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster.

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays.

Mutations induced in Drosophila spermatozoa at the alcohol dehydrogenase Adh locus by 1-ethyl-1-nitrosourea (ENU) were compared to X-ray-induced mutations using genetic tests for complementation, southern blotting, western blotting and northern blotting. 8 of 10 ENU-induced mutations complemented all known adjacent loci and were presumed to be intragenic. In contrast, 8 of 30 X-ray-induced mutations were intragenic. Southern blot analysis showed that 2 of 7 intragenic mutations induced by X-rays were altered at the Adh locus, whereas all 8 intragenic ENU mutants appeared normal. Western blot analysis showed 4 of 7 intragenic mutants induced by X-rays produced a detectable polypeptide; 1 of the 4 had normal molecular weight and charge. In contrast, 7 of the 8 intragenic mutants induced by ENU produced a polypeptide of normal molecular weight and charge. One ENU and two X-ray-induced mutants, which had normal southern blots and no detectable polypeptide, produced normal molecular weight mRNA by northern blots. The interpretation of these results is that in spermatozoa X-rays induce primarily deletions that either produce deficiencies of the Adh locus or nonsense mutations within the locus, whereas ENU induces primarily missense mutations. This forward mutation assay based on loss of enzymatic activity efficiently recovered a broad spectrum of mutations ranging from missense to intragenic deletions and multi-locus deficiencies. Only 3 of these 40 mutations produced a polypeptide detectable as an electrophoretic variant.