Effects of insulin therapy on weight gain and fat distribution in the HF/HS-STZ rat model of type 2 diabetes.

BACKGROUND/OBJECTIVES: Insulin therapy is required for many patients with the obesity-related disorder type 2 diabetes, but is also associated with weight gain. The specific location of adipose tissue location matters to cardiovascular disease (CVD) risk. We investigated effects of exogenous insulin on fat distribution in the high-fat/high-sucrose fed rat treated with streptozotocin (HF/HS-STZ) rat model of type 2 diabetes. We also examined effects of insulin therapy on circulating CVD markers, including adiponectin, triglycerides (TGs), total cholesterol and high-density lipoprotein. SUBJECTS/METHODS: Male SD rats were HF/HS fed for 5 weeks followed by STZ treatment to mimic the hallmarks of human obesity-associated insulin resistance followed by hyperglycemia. Magnetic resonance imaging and computed tomography were used to determine total fat, abdominal fat distribution and liver fat before and after insulin therapy in HF/HS-STZ rats. HbA1c%, TGs, cholesterol, high-density lipoprotein and adiponectin were analyzed by conventional methods adapted for rats. RESULTS: Insulin therapy lowered HbA1c (P<0.001), increased body weight (P<0.001), increased lean mass (P<0.001) and led to a near doubling of total fat mass (P<0.001), with the highest increase in subcutaneous adipose tissue as compared with visceral adipose tissue (P<0.001). No changes in liver fat were observed after insulin therapy, whereas plasma TG and cholesterol levels were decreased (P<0.001, P<0.01), while high-density lipoprotein (HDL) and adiponectin levels were elevated (P<0.01, P<0.001). CONCLUSIONS: Using the HF/HS-STZ rat as an animal model for type 2 diabetes, we find that insulin therapy modulates fat distribution. Specifically, our data show that insulin has a relatively positive effect on CVD-associated parameters, including abdominal fat distribution, lean body mass, adiponectin, TGs and HDL in HF/HS-STZ rats, despite a modest gain in weight.

Open flow microperfusion: pharmacokinetics of human insulin and insulin detemir in the interstitial fluid of subcutaneous adipose tissue.

AIMS: To compare the time profile of insulin detemir and human insulin concentrations in the interstitial fluid (ISF) of subcutaneous adipose tissue during constant i.v. infusion and to investigate the relationship between the pharmacokinetics of both insulin molecules in plasma and the ISF of subcutaneous adipose tissue. METHODS: During a 6-h hyperinsulinaemic-euglycaemic clamp (plasma glucose level 8 mmol/l) human insulin (21 and 42 pmol/min/kg) or insulin detemir (209 and 417 pmol/min/kg) were infused i.v. in eight rats per dose level. Open flow microperfusion (OFM) was used to continuously assess interstitial insulin concentrations in subcutaneous adipose tissue. RESULTS: At the lower infusion rate, insulin detemir appeared significantly later in the ISF than in the plasma (p < 0.05) and also appeared later in the ISF relative to human insulin (p < 0.005). CONCLUSIONS: By using OFM we were able to monitor albumin-bound insulin detemir directly in the ISF of subcutaneous tissue and confirm its delayed transendothelial passage to a peripheral site of action.

Sensitivity to nitrogen mustard relates to the ability of processing DNA damage in Chinese hamster ovary cells.

The hallmark of the excision repair pathways is the removal of DNA adducts by excision of the damaged nucleotides. In the course of repair, transient DNA strand breaks occur, which can be measured by the Comet assay. We have investigated the processing of DNA damage, mediated by nitrogen mustard, in wild-type AA8 Chinese hamster ovary cells, and in UV5, UV20 and UV41 DNA repair deficient cell lines. Whereas DNA repair could not be detected by unscheduled DNA synthesis at nitrogen mustard doses below 10 microM, processing of nitrogen mustard-mediated DNA damage was observed by the Comet assay at a 100-times lower concentration. Wild-type Chinese hamster ovary AA8 cells were able to process nitrogen mustard-mediated DNA damage within 4-24 hr depending on the dose of nitrogen mustard (0.1-10 microM). None of the repair-deficient cell lines was able to completely process the DNA damage induced by 10 microM nitrogen mustard. At nitrogen mustard doses that conferred 10% colony forming ability, the repair-deficient cells had an altered processing of nitrogen mustard-mediated DNA damage: In the AA8, UV20, and UV41 cells, the amplitude of strand breaks peaked early (within 4 hr), the level of strand breaks in the nitrogen mustard exposed UV20 and UV41 cells did not return to the baseline of the unexposed reference culture, and the peak in strand breaks in the UV5 cell line occurred after 4 hr. Our results indicate that the single cell gel electrophoresis (Comet) assay is suitable for assessing repair capability of DNA alkylations.

Proficient deoxyribonucleic acid repair of methylation damage in hamster ERCC-gene mutants.

Three major pathways, nucleotide excision repair (NER), base excision repair (BER) and O6-methylguanine-DNA methyltransferase (MGMT), are responsible for the removal of most adducts to DNA and thus for the survival of cells influenced by deoxyribonucleic acid (DNA) adduct-forming chemicals. We have evaluated host cell reactivation and cell survival of wild type Chinese hamster ovary cells and of mutants in the NER-genes ERCC1, ERCC2, and ERCC4 after treatment with the methylating compounds dimethylsulfate and methylnitrosourea. No effect of the three genes could be demonstrated, i.e., survival and host cell reactivation after methylation damage in the mutants and the wild type cells were similar. Gene-specific repair experiments confirmed the proficient removal of methyl lesions. We conclude that the three nucleotide excision repair genes are immaterial to the repair of methylation damage. This suggests that NER does not play a role in the removal of methylation in mammalian cells and that BER and MGMT are responsible for the survival of such cells, when they are challenged with methylation of DNA.

Ongoing activity of RNA polymerase II precludes chromatin collapse and DNA fragmentation in Chinese hamster ovary cells.

The role of ongoing RNA synthesis in chromatin organization in Chinese hamster ovary cells was examined upon exposure to the transcription inhibitor alpha-amanitin. Treatment with alpha-amanitin led to pleomorphic nuclei with chromatin heavily condensed and with the remaining ribonucleoprotein aggregated in large compact granular masses around the margins of the nuclear periphery. Concommitant with the changes in nuclei morphology transient focal dilatation of the rough endoplasmic reticulum was observed while other cytoplasmic organelles appeared structurally unaffected. The morphological changes occurred after complete inhibition of RNA polymerase II mediated transcription. The molecular integrity of isolated DNA was monitored in parallel with the structural analysis. Fragmentation of cellular DNA occurred in a time-dependent fashion and well after the complete inhibition of RNA synthesis. Characteristic oligonucleosomesized DNA fragments of about 187 base pairs in length was produced in a cotemporal time-dependent fashion. Our findings indicate that ongoing transcription and the structural state of chromatin are very closely integrated, and provide further evidence that RNA is a structural component of the nuclear matrix, which in turn is involved in keeping chromatin physically dispersed and decondensed.

The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16.

The aggregation phenotypes Agr+ and Agr- of Bacillus thuringiensis subsp. israelensis are correlated with a conjugation-like plasmid transfer and characterized by the formation of aggregates when the bacteria are socialized during exponential growth. We present evidence for the association of the Agr+ phenotype with the presence of the large (135-MDa) self-transmissible plasmid pXO16.

Ongoing activity of RNA polymerase II confers preferential repair of nitrogen mustard-induced N-alkylpurines in the hamster dihydrofolate reductase gene.

Recently, it has been demonstrated that nitrogen mustard-induced N-alkylpurines are excised rapidly from actively transcribing genes, while they persist longer in noncoding regions and in the genome overall. It was suggested that transcriptional activity is implicated as a regulatory element in the efficient removal of lesions. By treating cells or not with the transcription inhibitor alpha-amanitin, we have explored whether ongoing activity of RNA polymerase II was coordinately related to proficient repair of nitrogen mustard-induced alkylation products in the actively transcribed dihydrofolate reductase gene in the Chinese hamster ovary B11 cells. Nuclear run-off transcription analysis verified that alpha-amanitin completely and selectively inhibited transcription by RNA polymerase II. At the drug exposure examined, nitrogen mustard induced DNA damage capable of a complete transcription termination in the RNA polymerase II-transcribed dihydrofolate reductase gene and reduced 28S rDNA transcription by a factor of 7.9. The transcription activity did partially recover following reincubation in drug-free medium; this recovery was about 34 and 76% of ribosomal 28S gene transcripts and dihydrofolate reductase gene transcripts, respectively, after 6 h of repair incubation. alpha-Amanitin significantly inhibited the removal of nitrogen mustard-induced N-alkylpurines in the 5'-half of the essential, constitutively active dihydrofolate reductase gene, while no effect of alpha-amanitin was observed on the lesion removal from a noncoding region 3'-flanking to the gene and from the genome overall. In the actively transcribed gene region, about 77% of N-alkylpurines were removed 21 h following drug exposure of cells not treated with alpha-amanitin and about 47% in 21 h in alpha-amanitin treated cells. The global semiconservative replication seemed unaffected by the alpha-amanitin treatment. From these results we suggest that gene-specific repair of nitrogen mustard-induced N-alkylpurines is dependent on ongoing activity of the transcribing RNA polymerase II. The findings are discussed in terms of the current ideas about the mechanism of preferential DNA repair.

Complete nucleotide sequence of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3 and its correlation with biological properties.

The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp. israelensis has been determined. The circular DNA molecule was 7649 bp and had a G + C content of 35.1%. Twenty-two open reading frames larger than 50 codons were identified. Ten of these open reading frames are suggested to be protein coding regions. The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli. Even though the rep14-3 gene was expressed in E. coli the plasmid pTX14-3 was unable to replicate in this bacterium. The minimal region of the plasmid pTX14-3 required for replication in B. thuringiensis was identified. Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation. Extensive sequence homology with the B. thuringiensis subsp. thuringiensis plasmid pGI2 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3. A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa. 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity). The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation.

Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3, between strains of B. thuringiensis subsp. israelensis were examined. Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h. Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006. However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B. thuringiensis subsp. israelensis. A specific protease-sensitive coaggregation between strains of B. thuringiensis subsp. israelensis was found to be unambiguously correlated with plasmid transfer. Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies. Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution. Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented. Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell. The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid. No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.