Obesity-induced changes in lipid mediators persist after weight loss.

BACKGROUND: Obesity induces significant changes in lipid mediators, however, the extent to which these changes persist after weight loss has not been investigated. SUBJECTS/METHODS: We fed C57BL6 mice a high-fat diet to generate obesity and then switched the diet to a lower-fat diet to induce weight loss. We performed a comprehensive metabolic profiling of lipid mediators including oxylipins, endocannabinoids, sphingosines and ceramides in key metabolic tissues (including adipose, liver, muscle and hypothalamus) and plasma. RESULTS: We found that changes induced by obesity were largely reversible in most metabolic tissues but the adipose tissue retained a persistent obese metabolic signature. Prostaglandin signaling was perturbed in the obese state and lasting increases in PGD2, and downstream metabolites 15-deoxy PGJ2 and delta-12-PGJ2 were observed after weight loss. Furthermore expression of the enzyme responsible for PGD2 synthesis (hematopoietic prostaglandin D synthase, HPGDS) was increased in obese adipose tissues and remained high after weight loss. We found that inhibition of HPGDS over the course of 5 days resulted in decreased food intake in mice. Increased HPGDS expression was also observed in human adipose tissues obtained from obese compared with lean individuals. We then measured circulating levels of PGD2 in obese patients before and after weight loss and found that while elevated relative to lean subjects, levels of this metabolite did not decrease after significant weight loss. CONCLUSIONS: These results suggest that lasting changes in lipid mediators induced by obesity, still present after weight loss, may play a role in the biological drive to regain weight.

Omega-3 fatty acids reduce obesity-induced tumor progression independent of GPR120 in a mouse model of postmenopausal breast cancer.

Obesity and inflammation are both risk factors for a variety of cancers, including breast cancer in postmenopausal women. Intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of breast cancer, and also reduces obesity-associated inflammation and insulin resistance, but whether the two effects are related is currently unknown. We tested this hypothesis in a postmenopausal breast cancer model using ovariectomized, immune-competent female mice orthotopically injected with Py230 mammary tumor cells. Obesity, whether triggered genetically or by high-fat diet (HFD) feeding, increased inflammation in the mammary fat pad and promoted mammary tumorigenesis. The presence of tumor cells in the mammary fat pad further enhanced the local inflammatory milieu. Tumor necrosis factor-alpha (TNF-α) was the most highly upregulated cytokine in the obese mammary fat pad, and we observed that TNF-α dose-dependently stimulated Py230 cell growth in vitro. An ω-3 PUFA-enriched HFD (referred to as fish oil diet, FOD) reduced inflammation in the obese mammary fat pad in the absence of tumor cells and inhibited Py230 tumor growth in vivo. Although some anti-inflammatory effects of ω-3 PUFAs were previously shown to be mediated by the G-protein-coupled receptor 120 (GPR120), the FOD reduced Py230 tumor burden in GPR120-deficient mice to a similar degree as observed in wild-type mice, indicating that the effect of FOD to reduce tumor growth does not require GPR120 in the host mouse. Instead, in vitro studies demonstrated that ω-3 PUFAs act directly on tumor cells to activate c-Jun N-terminal kinase, inhibit proliferation and induce apoptosis. Our results show that obesity promotes mammary tumor progression in this model of postmenopausal breast cancer and that ω-3 PUFAs, independent of GPR120, inhibit mammary tumor progression in obese mice.

Mechanisms of human insulin resistance and thiazolidinedione-mediated insulin sensitization.

Cellular and tissue defects associated with insulin resistance are coincident with transcriptional abnormalities and are improved after insulin sensitization with thiazolidinedione (TZD) PPARgamma ligands. We characterized 72 human subjects by relating their clinical phenotypes with functional pathway alterations. We transcriptionally profiled 364 biopsies harvested before and after hyperinsulinemic-euglycemic clamp studies, at baseline and after 3-month TZD treatment. We have identified molecular and functional characteristics of insulin resistant subjects and distinctions between TZD treatment responder and nonresponder subjects. Insulin resistant subjects exhibited alterations in skeletal muscle (e.g., glycolytic flux and intramuscular adipocytes) and adipose tissue (e.g., mitochondrial metabolism and inflammation) that improved relative to TZD-induced insulin sensitization. Pre-TZD treatment expression of MLXIP in muscle and HLA-DRB1 in adipose tissue from insulin resistant subjects was linearly predictive of post-TZD insulin sensitization. We have uniquely characterized coordinated cellular and tissue functional pathways that are characteristic of insulin resistance, TZD-induced insulin sensitization, and potential TZD responsiveness.

Increased instability of human CTG repeat tracts on yeast artificial chromosomes during gametogenesis.

Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.

Cis-acting determinants affecting centromere function, sister-chromatid cohesion and reciprocal recombination during meiosis in Saccharomyces cerevisiae.

We have employed a system that utilizes homologous pairs of human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to assess the specific role(s) of conserved centromere DNA elements (CDEI, CDEII and CDEIII) in meiotic chromosome disjunction fidelity. Thirteen different centromere (CEN) mutations were tested for their effects on meiotic centromere function. YACs containing a wild-type CEN DNA sequence segregate with high fidelity in meiosis I (99% normal segregation) and in meiosis II (96% normal segregation). YACs containing a 31-bp deletion mutation in centromere DNA element II (CDEII delta 31) in either a heterocentric (mutant/wild type), homocentric (mutant/mutant) or monosomic (mutant/--) YAC pair configuration exhibited high levels (16-28%) of precocious sister-chromatid segregation (PSS) and increased levels (1-6%) of nondisjunction meiosis I (NDI). YACs containing this mutation also exhibit high levels (21%) of meiosis II nondisjunction. Interestingly, significant alterations in homolog recombination frequency were observed in the exceptional PSS class of tetrads, suggesting unusual interactions between prematurely separated sister chromatids and their homologous nonsister chromatids. We also have assessed the meiotic segregation effects of rare gene conversion events occurring at sites located immediately adjacent to or distantly from the centromere region. Proximal gene conversion events were associated with extremely high levels (60%) of meiosis I segregation errors (including both PSS and NDI), whereas distal events had no apparent effect. Taken together, our results indicate a critical role for CDEII in meiosis and underscore the importance of maintaining sister-chromatid cohesion for proper recombination in meiotic prophase and for proper disjunction in meiosis I.

An implanted recombination hot spot stimulates recombination and enhances sister chromatid cohesion of heterologous YACs during yeast meiosis.

Heterologous yeast artificial chromosomes (YACs) do not recombine with each other and missegregate in 25% of meiosis I events. Recombination hot spots in the yeast Saccharomyces cerevisiae have previously been shown to be associated with sites of meiosis-induced double-strand breaks (DSBs). A 6-kb fragment containing a recombination hot spot/DSB site was implanted onto two heterologous human DNA YACs and was shown to cause the YACs to undergo meiotic recombination in 5-8% of tetrads. Reciprocal exchanges initiated and resolved within the 6-kb insert. Presence of the insert had no detectable effect on meiosis I nondisjunction. Surprisingly, the recombination hot spots acted in cis to significantly reduce precocious sister-chromatid segregation. This novel observation suggests that DSBs are instrumental in maintaining cohesion between sister chromatids in meiosis I. We propose that this previously unknown function of DSBs is mediated by the stimulation of sister-chromatid exchange and/or its intermediates.

Meiotic recombination and segregation of human-derived artificial chromosomes in Saccharomyces cerevisiae.

We have developed a system that utilizes human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to study factors that contribute to the fidelity of meiotic chromosome transmission. Since aneuploidy for the YACs does not affect spore viability, different classes of meiotic missegregation can be scored accurately in four-viable-spore tetrads including precocious sister separation, meiosis I nondisjunction, meiotic chromatid loss, and meiosis II nondisjunction. Segregation of the homologous pair of 360-kilobase marker YACs was shown to occur with high fidelity in the first meiotic division and was associated with a high frequency of recombination within the human DNA segment. By using this experimental system, a series of YAC deletion derivatives ranging in size from 50 to 225 kilobases was analyzed to directly assess the relationship between meiotic recombination and meiosis I disjunction in a genotypically wild-type background. The relationship between physical distance and recombination frequency within the human DNA segment was measured to be comparable to that of endogenous yeast chromosomal DNA--ranging from less than 2.0 to 7.7 kilobases/centimorgan. Physical analysis of recombinant chromosomes detected no unequal crossing-over at dispersed repetitive elements distributed along the YACs. Recombination between YACs containing unrelated DNA segments was not observed. Furthermore, the segregational data indicated that meioses in which YAC pairs failed to recombine exhibited dramatically increased levels of meiosis I missegregation, including both precocious sister chromatid separation and nondisjunction.