Treatment with TUG891, a free fatty acid receptor 4 agonist, restores adipose tissue metabolic dysfunction following chronic sleep fragmentation in mice.

BACKGROUND: Sleep fragmentation (SF), a frequent occurrence in multiple sleep and other diseases leads to increased food intake and insulin resistance via increased macrophage activation and inflammation in visceral white adipose tissue (VWAT). Free fatty acid receptor 4 (FFA4) is reduced in pediatric sleep apnea patients and FFA4 agonists have been proposed in the treatment of obesity and metabolic dysfunction. METHODS: Male mice were subjected to SF exposures for 6 weeks, and treated during the last 2 weeks with either TUG891, a potent and selective FFA4 agonist, or vehicle (Veh). Glucose and insulin tolerance tests and VWAT insulin sensitivity tests were conducted (phosphorylated Akt/total Akt), along with flow cytometric assessments of VWAT macrophage polarity, and T-cell lymphocyte subsets. RESULTS: SF-TUG891 mice showed reduction in food consumption, weight gain and VWAT mass. Furthermore, TUG891 treatment ameliorated glucose tolerance test and insulin tolerance test responses and increased VWAT p-Akt/Akt responses to insulin. Increases in M1/M2 macrophages and decreased Treg counts in VWAT associated with SF were markedly improved by TUG891, and VWAT macrophages from TUG891-treated mice had markedly attenuated insulin resistance effects on naïve cultured adipocytes. CONCLUSIONS: Treatment with an FFA4 agonist reverses SF-induced food intake increases and gains in body weight, and significantly attenuates VWAT inflammation and insulin resistance. Thus, interventional dietary or pharmaceutical strategies aimed at increasing FFA4 activity may serve as potentially useful adjunctive therapies for sleep disorders accompanied by metabolic morbidity.

Epigenomic profiling in visceral white adipose tissue of offspring of mice exposed to late gestational sleep fragmentation.

BACKGROUND: Sleep fragmentation during late gestation (LG-SF) is one of the major perturbations associated with sleep apnea and other sleep disorders during pregnancy. We have previously shown that LG-SF induces metabolic dysfunction in offspring mice during adulthood. OBJECTIVES: To investigate the effects of late LG-SF on metabolic homeostasis in offspring and to determine the effects of LG-SF on the epigenome of visceral white adipose tissue (VWAT) in the offspring. METHODS: Time-pregnant mice were exposed to LG-SF or sleep control during LG (LG-SC) conditions during the last 6 days of gestation. At 24 weeks of age, lipid profiles and metabolic parameters were assessed in the offspring. We performed large-scale DNA methylation analyses using methylated DNA immunoprecipitation (MeDIP) coupled with microarrays (MeDIP-chip) in VWAT of 24-week-old LG-SF and LG-SC offspring (n=8 mice per group). Univariate multiple-testing adjusted statistical analyses were applied to identify differentially methylated regions (DMRs) between the groups. DMRs were mapped to their corresponding genes, and tested for potential overlaps with biological pathways and gene networks. RESULTS: We detected significant increases in body weight (31.7 vs 28.8 g; P=0.001), visceral (642.1 vs 497.0 mg; P=0.002) and subcutaneous (293.1 vs 250.1 mg; P=0.001) fat mass, plasma cholesterol (110.6 vs 87.6 mg dl(-1); P=0.001), triglycerides (87.3 vs 84.1 mg dl(-1); P=0.003) and homeostatic model assessment-insulin resistance values (8.1 vs 6.1; P=0.007) in the LG-SF group. MeDIP analyses revealed that 2148 DMRs (LG-SF vs LG-SC; P<0.0001, model-based analysis of tilling-arrays algorithm). A large proportion of the DMR-associated genes have reported functions that are altered in obesity and metabolic syndrome, such as Cartpt, Akt2, Apoe, Insr1 and so on. Overrepresented pathways and gene networks were related to metabolic regulation and inflammatory response. CONCLUSIONS: Our findings show a major role for epigenomic regulation of pathways associated with the metabolic processes and inflammatory responses in VWAT. LG-SF-induced epigenetic alterations may underlie increases in the susceptibility to obesity and metabolic syndrome in the offspring.

Sleep fragmentation promotes NADPH oxidase 2-mediated adipose tissue inflammation leading to insulin resistance in mice.

BACKGROUND: Short sleep has been implicated in higher risk of obesity in humans, and is associated with insulin resistance. However, the effects of fragmented sleep (SF) rather than curtailed sleep on glucose homeostasis are unknown. METHODS: Wild-type and NADPH oxidase 2 (Nox2) null male mice were subjected to SF or sleep control conditions for 3 days to 3 weeks. Systemic and visceral adipose tissue (VAT) insulin sensitivity tests, glucose tolerance test, fluorescence-activated cell sorting and immunohistochemistry for macrophages and its sub-types (M1 and M2), and Nox expression and activity were examined. RESULTS: Here we show that SF in the absence of sleep curtailment induces time-dependent insulin resistance, in vivo and also in vitro in VAT. Oxidative stress pathways were upregulated by SF in VAT, and were accompanied by M1 macrophage polarization. SF-induced oxidative stress, inflammation and insulin resistance in VAT were completely abrogated in genetically altered mice lacking Nox2 activity. CONCLUSIONS: These studies imply that SF, a frequent occurrence in many disorders and more specifically in sleep apnea, is a potent inducer of insulin resistance via activation of oxidative stress and inflammatory pathways, thereby opening the way for therapeutic strategies.

Effects of late gestational high-fat diet on body weight, metabolic regulation and adipokine expression in offspring.

AIMS/HYPOTHESIS: Gestational exposures such as dietary changes can alter offspring phenotype through epigenetic modifications and promote increased risk for specific diseases, such as metabolic syndrome. We hypothesized that high-fat diet (HFD) during late gestation would lead increased risk for insulin resistance and hyperlipidemia via associated epigenetic alterations in tissue adipocytokine genes. METHODS: Offspring mice of mothers fed a HFD during late gestation (HFDO) were weighed and their food intake measured weekly till age 20 weeks at which time glucose and insulin tolerance tests, plasma lipid and adipocytokine levels were assessed, as well as mRNA expression in visceral fat. Adipocytokine gene methylation levels in visceral fat, liver and muscle were also assayed. RESULTS: HFDO mice had increased weight accrual and food intake, and exhibited insulin resistance, hyperlipidemia and hyperleptinemia, as well as hypoadiponectinemia. Furthermore, increased methylation of adiponectin and leptin receptor, and decreased methylation of leptin genes with unchanged glucagon-like peptide-1 methylation patterns emerged in HFDO mice. CONCLUSIONS: Taken together, late gestational HFD induces increased risk of metabolic syndrome in the progeny, which is coupled with hypoadiponectinemia as well as with leptin resistance, and concomitant presence of selective tissue-based epigenetic changes among adipocytokine genes.

Deciphering the lithium transcriptome: microarray profiling of lithium-modulated gene expression in human neuronal cells.

The mechanisms underlying lithium's therapeutic efficacy in the chronic treatment of bipolar disorder are not clearly understood. Useful insights can be obtained by identifying genes that are differentially regulated during chronic lithium treatment. Toward this end, we have used microarray technology to identify mRNAs that are differentially expressed in a human neuronal cell line that has been continuously maintained in therapeutic levels of lithium for 33 days. Significantly, unlike other transcriptomes where predominantly rodent cells were used and a limited number of genes probed, we have used human cells probed with more extensive 44,000 gene microarrays. A total of 671 differentially regulated transcripts, after correcting for false discovery rates, were identified, of which 347 and 324, respectively, were found to be up- and downregulated. Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, was the most upregulated while tribbles homolog 3 (TRB3), a pro-apoptotic protein, was the most downregulated, implying a beneficial effect of lithium on neuronal cells. Several of the most highly regulated genes are novel, uncharacterized and encode proteins of unknown function. Differentially expressed genes associated with phosphoinositide metabolism include those encoding phosphatidyl inositol 4-phosphate 5-kinase type II alpha (PIP5K2A), WD repeat domain, phosphoinositide interacting 1 protein (WIPI49), tribbles homolog 3 (TRB3) and sorting nexin 14 (SNX14). A protein interactome using some of the saliently regulated genes identified protein kinase C (PKC) as a major target for lithium action while a global analysis of all 671 differentially expressed genes identified the mitogen-activated protein kinase pathway as the most regulated. The list of highly regulated genes, besides encoding putative targets for antimanic agents, should prove useful in defining novel pathways, or to better understand the mechanisms, underlying the mood stabilization process.

Characterization of elongation factor-1A (eEF1A-1) and eEF1A-2/S1 protein expression in normal and wasted mice.

The eEF1Alpha-2 gene (S1) encodes a tissue-specific isoform of peptide elongation factor-1A (eEF1A-1); its mRNA is expressed only in brain, heart, and skeletal muscle, tissues dominated by terminally differentiated, long-lived cells. Homozygous mutant mice exhibit muscle wasting and neurodegeneration, resulting in death around postnatal day 28. eEF1Alpha-2/S1 protein shares 92% identity with eEF1A-1; because specific antibodies for each were not available previously, it was difficult to study the developmental expression patterns of these two peptide elongation factors 1A in wasted and wild-type mice. We generated a peptide-derived antiserum that recognizes the eEF1Alpha-2/S1 isoform and does not cross-react with eEF1A-1. We characterized the expression profiles of eEF1A-1 and eEF1A-2/S1 during development in wild-type (+/+), heterozygous (+/wst), and homozygous (wst/wst) mice. In wild-type and heterozygous animals, eEF1A-2/S1 protein is present only in brain, heart, and muscle; the onset of its expression coincides with a concomitant decrease in the eEF1A-1 protein level. In wasted mutant tissues, even though eEF1A-2/S1 protein is absent, the scheduled decline of eEF1A-1 occurs nonetheless during postnatal development, as it does in wild-type counterparts. In the brain of adult wild-type mice, the eEF1A-2/S1 isoform is localized in neurons, whereas eEF1A-1 is found in non-neuronal cells. In neurons prior to postnatal day 7, eEF1A-1 is the major isoform, but it is later replaced by eEF1A-2/S1, which by postnatal day 14 is the only isoform present. The postdevelopmental appearance of eEF1A-2/S1 protein and the decline in eEF1A-1 expression in brain, heart, and muscle suggest that eEF1A-2/S1 is the adult form of peptide elongation factor, whereas its sister is the embryonic isoform, in these tissues. The absence of eEF1A-2/S1, as well as the on-schedule development-dependent disappearance of its sister gene, eEF1A, in wst/wst mice may result in loss of protein synthesis ability, which may account for the numerous defects and ultimate fatality seen in these mice.

Toxin injury-dependent switched expression between EF-1 alpha and its sister, S1, in rat skeletal muscle.

Elongation factor-1 alpha, (EF-1 alpha), a translation factor involved in peptide chain elongation, is found ubiquitously in all cells. Previously, we identified a highly homologous EF-1 alpha sister gene, S1, whose transcript is found in only three tissues: brain, heart, and muscle, where the tissue-specific expression of S1 is caused by its exclusive presence in cells such as neurons and myocytes. Using sequence-specific synthetic peptides, we have recently produced polyclonal antibodies that can distinguish the protein product of EF-1 alpha from that of its sister, S1. Results of Western blotting show that these two proteins appear in S1-positive muscle tissue in inverse relationship, i.e., when S1 protein is in abundance, EF-1 alpha protein is in contrast in low quantity, and vice versa. During early embryonic stages, EF-1 alpha is the predominant protein species, whereas S1 is hardly detectable. This high EF-1 alpha versus low S1 protein presence undergoes a switch in that by postnatal day 14, EF-1 alpha is scarce whereas S1 is abundant; thus, there is a development-dependent shift of EF-1 alpha/S1 ratio from high to low, and the low EF-1 alpha/S1 ratio is maintained in adulthood. In this report, we describe the reversal of the EF-1 alpha/S1 ratio from low to high during muscle injury (experimentally induced by Marcaine injection), and a return to the original low ratio once the injury is repaired by regeneration. In this injury condition, EF-1 alpha is rapidly upregulated immediately after the Marcaine treatment, possibly reflecting an injury-dependent response of regeneration. The increase of EF-1 alpha corresponds with a decrease of S1 protein presence, thus resulting in a change of EF-1 alpha/S1 ratio from low to high. However, the high EF-1 alpha/S1 ratio eventually reverts to low, when regeneration-associated proliferation ceases, and fully differentiated myotubes are reestablished in the injured cells. This result shows that: (1) a high EF-1 alpha/S1 ratio is an early molecular diagnostic marker for injury-elicited regeneration; and (2) when injury repair is accomplished, there is a reversion to the low EF-1 alpha/S1 ratio, reflecting the restoration of the muscle fiber to the preinjury functional status. Results presented here not only show that a high EF-1 alpha/S1 ratio is a molecular marker for injured muscle, but also reveal the underpinning translational regulation in muscle during injury.