New insights into adipocyte-specific leptin gene expression

The adipocyte-derived hormone leptin is a critical regulator of many physiological functions, ranging from satiety to immunity. Surprisingly, very little is known about the transcriptional pathways that regulate adipocyte-specific expression of leptin. In a recent published study, we pursued a strategy integrating BAC transgenic reporter mice, in vitro reporter assays, and chromatin state mapping to locate an adipocyte-specific cis-element upstream of the LEP gene in human fat cells. Quantitative proteomics (stable isotope labeling by amino acids in cell culture, SILAC) with affinity enrichment of protein-DNA complexes identified the transcription factor FOSL2 as a specific binder to the identified region. We confirmed that FOSL2 is an important regulator of LEP gene expression in vitro and in vivo using cell culture models and genetic mouse models. In this commentary, we discuss the transcriptional regulation of LEP gene expression, our strategy to identify an adipocyte-specific cis-regulatory element and the transcription factor(s) responsible for LEP gene expression. We also discuss our data on FOSL2 and leptin levels in physiology and pathophysiology. We speculate on unanswered questions and future directions

Human NK Cell Subset Functions Are Differentially Affected by Adipokines

Background: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Exercise-induced CITED4 expression is necessary for regional remodeling of cardiac microstructural tissue helicity

Both exercise-induced molecular mechanisms and physiological cardiac remodeling have been previously studied on a whole heart level. However, the regional microstructural tissue effects of these molecular mechanisms in the heart have yet to be spatially linked and further elucidated. We show in exercised mice that the expression of CITED4, a transcriptional co-regulator necessary for cardioprotection, is regionally heterogenous in the heart with preferential significant increases in the lateral wall compared with sedentary mice. Concordantly in this same region, the heart's local microstructural tissue helicity is also selectively increased in exercised mice. Quantification of CITED4 expression and microstructural tissue helicity reveals a significant correlation across both sedentary and exercise mouse cohorts. Furthermore, genetic deletion of CITED4 in the heart prohibits regional exercise-induced microstructural helicity remodeling. Taken together, CITED4 expression is necessary for exercise-induced regional remodeling of the heart's microstructural helicity revealing how a key molecular regulator of cardiac remodeling manifests into downstream local tissue-level changes. Expression of transcription factor CITED4 is necessary for exercise-induced regional remodeling of the heart's microstructural helicity, revealing how a key molecular regulator of cardiac remodeling mediates local tissue-level changes

Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging

SummaryEver since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible

Phosphorylation Provides a Negative Mode of Regulation for the Yeast Rab GTPase Sec4p

The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p

Human NK cell subset functions are differentially affected by adipokines.

BACKGROUND: Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host's primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. METHODS: Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). RESULTS: FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56(dim) NK cells. The production of GzmA in CD56(bright) NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. CONCLUSION: Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation

Altered NK cell function in obese healthy humans

Background: Obesity is associated with an elevated risk for several types of cancer and thus a major health hazard. However, the mechanism between overweight and cancer susceptibility is still elusive. Leptin, mainly produced by adipocytes links food intake and energy expenditure. In addition, recent studies have shown an immunomodulatory impact of leptin on NK cells. The purpose of the present study was to investigate whether leptin stimulation of NK cells from obese humans leads to altered functions as compared to NK cells from lean subjects. On the basis of body mass index 20 healthy individuals were classified in two groups: normal weight (30 kg/m2). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples. We used flow cytometry to assess differences in phenotype and activity markers (CD107a, CD178 and TRAIL) of PBMCs between both groups. Furthermore, we determined after short-term in vitro leptin stimulation the phosphorylation of JAK2, downstream target of the intracellular signaling cascade of the leptin receptor, by Western Blotting and numbers of NK-cell-tumor-cell-conjugates as well as Granzyme+ and IFN-γ+ NK cells by flow cytometry. Finally, the proliferative capacity of control and long-term (7 days) leptin-stimulated NK cells was examined. Results: As opposed to similar NK cell counts, the number of CD3+CD56+ cells was significantly lower in obese compared to lean subjects. Human NK cells express the leptin receptor (Ob-R). For further determination of Ob-R, intracellular target proteins of PBMCs were investigated by Western Blotting. Phosphorylation of JAK2 was lower in obese as compared to normal weight subjects. Furthermore, significantly lower levels of TNF-related apoptosis-inducing ligand (TRAIL) as an NK cell functional marker in obese subjects were found. In vitro leptin stimulation resulted in a higher production of interferon-γ in NK cells of normal weight subjects. Interestingly, long-term leptin stimulation had no significant influence on numbers of proliferating NK cells. Conclusions: NK cells from obese healthy humans show functional deficits and altered responses after in vitro leptin challenge

Obesity and the associated mediators leptin, estrogen and IGF-I enhance the cell proliferation and early tumorigenesis of breast cancer cells

Breast cancer continues to be a major cause of cancer deaths in women. Estrogen, which is also produced by the adipose tissue, is held responsible for the elevated risk of breast cancer in obese women. However, the adipose tissue secrets hormones and adipokines such as leptin and IGF-I and these substances could also contribute to an increased breast cancer risk for obese women. In this study, the impact of obesity on cell proliferation was investigated. The carcinogen 7, 12, dimethylbenz[a]anthracene (DMBA) was administered to normal weight and diet-induced obese female Sprague-Dawley rats. Cell proliferation was evaluated by immunohistological staining of BrdU-incorporation. In the mammary glands and inguinal lymphatic nodes of the obese rats, cell proliferation was significantly increased, indicating a significant influence of obesity on breast cancer. Effects of leptin, estrogen, and IGF-I on the proliferation of MCF-7 cells in vitro were assessed using an MTT assay. Cell culture experiments demonstrated a mitogenic role of these three mediators on cell proliferation. Our data demonstrate a stimulative effect of substances produced by the adipose tissue on breast cancer. Body weight specific cell proliferation suggests that obesity-related adipokines and mediators enhance cell proliferation and increase the risk for breast cancer

PP, PYY and NPY : synthesis, storage, release and degradation

Peptides of the NPY family are synthesized as large precursor molecules in the endoplasmatic reticulum (ER), where posttranslational modification takes place, and from where they are translocated to the Golgi apparatus. After several structural and functional adjustments, two types of mature vesicle - large dense core vesicles and synaptic vesicles - serve as a storage depot. Notably, the expression of a gene for a peptide from the NPY family is not sufficient to ensure the production of mature pep tides since several posttranslational steps are specifically involved and these steps themselves are subjected to specific regulatory processes. Similarly, after exocytotic release of NPY-like peptides, their local action depends on their concentration, their different receptor selectivity and the local expression of the different Y-receptors. Another major player in this complex network is found in the action of specific peptidases influencing half-life and receptor selectivity. At least aminopeptidase P, dipeptidylpeptidase IV-like enzymes, specific endopeptidases, like meprin or neprilysin-like enzymes, and post-arginine hydrolyzing endoproteases are cleaving enzymes for NPY-like peptides. Due to a striking change of receptor specificity after N-terminal cleavage of NPY-like peptides, the development of inhibitors for NPY, PYY and PP cleaving peptidases is a complementary approach to the development of Y-receptor agonists or antagonists. In this chapter, we summarize key findings about synthesis, storage, release and localization of NPY family peptides, add recent findings on their degradation by specific enzymes and discuss implications for the interpretation of studies in future research