Peculiar distribution of adipose tissue in patients with congenital generalized lipodystrophy.

Congenital generalized lipodystrophy (CGL) is a rare genetic disease with extreme paucity of fat from birth which is believed to be generalized, involving the whole body. Affected patients are characterized by severe insulin resistance. Sites of adipose tissue distribution in patients with CGL have not been studied systematically. Therefore, the fat distribution in three women (17-20 yr old) with CGL was investigated. Determination of body composition by underwater volume displacement suggested the complete absence of body fat (range, -3 to -7%; normal, 15-25%). Whole body magnetic resonance imaging, however, detected fat in particular anatomical sites, namely in orbits, palms and soles, and periarticular and epidural regions. Some fat was also localized in the tongue, breasts, vulva, and buccal area. Fat in other subcutaneous areas, intraabdominal and intrathoracic regions, and bone marrow was essentially absent. Thus, patients with CGL do not have a complete absence of body fat; of interest, fat is present in those sites where adipose tissue may be serving mainly a mechanical function. Patients with CGL, therefore, provided a unique opportunity to identify the various sites of localization of "mechanical" adipose tissue in the human body. Our study suggests that the genetic defect in CGL results in poor growth and development of metabolically active adipose tissue, whereas mechanical adipose tissue is well preserved.     

Regulation of neutrophil and eosinophil secondary granule gene expression by transcription factors C/EBP epsilon and PU.1

In the bone marrow of C/EBP epsilon(-/-) mice, expression of neutrophil secondary and tertiary granule mRNAs is absent for lactoferrin (LF), neutrophil gelatinase (NG), murine cathelin-like protein (MCLP), and the cathelin B9; it is severely reduced for neutrophil collagenase (NC) and neutrophil gelatinase-associated lipocalin (NGAL). In addition, the expression of eosinophil granule genes, major basic protein (MBP), and eosinophil peroxidase (EPX) is absent. These mice express C/EBP alpha, C/EBP beta, and C/EBP delta in the bone marrow at levels similar to those of their wild-type counterparts, suggesting a lack of functional redundancy among the family in vivo. Stable inducible expression of C/EBP epsilon and C/EBP alpha in the murine fibroblast cell line NIH 3T3 activated expression of mRNAs for B9, MCLP, NC, and NGAL but not for LF. In transient transfections of C/EBP epsilon and C/EBP alpha, B9 was strongly induced with weaker induction of the other genes. C/EBP beta and C/EBP delta proteins weakly induced B9 expression, but C/EBP delta induced NC expression more efficiently than the other C/EBPs. The expression of MBP was inefficiently induced by C/EBP epsilon alone and weakly induced with C/EBP epsilon and GATA-1, but the addition of PU.1 resulted in a striking cooperative induction of MBP in NIH 3T3 cells. Mutation of a predicted PU.1 site in the human MBP promoter-luciferase reporter construct abrogated the response to PU.1. Gel-shift analysis demonstrated binding of PU.1 to this site. MBP and EPX mRNAs were absent in a PU.1-null myeloid cell line established from the embryonic liver of PU.1(-/-) mice. Restitution of PU.1 protein expression restored MBP and EPX protein expression. This study demonstrates that C/EBP epsilon is essential and sufficient for the expression of a particular subset of neutrophil secondary granule genes. Furthermore, it indicates the importance of PU.1 in the cooperative activation of eosinophil granule genes.     

Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.

C3, the third component of complement, is critical in the host immune response in that it is involved in both the classical and alternative pathways of complement activation. We have previously shown that a region (bp -127 to -70) within the C3 promoter is indispensable for conferring interleukin 1 (IL-1) responsiveness to this gene. A sequence comparison reveals two CCAAT/enhancer binding protein (C/EBP) consensus sequences, basic DNA binding region and leucine zippers 1 and 2 (bZIP1 and bZIP2), within this region. Site-directed mutagenesis of the more 3' C/EBP site (bZIP1) in the C3 promoter significantly reduced the basal level of expression and the IL-1 responsiveness of the reporter gene, whereas mutation in the second, more 5', C/EBP consensus sequence (bZIP2) had a minimal effect on basal expression and IL-1 inducibility. Electrophoretic-mobility-shift assays, with and without antibodies to the different C/EBP proteins that "supershift" protein-DNA complexes, demonstrated that proteins binding at the 3' C/EBP site formed several complexes. Antibodies to C/EBP alpha supershifted the majority of complexes formed with extracts from control cells. Antibodies directed against C/EBP delta supershifted the major IL-1-inducible complexes. Western immunoblot analyses showed that the level of C/EBP delta protein was increased dramatically in the nuclei of Hep 3B2 cells after 4 h of IL-1 treatment. When Hep 3B2 cells were cotransfected with a C/EBP delta expression vector and a construct with a C3 promoter and a reporter gene, C/EBP delta was able to trans-activate the C3 promoter in an IL-1-responsive manner. The data strongly suggest that C/EBP delta is the major protein responsible for regulating the acute-phase expression of the human C3 gene.     

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

Obesity and the metabolic syndrome have evolved to be major health issues throughout the world. Whether loss of genome integrity contributes to this epidemic is an open question. DNA polymerase η (pol η), encoded by the xeroderma pigmentosum (XP-V) gene, plays an essential role in preventing cutaneous cancer caused by UV radiation-induced DNA damage. Herein, we demonstrate that pol η deficiency in mice (pol η^−/−) causes obesity with visceral fat accumulation, hepatic steatosis, hyperleptinemia, hyperinsulinemia, and glucose intolerance. In comparison to WT mice, adipose tissue from pol η^−/− mice exhibits increased DNA damage and a greater DNA damage response, indicated by up-regulation and/or phosphorylation of ataxia telangiectasia mutated (ATM), phosphorylated H₂AX (γH₂AX), and poly[ADP-ribose] polymerase 1 (PARP-1). Concomitantly, increased cellular senescence in the adipose tissue from pol η^−/− mice was observed and measured by up-regulation of senescence markers, including p53, p16^Ink4a, p21, senescence-associated (SA) β-gal activity, and SA secretion of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) as early as 4 wk of age. Treatment of pol η^−/− mice with a p53 inhibitor, pifithrin-α, reduced adipocyte senescence and attenuated the metabolic abnormalities. Furthermore, elevation of adipocyte DNA damage with a high-fat diet or sodium arsenite exacerbated adipocyte senescence and metabolic abnormalities in pol η^−/− mice. In contrast, reduction of adipose DNA damage with N-acetylcysteine or metformin ameliorated cellular senescence and metabolic abnormalities. These studies indicate that elevated DNA damage is a root cause of adipocyte senescence, which plays a determining role in the development of obesity and insulin resistance.The human genome is constantly challenged by exogenous and endogenous DNA damaging agents. To ensure genome integrity, human cells have faithful DNA replication machinery and DNA repair systems that are coordinated by DNA damage response networks. In response to different extents or types of DNA lesions, the DNA damage response activates appropriate cellular responses, including transient or permanent (senescence) cell cycle arrest, or apoptosis, to minimize the detrimental effects of DNA lesions (1). Reduction or deficiency in DNA repair/replication enzyme activity is well documented to increase vulnerability for the development of cancer, neurodegenerative diseases, and aging (2). In addition, defective DNA repair enzymes are associated with the metabolic symptom; for example, DNA glycosylase (Neil1)- and OGG1-deficient mice are obese (3–5), and nucleotide excision repair protein ERCC1-XPF deficiency causes lipodystrophy (6). Furthermore, DNA damage response protein ataxia telangiectasia mutated (ATM) suppresses JNK activity through p53 signaling and mediates an antioxidant action that has been suggested to be relevant to the metabolic syndrome (7). Nucleotide excision repair XP-A protein may affect metabolism by altering mitochondrial function (8). To date, the mechanisms that link genome instability and metabolic dysregulation have not been elucidated.DNA polymerase η (pol η) is a specialized lesion bypass polymerase that faithfully replicates across UV-induced cyclobutane pyrimidine dimers (9) to rescue stalled DNA replication forks from potential breakages and mutations. Defects in the gene encoding pol η produce a variant form of the autosomal recessive disease xeroderma pigmentosum (XP-V) (9). Patients with XP-V are highly sensitive to sunlight and prone to cutaneous cancer (9). In addition to skin, pol η is expressed in most tissues (10). The expression of pol η correlates with the effectiveness of anticancer therapeutic agents that exert their activity by introducing DNA lesions that block the progression of DNA replication (11). In addition to exogenous introduced DNA lesions, pol η contributes to genomic stability during unperturbed DNA replication (12) and replicates across reactive oxygen species (ROS)-induced oxidative DNA lesions 8-oxoG (7,8-dihydro-8-oxoguanine), thymine glycol, and lipid peroxidation DNA adducts generated during endogenous metabolic processes (13, 14). ROS generated from endogenous metabolism or exogenous sources has been associated with cancer, aging, and the metabolic syndrome.Therefore, the initial hypothesis for our studies was that pol η contributes to reduce tumorigenesis by managing oxidative DNA lesions in other organs. However, in a 2-y study, no increase in the incidence of tumors of any type was found with pol η KO (pol η^−/−) mice (15). Instead, we found pol η^−/− mice develop metabolic abnormalities that induce obesity.     

Histological and molecular features of lipomatous and nonlipomatous adipose tissue in familial partial lipodystrophy caused by LMNA mutations

Objectives Type 2 familial partial lipodystrophy (FPLD2) is a rare adipose tissue (AT) disease caused by mutations in LMNA, in which lipomas appear occasionally. In this study, we aimed to histologically characterize FPLD2‐associated lipomatosis and study the expression of genes and proteins involved in cell cycle control, mitochondrial function, inflammation and adipogenesis. Design and patients One lipoma and perilipoma fat from each of four subjects with FPLD2 and 10 control subjects were analysed by optical microscopy. The presence of inflammatory cells was evaluated by immunohistochemistry. Real‐time RT‐PCR and Western blot were used to evaluate gene and protein levels. Results Adipocytes from lipodystrophic patients were significantly larger than those of controls, in both the lipomas and perilipoma fat. Lipodystrophic AT exhibited CD68⁺ macrophages and CD3⁺ lymphocytes infiltration. TP53 expression was reduced in all types of lipomas. At protein level, C/EBPβ, p53 and pRb were severely disturbed in both lipodystrophic lipomas and perilipoma fat coming from lipoatrophic areas, whereas the expression of CEBPα was normal. Mitochondrial function genes were less expressed in lipoatrophic fat. In both lipomas and perilipoma fat from lipoatrophic areas, the expression of adipogenes was lower than controls. Conclusions Even in lipomas, the adipogenic machinery is impaired in lipodystrophic fat coming from lipoatrophic regions in FPLD2, although the histological phenotype is near‐normal, exhibiting low‐grade inflammatory features. Our results suggest that the p53 pathway and some adipogenic proteins, such as CEBPα, could contribute to the maintenance of this near normal phenotype in the remnant AT present in these patients.