PBOV1 is a known human protein-coding gene with an uncharacterized function. We have previously found that PBOV1 lacks orthologs in non-primate genomes and is expressed in a wide range of tumor types. Here we report that PBOV1 protein-coding sequence is human-specific and has originated de novo in the primate evolution through a series of frame-shift and stop codon mutations. We profiled PBOV1 expression in multiple cancer and normal tissue samples and found that it was expressed in 19 out of 34 tumors of various origins but completely lacked expression in any of the normal adult or fetal human tissues. We found that, unlike the cancer/testis antigens that are typically controlled by CpG island-containing promoters, PBOV1 was expressed from a GC-poor TATA-containing promoter which was not influenced by CpG demethylation and was inactive in testis. Our analysis of public microarray data suggests that PBOV1 activation in tumors could be dependent on the Hedgehog signaling pathway. Despite the recent de novo origin and the lack of identifiable functional signatures, a missense SNP in the PBOV1 coding sequence has been previously associated with an increased risk of breast cancer. Using publicly available microarray datasets, we found that high levels of PBOV1 expression in breast cancer and glioma samples were significantly associated with a positive outcome of the cancer disease. We also found that PBOV1 was highly expressed in primary but not in recurrent high-grade gliomas, suggesting the presence of a negative selection against PBOV1-expressing cancer cells. Our findings could contribute to the understanding of the mechanisms behind de novo gene origin and the possible role of tumors in this process.

The transient activation of inflammatory networks is required for adipose tissue remodeling including the "browning" of white fat in response to stimuli such as 3-adrenergic receptor activation. In this process, white adipose tissue acquires thermogenic characteristics through the recruitment of so-called beige adipocytes. We investigated the downstream signaling pathways impinging on adipocyte progenitors that promote de novo formation of adipocytes. We showed that the Jak family of kinases controlled TGF signaling in the adipose tissue microenvironment through Stat3 and thereby adipogenic commitment, a function that was required for beige adipocyte differentiation of murine and human progenitors. Jak/Stat3 inhibited TGF signaling to the transcription factors Srf and Smad3 by repressing local Tgfb3 and Tgfb1 expression before the core transcriptional adipogenic cascade was activated. This pathway cross-talk was triggered in stromal cells by ATGL-dependent adipocyte lipolysis and a transient wave of IL-6 family cytokines at the onset of adipose tissue remodeling induced by 3-adrenergic receptor stimulation. Our results provide insight into the activation of adipocyte progenitors and are relevant for the therapeutic targeting of adipose tissue inflammatory pathways.

ObjectiveExtracellular matrix remodeling is required for adipose expansion under increased caloric intake. In turn, inhibited expandability due to aberrant collagen deposition promotes insulin resistance and progression towards the metabolic syndrome. An emerging role for the small leucine-rich proteoglycan Lumican in metabolically driven nonalcoholic fatty liver disease sparks an interest in further understanding its role in diet-induced obesity and metabolic complications.MethodsWhole body ablation of Lumican (Lum−/−) gene and adeno-associated virus-mediated over-expression were used in combination with control or high fat diet to assess energy balance, glucose homeostasis as well as adipose tissue health and remodeling.ResultsLumican was found to be particularly enriched in the stromal cells isolated from murine gonadal white adipose tissue. Likewise murine and human visceral fat showed a robust increase in Lumican as compared to fat from the subcutaneous depot. Lumican null female mice exhibited moderately increased fat mass, decreased insulin sensitivity and increased liver triglycerides in a diet-dependent manner. These changes coincided with inflammation in adipose tissue and no overt effects in adipose expandability, i.e. adipocyte formation and hypertrophy. Lumican over-expression in visceral fat and liver resulted in improved insulin sensitivity and glucose clearance.ConclusionsThese data indicate that Lumican may represent a functional link between the extracellular matrix, glucose homeostasis, and features of the metabolic syndrome.

Objective The susceptibility to abdominal obesity and the metabolic syndrome is determined to a substantial extent during childhood and adolescence, when key adipose tissue characteristics are established. Although the general impact of postnatal nutrition is well known, it is not clear how specific dietary components drive adipose tissue growth and how this relates to the risk of metabolic dysfunction in adulthood. Methods Adipose tissue growth including cell proliferation was analyzed in juvenile mice upon dietary manipulation with in vivo nucleotide labeling. The proliferative response of progenitors to specific fatty acids was assayed in primary cultures. Long-term metabolic consequences were assessed through transient dietary manipulation post-weaning with a second obesogenic challenge in adulthood. Results Dietary lipids stimulated adipose tissue progenitor cell proliferation in juvenile mice independently of excess caloric intake and calorie-dependent adipocyte hypertrophy. Excess calories increased mitogenic IGF-1 levels systemically, whereas palmitoleic acid was able to enhance the sensitivity of progenitors to IGF-1, resulting in synergistic stimulation of proliferation. Early transient consumption of excess lipids promoted hyperplastic adipose tissue expansion in response to a second dietary challenge in adulthood and this correlated with abdominal obesity and hyperinsulinemia. Conclusions Dietary lipids and calories differentially and synergistically drive adipose tissue proliferative growth and the programming of the metabolic syndrome in childhood.

Appropriate cell models are necessary for the investigation of thermogenic beige adipocyte differentiation from progenitor cells. Here, we describe a primary cell culture method that is based on defined progenitor cells from murine white adipose tissue and aims at minimizing confounding factors including cell heterogeneity and nonphysiological differentiation inducers. Adipocyte progenitor cells are enriched by immuno-magnetic separation, expanded minimally, and induced for beige adipocyte differentiation with carbaprostacyclin, a stable analogue of the endogenous mediator PGI.

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5130 EPC is not very well defined population. These cells have either angiopoietic or angiostimulating function, the latter seems to be more relevant. It is well known that Mobilization of EPC to peripheral blood is decreased in diabetic rats. From the opposite, ischemia proved to stimulate mobilization. That is why, we decided to study EPC in diabetic patients with and without PAD Patients and methods 40 pts with diabetes(type I- 4, type II-36) were studied. Duration of diabetes -1-46 years. The duration of more than 10 years was in 57% patients. Age- 49-73 years(mean age- 62 years). The patients were divided into two groups- diabetic foot without PAD(25 pts) – Group A, and diabetic foot with PAD- group B. PAD was diagnosed with sonography, duplex sonography and Rx arteriography. Normal volunteers were use as controls(mean age 51.6+2.0) CD34+ and CD133+ cells were numerated with CytoFlow(BD). 5 Day CFU-Hill Colony Assay- non-adherent mononuclear cells (MNCs) was used to study EPC In this method, peripheral blood MNCs are plated on fibronectin-coated dishes (6-well). After a 48 h pre-plating step to deplete the sample of adherent macrophages and mature endothelial cells, the non-adherent cells are removed and re-plated on fibronectin-coated dishes (24-well). Unique colonies that are formed in the 5 Day CFU-Hill Colony Assay are referred to as colony-forming unit-Hill colonies (CFU-Hill colonies) or colony-forming unit-ECs (CFU-ECs). Count the number of colonies per well for each sample. CFU-Hill colonies are defined as a central core of round cells with radiating elongated spindle-like cells at the periphery. Colonies without the CFU-Hill morphology may also be present but are not scored as CFU-Hill colonies. CFU-Hill Colonies are fixed with methanol and stained with a Giemsa solution. Plasma concentration of VEGF was studied by ELISA Results EPC colony forming ability in group A was 4.0+ 0.7, in group B-27.4+3.9. The differences between these two groups is highly significant(p 0.001). Low EPC level in diabetic neuropathy is probably related to decreased mobilization(G.Fadini e.a.2006). Rather unusual was higher EPC level in group B. It was shown that age-related decrease in EPC is due to decline in HIF-1 signaling(M. Hoenig e.a 2008). Probably, diabetic defect of mobilization is partly overcome with ischemia induced up-regulation of HIF-1 and VEGF. EPC in group B patients does not differ from controls(26.1+6.5). There were now correlations between EPC number and the number of either CD34+, CD133+, or KDR+ cells. We also failed to find any correlations between VEGF and EPC Conlusion EPC number in peripheral blood in diabetic patients without PAD is severely decreased. Diabetic patients with PAD have near normal EPC number. Thus, ischemia in diabetic patients is unable adequately mobilize EPC Disclosures No relevant conflicts of interest to declare.