We identified all of the positive oligonucleotides with the threshold values R ¼ 13 and D ¼ 12Q (ref. 21). R and D are threshold values for the ratio and the difference between perfect match intensity and mismatch intensity, respectively. Thus varying these values gives different measures of sensitivity and specificity. We then used BLAST to identify conserved blocks that corresponded to at least two positive oligonucleotides so as to reduce the number of false positives.

Aim/hypothesis The distinct metabolic properties of visceral and subcutaneous adipocytes may be due to inherent characteristics of the cells that are resident in each fat depot. To test this hypothesis, human adipocytes were differentiated in vitro from precursor stromal cells obtained from visceral and subcutaneous fat depots and analysed for genetic, biochemical and metabolic endpoints. Methods Stromal cells were isolated from adipose tissue depots of nondiabetic individuals. mRNA levels of adipocytespecific proteins were determined by real-time RT-PCR.Insulin signalling was evaluated by immunoblotting with specific antibodies. Glucose transport was measured by a 2-deoxy-glucose uptake assay. Adiponectin secretion in the adipocyte-conditioned medium was determined by a specific RIA. Results With cell differentiation, mRNA levels of PPARG, C/EBPα (also known as CEBPA), AP2 (also known as GTF3A), GLUT4 (also known as SLC2A4) were markedly upregulated, whereas GLUT1 (also known as SLC2A1) mRNA did not change. However, expression of C/EBPα, AP2 and adiponectin was higher in subcutaneous than in visceral adipocytes. By contrast, adiponectin was secreted at threefold higher rates by visceral than by subcutaneous adipocytes while visceral adipocytes also showed two-to threefold higher insulin-stimulated glucose uptake. Insulininduced phosphorylation of the insulin receptor, IRS proteins, Akt and extracellular signal-regulated kinase-1/2 was more rapid and tended to decrease at earlier time-points in visceral than in subcutaneous adipocytes. Conclusions/interpretation Subcutaneous and visceral adipocytes, also when differentiated in vitro from precursor stromal cells, retain differences in gene expression, adiponectin secretion, and insulin action and signalling. Thus, the precursor cells that reside in the visceral and subcutaneous fat depots may already possess inherent and specific metabolic characteristics that will be expressed upon completion of the differentiation programme.

Natural antisense transcripts (NATs) are a class of genes whose role in controlling gene expression is becoming more and more relevant. We describe the identification of eight novel mouse NATs associated with transcription factors (Pax6, Pax2, Six3, Six6, Otx2, Crx, Rax and Vax2) that play an important role in eye development and function. These newly identified NATs overlap with the mature processed mRNAs or with the primary unprocessed transcript of their corresponding sense genes, are predicted to represent either protein coding or non-coding RNAs and undergo extensive alternative splicing. Expression studies, by both RT-PCR and RNA in situ hybridization, demonstrate that most of these NATs, similarly to their sense counterparts, display a specific or predominant expression in the retina, particularly at postnatal stages. We found a significant reduction of the expression levels of one of these NATs, Vax2OS (Vax2 opposite strand) in a mouse mutant carrying the inactivation of Vax2, the corresponding sense gene. In addition, we overexpressed another NAT, CrxOS, in mouse adult retina using adeno-associated viral vectors and we observed a significant decrease in the expression levels of the corresponding sense gene, Crx. These results suggest that these transcripts are functionally related to their sense counterparts and may play an important role in regulating the molecular mechanisms that underlie eye development and function in both physiological and pathological conditions.

Background/Objectives: The histone deacetylases SIRT1 and SIRT2 have been shown to be involved in the differentiation of rodent adipocyte precursors. In light of the differences in gene expression and metabolic function of visceral (V) and subcutaneous (S) adipose tissue (AT) and their resident cells, the aim of this study was to investigate the role of SIRT1 and SIRT2 in the differentiation of adipose stem cells (ASCs) isolated from SAT and VAT biopsies of non-diabetic subjects with varying levels of BMI.Methods: Human ASCs were isolated from paired SAT and VAT biopsies obtained from 83 of non-diabetic subjects and 92 obese individuals.Results: Visceral but not subcutaneous ASCs from obese subjects showed an intrinsic increase in both adipogenesis and lipid accumulation when compared to ASCs from nonobese subjects, and this was associated with reduced SIRT1 and SIRT2 mRNA and protein levels. Moreover, adipose tissue mRNA levels of SIRT1 and SIRT2 showed an inverse correlation with BMI in the visceral but not subcutaneous depot. Overexpression of SIRT1 or SIRT2 in visceral ASCs from obese subjects resulted in inhibition of adipocyte differentiation, whereas knockdown of SIRT1 or SIRT2 in visceral ASCs from non-obese subjects enhanced this process. Changes in SIRT1 or SIRT2 expression and adipocyte differentiation were paralleled by corresponding changes in PPARG, CEBPA and other genes marking terminal adipocyte differentiation. Conclusions:These observations indicate that reduced SIRT1 and SIRT2 expression in visceral ASCs may promote visceral adipose tissue expansion in human obesity by enhancing the differentiation capacity of these adipocyte precursors.

IGF-I regulates bone acquisition and maintenance, even though the cellular targets and signaling pathways responsible for its action in human bone cells are poorly understood. Whether abnormalities in IGF-I action and signaling occur in human osteoblasts under conditions of net bone loss has not been determined. Herein we carried out a comparative analysis of IGF-I signaling in primary cultures of human osteoblasts from osteoporotic and control donors. In comparison with control cells, osteoporotic osteoblasts showed increased tyrosine phosphorylation of the IGF-I receptor in the basal state and blunted stimulation of receptor phosphorylation by IGF-I. Augmentation of basal IGF-I receptor phosphorylation was associated with coordinate increases in basal tyrosine phosphorylation of insulin receptor substrate (IRS)-2 and activation of Erk, which were also minimally responsive to IGF-I stimulation. By contrast, phosphorylation levels of IRS-1, Akt, and glycogen synthase kinase-3 were similar in the basal state in control and osteoporotic osteoblasts and showed marked increases after IGF-I stimulation in both cell populations, even though these responses were significantly lower in the osteoporotic osteoblasts. The IGF-I signaling abnormalities in osteoporotic osteoblasts were associated with reduced DNA synthesis both under basal conditions and after stimulation with IGF-I. Interestingly, treatment of the osteoporotic osteoblasts with the MAPK kinase inhibitor PD098059 reduced the elevated levels of Erk phosphorylation and increased basal DNA synthesis. Collectively, our data show that altered osteoblast proliferation in human osteoporosis may result from dysregulation of IGF-I receptor signaling, including constitutive activation of the IRS-2/Erk signaling pathway, which becomes unresponsive to IGF-I, and defective induction of the IRS-1/Akt signaling pathway.