Highlights d Caloric restoration induces differential activity in the paraventricular hypothalamus d Sensory detection of food rapidly regulates PVH Glp1r and PVH Crh subpopulations d Stimulation of PVH Glp1r neurons bidirectionally orchestrates feeding behavior d Chronic perturbation of PVH Glp1r or PVH Mc4r populations induces obesity

Figure 1Derivation and characterization of cloned piglets from piPSCs. (A) Preimplantation and post-implantation development of the cloned embryos from piPSCs. Embryos at two-cell (a), four-cell (b), eight-cell (c), blastocyst stages (d, e) and two 36 day-old cloned fetuses (f) are shown. Scale bars are 100 μm. (B) The morphology and fluorescence of the hooves (left), tails (middle) and fibroblasts (right) of the 36 day-old embryos. Scale bars are 100 μm. (C) The morphology, fluorescence and hematoxylin/eosin-stained sections of tissues from piglet 00536-3#. Scale bars are 100 μm. (D) Cloned piglets. 00507-4# from differentiated iPF4-2 cell, 4 days old; 227-1#, 2#, 3# from undifferentiated iPF4-2, 2 days old. (E) Porcine ear fibroblasts (PEFs) from 00507-4#, EGFP positive. Scale bars are 100 μm. (F) PCR demonstrating genomic integration of Oct4, Sox2, and EGFP using tissues of the cloned fetuses and piglets. PEF, the original fibroblasts used to create iPF4-2. 00518, 00536, 00507, 227, foster mothers. 00518-1#, 00518-2#, the cloned fetuses derived from differentiated iPF4-2 cells. 00536-3# , 00507-4#, the cloned piglets derived from iPF4-2-differentiated cells. 227-1#~4#, the HMC piglets derived from iPF4-2. (G) Microsatellite analysis of the donor piPSC line iPF4-2, cloned fetuses and piglets. 00518, 00536, 00507, 227, foster mothers; 00518-1# and 00518-2#, cloned fetuses; 00536-3# and 00507-4#, the cloned piglets from differentiated iPF4-2 cells; 227-1#~4#, the cloned piglets derived from the Scriptaid-treated NT embryos from iPF4-2 cells.www.cell-research.com | Cell Research Nana Fan et al. 165npg

To assess the potential of tumor-associated, alternatively spliced gene products as a source of biomarkers in biological fluids, we have analyzed a large data set of mass spectra derived from the plasma proteome of a mouse model of human pancreatic ductal adenocarcinoma. MS/MS spectra were interrogated for novel splice isoforms using a nonredundant database containing an exhaustive three-frame translation of Ensembl transcripts and gene models from ECgene. This integrated analysis identified 420 distinct splice isoforms, of which 92 did not match any previously annotated mouse protein sequence. We chose seven of those novel variants for validation by reverse transcription-PCR. The results were concordant with the proteomic analysis. All seven novel peptides were successfully amplified in pancreas specimens from both wild-type and mutant mice. Isotopic labeling of cysteine-containing peptides from tumor-bearing mice and wild-type controls enabled relative quantification of the proteins. Differential expression between tumor-bearing and control mice was notable for peptides from novel variants of muscle pyruvate kinase, malate dehydrogenase 1, glyceraldehyde-3-phosphate dehydrogenase, proteoglycan 4, minichromosome maintenance, complex component 9, high mobility group box 2, and hepatocyte growth factor activator. Our results show that, in a mouse model for human pancreatic cancer, novel and differentially expressed alternative splice isoforms are detectable in plasma and may be a source of candidate biomarkers. [Cancer Res 2009;69(1):300-9]

Growth hormone (GH) excess results in structural and functional changes in the kidney and is implicated as a causative factor in the development of diabetic nephropathy (DN). Glomerular podocytes are the major barrier to the filtration of serum proteins, and altered podocyte function and/or reduced podocyte number is a key event in the pathogenesis of DN. We have previously shown that podocytes are a target for GH action. To elucidate the molecular basis for the effects of GH on the podocyte, we conducted microarray and RT-quantitative PCR analyses of immortalized human podocytes and identified zinc finger E-box-binding homeobox 2 (ZEB2) to be up-regulated in a GH dose-and time-dependent manner. We established that the GH-dependent increase in ZEB2 levels is associated with increased transcription of a ZEB2 natural antisense transcript required for efficient translation of the ZEB2 transcript. GH down-regulated expression of E-and P-cadherins, targets of ZEB2, and inhibited E-cadherin promoter activity. Mutation of ZEB2 binding sites on the E-cadherin promoter abolished this effect of GH on the E-cadherin promoter. Whereas GH increased podocyte permeability to albumin in a paracellular albumin influx assay, shRNA-mediated knockdown of ZEB2 expression abrogated this effect. We conclude that GH increases expression of ZEB2 in part by increasing expression of a ZEB2 natural antisense transcript. GH-dependent increase in ZEB2 expression results in loss of P-and E-cadherins in podocytes and increased podocyte permeability to albumin. Decreased expression of P-and E-cadherins is implicated in podocyte dysfunction and epithelial-mesenchymal transition observed in DN. We speculate that the actions of GH on ZEB2 and P-and E-cadherin expression play a role in the pathogenesis of microalbuminuria of DN. Pituitary growth hormone (GH)5 is essential for postnatal growth in mammals. In addition to growth, GH affects the metabolism of fat, protein, and carbohydrate (1). GH exerts these actions both by its direct effect on target organs and by stimulating the production of insulin-like growth factor-1 (IGF-1). At the tissue level, these pleiotropic actions of GH result from the interaction of GH with a specific cell surface receptor, the GH receptor (GHR). Whereas the GHR is ubiquitously expressed, the role and effects of GH have been most intensely investigated in organs and tissues such as liver, bone, muscle, and adipocytes in which GHR expression is substantial and are thus considered canonical targets of GH action. However, recent reports have highlighted the biological effects and physiological relevance of GH action in non-canonical targets such as the blastocyst (2), colonic epithelial cells (3), cardiomyocytes (4), and neurons (5).GH excess in both the human (acromegaly) and in transgenic animal models is characterized by significant structural and functional changes in the kidney (6 -8). An overactive GH/ GHR axis is implicated as a causative factor in the development of diabetic nephropathy (9 -11). Our previous study...

The cellular and molecular basis of growth hormone (GH) actions on the heart remain poorly defined, and it is unclear whether GH effects on the myocardium are direct or mediated at least in part via insulin-like growth factor (IGF-1). Here, we demonstrate that the cultured neonatal cardiomyocyte is not an appropriate model to study the effects of GH because of artifactual loss of GH receptors (GHRs). To circumvent this problem, rat neonatal cardiomyocytes were infected with a recombinant adenovirus expressing the murine GHR. Functional integrity of GHR was suggested by GH-induced activation of the cognate JAK2/STAT5, MAPK, and Akt intracellular pathways in the cells expressing GHR. Although exposure to GH resulted in a significant increase in the size of the cardiomyocyte and increased expression of c-fos, myosin light chain 2, and skeletal ␣-actin mRNAs, there were no significant changes in IGF-1 or atrial natriuretic factor mRNA levels in response to GH stimulation. In this model, GH increased incorporation of leucine, uptake of palmitic acid, and abundance of fatty acid transport protein mRNA. In contrast, GH decreased uptake of 2-deoxy-D-glucose and levels of Glut1 protein. Thus, in isolated rat neonatal cardiomyocytes expressing GHR, GH induces hypertrophy and causes alterations in cellular metabolic profile in the absence of demonstrable changes in IGF-1 mRNA, suggesting that these effects may be independent of IGF-1.Several observations implicate a role for growth hormone (GH) 1 in modulation of cardiac structure and function (1). Patients with excess endogenous GH (i.e. acromegaly) suffer from cardiac complications including biventricular hypertrophy, impaired diastolic filling, and decreased cardiac performance on effort due to diastolic and systolic dysfunction (2). Patients with chronic GH deficiency also show cardiac abnormalities; in general, the data support the presence of a hypokinetic cardiac syndrome in patients with GH deficiency that can be reversed with GH replacement therapy (3-5). Fazio et al. (6) reported that GH therapy in patients with idiopathic dilated cardiomyopathy was associated with significant improvement in left ventricular ejection fraction, isovolumic relaxation time, and efficiency of myocardial energy utilization. Subsequent to these landmark findings, some studies have supported a beneficial effect of exogenous GH on cardiac function (7), whereas other investigators were unable to demonstrate salutary effects of GH on cardiac function in patients with heart failure (8).A particularly well studied animal model is that of the transplanted GH-secreting pituitary tumor cell line, GH 3. In this model of GH excess, there is increased myocardial contractility and calcium sensitivity of myocardial contractile proteins (1). Similarly, normal rats given recombinant GH show an increase in left ventricular mass, as well as an increase in several aspects of cardiac performance (9). In the rodent model of myocardial infarction, administration of GH results in improvements in myocardial ...