Adenovirus-mediated transient expression of the pancreatic duodenal homeobox transcription factor Pdx1 in mouse liver activates pancreatic endocrine and exocrine genes, the latter reportedly resulting in severe hepatitis. Expression of a super-active form of Pdx1 or Pdx1-VP16 selectively transdifferentiates hepatic WB cells into functional pancreatic beta-like insulin-producing cells, without evidence of exocrine differentiation. No study has systematically compared the transdifferentiation efficiency of Pdx1 and Pdx1-VP16 at the cellular and molecular level. Comparisons can be ambiguous when vectors harboring a transcription factor cDNA have differing extents and duration of gene expression. In view of the remarkable capacity of lentiviral vector (LV) for delivering and integrating transgene into both dividing and nondividing cells, we transduced rat hepatic stem cell-like WB cells with LV-Pdx1 or LV-Pdx1-VP16, and then used the limiting-dilution technique to clone singlecell-derived cell lines that stably express either Pdx1 or Pdx1-VP16. With these cell lines, we studied: (a) the expression of Pdx1 or Pdx1-VP16 protein by Western blotting and immunocytochemistry; (b) the repertoire of long-term expression of Pdx1-or Pdx1-VP16-induced pancreatic gene expression using RT-PCR methods; and (c) their capacity to serve as beta-cell surrogates in restoring euglycemia in streptozotocin-treated diabetic mice. We found that cell lines expressing either Pdx1 or Pdx1-VP16 long-term exhibited similar profiles for expression of genes related to pancreatic development and beta-cell function, and reversed hyperglycemia in diabetic mice. We also examined short-term expression of Pdx1 or Pdx1-VP16, and the results demonstrated that expression of Pdx1-VP16 is more efficient in initiating liver-to-endocrine pancreas transdifferentiation. Our findings demonstrate: (a) that the LV system is highly effective in producing persistent expression of Pdx1 or Pdx1-VP16 in WB hepatic cells; and (b) long-term, persistent expression of either Pdx1 or Pdx1-VP16 is similarly effective in converting hepatic stem cells into pancreatic endocrine precursor cells that, upon transplantation into diabetic mice, become functional insulin-producing cells and restore euglycemia. Laboratory Investigation (2006) 86, 83-93.

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, ␣-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic ␣-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyeride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyeride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.adult-onset obesity; proopiomelanocortin overexpression; gene delivery; brain stem THE BRAIN MELANOCORTIN PATHWAY is a key leptin target in the central nervous system and plays an essential role in the homeostatic regulation of body weight (6,8,28). Melanocortins are peptides cleaved from a common precursor, proopiomelanocortin (POMC). Rodents with POMC deficiency and humans with POMC mutations are hyperphagic and obese (15,35). The contribution of the central melanocortin system on the regulation of food intake and body weight has been attributed primarily to hypothalamic POMC neurons in the arcuate nucleus (ARC), which produce alpha-melanocyte-stimulating hormone (␣-MSH), the principal central melanocortin. ␣-MSH and its analog, melanotan II (MTII), inhibit food intake and enhance energy expenditure mainly through activation of melanocortin 3 (MC3R) and 4 (MC4R) receptors in the hypothalamus (5). However, the central melanocortin system is not limited to the hypothalamus. POMC neurons and ␣-MSH are both found within the commissural region of the nucleus of the solitary tract (NTS) in the brain stem (11,24) where MC4Rs are also expressed (14,22). Thus melanocortin signaling within the NTS may contr...

Short-term pharmacological melanocortin activation deters diet-induced obesity (DIO) effectively in rodents. However, whether central pro-opiomelanocortin (POMC) gene transfer targeted to the hypothalamus or hindbrain nucleus of the solitary track (NTS) can combat chronic dietary obesity has not been investigated. Four-week-old Sprague Dawley rats were fed a high fat diet for five months, and then injected with either the POMC or control vector into the hypothalamus or NTS, and body weight and food intake recorded for 68 days. Insulin sensitivity, glucose metabolism and adrenal indicators of central sympathetic activation were measured, and voluntary wheel running (WR) assessed. Whereas the NTS POMC-treatment decreased cumulative food consumption and caused sustained weight reduction over 68 days, the hypothalamic POMC-treatment did not alter cumulative food intake and produced weight loss only in the first 25 days. At death, only the NTS-POMC rats had a significant decrease in fat mass. They also displayed enhanced glucose tolerance, lowered fasting insulin and increased QUICK value, and elevated adrenal indicators of central sympathetic activation. Moreover, the NTS-POMC animals exhibited a near 20% increase in distance ran relative to the respective controls, but the ARC-POMC rats did not. In conclusion, POMC gene transfer to the NTS caused modest anorexia, persistent weight loss, improved insulin sensitivity, and increased propensity for WR in DIO rats. These metabolic improvements may involve stimulation of energy expenditure via centrally regulated sympathetic outflow. The similar POMC treatment in the hypothalamus had minimal long-term physiological or metabolic impact. Thus, melanocortin activation in the brainstem NTS region effectively ameliorates chronic dietary obesity whilst that in the hypothalamus fails to do so.

Pro-opiomelanocortin (POMC) neurons are identified in two brain sites, the arcuate nucleus (ARC) of the hypothalamus and nucleus of the solitary tract (NTS) in brainstem. Earlier pharmacological and POMC gene transfer studies demonstrate melanocortin activation in either site alone improves insulin sensitivity and reduces obesity. The present study, for the first time, investigated the long-term efficacy of POMC gene transfer concurrently into both sites in the regulation of energy metabolism in aged F344xBN rats bearing adult-onset obesity. Pair feeding was included to reveal food-independent POMC impact on energy expenditure. We introduced adeno-associated virus encoding either POMC or green fluorescence protein to the two brain areas in 22-month-old rats, then recorded food intake and body weight, assessed oxygen consumption, serum leptin, insulin and glucose, tested voluntary wheel running, analyzed POMC expression, and examined fat metabolism in brown and white adipose tissues. POMC mRNA was significantly increased in both the hypothalamus and NTS region at termination. Relative to pair feeding, POMC caused sustained weight reduction and additional fat loss, lowered fasting insulin and glucose, and augmented white fat hormone-sensitive lipase activity and brown fat uncoupling protein 1 level. By wheel running assessment, the POMC-animals ran twice the distance as the control or pair-fed rats. Thus, the dual-site POMC treatment ameliorated adult-onset obesity effectively, involving a moderate hypophagia lasting ~ 60 days, enhanced lipolysis and thermogenesis, and increased physical activity in the form of voluntary wheel running. The latter finding provides a clue for countering age-related decline in physical activity.