Leucine acts in the brain to suppress food intake but does not function as a physiological signal of low dietary protein.

Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 µg) was sufficient to suppress food intake. Isocaloric low- (9% protein energy; LP) or normal- (18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P < 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.

Impaired branched chain amino acid metabolism alters feeding behavior and increases orexigenic neuropeptide expression in the hypothalamus.

Elevation of dietary or brain leucine appears to suppress food intake via a mechanism involving mechanistic target of rapamycin, AMPK, and/or branched chain amino acid (BCAA) metabolism. Mice bearing a deletion of mitochondrial branched chain aminotransferase (BCATm), which is expressed in peripheral tissues (muscle) and brain glia, exhibit marked increases in circulating BCAAs. Here, we test whether this increase alters feeding behavior and brain neuropeptide expression. Circulating and brain levels of BCAAs were increased two- to four-fold in BCATm-deficient mice (KO). KO mice weighed less than controls (25·9 vs 20·4 g, P<0·01), but absolute food intake was relatively unchanged. In contrast to wild-type mice, KO mice preferred a low-BCAA diet to a control diet (P<0·05) but exhibited no change in preference for low- vs high-protein (HP) diets. KO mice also exhibited low leptin levels and increased hypothalamic Npy and Agrp mRNA. Normalization of circulating leptin levels had no effect on either food preference or the increased Npy and Agrp mRNA expression. If BCAAs act as signals of protein status, one would expect reduced food intake, avoidance of dietary protein, and reduction in neuropeptide expression in BCATm-KO mice. Instead, these mice exhibit an increased expression of orexigenic neuropeptides and an avoidance of BCAAs but not HP. These data thus suggest that either BCAAs do not act as physiological signals of protein status or the loss of BCAA metabolism within brain glia impairs the detection of protein balance.

The effect of wild type P53 gene transfer on growth properties and tumorigenicity of PANC-1 tumor cell line.

BACKGROUND: The p53 protein function is essential for the maintenance of the nontumorigenic cell phenotype. Pancreatic tumor cells show a very high frequency of p53 mutation. To determine if restoration of wild type p53 function can be used to eliminate the tumorigenic phenotype in these cells, pancreatic tumor cell lines, PANC-1 and HTB80, differing in p53 status were stably transfected with exogenous wild type p53 gene. METHODS: The transfection was performed using Polybrene/DMSO-Assisted Gene Transfer method. The wild type p53 gene integration into genomic DNA was detected by Southern blot and PCR. Furthermore, the expression of wild type p53 protein was detected in selected clones by immunohistochemistry and Western blot. RESULTS: While HTB80 cell line failed to produce a stable p53 expressing clone, the PANC-1 cells produced stable lines. Following characterization of clones, the growth rate and tumorigenicity of PANC-1 wild type p53 clones were compared to the control cells. Our data showed that the expression of wild type p53 decreased the growth rate of PANC-1 cells. It was also observed that the expression of wild type p53 in PANC-1 cells suppressed its potential for tumor formation in nude mice, completely, while the parental line leads to the formation of a relatively large tumor. CONCLUSION: Our results suggest that gene therapy based on restoration of wild type p53 protein function in pancreatic tumor cells with high amount of mutant p53 is a feasible option in pancreatic cancer treatment.

Restoration of p53 functions suppresses tumor growth of pancreatic cells with different p53 status.

Pancreatic tumor cells show a very high frequency of p53 mutation. Our aim in this study was to determine if the restoration of wild-type p53 function could be used to eliminate the tumorigenic phenotype in these cells. Pancreatic tumor cell lines, CRL1420, which contains elevated levels of mutant p53, and CRL1682, with no detectable p53 protein, were stably transfected with the exogenous wild-type p53 gene. The growth rate and tumorigenicity in nude mice of wild-type p53 expressing clones were measured. Our data showed that the expression of wild-type p53 decreased the growth rate of CRL1420 and completely suppressed its potential for tumor formation in nude mice. Moreover, the size of the tumor formed in nude mice by CRL1682 was reduced drastically. G1 arrest as a possible cause for tumor suppression was investigated by flowcytometry. Neither of the cell lines irrespective of the status of p53 was arrested at G1 in response to x-irradiation. Thus, our results provide functional evidence that the deletion or mutational inactivation of the p53 gene represents an important step in the tumorigenicity of pancreatic cancer. Furthermore, the extent of the restoration of p53 function by introduction of the p53 gene depends on both the cell type and the cell settings (in vitro or in vivo conditions).