The insulin receptor translocates to the nucleus to regulate cell proliferation in liver.

UNLABELLED: Insulin's metabolic effects in the liver are widely appreciated, but insulin's ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca(2+) signals within the nucleus regulate cell proliferation, we investigated whether insulin's mitogenic effects result from activation of Ca(2+)-signaling pathways by IRs within the nucleus. Insulin-induced increases in Ca(2+) and cell proliferation depended upon clathrin- and caveolin-dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,-trisphosphate (InsP3) in the nucleus, whereas insulin's metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus. CONCLUSION: These findings provide evidence that insulin's mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3 -dependent Ca(2+)-signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease.

Cloning and expression of a functional core streptavidin in Pichia pastoris: strategies to increase yield.

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min(-1) , and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L(-1) ). These parameters yielded 4.0 g L(-1) of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L(-1) of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

UNLABELLED: Subcellular Ca(2+) signals control a variety of responses in the liver. For example, mitochondrial Ca(2+) (Ca(mit)(2+)) regulates apoptosis, whereas Ca(2+) in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca(mit)(2+) also affects liver regeneration. The Ca(2+)-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin-mitochondrial targeting sequence-green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca(2+) signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2-associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca(mit)(2+) buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. CONCLUSION: Together, these results reveal an essential role for Ca(mit)(2+) in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis.