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Zhou, Yun-Ping, John C. Pena, Michael W. Roe, Anshu Mittal, Matteo Levisetti, Aaron C. Baldwin, William Pugh, Diane Ostrega, Noreen Ahmed, Vytautas P. Bindokas, Louis H. Philipson, Douglas Hanahan, Craig B. Thompson, and Kenneth S. Polonsky. Overexpression of Bcl-[x.sub.L] in [Beta]-cells prevents cell death but impairs mitochondrial signal for insulin secretion. Am. J. Physiol. Endocrinol. Metab. 278: E340--E351, 2000.--To study effects of Bcl-[x.sub.L] in the pancreatic [Beta]-cell, two transgenic lines were produced using different forms of the rat insulin promoter. Bcl-[x.sub.L] expression in [Beta]-cells was increased 2- to 3-fold in founder (Fd) 1 and over 10-fold in Fd 2 compared with littermate controls. After exposure to thapsigargin (10 [micro]M for 48 h), losses of cell viability in islets of Fd 1 and Fd 2 Bcl-[x.sub.L] transgenic mice were significantly lower than in islets of wild-type mice. Unexpectedly, severe glucose intolerance was observed in Fd 2 but not Fd 1 Bcl-[x.sub.L] mice. Pancreatic insulin content and islet morphology were not different from control in either transgenic line. However, Fd 2 Bcl-[x.sub.L] islets had impaired insulin secretory and intracellular free [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) responses to glucose and KCL. Furthermore, insulin and [[[Ca.sup.2+]].sub.i] responses to pyruvate methyl ester (PME) were similarly reduced as glucose in Fd 2 Bcl-[x.sub.L] islets. Consistent with a mitochondrial defect, glucose oxidation, but not glycolysis, was significantly lower in Fd 2 Bcl-[x.sub.L] islets than in wild-type islets. Glucose-, PME-, and [Alpha]-ketoisocaproate-induced hyperpolarization of mitochondrial membrane potential, NAD(P)H, and ATP production were also significantly reduced in Fd 2 Bcl-[x.sub.L] islets. Thus, although Bcl-[x.sub.L] promotes [Beta]-cell survival, high levels of expression of Bcl-[x.sub.L] result in reduced glucose-induced insulin secretion and hyperglycemia due to a defect in mitochondrial nutrient metabolism and signaling for insulin secretion. apoptosis; calcium; islets of Langerhans; mitochondria

Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 microm circle plasmid (2 microm). Here we show that accumulation of 2 microm in the SUMO pathway mutants siz1Delta siz2Delta, slx5Delta, and slx8Delta is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 microm. Characterization of this species from siz1Delta siz2Delta showed that it contains tandem copies of the 2 mum sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 microm-encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 microm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir(o) strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 microm amplification.