Chronic hyperglycemia is toxic to pancreatic ␤-cells, impairing cellular functioning as observed in type 2 diabetes; however, the mechanism underlying ␤-cell dysfunction and the resulting apoptosis via glucose toxicity are not fully characterized. Here, using MIN6N8 cells, a mouse pancreatic ␤-cell line, we show that chronic exposure to high glucose increases cell death mediated by Bax oligomerization, cytochrome C release, and caspase-3 activation. During apoptosis, glucokinase (GCK) expression decreases in high-glucose-treated cells, concomitant with a decrease in cellular ATP production and insulin secretion. Moreover, exposure to a chronically high dose of glucose decreases interactions between GCK and mitochondria with an increase in Bax binding to mitochondria and cytochrome C release. These events are prevented by GCK overexpression, and phosphorylation of proapoptotic Bad proteins in GCK-overexpressing cells is prolonged compared with Neo-transfected cells. Similar results are obtained using primary islet cells. Collectively, these data demonstrate that ␤-cell apoptosis from exposure to chronic high glucose occurs in relation to lowered GCK expression and reduced association with mitochondria. Our results show that this may be one mechanism by which glucose is toxic to ␤-cells and suggests a novel approach to prevent and treat diabetes by manipulating Bax-and GCK-controlled signaling to promote apoptosis or proliferation. Diabetes 54:2602-2611, 2005

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a ϳ400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

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Expression of adiponectin decreases with obesity and insulin resistance. At present, the mechanisms responsible for negatively regulating adiponectin expression in adipocytes are poorly understood. In this investigation, we analyzed the effects of 5 serial deletion constructs on the murine adiponectin promoter. Here, we identified the repressor region located between ؊472 and ؊313 bp of the promoter. Removal of the putative nuclear factor of activated T-cells (NFATs) binding site increased the promoter activity, and overexpression of NFATc4 reduced the promoter activity. Treatment with the calcium ionophore A23187, an activator of NFAT, reduced mRNA as well as promoter activity. The binding of NFATc4 to the promoter was associated with increased recruitment of histone deacetylase 1 and reduced acetylation of histone H3 at the promoter site. In addition, binding of activating transcription factor 3 (ATF3) to the putative activator protein-1 site located adjacent to the NFAT binding site also repressed the promoter activity. Treatment with thapsigargin, an inducer of ATF3, reduced both mRNA and promoter activity. Importantly, the binding activities of NFATc4 and ATF3, increased significantly in white adipose tissues of ob/ob and db/db mice compared with controls. Taken together, this study demonstrates for the first time that NFATc4 and ATF3 function as negative regulators of adiponectin gene expression, which may play critical roles in downregulating adiponectin expression in obesity and type 2 diabetes.

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Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is signi®cantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.

The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.Although mitochondria can synthesize a small subset of proteins on their own, the vast majority have to be imported after their synthesis in the cytosol (11,23,36,40). The precursor proteins are transported through specific translocase complexes residing in the outer and inner membranes (21, 37). However, the relatively small inner diameter of the translocation channels presents a major constraint for the import process (17, 49). Experimental evidence indicates that preproteins destined for the mitochondrial matrix cross the membranes in an extended conformation (42). Posttranslational import of mitochondrial preproteins therefore requires the unfolding of folded preprotein domains prior to or during the translocation reaction.Two mitochondrial energy sources are responsible for both vectorial movement of the precursor polypeptide in the translocation channel and unfolding of preproteins. The insertion of the amino-terminal segment of the preprotein into the inner membrane is dependent on the electric potential (⌬) across the inner membrane. It is thought that the potential exerts an electrophoretic force on the positively charged N-terminal targeting sequence (29). In addition, the ⌬ has been shown to be involved in the function of the TIM23 protein complex (2). The full translocation of the polypeptide chain requires a second energy source, the hydrolysis of matrix ATP. The enzymatic machinery that couples preprotein translocation and ATP hydrolysis is provided by the import motor complex, which is located at the inner face of the inner membrane in the direct vicinity of the TIM23 translocation channel (36,39,48,55). Its core component is the main mitochondrial chaperone...