Chronic free fatty acid (FFA) exposure induces pancreatic beta-cell death, which may contribute to the development of type 2 diabetes. The mechanisms involved in FFA-induced cell death are not completely understood. Here we have investigated the effect of FFA on endoplasmic reticulum (ER) stress pathways in INS-1 pancreatic beta-cells. INS-1 cells exposed to palmitate for 16-24 h under serum-free conditions showed marked apoptosis and increased protein levels of phosphorylated eukaryotic translation initiation factor 2alpha (eIF2alpha), activating transcription factor 4 (ATF4), X box-binding protein 1 (XBP-1), and C/EBP homologous transcription factor (CHOP) compared with control cells. The CHOP transcription factor has been implicated in mediating ER stress-induced apoptosis. Unexpectedly, the levels of the ER chaperone proteins Grp78/BiP and PDI were not affected by palmitate treatment, suggesting that the cell protective aspects of the unfolded protein response (UPR) are not up-regulated by palmitate. Palmitate-treated cells had markedly altered distribution of ER chaperones and altered ER morphology, suggesting that accumulation of misfolded proteins might trigger the ER stress response. In contrast, oleate treatment did not significantly induce the UPR pathways, nor was it as detrimental to INS-1 beta-cells. The results suggest that activation of the UPR may significantly contribute to palmitate- but not oleate-induced pancreatic beta-cell death.

The endoplasmic reticulum (ER) is the cellular site of newly synthesized secretory and membrane proteins. Such proteins must be properly folded and posttranslationally modified before exit from the organelle. Proper protein folding and modification requires molecular chaperone proteins as well as an ER environment conducive for these reactions. When ER lumenal conditions are altered or chaperone capacity is overwhelmed, the cell activates signaling cascades that attempt to deal with the altered conditions and restore a favorable folding environment. Such alterations are referred to as ER stress, and the response activated is the unfolded protein response (UPR). When the UPR is perturbed or not sufficient to deal with the stress conditions, apoptotic cell death is initiated. This review will examine UPR signaling that results in cell protective responses, as well as the mechanisms leading to apoptosis induction, which can lead to pathological states due to chronic ER stress.

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC 50 similar to that for inhibition of p38 MAP kinase (0.6 M). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

Diabetes-induced oxidative stress can lead to protein misfolding and degradation by the ubiquitin-proteasome system. This study examined protein ubiquitination in pancreatic sections from Zucker diabetic fatty rats. We observed large aggregates of ubiquitinated proteins (Ubproteins) in insulin-expressing ␤-cells and surrounding acinar cells. The formation of these aggregates was also observed in INS1 832/13 ␤-cells after exposure to high glucose (30 mmol/l) for 8 -72 h, allowing us to further characterize this phenotype. Oxidative stress induced by aminotriazole (ATZ) was sufficient to stimulate Ub-protein aggregate formation. Furthermore, the addition of the antioxidants N-acetyl cysteine (NAC) and taurine resulted in a significant decrease in formation of Ub-protein aggregates in high glucose. Puromycin, which induces defective ribosomal product (DRiP) formation was sufficient to induce Ub-protein aggregates in INS1 832/13 cells. However, cycloheximide (which blocks translation) did not impair Ub-protein aggregate formation at high glucose levels, suggesting that long-lived proteins are targeted to these structures. Clearance of Ub-protein aggregates was observed during recovery in normal medium (11 mmol/l glucose). Despite the fact that 20S proteasome was localized to Ub-protein aggregates, epoxomicin treatment did not affect clearance, indicating that the proteasome does not degrade proteins localized to these structures. The autophagy inhibitor 3MA blocked aggregate clearance during recovery and was sufficient to induce their formation in normal medium. Together, these findings demonstrate that diabetes-induced oxidative stress induces ubiquitination and storage of proteins into cytoplasmic aggregates that do not colocalize with insulin. Autophagy, not the proteasome, plays a key role in regulating their formation and degradation. To our knowledge, this is the first demonstration that autophagy acts as a defense to cellular damage incurred during diabetes. Diabetes 56:930 -939, 2007

Weight-neutral treatment with a glucagon-like peptide-1 analog activates several cardioprotective pathways, prevents HFD-induced insulin resistance and inflammation, reduces monocyte vascular adhesion, and improves cardiac function in vivo by activating AMP-activated protein kinase. These data support a role for glucagon-like peptide-1 analogs in limiting the cardiovascular risks of obesity.

sure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic ␤-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic ␤-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced ␤-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in ␤-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate. endoplasmic reticulum; lipotoxicity; palmitate LIPOTOXICITY CONTRIBUTES to ␤-cell dysfunction during the development of type 2 diabetes (19,42,48). Chronically elevated levels of free fatty acids (FFAs), particularly saturated FFAs such as palmitate, have been shown to be cytotoxic to pancreatic ␤-cells (8, 10, 24, 30 -33, 41, 46, 50). The monounsaturated FFA oleate on the other hand has been shown to not cause apoptosis of ␤-cells in some studies (16,17,31,32,35), while other studies (11,24,27,33,50) found that it does, particularly those that used the INS-1 ␤-cell line.Palmitate-induced cytotoxicity can result from multiple mechanisms, such as increases in reactive oxygen species (8,18,33), ceramide, and nitric oxide (NO) levels (30, 45) and mitochondrial perturbations (8,18,33). In addition, recent studies (23-25) have shown that endoplasmic reticulum (ER) stress pathways are activated by chronic palmitate exposure.ER stress is caused when the protein folding capacity of the ER is not sufficient to deal with protein folding demands or when an excess of misfolded or aggregated proteins accumulate. Such conditions activate the unfolded protein response (UPR) that attempts to reduce the amount of new protein synthesis, increase folding capacity, and degrade terminally misfolded proteins (20, 53). Prolonged ER stress can lead to apoptosis induction (37, 53) and thus may contribute to palmitate-induced ␤-c...

Insulin stimulates glucose transport in muscle and fat cells by inducing translocation of GLUT4 glucose transporters from a storage site to the cell surface. The mechanism of this translocation and the identity of the storage site are unknown, but it has been hypothesized that transporters recycle between an insulin-sensitive pool, endosomes, and the cell surface. Upon cell homogenization and fractionation, the storage site migrates with light microsomes (LDM) separate from the plasma membrane fraction (PM). Cellubrevin is a recently identified endosomal protein that may be involved in the reexocytosis of recycling endosomes. Here we describe that cellubrevin is expressed in 3T3-L1 adipocytes and is more abundant in the LDM than in the PM. Cellubrevin was markedly induced during differentiation of 3T3-L1 fibroblasts into adipocytes, in parallel with GLUT4, and the development of insulin regulated traffic. In response to insulin, the cellubrevin content decreased in the LDM and increased in the PM, suggesting translocation akin to that of the GLUT4 glucose transporter. Vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin-II, a protein associated with regulated exocytosis in secretory cells, also redistributed in response to insulin. Both cellubrevin and VAMP-2 were susceptible to cleavage by tetanus toxin. Immunopurified GLUT4-containing vesicles contained cellubrevin and VAMP-2, and immunopurified cellubrevin-containing vesicles contained GLUT4 protein, but undiscernible amounts of VAMP-2. These observations suggest that cellubrevin and VAMP-2 are constituents of the insulin-regulated pathway of membrane traffic. These results are the first demonstration that cellubrevin is present in a regulated intracellular compartment. We hypothesize that cellubrevin and VAMP-2 may be present in different subsets of GLUT4-containing vesicles.