Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1

In pancreatic beta cells, the endoplasmic reticulum (ER) is an important site for insulin biosynthesis and the folding of newly synthesized proinsulin. Here, we show that IRE1alpha, an ER-resident protein kinase, has a crucial function in insulin biosynthesis. IRE1alpha phosphorylation is coupled to insulin biosynthesis in response to transient exposure to high glucose; inactivation of IRE1alpha signaling by siRNA or inhibition of IRE1alpha phosphorylation hinders insulin biosynthesis. IRE1 activation by high glucose does not accompany XBP-1 splicing and BiP dissociation but upregulates its target genes such as WFS1. Thus, IRE1 signaling activated by transient exposure to high glucose uses a unique subset of downstream components and has a beneficial effect on pancreatic beta cells. In contrast, chronic exposure of beta cells to high glucose causes ER stress and hyperactivation of IRE1, leading to the suppression of insulin gene expression. IRE1 signaling is therefore a potential target for therapeutic regulation of insulin biosynthesis.     

PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress

The accumulation of unfolded proteins elicits a cellular response that triggers both pro-survival and pro-apoptotic signaling events. PERK-dependent activation of NF-E2-related factor-2 (Nrf2) is critical for survival signaling during this response; however, the mechanism whereby Nrf2 confers a protective advantage to stressed cells remains to be defined. We now demonstrate that Nrf2 activation contributes to the maintenance of glutathione levels, which in turn functions as a buffer for the accumulation of reactive oxygen species during the unfolded protein response. The deleterious effects of Nrf2 or PERK deficiencies could be attenuated by the restoration of cellular glutathione levels or Nrf2 activity. In addition, the inhibition of reactive oxygen species production attenuated apoptotic induction following endoplasmic reticulum stress. Our data suggest that perturbations in cellular redox status sensitize cells to the harmful effects of endoplasmic reticulum stress, but that other factors are essential for apoptotic commitment.     

PDK1 deficiency in POMC-expressing cells reveals FOXO1-dependent and -independent pathways in control of energy homeostasis and stress response

Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.     

Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents

The endogenous lipid signaling agent oleoylethanolamide (OEA) has recently been described as a peripherally acting agent that reduces food intake and body weight gain in rat feeding models. This paper presents evidence that OEA is an endogenous ligand of the orphan receptor GPR119, a G protein-coupled receptor (GPCR) expressed predominantly in the human and rodent pancreas and gastrointestinal tract and also in rodent brain, suggesting that the reported effects of OEA on food intake may be mediated, at least in part, via the GPR119 receptor. Furthermore, we have used the recombinant receptor to discover novel selective small-molecule GPR119 agonists, typified by PSN632408, which suppress food intake in rats and reduce body weight gain and white adipose tissue deposition upon subchronic oral administration to high-fat-fed rats. GPR119 therefore represents a novel and attractive potential target for the therapy of obesity and related metabolic disorders.     

Nck-dependent activation of extracellular signal-regulated kinase-1 and regulation of cell survival during endoplasmic reticulum stress

In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44(MAPK)/extracellular signal-regulated kinase-1 (ERK-1), and p38(MAPK) through IRE1alpha-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival.     

Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes

The serine/threonine kinase Akt2 has been implicated in insulin-regulated glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. However, it remains unclear whether activation of Akt2 is sufficient since a role for alternate signaling pathways has been proposed. Here we have engineered 3T3-L1 adipocytes to express a rapidly inducible Akt2 system based on drug-inducible heterodimerization. Addition of the dimerizer rapalog resulted in activation of Akt2 within 5 min, concomitant with phosphorylation of the Akt substrates AS160 and GSK3. Comparison with insulin stimulation revealed that the level of Akt2 activity observed with rapalog was within the physiological range, reducing the likelihood of off-target effects. Transient activation of Akt2 also increased glucose transport and GLUT4 translocation to the plasma membrane. These results show that activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes to an extent similar to insulin.     

SirT1 inhibition reduces IGF-I/IRS-2/Ras/ERK1/2 signaling and protects neurons

Sirtuins are known to protect cells and extend life span, but our previous studies indicated that S. cerevisiae Sir2 can also increase stress sensitivity and limit life-span extension. Here we provide evidence for a role of the mammalian Sir2 ortholog SirT1 in the sensitization of neurons to oxidative damage. SirT1 inhibition increased acetylation and decreased phosphorylation of IRS-2; it also reduced activation of the Ras/ERK1/2 pathway, suggesting that SirT1 may enhance IGF-I signaling in part by deacetylating IRS-2. Either the inhibition of SirT1 or of Ras/ERK1/2 was associated with resistance to oxidative damage. Markers of oxidized proteins and lipids were reduced in the brain of old SirT1-deficient mice, but the life span of the homozygote knockout mice was reduced under both normal and calorie-restricted conditions. These results are consistent with findings in S. cerevisiae and other model systems, suggesting that mammalian sirtuins can play both protective and proaging roles.     

Loss of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease

The Akt signaling pathway controls several cellular functions in the cardiovascular system; however, its role in atherogenesis is unknown. Here, we show that the genetic ablation of Akt1 on an apolipoprotein E knockout background (ApoE^−/−Akt1^−/−) increases aortic lesion expansion and promotes coronary atherosclerosis. Mechanistically, lesion formation is due to the enhanced expression of proinflammatory genes and endothelial cell and macrophage apoptosis. Bone marrow transfer experiments showing that macrophages from ApoE^−/−Akt1^−/− donors were not sufficient to worsen atherogenesis when transferred to ApoE^−/− recipients suggest that lesion expansion in the ApoE^−/−Akt1^−/− strain might be of vascular origin. In the vessel wall, the loss of Akt1 increases inflammatory mediators and reduces eNOS phosphorylation, suggesting that Akt1 exerts vascular protection against atherogenesis. The presence of coronary lesions in ApoE^−/−Akt1^−/− mice provides a new model for studying the mechanisms of acute coronary syndrome in humans.     

Differences in endoplasmic reticulum stress signalling kinetics determine cell survival outcome through activation of MKP-1

Endoplasmic reticulum (ER) stress signalling pathways are involved in various alterations of the central nervous system such as neurodegenerative diseases or ischemia. The current mechanisms linking ER stress activation to neuronal cell fate upon chronic or acute stresses remain however to be fully understood. Recent studies have associated ER stress severity and the relative activation levels of certain output pathways to influence cell-fate decisions. In the present report, to further test the impact of ER stress severity on neuronal survival, we designed an experimental system recapitulating acute and chronic stress in cerebellar granule neurons (CGNs) and c17.2 mouse neural stem cells (NSCs). Two well characterized ER stress inducers, tunicamycin (TM) and dithiothreitol (DTT), were used to induce “slow motion” and “fast motion” stresses, respectively. We show that the duration of JNK activation is critical for cell survival upon ER stress. TM-induced transient JNK activation is a protective event in CGNs and c17.2 NSCs via the phosphorylation of BAD, while DTT-induced prolonged JNK activation mediates pro-apoptotic signalling. In addition, we demonstrate that ER stress mediated MKP-1/DUSP1 expression regulates JNK activation kinetics. MKP-1 phosphorylation and protein expression level are differentially altered upon TM and DTT treatment. Increased MKP-1 protein stability via its phosphorylation on ser359 induced by TM accounts for transient JNK activation and the resulting cell survival in CGNs and c17.2 NSCs subjected to ER stress. These results suggest that MKP-1 plays a pivotal role in ER stress-induced cell apoptosis through regulating JNK-BAD signalling.     

IRS1-independent defects define major nodes of insulin resistance

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.     

Cannabinoid Receptor Type 1 Protects against Age- Related Osteoporosis by Regulating Osteoblast and Adipocyte Differentiation in Marrow Stromal Cells

Age-related osteoporosis is characterized by reduced bone formation and accumulation of fat in the bone marrow compartment. Here, we report that the type 1 cannabinoid receptor (CB1) regulates this process. Mice with CB1 deficiency (CB1^−/−) had increased peak bone mass due to reduced bone resorption, but developed age-related osteoporosis with reduced bone formation and accumulation of adipocytes in the bone marrow space. Marrow stromal cells from CB1^−/− mice had an enhanced capacity for adipocyte differentiation, a reduced capacity for osteoblast differentiation, and increased expression of phosphorylated CREB (pCREB) and PPARγ. Pharmacological blockade of CB1 receptors stimulated adipocyte differentiation, inhibited osteoblast differentiation, and increased cAMP and pCREB in osteoblast and adipocyte precursors. The CB1 receptor is therefore unique in that it regulates peak bone mass through an effect on osteoclast activity, but protects against age-related bone loss by regulating adipocyte and osteoblast differentiation of bone marrow stromal cells.     

JNK1 in hematopoietically derived cells contributes to diet-induced inflammation and insulin resistance without affecting obesity

Obesity-induced insulin resistance is a major factor in the etiology of type 2 diabetes, and Jun kinases (JNKs) are key negative regulators of insulin sensitivity in the obese state. Activation of JNKs (mainly JNK1) in insulin target cells results in phosphorylation of insulin receptor substrates (IRSs) at serine and threonine residues that inhibit insulin signaling. JNK1 activation is also required for accumulation of visceral fat. Here we used reciprocal adoptive transfer experiments to determine whether JNK1 in myeloid cells, such as macrophages, also contributes to insulin resistance and central adiposity. Our results show that deletion of Jnk1 in the nonhematopoietic compartment protects mice from high-fat diet (HFD)-induced insulin resistance, in part through decreased adiposity. By contrast, Jnk1 removal from hematopoietic cells has no effect on adiposity but confers protection against HFD-induced insulin resistance by decreasing obesity-induced inflammation.     

Autophagy is activated for cell survival after endoplasmic reticulum stress

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 “dots”), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.     

Hypoxia triggers endothelial endoplasmic reticulum stress and apoptosis via induction of VLDL receptor

Endothelial cells express very low density lipoprotein receptor (VLDLr). Beyond the function as peripheral lipoprotein receptor, other roles of VLDLr in endothelial cells have not been completely unraveled. In the present study, human umbilical vein endothelial cells were subjected to hypoxia, and VLDLr expression, endoplasmic reticulum (ER) stress, and apoptosis were assessed. Hypoxia triggered endothelial ER stress and apoptosis, and induced VLDLr expression. Silencing or stabilization of HIF-1α reduced and enhanced VLDLr expression, respectively. HIF-1α affected vldlr promoter activity by interacting with a hypoxia-responsive element (HRE). Knockdown or overexpression of VLDLr alleviated and exacerbated hypoxia-induced ER stress and apoptosis, respectively. Thus, hypoxia induces VLDLr expression through the interaction of HIF-1α with HRE at the vldlr promoter. VLDLr then mediates ER stress and apoptosis.