Ulupi S. Jhala

Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells.

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.

COP1 Functions as a FoxO1 Ubiquitin E3 Ligase to Regulate FoxO1-mediated Gene Expression

COP1 is a Ring-Finger E3 ubiquitin ligase that is involved in plant development, mammalian cell survival, growth, and metabolism. Here we report that COP1, whose expression is enhanced by insulin, regulates FoxO1 protein stability. We found that in Fao hepatoma cells, ectopic expression of COP1 decreased, whereas knockdown of COP1 expression increased the level of endogenous FoxO1 protein without impacting other factors such as C/EBPα and CREB (cAMP-response element-binding protein). We further showed that COP1 binds FoxO1, enhances its ubiquitination, and promotes its degradation via the ubiquitin-proteasome pathway. To determine the biological significance of COP1-mediated FoxO1 protein degradation, we have examined the impact of COP1 on FoxO1-mediated gene expression and found that COP1 suppressed FoxO1 reporter gene as well as FoxO1 target genes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two key targets for FoxO1 in the regulation of gluconeogenesis, with corresponding changes of hepatic glucose production in Fao cells. We suggest that by functioning as a FoxO1 E3 ligase, COP1 may play a role in the regulation of hepatic glucose metabolism.

Glucose Regulates Steady-state Levels of PDX1 via the Reciprocal Actions of GSK3 and AKT Kinases

The pancreatic beta cell is sensitive to even small changes in PDX1 protein levels; consequently, Pdx1 haploinsufficiency can inhibit beta cell growth and decrease insulin biosynthesis and gene expression, leading to compromised glucose-stimulated insulin secretion. Using metabolic labeling of primary islets and a cultured β cell line, we show that glucose levels modulate PDX1 protein phosphorylation at a novel C-terminal GSK3 consensus that maps to serines 268 and 272. A decrease in glucose levels triggers increased turnover of the PDX1 protein in a GSK3-dependent manner, such that PDX1 phosphomutants are refractory to the destabilizing effect of low glucose. Glucose-stimulated activation of AKT and inhibition of GSK3 decrease PDX1 phosphorylation and delay degradation. Furthermore, direct pharmacologic inhibition of AKT destabilizes, and inhibition of GSK3 increases PDX1 protein stability. These studies define a novel functional role for the PDX1 C terminus in mediating the effects of glucose and demonstrate that glucose modulates PDX1 stability via the AKT-GSK3 axis.

Proteomic analysis of protein palmitoylation in adipocytes.

Protein palmitoylation, by modulating the dynamic interaction between protein and cellular membrane, is involved in a wide range of biological processes, including protein trafficking, sorting, sub-membrane partitioning, protein-protein interaction and cell signaling. To explore the role of protein palmitoylation in adipocytes, we have performed proteomic analysis of palmitoylated proteins in adipose tissue and 3T3-L1 adipocytes and identified more than 800 putative palmitoylated proteins. These include various transporters, enzymes required for lipid and glucose metabolism, regulators of protein trafficking and signaling molecules. Of note, key proteins involved in membrane translocation of the glucose-transporter Glut4 including IRAP, Munc18c, AS160 and Glut4, and signaling proteins in the JAK-STAT pathway including JAK1 and 2, STAT1, 3 and 5A and SHP2 in JAK-STAT, were palmitoylated in cultured adipocytes and primary adipose tissue. Further characterization showed that palmitoylation of Glut4 and IRAP was altered in obesity, and palmitoylation of JAK1 played a regulatory role in JAK1 intracellular localization. Overall, our studies provide evidence to suggest a novel and potentially regulatory role for protein palmitoylation in adipocyte function.

Specification of hepatopancreas progenitors in zebrafish by hnf1ba and wnt2bb

Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating β-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and β-cell development, and suggest a new mechanism for hepatopancreatic specification.

Lysine 63-linked ubiquitination modulates mixed lineage kinase-3 interaction with JIP1 scaffold protein in cytokine-induced pancreatic β cell death.

Background: Cytokines activate MLK3 to compromise mitochondrial integrity and promote pancreatic β cell death. Results: Results suggest a model in which IL-1β/TRAF6-mediated Lys-63-linked ubiquitination of MLK3 dissociates monomeric MLK3 from JIP1 for subsequent dimerization and activation. Conclusion: IL-1β-stimulated Lys-63-linked ubiquitination activates MLK3, most likely by altering the dynamics of MLK-JNK-JIP module. Significance: These findings can be exploited for therapeutic intervention in diabetes. The mixed lineage kinase MLK3 plays a crucial role in compromising mitochondrial integrity and functions as a proapoptotic competence factor in the early stages of cytokine-induced pancreatic β cell death. In an effort to identify mechanisms that regulate MLK3 activity in β cells, we discovered that IL-1β stimulates Lys-63-linked ubiquitination of MLK3 via a conserved, TRAF6-binding peptapeptide motif in the catalytic domain of the kinase. TRAF6-mediated ubiquitination was required for dissociation of inactive monomeric MLK3 from the scaffold protein IB1/JIP1, facilitating the subsequent dimerization, autophosphorylation, and catalytic activation of MLK3. Inability to ubiquitinate MLK3, or the presence of A20, an upstream Lys-63-linked deubiquitinase, strongly curtailed the ability of MLK3 to affect the proapoptotic translocation of BAX in cytokine-stimulated pancreatic β cells, an early step in the progression toward β cell death. These studies suggest a novel mechanism for MLK3 activation and provide new clues for therapeutic intervention in promoting β cell survival.

Correction: Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells.

The authors would like to clarify that the controls previously depicted in Figs ​Figs4E4E and ​and7A7A were for different experiments and were included in error. Fig 4 UCP2 is Up-Regulated in Sirt1 Knockdown Cells and in Sirt1 KO Mice. Fig 7 UCP2 mRNA or Protein Levels in Fed or Starved Wild-Type Mice. The correct control for Fig 4E was located and used to prepare a corrected figure. The correct control for the original Fig 7A could not be located; this panel has therefore been removed after a careful assessment and investigation determined that the result for which original Fig 7A was cited is supported elsewhere in this article, and that removal of this panel does not affect the conclusions of the paper. We have also taken this opportunity to provide new versions of several figures (Figs ​(Figs4,4, ​,5,5, ​,6,6, ​,7)7) in which gel/blot splices and a non-linear level adjustment were made but were not previously indicated or declared, or to replace incorrectly spliced gels/blots with the un-spliced originals. We also take the opportunity to correct two errors in the legend to Fig 6, first to remove a redundant and incorrect sentence, and second to address incorrect description of p values. Fig 5 Sirt1 Binds at the UCP2 Promoter and Represses the Gene. Fig 6 Knockdown of UCP2 in Sirt1 Knockdown Cells Restores Glucose-Induced Insulin Secretion. The text in the Results section titled “UCP2 Levels Increase in Food-Deprived Mice” has been edited to accommodate the removal of the original Fig 7A and the relabeling of Fig 7B, 7C and 7D as Fig 7A, 7B and 7C, respectively. The corrected text and Figs ​Figs4,4, ​,5,5, ​,66 and ​and77 are provided here.

Loss of TRB3 alters dynamics of MLK3-JNK signaling and inhibits cytokine-activated pancreatic beta cell death.

Background: MLK3 induces TRB3 to inhibit AKT and compromise beta cell survival. Results: AKT regulates novel phosphorylation, ubiquitination, and degradation of MLK3, explaining why TRB3−/− islets display attenuated MLK3/JNK activation and beta cell death. Conclusion: TRB3 functionally cooperates with the JNK module to fine-tune inflammatory signaling in beta cells. Significance: Even modest modulation of signaling dynamics can alter inflammatory outcomes. Disabling cellular defense mechanisms is essential for induction of apoptosis. We have previously shown that cytokine-mediated activation of the MAP3K MLK3 stabilizes TRB3 protein levels to inhibit AKT and compromise beta cell survival. Here, we show that genetic deletion of TRB3 results in basal activation of AKT, preserves mitochondrial integrity, and confers resistance against cytokine-induced pancreatic beta cell death. Mechanistically, we find that TRB3 stabilizes MLK3, most likely by suppressing AKT-directed phosphorylation, ubiquitination, and proteasomal degradation of MLK3. Accordingly, TRB3−/− islets show a decrease in both the amplitude and duration of cytokine-stimulated MLK3 induction and JNK activation. It is well known that JNK signaling is facilitated by a feed forward loop of sequential kinase phosphorylation and is reinforced by a mutual stabilization of the module components. The failure of TRB3−/− islets to mount an optimal JNK activation response, coupled with the ability of TRB3 to engage and maintain steady state levels of MLK3, recasts TRB3 as an integral functional component of the JNK module in pancreatic beta cells.