Hepatic FoxO1 Integrates Glucose Utilization and Lipid Synthesis through Regulation of Chrebp O-Glycosylation

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis.     

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life ^1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known^6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory ^8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.     

Through the looking glass – a new world of proteins enabled by chemical synthesis

‘Chemical ligation’ – the regioselective and chemoselective covalent condensation of unprotected peptide segments – has enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester‐linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high‐resolution X‐ray crystallography. A ‘glycoprotein mimetic’ of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. d ‐Protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the d ‐enantiomers and l ‐enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear‐loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi‐racemate with the d ‐form of the native protein molecule. We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor‐A; total synthesis of the mirror‐image protein will be used to systematically develop d ‐protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Morphology of lymphatics of the mammalian heart with special reference to the architecture and distribution of the subepicardial lymphatic system

The subepicardial lymphatic system in the rat and dog heart has been investigated by means of scanning electron microscopy. Following application of hydrogen peroxide, the epicardium was removed with a forceps under a dissecting microscope. The subepicardial region contained a well-developed lymphatic system which consisted of the main lymphatic trunks and lymphatic capillaries. The lymphatic trunks of large diameters ran from the apex of the heart to its base. The subepicardial lymphatic capillaries were ramified and anastomosed with each other to form a relatively dense network which extended over the entire surface of both ventricles. These networks joined the main lymphatic trunks. Further, some similar networks were connected with the underlying myocardial lymphatic capillaries.     

Structure and properties of oligonucleotide analogs having phosphoramidate linkages

The oligothymidylate analogs, having several stereo regular phosphoramidate linkages, were synthesized. Melting temperatures(Tm) of complexes of the analogs and poly(dA) were measured by spectroscopic method. The abilities of the analogs to form the complexes with poly(dA) depended on their P-chirality of their modified linkages: one of the chiral isomers formed stable complexes, but another isomer formed less stable complexes.     

Phosphorylation of myocardial fructose-6-phosphate,2-kinase: fructose-2,6-bisphosphatase by cAMP-dependent protein kinase and protein kinase C. Activation by phosphorylation and amino acid sequences of the phosphorylation sites

Phosphorylation of pure fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase from bovine heart by cAMP-dependent protein kinase and protein kinase C was investigated. The major enzyme form (subunit Mr of 58,000) was rapidly phosphorylated by both cAMP-dependent protein kinase and protein kinase C, incorporating 0.8 and 1.0 mol/mol of subunit, respectively. The rate of phosphorylation of the heart enzyme by cAMP-dependent protein kinase was 10 times faster than that of the rat liver enzyme. The minor enzyme (subunit Mr of 54,000), however, was phosphorylated only by protein kinase C and was phosphorylated much more slowly with a phosphate incorporation of less than 0.1 mol/mol of subunit. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C activated the enzyme, but each phosphorylation affected different kinetic parameters. Phosphorylation by cAMP-dependent protein kinase lowered the Km value for fructose 6-phosphate from 87 to 42 microM without affecting the Vmax, whereas the phosphorylation by protein kinase C increased the Vmax value from 55 to 85 milliunits/mg without altering the Km value. The phosphorylated peptides were isolated, and their amino acid sequences were determined. The phosphorylation sites for both cAMP-dependent protein kinase and protein kinase C were located in a single peptide whose sequence was Arg-Arg-Asn-Ser-(P)-Phe-Thr-Pro-Leu-Ser-Ser-Ser-Asn-Thr(P)-Ile-Arg-Arg-Pro. The seryl residue nearest the N terminus was the residue specifically phosphorylated by cAMP-dependent protein kinase, whereas the threonine residue nearest the C terminus was phosphorylated by protein kinase C.