Skeletal Muscle–Specific Deletion of Lipoprotein Lipase Enhances Insulin Signaling in Skeletal Muscle but Causes Insulin Resistance in Liver and Other Tissues

OBJECTIVE—Skeletal muscle–specific LPL knockout mouse (SMLPL−/−) were created to study the systemic impact of reduced lipoprotein lipid delivery in skeletal muscle on insulin sensitivity, body weight, and composition

The Extended Cleavage Specificity of Human Thrombin

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times

DE INVLOED VAN TARIEFSWIJZIGINGEN OP DEN VERKEERSOMVANG BIJ HET AMSTERDAMSCHE TRAMBEDRIJF (Inhoud juli 1932 no. 7)

DE INVLOED VAN TARIEFSWIJZIGINGEN OP DEN VERKEERSOMVANG BIJ HET AMSTERDAMSCHE TRAMBEDRIJF (Inhoud juli 1932 no. 7

Nicotinamide Promotes Adipogenesis in Umbilical Cord-Derived Mesenchymal Stem Cells and Is Associated with Neonatal Adiposity: The Healthy Start BabyBUMP Project

<div><p>The cellular mechanisms whereby excess maternal nutrition during pregnancy increases adiposity of the offspring are not well understood. However, nicotinamide (NAM), a fundamental micronutrient that is important in energy metabolism, has been shown to regulate adipogenesis through inhibition of SIRT1. Here we tested three novel hypotheses: 1) NAM increases the adipogenic response of human umbilical cord tissue-derived mesenchymal stem cells (MSCs) through a SIRT1 and PPARγ pathway; 2) lipid potentiates the NAM-enhanced adipogenic response; and 3) the adipogenic response to NAM is associated with increased percent fat mass (%FM) among neonates. MSCs were derived from the umbilical cord of 46 neonates born to non-obese mothers enrolled in the Healthy Start study. Neonatal %FM was measured using air displacement plethysmography (Pea Pod) shortly after birth. Adipogenic differentiation was induced for 21 days in the 46 MSC sets under four conditions, +NAM (3mM)/–lipid (200 μM oleate/palmitate mix), +NAM/+lipid, –NAM/+lipid, and vehicle-control (–NAM/–lipid). Cells incubated in the presence of NAM had significantly higher PPARγ protein (+24%, p <0.01), FABP4 protein (+57%, p <0.01), and intracellular lipid content (+51%, p <0.01). Lipid did not significantly increase either PPARγ protein (p = 0.98) or FABP4 protein content (p = 0.82). There was no evidence of an interaction between NAM and lipid on adipogenic response of PPARγ or FABP4 protein (p = 0.99 and p = 0.09). In a subset of 9 MSC, SIRT1 activity was measured in the +NAM/-lipid and vehicle control conditions. SIRT1 enzymatic activity was significantly lower (-70%, p <0.05) in the +NAM/-lipid condition than in vehicle-control. In a linear model with neonatal %FM as the outcome, the percent increase in PPARγ protein in the +NAM/-lipid condition compared to vehicle-control was a significant predictor (β = 0.04, 95% CI 0.01–0.06, p <0.001). These are the first data to support that chronic NAM exposure potentiates adipogenesis in human MSCs <i>in-vitro</i>, and that this process involves PPARγ and SIRT1.</p></div

Effects of NAM (3mM) and lipid (200uM) during adipose differentiation on SIRT1 protein and enzyme activity.

<p>SIRT1 protein content in MSCs measured by ICE assay at day 21 in all four treatment conditions in the full experimental cohort of 46 cell sets (A). A representative sample of 9 sets of cells from the 46 experimental sets was used for SIRT1 enzyme activity at day 21 in vehicle-control and NAM conditions only (B). *p<0.05 for t-test of NAM vs. vehicle-control; #p<0.05 for main effect of NAM regardless of lipid treatment using MANOVA.</p

Scatter plots depicting the correlation between neonatal adiposity and the percent change of PPARγ (A) and FABP4 (B) protein content at day 21 between vehicle-control and NAM only conditions (N = 46).

<p>Scatter plots depicting the correlation between neonatal adiposity and the percent change of PPARγ (A) and FABP4 (B) protein content at day 21 between vehicle-control and NAM only conditions (N = 46).</p

Effects of NAM (3mM) incubation during adipocyte cell differentiation in human MSC.

<p>Cells were harvested at day 9 of differentiation and mRNA expression of PPARγ (A), SIRT1 enzyme activity (B), and acetylation of SIRT1 protein targets, PPARγ (C) and β-catenin (D), and mRNA expression of NAMPT (E) in the vehicle-control and NAM only conditions as described in Methods. N = 9 per group. *p<0.05 vs. vehicle-control.</p