Multiorgan Systems Study Reveals Igfbp7 as a Suppressor of Gluconeogenesis after Gastric Bypass Surgery.

Roux-en-Y gastric bypass (RYGB) surgery reduces weight in obese patients. A marked decrease in blood glucose levels occurs before weight loss; however, key molecules that improve the glycemic profile remain largely unknown. Using a murine RYGB surgery model, we performed multiorgan proteomics and bioinformatics to monitor the proteins and molecular pathways that change in this early glycemic response. Multiplexed proteomic kinetics data analysis revealed that the Roux limb, biliopancreatic limb, liver, and pancreas each exhibited unique temporal and molecular responses to the RYGB surgery. In addition, protein-protein network analysis indicated that the changes to the microbial environment in the intestine may play a crucial role in the beneficial effects of RYGB surgery. Furthermore, insulin-like growth factor binding protein 7 (Igfbp7) was identified as an early induced protein in the Roux limb. Known secretory properties of Igfbp7 prompted us to further investigate its role as a remote organ regulator of glucose metabolism. Igfbp7 overexpression decreased blood glucose levels in diet-induced obese mice and attenuated gluconeogenic gene expression in the liver. Secreted Igfbp7 appeared to mediate these beneficial effects. These results demonstrate that organs responded differentially to RYGB surgery and indicate that Igfbp7 may play an important role in improving blood glucose levels.

Dynamin-Related Protein 1 Inhibition Attenuates Cardiovascular Calcification in the Presence of Oxidative Stress.

RATIONALE: Mitochondrial changes occur during cell differentiation and cardiovascular disease. DRP1 (dynamin-related protein 1) is a key regulator of mitochondrial fission. We hypothesized that DRP1 plays a role in cardiovascular calcification, a process involving cell differentiation and a major clinical problem with high unmet needs. OBJECTIVE: To examine the effects of osteogenic promoting conditions on DRP1 and whether DRP1 inhibition alters the development of cardiovascular calcification. METHODS AND RESULTS: DRP1 was enriched in calcified regions of human carotid arteries, examined by immunohistochemistry. Osteogenic differentiation of primary human vascular smooth muscle cells increased DRP1 expression. DRP1 inhibition in human smooth muscle cells undergoing osteogenic differentiation attenuated matrix mineralization, cytoskeletal rearrangement, mitochondrial dysfunction, and reduced type 1 collagen secretion and alkaline phosphatase activity. DRP1 protein was observed in calcified human aortic valves, and DRP1 RNA interference reduced primary human valve interstitial cell calcification. Mice heterozygous for Drp1 deletion did not exhibit altered vascular pathology in a proprotein convertase subtilisin/kexin type 9 gain-of-function atherosclerosis model. However, when mineralization was induced via oxidative stress, DRP1 inhibition attenuated mouse and human smooth muscle cell calcification. Femur bone density was unchanged in mice heterozygous for Drp1 deletion, and DRP1 inhibition attenuated oxidative stress-mediated dysfunction in human bone osteoblasts. CONCLUSIONS: We demonstrate a new function of DRP1 in regulating collagen secretion and cardiovascular calcification, a novel area of exploration for the potential development of new therapies to modify cellular fibrocalcific response in cardiovascular diseases. Our data also support a role of mitochondrial dynamics in regulating oxidative stress-mediated arterial calcium accrual and bone loss.

Cystathionine γ-lyase accelerates osteoclast differentiation: identification of a novel regulator of osteoclastogenesis by proteomic analysis.

OBJECTIVE: Clinical evidence has linked vascular calcification in advanced atherosclerotic plaques with overt cardiovascular disease and mortality. Bone resorbing monocyte-derived osteoclast-like cells are sparse in these plaques, indicating that their differentiation capability could be suppressed. Here, we seek to characterize the process of osteoclastogenesis by identifying novel regulators and pathways, with the aim of exploring possible strategies to reduce calcification. APPROACH AND RESULTS: We used a quantitative mass spectrometry strategy, tandem mass tagging, to quantify changes in the proteome of osteoclast-like cells differentiated from RAW264.7 cells in response to, receptor activator of nuclear factor κ-B ligand induction, a common in vitro model for osteogenesis. More than 4000 proteins were quantified, of which 138 were identified as novel osteoclast-related proteins. We selected 5 proteins for subsequent analysis (cystathionine γ-lyase [Cth/CSE], EGF-like repeat and discoidin I-like domain-containing protein 3, integrin α FG-GAP repeat containing 3, adseverin, and serpinb6b) and show that gene expression levels are also increased. Further analysis of the CSE transcript profile reveals an early onset of an mRNA increase. Silencing of CSE by siRNA and dl-propargylglycine, a CSE inhibitor, attenuated receptor activator of nuclear factor κ-B ligand-induced tartrate-resistant acid phosphatase type 5 activity and pit formation, suggesting that CSE is a potent inducer of calcium resorption. Moreover, knockdown of CSE suppressed expression of osteoclast differentiation markers. CONCLUSIONS: Our large-scale proteomics study identified novel candidate regulators or markers for osteoclastogenesis and demonstrated that CSE may act in early stages of osteoclastogenesis.

PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation.

Despite the global impact of macrophage activation in vascular disease, the underlying mechanisms remain obscure. Here we show, with global proteomic analysis of macrophage cell lines treated with either IFNγ or IL-4, that PARP9 and PARP14 regulate macrophage activation. In primary macrophages, PARP9 and PARP14 have opposing roles in macrophage activation. PARP14 silencing induces pro-inflammatory genes and STAT1 phosphorylation in M(IFNγ) cells, whereas it suppresses anti-inflammatory gene expression and STAT6 phosphorylation in M(IL-4) cells. PARP9 silencing suppresses pro-inflammatory genes and STAT1 phosphorylation in M(IFNγ) cells. PARP14 induces ADP-ribosylation of STAT1, which is suppressed by PARP9. Mutations at these ADP-ribosylation sites lead to increased phosphorylation. Network analysis links PARP9-PARP14 with human coronary artery disease. PARP14 deficiency in haematopoietic cells accelerates the development and inflammatory burden of acute and chronic arterial lesions in mice. These findings suggest that PARP9 and PARP14 cross-regulate macrophage activation.

mIMT-visHTS: A novel method for multiplexing isobaric mass tagged datasets with an accompanying visualization high throughput screening tool for protein profiling.

Isobaric mass tagging (IMT) methods enable the analysis of thousands of proteins simultaneously. We used tandem mass tagging reagents (TMT™) to monitor the relative changes in the proteome of the mouse macrophage cell line RAW264.7 at the same six time points after no stimulation (baseline phenotype), stimulation with interferon gamma (pro-inflammatory phenotype) or stimulation with interleukin-4 (anti-inflammatory phenotype). The combined TMT datasets yielded nearly 12,000 protein profiles for comparison. To facilitate this large analysis, we developed a novel method that combines or multiplexes the separate IMT (mIMT) datasets into a single super dataset for subsequent model-based clustering and co-regulation analysis. Specially designed visual High Throughput Screening (visHTS) software screened co-regulated proteins. visHTS generates an interactive and visually intuitive color-coded bullseye plot that enables users to browse the cluster outputs and identify co-regulated proteins.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase--effect of cMOAT deficiency on hepatobiliary excretion in rats and of mdr1a/b gene disruption on tissue distribution in mice.

Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase. In the current study, to elucidate the hepatobiliary excretion of pitavastatin, we investigated the plasma concentration and biliary excretion of (14)C-pitavastatin in EHBR. We also evaluated the distribution of pitavastatin in mdr1a/b knockout mice by whole body autoradiography and quantitative radioassay. In view of the widespread clinical use of pitavastatin and the importance of drug-drug interaction, the inhibitory effect on Pgp-mediated activation of ATPase was also investigated. No marked difference was observed in the plasma concentration and biliary excretion of radioactivity between SDR and EHBR after dosing of (14)C-pitavastatin. Little radioactive transfer into the brain was detected in mdr1a/b knockout mice and the ATPase activity of human Pgp was negligible in the presence of pitavastatin. Moreover, no inhibitory effect on the Pgp-mediated activation of ATPase by verapamil was found in the presence of pitavastatin over a wide concentration range. These results indicated that a cMOAT and Pgp-mediated transport mechanism did not play a major role in the distribution of pitavastatin.

Interaction between fibrates and statins--metabolic interactions with gemfibrozil.

An in vitro study was carried out in order to examine the metabolic basis of the interaction between fibrates and statins. Metabolic inhibition of statins was noted in the presence of gemfibrozil. However, increase in the unchanged form was fairly small for pitavastatin, compared with other statins. Several CYP enzymes were shown to be principally responsible for the metabolism of gemfibrozil in contrast to other fibrates. In the presence of gemfibrozil, a focal point was obtained in Dixon plots, demonstrating that there was inhibition of CYP2C8-, CYP2C9- and CYP3A4-mediated metabolism. We propose that the increase of plasma concentration caused by co-administration of gemfibrozil and statins is at least partially due to CYP-mediated inhibition.

Metabolic fate of pitavastatin (NK-104), a new inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase. Effects on drug-metabolizing systems in rats and humans.

Pitavastatin (CAS 147526-32-7, NK-104) is a new and very potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and has been approved for treatment of hyperlipoproteinaemia. Pitavastatin has been studied for its effects on hepatic microsomal drug metabolism in rats, and the activities of several drug-metabolizing enzymes have been measured. No induction of the drug metabolizing enzymes (aniline hydroxylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and UDP-glucuronic acid transferase) was found in the pitavastatin group compared to the control after the multiple administrations of pitavastatin at the dosage of 1-10 mg/kg per day for 7 days. Based on several different in vitro approaches, it is concluded that CYP2C9 is the enzyme responsible for the metabolism of pitavastatin and no metabolite is present in renal and intestinal microsomes. The CYP2C9 polymorphism was not involved in the pitavastatin metabolism. No inhibitory effect in CYP-mediated metabolism was detected on the tolbutamide 4-hydroxylation (CYP2C9) and testosterone 6 beta-hydroxylation (CYP3A4) in the presence of pitavastatin. The results suggested that pitavastatin did not affect the drug-metabolizing systems.

Studies on the interaction between fibrates and statins using human hepatic microsomes.

To gain a better understanding of the mechanism of drug-drug interaction between fibrates and statins, several in vitro experiments were performed. On coincubation with several fibrates, pitavastatin (CAS 147526-32-7) did not displace fibrates from their protein binding in human plasma. The presence of gemfibrozil (CAS 25812-30-0) inhibited the metabolism of statins (cerivastatin (CAS 145599-86-6) and atorvastatin (CAS 134523-00-5)) remarkably. However, the increase of the unchanged form was fairly small for pitavastatin. The metabolic profile of gemfibrozil was also investigated. The cytochrome P (CYP) enzyme CYP2C9 plays a major role in the metabolism of gemfibrozil. Gemfibrozil showed a high affinity for CYP enzymes and a relatively high metabolism velocity. Moreover, several inhibitory effects of gemfibrozil on CYP-mediated metabolism were detected--in contrast to other fibrates. Although the mechanism of the drug-drug interaction was not completely clarified, it is suggested that the increase of plasma concentration caused by the co-administration of gemfibrozil and statins is at least partially due to the inhibition of the CYP-mediated metabolism.

Total synthesis of spicamycin.

The first total synthesis of one of the spicamycin congeners, SPM VIII (3), is described. A preliminary model study for construction of the characteristic N-glycoside linkage in spicamycin using tetra-O-benzyl-beta-D-mannopyranosylamine (13) and halopurines 5 revealed that Pd-catalyzed conditions successfully provided the coupling products 14 and 15 in good yields. It was also shown that thermal anomerization of the N-glycosides easily occurred, which resulted in the predominant formation of the beta-anomer as the thermodynamically favored compound, and the activation energy of anomerization of 15 was estimated to be ca. 30 kcal/mol. The novel aminoheptose unit of spicamycin 6 was prepared stereoselectively by carbon elongation of an acyclic aldehyde, prepared by ring cleavage reaction of a highly functionalized cyclohexane derived from naturally abundant myo-inositol. The Pd-catalyzed coupling reaction of the beta-heptopyranosylamine 6 with protected 6-chloropurine 5d, followed by deprotection, provided spicamycin amino nucleoside 2, whose condensation with dodecanoylglycine completed the total synthesis of 3. This study confirmed the proposed unique structure of a novel nucleoside antibiotic.