Mandarino, Lawrence J.

The mechanisms of metabolic improvements after Roux-en-Y gastric bypass (RYGB) surgery are not entirely clear. Therefore, the aim of our study was to investigate the role of obesity and RYGB on the human skeletal muscle proteome. Basal muscle biopsies were obtained from seven obese (BMI >40 kg/m2) female subjects (45.1 ± 3.6 years) pre- and 3 months post-RYGB, and euglycemic-hyperinsulinemic clamps were used to assess insulin sensitivity. Four age-matched (48.5 ± 4.7 years) lean (BMI <25 kg/m2) females served as control subjects. We performed quantitative mass spectrometry and microarray analyses on protein and RNA isolated from the muscle biopsies. Significant improvements in fasting plasma glucose (104.2 ± 7.8 vs. 86.7 ± 3.1 mg/dL) and BMI (42.1 ± 2.2 vs. 35.3 ± 1.8 kg/m2) were demonstrated in the pre- versus post-RYGB, both P < 0.05. Proteomic analysis identified 2,877 quantifiable proteins. Of these, 395 proteins were significantly altered in obesity before surgery, and 280 proteins differed significantly post-RYGB. Post-RYGB, 49 proteins were returned to normal levels after surgery. KEGG pathway analysis revealed a decreased abundance in ribosomal and oxidative phosphorylation proteins in obesity, and a normalization of ribosomal proteins post-RYGB. The transcriptomic data confirmed the normalization of the ribosomal proteins. Our results provide evidence that obesity and RYGB have a dynamic effect on the skeletal muscle proteome.

Abstract Immunomodulatory drugs (IMiDs) are therapeutically active compounds that bind to the E3 ubiquitin ligase substrate recruiter cereblon (CRBN) and induce many cellular responses, including cytotoxicity. We had found that the expression of CRBN in multiple myeloma (MM) cells provided the basis for their responses to IMiDs. In this report, we have identified argonaute 2 (AGO2) as a new member of CRBN-downstream binding protein and found that: 1) the steady-state levels of CRBN in IMiD-sensitive cells are significantly higher than in IMiD-resistant cells; 2) the steady-state levels of AGO2 in IMiD-sensitive cells are significantly less compared to IMiD-resistant cells, suggesting that CRBN plays a role in regulating AGO2. AGO2 is a component of microRNA (miRNA)-induced silencing complexes and plays an important role in miRNA maturation and function. The matured miRNAs are considered as key regulators of almost all cellular pathways. Treatment of IMiD-sensitive MM cells with lenalidomide, an IMiD, significantly increased CRBN, decreased both AGO2 and miRNAs and inhibited cell growth. In contrast, although treatment of IMiD-resistant MM cells with lenalidomide moderately increased CRBN and decreased both AGO2 and miRNAs, this treatment did not significantly inhibit cell growth. However, silencing of AGO2 with small hairpin RNA significantly decreased both AGO2 and miRNAs and inhibited cell growth regardless of whether the cells are sensitive or resistant to IMiDs. Therefore, AGO2 could be considered as a new drug target for overcoming IMiDs resistance in MM cells. Citation Format: Qinqin Xu, Yuexian Hou, Paul Langlais, Patrick Erickson, James Zhu, Changxin Shi, Moulun Luo, Yuanxiao Zhu, Lawrence Mandarino, Keith Stewart, Xiu-Bao Chang. Expression of CRBN binding protein AGO2 plays important roles for myeloma cell growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3625. doi:10.1158/1538-7445.AM2015-3625

ObjectiveTo examine associations between circulating levels of the bone‐derived protein osteocalcin (OC) and type 2 diabetes (T2D) risk in Latino children and adults.MethodsSerum OC was measured in 136 children and 531 adults who had the following T2D risk factors assessed, body mass index (BMI), Hemoglobin A1c (HbA1c), fasting and 2‐hour glucose during an oral glucose tolerance test.ResultsOC was significantly higher in children than adults (209.0 ± 12.1 vs. 41.0 ± 0.9 ng/ml, p<0.0001). In adults, OC was inversely associated (all p<0.001) with BMI (r=‐0.2), HbA1c (r=‐0.2), fasting glucose (r=‐0.16), and 2‐hour glucose (r=‐0.21), while there were no significant associations in children. There was a stepwise decrease in OC with increasing dysglycemia in adults, normoglycemic (44.1 ± 1.3 ng/ml), prediabetic (39.3 ± 1.3 ng/ml), and T2D (31.8 ± 1.2 ng/ml), (p<0.0001), whereas there were no differences between normal and prediabetic youth (195.7 ± 16.1 vs. 194.7 ± 25.8 ng/ml, p=0.3).ConclusionsOC was inversely associated with T2D risk in Latino adults; however, this pattern was not observed in children. Am. J. Hum. Biol. 27:859–861, 2015. © 2015 Wiley Periodicals, Inc.

Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr372 resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr372 eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr372 to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr372 as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.

Abstract Motivation: Modern techniques have produced many sequence annotation databases and protein structure portals, but these Web resources are rarely integrated in ways that permit straightforward exploration of protein functional residues and their co-localization. Results: We have created the AMASS database, which maps 1D sequence annotation databases to 3D protein structures with an intuitive visualization interface. Our platform also provides an analysis service that screens mass spectrometry sequence data for post-translational modifications that reside in functionally relevant locations within protein structures. The system is built on the premise that functional residues such as active sites, cancer mutations and post-translational modifications within proteins may co-localize and share common functions. Availability and implementation: AMASS database is implemented with Biopython and Apache as a freely available Web server at amass-db.org. Contact: clinton.mielke@gmail.com

ObjectiveLifestyle intervention can improve insulin sensitivity in obese youth, yet few studies have examined the molecular signatures associated with these improvements. Therefore, the purpose of this study was to explore gene expression changes in whole blood that are associated with intervention‐induced improvements in insulin sensitivity.MethodsFifteen (7M/8F) overweight/obese (BMI percentile = 96.3 ± 1.1) Latino adolescents (15.0 ± 0.9 years) completed a 12‐week lifestyle intervention that included weekly nutrition education and 180 minutes of moderate–vigorous exercise per week. Insulin sensitivity was estimated by an oral glucose tolerance test and the Matsuda Index. Global microarray analysis profiling from whole blood was performed to examine changes in gene expression and to explore biological pathways that were significantly changed in response to the intervention.ResultsA total of 1,459 probes corresponding to mRNA transcripts (717 up, 742 down) were differentially expressed with a fold change ≥1.2. These genes were mapped within eight significant pathways identified, including insulin signaling, type 1 diabetes, and glycerophospholipid metabolism. Participants with increased insulin sensitivity exhibited five times the number of significant genes altered compared with nonresponders (1,144 vs. 230).ConclusionsThese findings suggest that molecular signatures from whole blood are associated with lifestyle‐induced health improvements among high‐risk Latino youth.

Type A insulin resistance, associated with acanthosis nigricans and menstrual irregularity, has been ascribed to a decreased concentration of insulin receptors. We now report four affected females from one family, a mother and three daughters (including identical twins) who appear to have the type A syndrome. Two of the kindred had no apparent ovarian dysfunction, while the other two had hyperprolactinemia without other findings of polycystic ovary disease, suggesting a genetic disease with variable penetrance. All had normal erythrocyte and monocyte insulin binding. Insulin dose-response studies to assess glucose metabolism and insulinsensitivity were performed in the affected twins. The dose response to insulin was shifted to the right with a decrease in maximal response. These results are consistent with a postbinding defect in insulin action in these patients.