Bariatric Surgery vs Lifestyle Intervention for Diabetes Treatment: 5-Year Outcomes From a Randomized Trial.

CONTEXT: Questions remain about bariatric surgery for type 2 diabetes mellitus (T2DM) treatment. OBJECTIVE: Compare the remission of T2DM following surgical or nonsurgical treatments. DESIGN, SETTING, AND PARTICIPANTS: Randomized controlled trial at the University of Pittsburgh, in the United States. Five-year follow-up from February 2015 until June 2016. INTERVENTIONS: 61 participants with obesity and T2DM who were initially randomized to either bariatric surgical treatments (Roux-en-Y gastric bypass [RYGB] or laparoscopic adjustable gastric banding [LAGB]) or an intensive lifestyle weight loss intervention (LWLI) program for 1 year. Lower level lifestyle weight loss interventions (LLLIs) were then delivered for 4 years. MAIN OUTCOMES AND MEASURES: Diabetes remission assessed at 5 years. RESULTS: The mean age of the patients was 47 ±â€…6.6 years, 82% were women, and 21% African American. Mean hemoglobin A1c level 7.8% ±â€…1.9%, body mass index (BMI) 35.7 ±â€…3.1 kg/m2, and 26 participants (43%) had BMI < 35 kg/m2. Partial or complete T2DM remission was achieved by 30% (n = 6) of RYGB, 19% (n = 4) of LAGB, and no LWLI participants (P = .0208). At 5 years those in the RYGB group had the largest percentage of individuals (56%) not requiring any medications for T2DM compared with those in the LAGB (45%) and LWLI (0%) groups (P = .0065). Mean reductions in percent body weight at 5 years was the greatest after RYGB 25.2% ±â€…2.1%, followed by LAGB 12.7% ±â€…2.0% and lifestyle treatment 5.1% ±â€…2.5% (all pairwise P < .01). CONCLUSIONS: Surgical treatments are more effective than lifestyle intervention alone for T2DM treatment.

Retropharyngeal Parathyroid Glands: Important Differences.

INTRODUCTION: In primary hyperparathyroidism (PHPT), parathyroid ectopia is seen in up to 22% leading to more difficult surgery. We aimed to determine the rate and characteristics of retropharyngeal (RP) parathyroid glands. METHODS: A prospective database was queried for patients with sporadic PHPT who had surgery from 1997 to 2016. The data of RP patients were compared to those who had surgery for sporadic PHPT over the same time period with hyperfunctioning parathyroids in anatomically normal positions (N). RESULTS: RP glands occurred in 47/3006 (1.6%) patients and were more common at reoperative than initial surgery (5.5 vs 1.4%, p < 0.01). RP patients had prior failed surgery more often than N patients (17 vs 3.1%, p < 0.01). Preoperative calcium levels (p = 0.06), PTH levels (p = 0.15), and mean gland weights (p = 0.07) were similar among groups. For RP glands, ultrasound imaging was negative in all but one patient, while 99mTc-sestamibi accurately indicated a posterior midline position in only 13/47 (28%) and was negative in 21%. All RP glands were anatomically superior. RP patients more often required > 1 post-resection intraoperative PTH level (36 vs 21%, p = 0.02). Failure due to persistent PHPT was more likely in RP patients (4.7 vs 2.1%, p = 0.2). CONCLUSION: In PHPT, hyperfunctioning RP glands are seen in 1.6% of cases and often associated with initial failure (17%). At reoperation, RP ectopia is 4X more common. RP glands are associated with a high rate of negative imaging, but imaging results suggestive of a midline abnormality can guide exploration. The RP space should be evaluated prior to ending an otherwise unfruitful surgery.

Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.

Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.

Low molecular weight protein enrichment on mesoporous silica thin films for biomarker discovery.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.(1-3) The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.(4,5) Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.(6) Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.(7-9) Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.(10,11) Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

Monitoring the progression of metastatic breast cancer on nanoporous silica chips.

Breast cancer accounted for 15 per cent of total cancer deaths in female patients in 2010. Although significant progress has been made in treating early-stage breast cancer patients, there is still no effective therapy targeting late-stage metastatic breast cancers except for the conventional chemotherapy interventions. Until effective therapy for later-stage cancers emerges, the identification of biomarkers for the early detection of tumour metastasis continues to hold the key to successful management of breast cancer therapy. Our study concentrated on the low molecular weight (LMW) region of the serum protein and the information it contains for identifying biomarkers that could reflect the ongoing physiological state of all tissues. Owing to technical difficulties in harvesting LMW species, studying these proteins/peptides has been challenging until now. In our study, we have recently developed nanoporous chip-based technologies to separate small proteins/peptides from the large proteins in serum. We used nanoporous silica chips, with a highly periodic nanostructure and uniform pore size distribution, to isolate LMW proteins and peptides from the serum of nude mice with MDA-MB-231 human breast cancer lung metastasis. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and biostatistical analysis, we were able to identify protein signatures unique to different stages of cancer development. The approach and results reported in this study possess a significant potential for the discovery of proteomic biomarkers that may significantly enhance personalized medicine targeted at metastatic breast cancer.

ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins.

Although the evidence linking apoA-IV expression and triglyceride (TG)-rich lipoprotein assembly and secretion is compelling, the intracellular mechanisms by which apoA-IV could modulate these processes remain poorly understood. We therefore examined the functional impact of apoA-IV expression on endogenous apoB, TG, and VLDL secretion in stably transfected McA-RH7777 rat hepatoma cells. Expression of apoA-IV modified with the endoplasmic reticulum (ER) retention signal KDEL (apoA-IV-KDEL) dramatically decreased both the rate and efficiency of endogenous apoB secretion, suggesting a presecretory interaction between apoA-IV-KDEL and apoB or apoB-containing lipoproteins. Expression of native apoA-IV using either a constitutive or tetracycline-inducible promoter delayed the initial rate of apoB secretion and reduced the final secretion efficiency by ∼40%. However, whereas apoA-IV-KDEL reduced TG secretion by 75%, expression of native apoA-IV caused a 20-35% increase in TG secretion, accompanied by a ∼55% increase in VLDL-associated apoB, an increase in the TG:phospholipid ratio of secreted d < 1.006 lipoproteins, and a 10.1 nm increase in peak VLDL(1) particle diameter. Native apoA-IV expression had a negligible impact on expression of the MTP gene. These data suggest that by interacting with apoB in the secretory pathway, apoA-IV alters the trafficking kinetics of apoB-containing TG-rich lipoproteins through cellular lipidation compartments, which in turn, enhances particle expansion and increases TG secretion.

Reduced eukaryotic initiation factor 2Bepsilon-subunit expression suppresses the transformed phenotype of cells overexpressing the protein.

Eukaryotic initiation factor 2B (eIF2B), a five-subunit guanine nucleotide exchange factor, plays a key role in the regulation of mRNA translation. Expression of its epsilon-subunit is specifically up-regulated in certain conditions associated with increased cell growth. Therefore, the purpose of the present study was to examine the effect of repressing eIF2Bepsilon expression on growth rate, protein synthesis, and other characteristics of two tumorigenic cell lines that display up-regulated expression of the epsilon-subunit. Experiments were designed to compare spontaneously transformed fibroblasts to transformed mouse embryonic fibroblasts infected with a lentivirus containing a short hairpin RNA directed against eIF2Bepsilon. Cells expressing the short hairpin RNA displayed a reduction in eIF2Bepsilon abundance to 30% of the value observed in uninfected transformed mouse embryonic fibroblasts, with no change in the expression of any of the other four subunits. The repression of eIF2Bepsilon expression was accompanied by reductions in guanine nucleotide exchange factor activity and global rates of protein synthesis. Moreover, repressed eIF2Bepsilon expression led to marked reductions in cell growth rate in culture, colony formation in soft agar, and tumor progression in nude mice. Similar results were obtained in MCF-7 human breast cancer cells in which eIF2Bepsilon expression was repressed through transient transfection with a small interfering RNA directed against the epsilon-subunit. Overall, the results support a role for eIF2Bepsilon in the regulation of cell growth and suggest that it might represent a therapeutic target for the treatment of human cancer.

Activation of the mammalian target of rapamycin complex 1 is both necessary and sufficient to stimulate eukaryotic initiation factor 2Bvarepsilon mRNA translation and protein synthesis.

In a previous study we demonstrated a requirement for activation of mTORC1 in the stimulation of eIF2Bepsilon mRNA translation in skeletal muscle in response to resistance exercise. Although that study established the necessity of mTORC1 activation, the experimental model used did not lend itself readily to address the question of whether or not mTORC1 activation was sufficient to produce the response. Therefore, the present study was designed to address the sufficiency of mTORC1 activation, using cultures of Rat2 fibroblasts in which mTORC1 signaling was repressed by serum/leucine-depletion and stimulated by repletion of leucine and/or IGF-1. Repletion with leucine and IGF-1 caused a shift of eIF2Bepsilon mRNA into actively translating polysomes and a stimulation of new eIF2Bepsilon protein synthesis, but had no effect on mRNAs encoding the other four eIF2B subunits. Stimulation of eIF2Bepsilon translation was reversed by pre-treatment with the mTORC1 inhibitor rapamycin. Exogenous overexpression of FLAG-Rheb, a proximal activator of mTORC1, also caused a re-distribution of eIF2Bepsilon mRNA into polysomes and a stimulation of eIF2Bepsilon protein synthesis. The stimulation of eIF2Bepsilon mRNA translation occurred in the absence of any effect on eIF2Bepsilon mRNA abundance. RNAi-mediated knockdown of eIF2Bepsilon resulted in reduced cellular proliferation, a result that phenocopied the known cytostatic effect of mTORC1 repression. Overall the results demonstrate that activation of mTORC1 is both necessary and sufficient to stimulate eIF2Bepsilon mRNA translation and that this response may represent a novel mechanism through which mTORC1 can affect mRNA translation initiation, rates of protein synthesis, and cellular growth/proliferation.

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size.

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB.

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion.

Structure and interfacial properties of human apolipoprotein A-V.

Apolipoprotein A-V (apoA-V), the newest member of the plasma apolipoprotein family, was recently discovered by comparison of the mouse and human genomes. Studies in rodents and population surveys of human apoA-V polymorphisms have noted a strong effect of apoA-V on plasma triglyceride levels. Toward the elucidation of the biologic function of apoA-V, we used spectroscopic and surface chemistry techniques to probe its structure and interfacial activity. Computer-assisted sequence analysis of apoA-V predicts that it is very hydrophobic, contains a significant amount of alpha-helical secondary structure, and probably is composed of discrete structural regions with varying degrees of lipid affinity. Fluorescence spectroscopy of recombinant human apoA-V provided evidence of tertiary folding, and light scattering studies indicated that apoA-V transforms dimyristoylphosphatidylcholine vesicles into discoidal complexes with an efficiency similar to that of apoA-I. Surface chemistry techniques revealed that apoA-V displays high affinity, low elasticity, and slow binding kinetics at hydrophobic interfaces, properties we propose may retard triglyceride-rich particle assembly. Metabolic labeling and immunofluorescence studies of COS-1 cells transfected with human apoA-V demonstrated that apoA-V is poorly secreted, remains associated with the endoplasmic reticulum, and does not traffic to the Golgi. Given that overexpression of the apoA-V gene lowers plasma triglycerides in mice, these data together suggest that apoA-V may function intracellularly to modulate hepatic VLDL synthesis and/or secretion.