Polypeptide Levels Research Articles

Free fatty acids, glicentin and glucose-dependent insulinotropic polypeptide as potential major determinants of fasting substrate oxidation

The selection of carbohydrates or fat to generate intracellular energy is thought to be crucial for long-term metabolic health. While most studies assess fuel selection after a metabolic challenge, the determinants of substrate oxidation in the fasted state remain largely unexplored. We therefore assessed the respiratory quotient by indirect calorimetry as a read-out for substrate oxidation following an overnight fast. This cross-sectional analysis consisted of 192 (92 women, 100 men) either lean or obese participants. Following an overnight fast, the respiratory quotient (RQ) was assessed, after which a 5-point 75-g oral glucose tolerance test was performed. Unlike glucose and insulin, fasting free fatty acids (FFA) correlated negatively with fasting RQ (p < 0.0001). Participants with high levels of the ketone body β-hydroxybutyric acid had significantly lower RQ values. Fasting levels of glucose-dependent insulinotropic polypeptide (GIP) and glicentin were positively associated with fasting RQ (all p ≤ 0.03), whereas GLP-1 showed no significant association. Neither BMI, nor total body fat, nor body fat distribution correlated with fasting RQ. No relationship between the RQ and diabetes or the metabolic syndrome could be observed. In the fasting state, FFA concentrations were strongly linked to the preferentially oxidized substrate. Our data did not indicate any relationship between fasting substrate oxidation and metabolic diseases, including obesity, diabetes, and the metabolic syndrome. Since glicentin and GIP are linked to fuel selection in the fasting state, novel therapeutic approaches that target these hormones may have the potential to modulate substrate oxidation.

Relationship between levels of serum gastric inhibitory polypeptide (GIP), soluble interleukin-2 receptor (sIL-2R), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and disease condition and prognosis of patients with severe acute pancreatitis.

To explore the relationship between levels of serum gastric inhibitory polypeptide (GIP), soluble interleukin-2 receptor (sIL-2R), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and the disease condition and prognosis in patients with severe acute pancreatitis (SAP). A total of 52 patients with SAP (SAP group) and 50 patients with mild acute pancreatitis (MAP group) admitted to Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China between April 2017 and December 2019 were included in the present study. A further 50 people who had received a healthy physical examination during the same period constituted the healthy control group. The levels of serum GIP, sIL-2R, and sTREM-1 were measured. The levels of serum GIP, sIL-2R, and sTREM-1 were compared among the SAP, MAP and healthy control groups, and the severity of disease (Ranson scoring system) was compared between the SAP and MAP groups. Pearson correlation analysis was used to analyze the correlation between the levels of serum GIP, sIL-2R, and sTREM-1 with the Ranson scores in the SAP group. A receiver operating characteristic (ROC) curve was drawn to evaluate the predictive efficacy of serum GIP, sIL-2R, and sTREM-1 on prognosis. The levels of serum GIP, sIL-2R, and sTREM-1 in the SAP and MAP groups were higher than those in the healthy control group, and the levels in the SAP group were higher than those in the MAP group. The Pearson correlation analysis showed that the levels of serum GIP, sIL-2R, and sTREM-1 in the SAP group were positively correlated with Ranson scores. The levels of serum GIP, sIL-2R, and sTREM-1 in the survival group were lower than those in the deceased group. The ROC curve showed that the best cut-off values of serum GIP, sIL-2R, and sTREM-1 in predicting prognostic survival were 167.040 pg/mL, 70.840 pg/mL, and 128.325 ng/mL, respectively. The levels of serum GIP, sIL-2R, and sTREM-1 are closely related to the severity of illness in patients with SAP and can be used as reference indicators for assessing the onset of SAP and predicting prognosis.

224-LB: A Study to Evaluate the Glucagon and Incretin Responses and Identify the Role of Extrapancreatic Glucagon in Patients with Fibrocalcific Pancreatic Diabetes

Aim: The aim of this study is to evaluate glucagon and incretin responses in patients with FCPD. Methods: Nine FCPD patients and six age- and body mass index-matched healthy controls were subjected to 75 g oral glucose tolerance test (OGTT) followed by isoglycemic intravenous glucose infusion (IIGI). Blood samples were analyzed for incretins and glucagon at nine pre-specified time-points over 3 h and the area under curve was calculated. Results: Glucagon was significantly higher during OGTT compared to IIGI in FCPD patients (98.8 ± 13 pg/ml vs. 63.4 ± 7 pg/ml, P = 0.03) and was also higher when compared to controls (98.8 ± 13 pg/ ml vs. 65.8 ± 18 pg/ml, P = ns). FCPD patients showed very low basal and stimulated c-peptide (0.43 ± 0.14 ng/ml and 1.09 ± 0.3 ng/ ml, respectively) and pancreatic polypeptide (12.3 ± 0.0pg/ml and 12.0 ± 0.6 pg/ml, respectively) levels. Glucagon-like peptide-1 (GLP-1) was significantly higher in cases on OGTT when compared to controls (44.5 ± 9.2 pM vs. 12.8 ± 7.5 pM, P = 0.02). Oxyntomodulin was also insignificantly higher in cases on OGTT when compared to controls (1252 ± 350 pg/ml vs. 859.8 ± 165 pg/ml, P = 0.43). GIP was lower in cases on IIGI when compared to controls (52.9 ± 23.9 pg/ml vs. 144.5 ± 36.1 pg/ml, P = 0.045) and there was blunted response on OGTT as well (106.8 ± 40.3 pg/ml vs. 557.8 ± 96.4 pg/ml, P = 0.003). Discussion: We found hyperglucagonemia in FCPD on OGTT, which was suppressed on IIGI. Increase in L-cell products: GLP-1 and oxyntomodulin and a good correlation between glucagon and GLP-1 during OGTT were suggestive of extrapancreatic glucagon production probably from L-cell. The blunted GIP could probably be due to: inadequate pancreatic enzyme supplements, a selective PC-2 enzyme up-regulation or a negative feedback regulation from extrapancreatic glucagon. Conclusion: Extrapancreatic glucagon does exist in FCPD and may contribute to postprandial hyperglycemia and lower GIP levels. Disclosure A. Shankar: None.

Auricular vagal nerve stimulation enhances gastrointestinal motility and improves interstitial cells of Cajal in rats treated with loperamide.

Gastrointestinal (GI) motility disorders affect a large proportion of the population with limited treatment options. The aims of this study were to investigate the potential of a non-invasive method of auricular vagal nerve stimulation (aVNS) for treating GI dysmotility and to explore possible mechanisms involving slow waves and interstitial cells of Cajal (ICC). Normal rats were treated daily with loperamide for 1 week and then treated, while still on daily loperamide, with aVNS/Sham-aVNS for another 1week. Gastric emptying (GE), small intestine transit (SIT), and GI slow waves were measured. The plasma level of pancreatic polypeptide (PP) and noradrenaline (NE) was assessed by ELISA. ICC in the gastric antrum were detected by immunohistochemistry. (a) aVNS significantly increased the percentage of normal GI slow waves (p<0.05 for both fasting and postprandial states, vs. Sham-aVNS) and accelerated GE (p<0.05, vs. Sham-aVNS) and SIT (p<0.05, vs. Sham-aVNS) impaired by loperamide. (b) aVNS increased plasma PP (p<0.01) and decreased plasma NE (p<0.01), compared with Sham-aVNS. (c) Gastric ICC was decreased by loperamide (p<0.01) but increased after aVNS (p<0.01, vs. Sham aVNS). Loperamide induces upper GI dysmotility. aVNS accelerates upper GI transit and improving pace-making activity mediated via the ICC. Non-invasive aVNS may have a therapeutic potential for upper GI dysmotility.

Genetically Encoding Light‐Responsive Protein‐Polymers Using Translation Machinery for the Multi‐Site Incorporation of Photo‐Switchable Unnatural Amino Acids

AbstractA general and versatile technology to engineer light‐responsive protein‐based biomaterials can enable the manipulation and interrogation of proteins, pathways, and cells, and it will assist the design of “smart” light‐responsive biomaterials. This study reports the evolution of chromosomal aminoacyl‐tRNA synthetases (aaRSs) for azobenzene‐bearing unnatural amino acids (uAAs) with up to ≈40‐fold increased protein production and improved fidelity, as compared with a previously described aaRS. The evolved translation systems enable efficient and accurate incorporation of up to 10 instances of the various light‐responsive uAAs in elastin‐like polypeptides (ELPs). Azobenzene‐containing ELPs are capable of isothermal, reversible, light‐mediated soluble‐to‐insoluble phase transition, with up to a 12 °C difference in the ELP transition temperature upon cis‐to‐trans azobenzene isomerization. Furthermore, the incorporation of azobenzene‐uAAs in ELP diblock‐copolymers enables the creation of light‐responsive self‐assembled nanostructures. Finally, light‐responsive resilin‐inspired polymers are also generated by multi‐site azobenzene‐incorporation. The translation machinery evolved in this study can be used for the multi‐site incorporation of azobenzene moieties at the polypeptide level and constitute a universal methodology for the design of light‐responsive proteins and additional families of protein‐based biomaterials with customized and tunable light‐responsive behavior.

Selective release of gastrointestinal hormones induced by an orally active GPR39 agonist

ObjectivesObesity is a complex disease associated with a high risk of comorbidities. Gastric bypass surgery, an invasive procedure with low patient eligibility, is currently the most effective intervention that achieves sustained weight loss. This beneficial effect is attributed to alterations in gut hormone signaling. An attractive alternative is to pharmacologically mimic the effects of bariatric surgery by targeting several gut hormonal axes. The G protein-coupled receptor 39 (GPR39) expressed in the gastrointestinal tract has been shown to mediate ghrelin signaling and control appetite, food intake, and energy homeostasis, but the broader effect on gut hormones is largely unknown. A potent and efficacious GPR39 agonist (Cpd1324) was recently discovered, but the in vivo function was not addressed. Herein we studied the efficacy of the GPR39 agonist, Cpd1324, on metabolism and gut hormone secretion. MethodsBody weight, food intake, and energy expenditure in GPR39 agonist-treated mice and GPR39 KO mice were studied in calorimetric cages. Plasma ghrelin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) levels were measured. Organoids generated from murine and human small intestine and mouse colon were used to study GLP-1 and PYY release. Upon GPR39 agonist administration, dynamic changes in intracellular GLP-1 content were studied via immunostaining and changes in ion transport across colonic mucosa were monitored in Ussing chambers. The G protein activation underlying GPR39-mediated selective release of gut hormones was studied using bioluminescence resonance energy transfer biosensors. ResultsThe GPR39 KO mice displayed a significantly increased food intake without corresponding increases in respiratory exchange ratios or energy expenditure. Oral administration of a GPR39 agonist induced an acute decrease in food intake and subsequent weight loss in high-fat diet (HFD)-fed mice without affecting their energy expenditure. The tool compound, Cpd1324, increased GLP-1 secretion in the mice as well as in mouse and human intestinal organoids, but not in GPR39 KO mouse organoids. In contrast, the GPR39 agonist had no effect on PYY or GIP secretion. Transepithelial ion transport was acutely affected by GPR39 agonism in a GLP-1- and calcitonin gene-related peptide (CGRP)-dependent manner. Analysis of Cpd1324 signaling properties showed activation of Gαq and Gαi/o signaling pathways in L cells, but not Gαs signaling. ConclusionsThe GPR39 agonist described in this study can potentially be used by oral administration as a weight-lowering agent due to its stimulatory effect on GLP-1 secretion, which is most likely mediated through a unique activation of Gα subunits. Thus, GPR39 agonism may represent a novel approach to effectively treat obesity through selective modulation of gastrointestinal hormonal axes.

Comprehensive efficacy of ipragliflozin on various conditioned type 2 diabetes compared with dipeptidyl peptidase-4 inhibitors and with both agents, based on a real-world multicenter trial

The effects of ipragliflozin, the first sodium-glucose co-transporter 2 inhibitors (SGLT2i) launched in Japan in 2014, and with dipeptidyl peptidase-4 inhibitors (DPP-4i) on glycemic control and metabolic changes were investigated comprehensively on various conditioned type 2 diabetes (T2DM) by evaluating various clinical parameters in a real-world setting. A total of 101 patients with T2DM aged 20-80years with 7.0% ≤ HbA1c < 10.0% were followed in this 52-week, open-label, prospective, real-world, multicenter study. HbA1c decreased significantly in all groups. In ipragliflozin using groups, body weight, waist circumference, blood pressure, HOMA-IR, AST, ALT, γ-GTP, uric acid and leptin levels decreased, in contrast, HDL-cholesterol, total ketone bodies, blood urea nitrogen, creatinine, RBC, hemoglobin and hematocrit levels increased, however, in DPP-4i sole group, no significant trends were observed in these parameters. Change in leptin positively correlated with insulin, while change in total ketone bodies inversely correlated with ALT in ipragliflozin using groups. Fasting active gastric inhibitory polypeptide levels decreased in ipragliflozin sole group. Glucagon showed no changes. No significant safety concerns were observed in this study. Ipragliflozin is useful and safe, showing some contrastive effects on several clinical parameters which are not shown with DPP-4i, resulting several clinical benefits. The co-administration of ipragliflozin and a DPP-4i has a better clinical outcome than either single-agent therapy.

Experimental data using candesartan and captopril indicate no double-edged sword effect in COVID-19.

The key link between renin–angiotensin system (RAS) and COVID-19 is ACE2 (angiotensin-converting enzyme 2), which acts as a double-edged sword, because ACE2 increases the tissue anti-inflammatory response but it is also the entry receptor for the virus. There is an important controversy on several drugs that regulate RAS activity and possibly ACE2, and are widely used, particularly by patients most vulnerable to severe COVID-19. In the lung of healthy rats, we observed that candesartan (an angiotensin type-1, AT1, receptor blocker; ARB) and captopril (an ACE inhibitor; ACEI) up-regulated expression of tissue ACE2 and RAS anti-inflammatory axis receptors (AT2 and Mas receptors). This effect was particularly pronounced in rats with metabolic syndrome (obesity, increased blood pressure and hyperglycemia) and aged rats. Treatment of cultures of human type-II pneumocytes with candesartan or captopril induced up-regulation of ACE2 expression in cells. Treatment with viral spike protein induced a decrease in full-length (i.e. transmembrane) ACE2, an increase in levels of a short intracellular ACE2 polypeptide and an increase in ADAM17 activity in cells, together with an increase in levels of soluble ACE2 and major proinflammatory cytokines in the culture medium. Spike protein-induced changes and levels of spike protein internalization in cells were inhibited by pretreatment with the above-mentioned drugs. The results suggest that these drugs increase ACE2 levels and promote the anti-inflammatory RAS axis in the lung. Furthermore, possible up-regulation of viral entry by the drug-induced increase in expression of transmembrane ACE2 is counteracted by additional mechanisms, particularly by drug-induced inhibition of ADAM17 activity.

Decreased Plasma Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Levels are Linked with Disease Severity in Patients with Liver Cirrhosis

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide that has been found to reduce liver inflammation and protect liver from steatosis. We aimed to measure the level of plasma PACAP in patients with liver cirrhosis and to correlate its level with disease severity and laboratory parameters. 113 patients with clinically diagnosed liver cirrhosis and 110 healthy individuals were selected. Child–Pugh score was performed to evaluate disease severity. Liver biopsy was performed using the Knodell modified histology activity index (KmHAI). The L3 skeletal muscle index (L3SMI) was calculated to evaluate the nutrition status. Plasma PACAP concentrations were detected using ELISA. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the sensitivity and specificity of PACAP for detecting the severity of liver cirrhosis. Plasma PACAP levels were found to be significantly downregulated in patients with liver cirrhosis compared with healthy controls. The case group included 38 Child–Pugh A patients, 39 Child–Pugh B patients, and 36 Child–Pugh C patients. Cirrhosis patients with Child–Pugh C had significantly lower plasma PACAP levels compared with those with Child–Pugh B and A. Cirrhosis patients with Child–Pugh B demonstrated markedly decreased plasma PACAP concentrations compared with those with Child–Pugh A. ROC curve analysis indicated that low level of plasma PACAP may act as a potential indicator for disease progression of liver cirrhosis determined by Child–Pugh classification at both compensation stage (Child–Pugh A developed to Child–Pugh B) and decompensation stage (Child–Pugh B developed to Child–Pugh C).In addition, plasma PACAP levels were inversely associated with KmHAI and ALT as well as AST levels. In both male and female patients, plasma PACAP levels were positively related to L3SMI score. Decreased plasma PACAP levels are linked with disease severity in patients with liver cirrhosis. Reduced plasma PACAP may serve as a biomarker for detection and assessment of the severity of liver cirrhosis.

Novel-iridoviral kinase induces mortality and reduces performance of green peach aphids (Myzus persicae) in transgenic Arabidopsis plants

Insect pests are a serious threat to agricultural productivity. Genetically-modified crops offer a promising alternative, and Bacillus thuringiensis endotoxin genes have played a major role in this respect. However, to overcome insect tolerance issues and to broaden the target range, it is critical to identify alternative insecticidal toxins working through novel mechanisms. In our previous study, a kinase from Chilo iridescent virus (CIV) that has insecticidal activity was identified and designated as Iridovirus Serine/Threonine Kinase (ISTK). A 35 kDa truncated form of ISTK, designated iridoptin, was obtained during expression and purification of ISTK in the yeast system. This yeast-expressed CIV toxin induced 50% mortality in cotton aphids and 100% mortality in green peach aphids (GPA). In this study, codon optimized ISTK gene and iridoptin fragment (designated oISTK and oIRI, respectively) were designed by altering the codon usage features that are seldom present in plant exons. Codon-optimized gene(s) cloned into plant expression vectors were used to stably transform Arabidopsis plants. PCR analysis of genomic DNA of transformed plants confirmed the presence of the DNA insert (oISTK/oIRI) in selected transgenic lines. Further screening was performed by selecting the PCR positive lines, which showed expression of respective toxins at the polypeptide level, using Western blot analyses. Codon-optimized gene constructs resulted in significant improvement in levels of expression of ISTK and iridoptin polypeptide and confirmed its stability in planta. The stable lines expressing either of the two toxin forms induced moderate to very high mortality in GPAs and significantly affected GPA development and fecundity.

Fasting oxyntomodulin, glicentin, and gastric inhibitory polypeptide levels are associated with activation of reward- and attention-related brain centres in response to visual food cues in adults with obesity: A cross-sectional functional MRI study.

Postprandial increases in gastrointestinal hormones are associated with reduced energy intake, partially through direct effects on the brain. However, it remains unknown whether the fasting levels of gastrointestinal hormones are associated with altered brain activity in response to visual food stimuli. We therefore performed a whole-brain regression cross-sectional analysis to assess the association between fasting brain activations according to functional magnetic resonance imaging, performed during viewing of highly desirable versus less desirable food images, with fasting levels of five gastrointestinal hormones (glucagon-like peptide [GLP]-1, GLP-2, oxyntomodulin, glicentin and gastric inhibitory polypeptide [GIP]) in 36 subjects with obesity. We observed that fasting blood levels of GIP were inversely associated with the activation of attention-related areas (visual cortices of the occipital lobe, parietal lobe) and of oxyntomodulin and glicentin with reward-related areas (insula, putamen, caudate for both, and additionally orbitofrontal cortex for glicentin) and the hypothalamus when viewing highly desirable as compared to less desirable food images. Future studies are needed to confirm whether fasting levels of oxyntomodulin, glicentin and GIP are associated with the activation of brain areas involved in appetite regulation and with energy intake in people with obesity.

Torsin ATPases influence chromatin interaction of the Torsin regulator LAP1.

The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.

Маркери ендотеліальної дисфункції як предиктори ускладненого перебігу гострого інфаркту міокарда у поєднанні з цукровим діабетом 2 типу

Today, the endothelium is considered to be a multifunctional organ and a non-specific marker in the pathogenesis of diseases of the cardiovascular system. The course of diseases of the cardiovascular system depends on the presence of comorbid pathology, where type 2 diabetes is one of the possible representations of such pathology. The purpose of the study was to determine the diagnostic role of marker of endothelial dysfunction in patients with acute myocardial infarction with concomitant type 2 diabetes mellitus with regard to the clinical course of comorbid conditions. Material and methods. The study design consisted of 120 patients. They were divided into 2 groups: Group 1 consisted of patients with acute myocardial infarction and concomitant type 2 diabetes (n=69), Group 2 consisted of patients with acute myocardial infarction without concomitant type 2 diabetes (n=51). Patients of both groups matched on age and sex (60 men (50%) and 60 women (50%); their average age was 66.35±0.91 years, р&lt;0,05). The control group consisted of 20 almost healthy people, among them 12 women (60%) and 8 men (40%) (average age was equal to 45.17±2.88 years). Examination of patients was conducted on the basis of Municipal Non-profit Enterprise "City Clinical Hospital No. 27" of Kharkiv City Council in the department of cardiology for patients with acute myocardial infarction. Diagnoses were determined according to the current criteria. All patients enrolled in the study signed a voluntary informed consent to participate in it. All patients underwent general clinical and instrumental examinations. The level of endothelial monocyte-activating polypeptide II in participants of the study was determined on the first day of acute myocardial infarction by using a commercial test system "Human Endothelial monocyte activating polepeptide II ELISA KIT". Results and discussion. According to the results of the study, in patients with acute myocardial infarction in combination with type 2 diabetes the levels of endothelial monocyte-activating polypeptide II was higher than in patients without concomitant type 2 diabetes by 1.65 times (p &lt;0.05). The course of the study revealed the level of endothelial monocyte-activating polypeptide II, which was marked by high risk of cardiogenic shock and acute left ventricular failure. Having analyzed the parameter of endothelial dysfunction in terms of mortality, the researchers made the following conclusion: the endothelial monocyte-activating polypeptide II values greater than 3.44 ng/ml were the predictor of cardiovascular death probability during 6 months. Conclusion. The results of analysis of endothelial function made with use of the marker of endothelial monocyte-activating polypeptide II in patients with acute myocardial infarction with concomitant type 2 diabetes showed that such indicator can be deemed as predictor in complicated comorbid conditions

Bone morphogenetic protein 2 increases insulin-like growth factor I and II transcripts and polypeptide levels in bone cell cultures.

Insulin-like growth factors (IGF) I and II are among the most prevalent growth factors secreted by bone cells and are presumed to act as autocrine regulators of bone formation. Certain growth factors, synthesized by skeletal cells and known to stimulate the replication but not the differentiated function of cells of the osteoblastic lineage, have been shown to inhibit skeletal IGF-I and II synthesis. We postulated that growth factors with limited mitogenic activity and with differentiation-inducing properties, such as bone morphogenetic protein (BMP) 2, have the opposite effect and enhance IGF-I and II synthesis. We tested the effects of BMP-2 on IGF-I and II mRNA expression and polypeptide concentrations in cultures of osteoblast-enriched (OB) cells from 22 day fetal rat calvariae. Steady-state IGF-I and II mRNA levels were determined by northern blot analysis, and IGF-I and II concentrations were determined in acidified and fractionated culture medium by a specific radioimmunoassay. After 24-48 h of treatment, BMP-2 at 3.3 nM increased IGF-I and II transcripts by up to twofold and polypeptide levels by up to fourfold. BMP-2 was a more potent stimulator of IGF-II synthesis, and it was active at doses as low as 0.03 nM for IGF-II mRNA and 0.3 nM for IGF-II protein, whereas a dose of 3.3 nM was required to observe the effect on IGF-I synthesis. The effects of BMP-2 on IGF-I and II transcripts and polypeptide levels were dependent on protein synthesis and decreased in the presence of cycloheximide at 3.6 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Normal levels of ribosome-associated chaperones cure two groups of [PSI+] prion variants

The yeast prion [PSI+] is a self-propagating amyloid of the translation termination factor, Sup35p. For known pathogenic prions, such as [PSI+], a single protein can form an array of different amyloid structures (prion variants) each stably inherited and with differing biological properties. The ribosome-associated chaperones, Ssb1/2p (Hsp70s), and RAC (Zuo1p (Hsp40) and Ssz1p (Hsp70)), enhance de novo protein folding by protecting nascent polypeptide chains from misfolding and maintain translational fidelity by involvement in translation termination. Ssb1/2p and RAC chaperones were previously found to inhibit [PSI+] prion generation. We find that most [PSI+] variants arising in the absence of each chaperone were cured by restoring normal levels of that protein. [PSI+] variants hypersensitive to Ssb1/2p have distinguishable biological properties from those hypersensitive to Zuo1p or Ssz1p. The elevated [PSI+] generation frequency in each deletion strain is not due to an altered [PIN+], another prion that primes [PSI+] generation. [PSI+] prion generation/propagation may be inhibited by Ssb1/2/RAC chaperones by ensuring proper folding of nascent Sup35p, thus preventing its joining amyloid fibers. Alternatively, the effect of RAC/Ssb mutations on translation termination and the absence of an effect on the [URE3] prion suggest an effect on the mature Sup35p such that it does not readily join amyloid filaments. Ssz1p is degraded in zuo1Δ [psi-] cells, but not if the cells carry any of several [PSI+] variants. Our results imply that prions arise more frequently than had been thought but the cell has evolved exquisite antiprion systems that rapidly eliminate most variants.

Pre-operative Predictors of Weight Loss and Weight Regain Following Roux-en-Y Gastric Bypass Surgery: a Prospective Human Study.

There are currently few pre-operative predictors of initial and long-term weight loss following bariatric surgery. We evaluated the role of pre-operative patient characteristics and baseline gut and adipose-derived hormones in predicting maximal total body weight loss (WLmax) and risk of weight regain (WR) after Roux-en-Y gastric bypass (RYGB) surgery. One hundred five adult patients undergoing primary RYGB were prospectively recruited. Baseline demographics were recorded and fasting plasma glucose, glycosylated hemoglobin (A1C), insulin, glucagon, leptin, active ghrelin, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) levels were measured on day of surgery. Our cohort had a mean age of 44.4 ± 13.0years, and initial BMI (body mass index) of 45.1 ± 6.7kg/m2 with mean post-operative follow-up of 40months. Eighty patients were female and 26 had type 2 diabetes mellitus (T2D). Average WLmax was 35.3 ± 7.4%. On univariate analysis, higher baseline fasting ghrelin, lower age, lower CRP (C-reactive protein), lower A1C, and negative T2D status were associated with greater WLmax (p < 0.05). Controlling for these variables using stepwise multivariate regression, only higher fasting ghrelin and younger age were associated significantly with greater WLmax (p < 0.05). In subgroup multivariate regression analysis of T2D patients, higher ghrelin and glucagon were significantly associated with greater WLmax. Following stepwise multivariate regression, lower initial BMI and lower glucagon were associated with greater WR (p < 0.05). Incorporation of baseline biological and hormonal markers may help in developing more accurate predictive models for weight loss following bariatric surgery that help inform patient counseling and decision-making.

Conversion of Neural Stem Cells into Functional Neuron-Like Cells by MicroRNA-218: Differential Expression of Functionality Genes.

Conversion of mesenchymal stem cells (MSC) into neuron-like cells (NLC) is a feasible cell therapy strategy for replacing lost neurons in neuronal disorders. In this study, adipose-derived MSC (ADMSC) were converted into neural stem cells (NSC) via neurosphere. The resulting NSC were then differentiated into NLC by transduction with microRNA-218, using a lentiviral vector. ADMSC, NSC, and NLC were first characterized by flow cytometry, RT-PCR, and immunocytochemistry. The functionality of the NLC was evaluated by qRT-PCR and patch clamp recording. Immunophenotyping of ADMSC showed their immunoreactivity to MSC markers CD90, CD73, CD105, and CD49d, but not to CD31 and CD45. RT-PCR results demonstrated the expression of nestin, neurogenin, neurod1, neurofilament light, and GAP43 genes in NSC while NLC expressed synaptophysin, neurofilament heavy, and GAP43. In addition, NSC morphology changed into multipolar with long processes after transduction with miR-218. Moreover, using qRT-PCR, the expression levels of miR-218 and functionality genes CACNA1C, SNAP25, KCNH1, KCNMA1, and SCN9A were significantly increased in NLC, compared with NSC, and ADMSC at 3weeks and 5months post-transduction. Furthermore, the generated NLC expressed significantly higher protein levels of neurofilament heavy polypeptide (NFh) and enolase 2 (Eno2) neuronal markers, compared with ADMSC and NSC. Finally, action potentials were successfully recorded by the generated NLC, using patch clamp. In summary, ADMSC-derived NSC differentiated into functional NLC by transduction with miR-218. The generated NLC expressed functional SNAP25, CACNA1C, KCNH1, KCNMA1, and SCN9A and produced an action potential, which provides useful insights into the generation of functional neuronal cells.

Decreased Serum Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Levels May Reflect Disease Severity in Patients with Non-traumatic Osteonecrosis of Femoral Head

To investigate serum pituitary adenylate cyclase activating polypeptide (PACAP) levels in relation to disease progression in patients diagnosed with non-traumatic osteonecrosis of femoral head (ONFH). A total of 102 non-traumatic ONFH patients and 95 healthy controls were enrolled in this study. Serum PACAP levels were examined using enzyme-linked immunosorbent assay (ELISA). Radiographic progression of ONFH was detected using the Association Research Circulation Osseous (ARCO) classification system. Harris hip score (HSS) and visual analogue scale (VAS) were analyzed to understand clinical severity. Serum levels of the ONFH marker IL-33 and β‐CTX were also detected. ROC curve analysis was performed to investigate the potential diagnostic value of serum PACAP in the radiographic progression of ONFH. Serum PACAP levels were significantly decreased in non-traumatic ONFH patients when compared to healthy controls. ARCO stage 4 showed markedly lower serum PACAP levels than stage 3. ARCO stage 3 had significantly decreased serum PACAP levels compared to stage 1/2. Serum PACAP levels were negatively correlated with the ARCO classification. In addition, serum PACAP levels were positively associated with HSS score and negatively related to VAS score. Last, PACAP levels were negatively correlated with serum IL-33 and positively associated with β-CTX. ROC curve analysis indicated that PACAP may serve as an early indicator for the radiographic progression of ONFH. Reduced serum PACAP levels may reflect disease severity in non-traumatic ONFH patients.

SAT-650 Novel Insights into the Entero-Insular Axis in Fibrocalcific Pancreatic Diabetes: An Isoglycemic Intravenous Glucose Infusion (IIGI) Study from India

In tropical countries including India, one of the common causes for young onset diabetes mellitus (DM) is “Fibro Calcific Pancreatic Diabetes” (FCPD) characterized by progressive pancreatic destruction. Despite this, glucagon has been found to be elevated in FCPD (1,2). The L-Cells in gut produce glucagon like peptide- 1 (GLP-1) and oxyntomodulin which are products of glucagon gene, thus raising probability of extra-pancreatic glucagon in FCPD. To test this hypothesis we performed 75grams oral glucose tolerance test (OGTT) followed by IIGI on separate days on nine FCPD and six healthy subjects. The latter procedure ensured matched glucose levels achieved during OGTT. Glucagon and incretins were measured at nine pre-specified time points. We found an increase in L-Cell products: GLP-1 (44.5±9.2pM vs. 12.4±4.5pM, p=0.02) and Oxyntomodulin (1252±350pg/ml vs. 859.8±165pg/ml, p=0.43) along with significant rise in glucagon during OGTT (98.8±13pg/ml vs. 63.4±7pg/ml, p=0.03) despite flat basal & stimulated C-peptide (0.43±0.14ng/ml and 1.09±0.3ng/ml, respectively) and Pancreatic polypeptide (12.3±0.0pg/ml and 14.7±1.7pg/ml, respectively) levels.Paradoxically, gastric inhibitory polypeptide (GIP) levels were low in FCPD (106.8±40.3pg/ml vs. 557.8±96.4pg/ml, p=0.003). We speculate that the hyperglucagonemia is extra-pancreatic (L-Cell) in origin and may also contribute to the dichotomous incretin response in FCPD.

Receptor-Mediated Bioassay Reflects Dynamic Change of Glucose-Dependent Insulinotropic Polypeptide by Dipeptidyl Peptidase 4 Inhibitor Treatment in Subjects With Type 2 Diabetes.

Objective: We recently observed a greater increase in plasma levels of bioactive glucose-dependent insulinotropic polypeptide (GIP) than glucagon-like peptide 1 (GLP-1) using the receptor-mediated bioassays in the subjects with normal glycemic tolerance (NGT) treated with dipeptidyl peptidase 4 (DPP-4) inhibitors, which may be unappreciated using conventional enzyme-linked immunosorbent assays (ELISAs) during oral glucose tolerance test. Thus, we determined incretin levels in addition to glucagon level using the bioassays in type 2 diabetes mellitus (T2DM) subjects with or without treatment of DPP-4 inhibitor, to evaluate whether these assays can accurately measure bioactivity of these peptides.Methods: We performed single meal tolerance test (MTT) by using a cookie meal (carbohydrate 75.0 g, protein 8.0 g, fat 28.5 g) in the subjects with NGT (n = 9), the subjects with T2DM treated without DPP-4 inhibitor (n = 7) and the subjects with T2DM treated with DPP-4 inhibitor (n = 10). All subjects fasted for 10–12 h before the MTT, and blood samples were collected at 0, 30, 60, and 120 min. We used the cell lines stably cotransfected with human-form GIP, GLP-1 or glucagon receptor, and a cyclic adenosine monophosphate–inducible luciferase expression construct for the bioassays. We measured active GIP, active GLP-1, and glucagon by the bioassays. To evaluate the efficacy of bioassay, we measured identical samples via ELISA kits.Results: During the single MTT study, postprandial active GIP bioassay levels of T2DM with DPP-4 inhibitor treatment were drastically higher than those of NGT and T2DM without DPP-4 inhibitor, although the DPP-4 inhibitor-treated group showed moderate increase of active GIPELISA and active GLP-1bioassay, while active GLP-1bioassay levels of T2DM subjects without DPP-4 inhibitor were comparable to those of NGT subjects. During the serial MTT, administration of DPP-4 inhibitor significantly increased active GIPbioassay levels, but not active GLP-1bioassay.Conclusions: In comparison to conventional ELISA, receptor-mediated bioassay reflects dynamic change of GIP polypeptide by DPP-4 inhibitor treatment in subjects with type 2 diabetes.