Development and Application of Metabolomics Techniques to Improve Understanding of Glucose and Fatty Acid Metabolism in ß-cells and their Role in Insulin Secretion.

The primary function of pancreatic beta cells is to secrete insulin in response to glucose metabolism. Decline in beta cell function contributes to the development of type 2 diabetes (T2D). Diabetic patients suffer from low levels of insulin and high levels of glucose and fatty acid in their blood. However, in pre-diabetes, before the decline in beta cell function and the development of T2D has occurred, beta cells secrete higher levels of insulin in blood. The mechanism of insulin secretion and hyperinsulemia are not yet fully understood. Metabolomics, a method first described in 1998, is a modern tool devised to monitor the dynamics of metabolites in biological samples. This technique provides valuable information which may aid understanding of how beta cells secrete insulin in response to different nutrients. This dissertation is focused on the use of metabolomics techniques to study insulin secretion in a beta cell line (INS-1 cells). We developed and optimized an analytical platform that allowed us to monitor glucose and fatty acid metabolism inside beta cells. We used stable-isotope labeled metabolic tracers that enabled us to probe nutrient flux in several pathways involved in insulin secretion. Using the developed platform we were able to better understand the mechanism(s) by which glucose can stimulate insulin secretion. Important pathways involved in insulin secretion were probed using labeled glucose and pharmacological agents. We applied metabolomics to understand why acute exposure to high fatty acid and glucose lead to the potentiation of insulin secretion. We showed that in beta cells, fatty acids increase glucose oxidation, while themselves being minimally oxidized. Using pharmacological agents and siRNA techniques, we showed a novel role for the free fatty acid receptor (FFAR1/GPR40) in controlling fatty acid and glucose metabolism. Finally, we tried to understand the mechanism of action of a new pharmacological agent (AICAR), which mimics starvation conditions within a cell. We showed that AMPK activation by AICAR treatment lead to beneficial effects on beta cells by reducing lipid mediators that are known to reduce apoptosis. However, as AICAR is a purine analog, we showed that it blocked pathways involved in nucleic acid synthesis.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/107168/1/mazzouny_1.pd

Strengthening of Edge and Corner Columns using Concrete Jackets

Strengthening of columns using concrete jackets depends on friction at the interface between them. So strengthening of edge and corner columns in only one story needs a large cross section area due to the shortage of friction length which leads to architectural issues. This research aims to study strengthening the edge and corner columns using a concrete Jacket in more than one story which increases the friction area between the Jacket and the original column. As a result, the load transferred from original column to the jacket will be increased. Thirteen models were done using ANSYS program to study the effect of various factors on the Jacket capacity such as the number of strengthened floors, the Jacket type (two sides or three sides), and whether there were shear connectors or not. The results showed that in the case of the edge and corner columns, it is preferable to strengthen the column by making a concrete Jacket on at least two or three floors to increase the surface area, which leads to increase the friction and thus increases the capacity of the strengthened column by an acceptable percentage. The results of ANSYS models were compared with the Indian code IS 15988 (2013) and the results were shown differently because the code equations depend on the presence of a full bond between the concrete column and the Jacket, which does not occur, but rather the load is transferred by friction between the Jacket and the original column. Doi: 10.28991/cej-2021-03091716 Full Text: PD

A validated stability indicating HPLC method for determination of sitagliptin

A comparative and stability-indicating reversed phase high performance liquid chromatographic study have been developed and validated for sitagliptin phosphate. The liquid chromatographic determination was achieved isocratically on Poroshell 120 EC-C18 (100 × 4.6 mm, i.d.; particle size, 2.7 µm), Pursuit 5PFP (150 × 4.6 mm, i.d.; particle size, 5 µm) and Chromolith performance RP-18e (100 × 4.6 mm, i.d.; macropore diameter, 2 µm) columns using a mobile phase consisting of methanol:water:triethylamine:acetic acid (60:40:0.1:0.1; v:v:v:v), at a flow rate 0.5 mL/min and UV detection at 268 nm. The method was linear over the concentration range of 100-1000 µg/mL (r = 0.9998) with a limit of detection and quantitation of 10 and 30 µg/mL, respectively. All the validation parameters and stability indicating study were studied on Poroshell 120 EC-C18 column, which achieved the best separation. The proposed method has been found to have the required accuracy, selectivity, sensitivity, and precision to assay sitagliptin phosphate in bulk form and in a pharmaceutical dosage form. Degradation products resulting from the stress studies did not interfere with the detection of sitagliptin phosphate that indicates that the assay are stability-indicating assay

N-Phenyl-2-(propan-2-yl­idene)­hydrazine­carboxamide

In the title compound, C10H13N3O, the hydrazinecarboxamide N—N—C(=O)—N unit is nearly planar [maximum deviation = 0.018 (2) Å] and is inclined at a dihedral angle of 8.45 (10)° with respect to the plane of the phenyl ring. The mol­ecular structure is stabilized by an intra­molecular C—H⋯O hydrogen bond which generates an S(6) ring motif. In the crystal, mol­ecules are linked into an inversion dimer by pairs of N—H⋯O and C—H⋯O hydrogen bonds

Synthesis and preliminary biological screening of certain 5-aralkyl pyrrolidine-3-carboxylic acids as anticonvulsants

Synthesis of a series of 5-aralkyl pyrrolidine-3-carboxylic acid derivatives namely, 1-acetyl-4-hydroxy-5-benzyl or 5-(4-alkoxy-benzyl)-pyrrolidine-3-carboxylic acids (3a-e), 1-H-4-hydroxy-5-benzyl or 5-(4-alkoxy-benzyl)-pyrrolidine-3-carboxylic acids (4a-e), 1-acetyl-5-benzyl or 5-(4-alkoxy-benzyl)-pyrrolidine-3-carboxylic acids (8a-e), 1-H-5-benzyl or 5-(4-alkoxy-benzyl)-pyrrolidine-3-carboxylic acids (9a-e) have been accomplished. The structures of the new compounds were assigned from IR, 1H NMR, 13C NMR and elemental analyses. Compounds 3a-e, 4a-e, 8a-e and 9a-e were biologically screened for their anticonvulsant potential using the subcutaneous pentylenetetrazole seizures (scPTZ) assay and Gabapentin as reference standard. The 1-H-4-hydroxy-5-benzyl or 5-(4-alkoxy-benzyl)-pyrrolidine-3-carboxylic acids (4a-e) showed the highest anticonvulsant activity. Compound 4b was found to be the most potent one which exhibited 100% protection

Design, synthesis, and biological profile of novel N-(5-aryl-1,3,4-thiadiazol-2-yl) hydrazinecarboxamides

New series of arylthiadiazole hydrazinecarboxamides (5a-e) have been synthesized by hydrazinolysis of carbamates (4a-e) and spectrally characterized. The new candidates have been screened for their anticonvulsant and immunomodulatory activities. Compound 5e was the most potent anticonvulsant candidate as it showed 100% protection against both maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) screens without neurotoxicity at 100 mg/kg (0.318 mmol/kg). With respect to immunomodulation, compounds 5a and 5d revealed immunostimulatory activity while compounds 5b, 5c, and 5e had immunosuppressive responses based on ELISA detection of IgM and IgG levels, counting the total mesenteric lymph nodes lymphocytes, and histo-pathological examinations

Anticonvulsant Potential of Certain New (2 E

Anticonvulsant potential and neurotoxicity of certain new imidazole-containing arylsemicarbazones 6a–p are reported. The test compounds 6a–p exhibited anticonvulsant activity mainly in the scPTZ screen. Compound 6p emerged as the most active surrogate displaying 100% protection at a dose level of 636 μmol/kg in the scPTZ screen without any neurotoxicity. The assigned (E)-configuration of the title compounds 6a–p was confirmed via single crystal X-ray structure of compound 6g

Disruption of beta cell acetyl-CoA carboxylase-1 in mice impairs insulin secretion and beta cell mass

Aims/hypothesis Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. Methods Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (βACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-βACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. Results βACC1KO and tmx-βACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in βACC1KO mice but not in tmx-βACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. Conclusions/interpretation Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood

Enantioselective HPLC separation of bioactive C5-chiral 2-pyrazolines on lux amylose-2 and lux cellulose-2: Comparative and mechanistic approaches

<p>Stereoselective analytical HPLC separations have been developed for a series of biologically active chiral 2-pyrazolines (<b>1-22</b>) to be used in monitoring their resolution reactions or to custom semipreparative HPLC separations prior to biological assessment of both enantiomers. Polysaccharide-based chiral stationary phases (CSPs), namely, Lux amylose-2 and cellulose-2, have been used. Both normal (<i>n</i>-hexane/ethanol) and polar organic (ethanol, methanol, acetonitrile, or mixtures thereof) elution modes were very beneficial for the achievement of baseline separations. The impact of various chemical moieties embedded in the structures of 2-pyrazolines <b>1-22</b> and the adopted stationary phases on chiral recognition has been investigated. A case of reversed order of elution following alterations in either stationary phase or elution mode has been observed. Our findings recommend that normal elution mode can be used for optimizing semipreparative HPLC methods whereas polar organic mobile phases (such as acetonitrile and ethanol) are more suited to stereoselective reactions monitoring, routine quality control work, or for pharmacological and toxicological assays. These results settle the implementation of polysaccharide-based CSPs using different elution modes and declare the practicality of such CSPs in stereoselective HPLC.</p