197 research outputs found
Polyomic profiling reveals significant hepatic metabolic alterations in glucagon-receptor (GCGR) knockout mice: implications on anti-glucagon therapies for diabetes
<p>Abstract</p> <p>Background</p> <p>Glucagon is an important hormone in the regulation of glucose homeostasis, particularly in the maintenance of euglycemia and prevention of hypoglycemia. In type 2 Diabetes Mellitus (T2DM), glucagon levels are elevated in both the fasted and postprandial states, which contributes to inappropriate hyperglycemia through excessive hepatic glucose production. Efforts to discover and evaluate glucagon receptor antagonists for the treatment of T2DM have been ongoing for approximately two decades, with the challenge being to identify an agent with appropriate pharmaceutical properties and efficacy relative to potential side effects. We sought to determine the hepatic & systemic consequence of full glucagon receptor antagonism through the study of the glucagon receptor knock-out mouse (Gcgr<sup>-/-</sup>) compared to wild-type littermates.</p> <p>Results</p> <p>Liver transcriptomics was performed using Affymetric expression array profiling, and liver proteomics was performed by iTRAQ global protein analysis. To complement the transcriptomic and proteomic analyses, we also conducted metabolite profiling (~200 analytes) using mass spectrometry in plasma. Overall, there was excellent concordance (R = 0.88) for changes associated with receptor knock-out between the transcript and protein analysis. Pathway analysis tools were used to map the metabolic processes in liver altered by glucagon receptor ablation, the most notable being significant down-regulation of gluconeogenesis, amino acid catabolism, and fatty acid oxidation processes, with significant up-regulation of glycolysis, fatty acid synthesis, and cholesterol biosynthetic processes. These changes at the level of the liver were manifested through an altered plasma metabolite profile in the receptor knock-out mice, e.g. decreased glucose and glucose-derived metabolites, and increased amino acids, cholesterol, and bile acid levels.</p> <p>Conclusions</p> <p>In sum, the results of this study suggest that the complete ablation of hepatic glucagon receptor function results in major metabolic alterations in the liver, which, while promoting improved glycemic control, may be associated with adverse lipid changes.</p
Quantitative Analysis of Serum Procollagen Type I C-Terminal Propeptide by Immunoassay on Microchip
BACKGROUND: Sandwich enzyme-linked immunosorbent assay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. METHODS AND FINDINGS: The microchip was made of cyclic olefin copolymer with straight microchannels that were 300 Β΅m wide and 100 Β΅m deep. For the construction of a sandwich ELISA for procollagen type I C-peptide (PICP), a biomarker for bone formation, we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-PICP antibody on the surface of the microchannel. After the infusion of the mixture of 2.0 Β΅l of peroxidase-labeled 2nd anti-PICP antibody and 0.4 Β΅l of sample to the microchannel and a 30-min incubation, the substrate for peroxidase was infused into the microchannel; and the luminescence intensity of each spot of 1st antibody was measured by CCD camera. A linear relationship was observed between PICP concentration and luminescence intensity over the range of 0 to 600 ng/ml (r(2)β=β0.991), and the detection limit was 4.7 ng/ml. Blood PICP concentrations of 6 subjects estimated from microchip were compared with results obtained by the conventional method. Good correlation was observed between methods according to simple linear regression analysis (R(2)β=β0.9914). The within-day and between-days reproducibilities were 3.2-7.4 and 4.4-6.8%, respectively. This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. CONCLUSION: This assay enabled us to determine serum PICP with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis
Methodology of calculation of construction and hydrodynamic parameters of a foam layer apparatus for mass-transfer processes
ΠΡΠΎΠΌΠΈΡΠ»ΠΎΠ²Π° ΡΠ΅Π°Π»ΡΠ·Π°ΡΡΡ ΠΌΠ΅ΡΠΎΠ΄Ρ ΡΡΠ°Π±ΡΠ»ΡΠ·Π°ΡΡΡ Π³Π°Π·ΠΎΡΡΠ΄ΠΈΠ½Π½ΠΎΠ³ΠΎ ΡΠ°ΡΡ Π΄ΠΎΠ·Π²ΠΎΠ»ΡΡ Π·Π½Π°ΡΠ½ΠΎ ΡΠΎΠ·ΡΠΈΡΠΈΡΠΈ Π³Π°Π»ΡΠ·Ρ Π·Π°ΡΡΠΎΡΡΠ²Π°Π½Π½Ρ ΠΏΡΠ½Π½ΠΈΡ
Π°ΠΏΠ°ΡΠ°ΡΡΠ² Ρ Π²ΡΠ΄ΠΊΡΠΈΠ²Π°Ρ Π½ΠΎΠ²Ρ ΠΌΠΎΠΆΠ»ΠΈΠ²ΠΎΡΡΡ ΡΠ½ΡΠ΅Π½ΡΠΈΡΡΠΊΠ°ΡΡΡ ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΡΡΠ½ΠΈΡ
ΠΏΡΠΎΡΠ΅ΡΡΠ² Π· ΠΎΠ΄Π½ΠΎΡΠ°ΡΠ½ΠΈΠΌ ΡΡΠ²ΠΎΡΠ΅Π½Π½ΡΠΌ ΠΌΠ°Π»ΠΎΠ²ΡΠ΄Ρ
ΠΎΠ΄Π½ΠΈΡ
ΡΠ΅Ρ
Π½ΠΎΠ»ΠΎΠ³ΡΠΉ. Π£ ΡΡΠ°ΡΡΡ Π²ΡΡΠ°Π½ΠΎΠ²Π»Π΅Π½Ρ ΠΎΡΠ½ΠΎΠ²Π½Ρ ΠΏΠ°ΡΠ°ΠΌΠ΅ΡΡΠΈ, ΡΠΎ Π²ΠΏΠ»ΠΈΠ²Π°ΡΡΡ Π½Π° Π³ΡΠ΄ΡΠΎΠ΄ΠΈΠ½Π°ΠΌΡΠΊΡ ΠΏΡΠ½Π½ΠΈΡ
Π°ΠΏΠ°ΡΠ°ΡΡΠ², ΡΠΎΠ·Π³Π»ΡΠ½ΡΡΡ ΠΎΡΠ½ΠΎΠ²Π½Ρ ΠΊΠΎΠ½ΡΡΡΡΠΊΡΡΡ ΡΠ° ΡΠ΅ΠΆΠΈΠΌΠΈ ΡΠΎΠ±ΠΎΡΠΈ ΠΏΡΠ½Π½ΠΈΡ
Π°ΠΏΠ°ΡΠ°ΡΡΠ². ΠΠΈΡΠ²Π»Π΅Π½ΠΎ Π·Π²'ΡΠ·ΠΎΠΊ Π³ΡΠ΄ΡΠΎΠ΄ΠΈΠ½Π°ΠΌΡΡΠ½ΠΈΡ
ΠΏΠ°ΡΠ°ΠΌΠ΅ΡΡΡΠ². Π ΠΎΠ·Π³Π»ΡΠ½ΡΡΠΎ Π³ΡΠ΄ΡΠΎΠ΄ΠΈΠ½Π°ΠΌΡΡΠ½Ρ Π·Π°ΠΊΠΎΠ½ΠΎΠΌΡΡΠ½ΠΎΡΡΡ ΠΏΡΠ½Π½ΠΎΠ³ΠΎ ΡΠ°ΡΡ. ΠΠΊΠ°Π·Π°Π½Ρ ΡΠ°ΠΊΡΠΎΡΠΈ, ΡΠΎ Π²ΠΏΠ»ΠΈΠ²Π°ΡΡΡ Π½Π° ΠΏΡΠΎΡΠ΅Ρ ΠΌΠ°ΡΠΎΠΎΠ±ΠΌΡΠ½Ρ, ΡΠΊ Π² Π³Π°Π·ΠΎΠ²ΡΠΉ, ΡΠ°ΠΊ Ρ Π² ΡΡΠ΄ΠΊΡΠΉ ΡΠ°Π·Π°Ρ
. ΠΡΠΎΠ²Π΅Π΄Π΅Π½ΠΈΠΉ Π°Π½Π°Π»ΡΠ· ΡΡΠ΄Ρ Π΄ΠΎΡΠ»ΡΠ΄ΠΆΠ΅Π½Ρ ΠΏΠΎΠΊΠ°Π·Π°Π², ΡΠΎ ΠΏΠ΅ΡΡΠΏΠ΅ΠΊΡΠΈΠ²Π½ΠΈΠΌ Π½Π°ΠΏΡΡΠΌΠΊΠΎΠΌ ΡΠ½ΡΠ΅Π½ΡΠΈΡΡΠΊΠ°ΡΡΡ ΠΏΡΠΎΡΠ΅ΡΡ ΠΌΠ°ΡΠΎΠΎΠ±ΠΌΡΠ½Ρ Ρ ΡΠΎΠ·ΡΠΎΠ±ΠΊΠ° Π°ΠΏΠ°ΡΠ°ΡΡΠ² Π· ΡΡΠΈΡΠ°Π·Π½ΠΈΠΌ ΠΏΡΠ΅Π²Π΄ΠΎΠ·ΡΡΠ΄ΠΆΠ΅Π½ΠΈΠΌ ΡΠ°ΡΠΎΠΌ Π·ΡΠΎΡΡΠ²Π°Π½ΠΎΡ Π½Π°ΡΠ°Π΄ΠΊΠΈ ΡΠΊΠ»Π°Π΄Π½ΠΈΡ
ΡΠΎΡΠΌ ΡΠ· ΡΡΡΡΠ°ΡΡΠΈΡ
ΠΌΠ°ΡΠ΅ΡΡΠ°Π»ΡΠ². ΠΡΠΆΠ΅, Π½Π΅ΠΎΠ±Ρ
ΡΠ΄Π½Π΅ ΠΏΡΠΎΠ²Π΅Π΄Π΅Π½Π½Ρ ΡΠΏΠ΅ΡΡΠ°Π»ΡΠ½ΠΈΡ
Π΄ΠΎΡΠ»ΡΠ΄ΠΆΠ΅Π½Ρ Π³ΡΠ΄ΡΠΎΠ΄ΠΈΠ½Π°ΠΌΡΡΠ½ΠΈΡ
ΡΠ΅ΠΆΠΈΠΌΡΠ² ΡΠΎΠ±ΠΎΡΠΈ Π°ΠΏΠ°ΡΠ°ΡΡ Π· ΡΡΡΡΠ°ΡΡΠΎΡ Π½Π°ΡΠ°Π΄ΠΊΠΎΡ Ρ Π²ΠΈΠ·Π½Π°ΡΠ΅Π½Π½ΡΠΌ ΠΏΠ°ΡΠ°ΠΌΠ΅ΡΡΡΠ², ΡΠΎ Π²ΠΏΠ»ΠΈΠ²Π°ΡΡΡ Π½Π° ΡΠ²ΠΈΠ΄ΠΊΡΡΡΡ ΠΏΠ΅ΡΠ΅Ρ
ΠΎΠ΄Ρ Π½Π°ΡΠ°Π΄ΠΊΠΈ Π· ΠΎΠ΄Π½ΠΎΠ³ΠΎ ΡΠ΅ΠΆΠΈΠΌΡ Π² ΡΠ½ΡΠΈΠΉ.Industrial implementation of the stabilization method of the gas-liquid layer can significantly expand the field of use of foaming apparatus and opens up new opportunities for intensifying technological processes with the simultaneous creation of low-waste technologies. The article establishes the basic parameters influencing the hydrodynamics of foam apparatus, considers the basic constructions and operating modes of foam apparatus. The connection of hydrodynamic parameters is revealed. The hydrodynamic laws of the foam layer are considered. The indicated factors affecting the process of mass transfer, both in the gas and in the liquid phases. The conducted analysis of a number of studies showed that the perspective direction of intensification of the mass transfer process is the development of apparatuses with a three-phase fluidized bed of an irrigated nozzle of complex forms with mesh materials
Inhibition of StearoylCoA Desaturase Activity Blocks Cell Cycle Progression and Induces Programmed Cell Death in Lung Cancer Cells
Lung cancer is the most frequent form of cancer. The survival rate for patients with metastatic lung cancer is βΌ5%, hence alternative therapeutic strategies to treat this disease are critically needed. Recent studies suggest that lipid biosynthetic pathways, particularly fatty acid synthesis and desaturation, are promising molecular targets for cancer therapy. We have previously reported that inhibition of stearoylCoA desaturase-1 (SCD1), the enzyme that produces monounsaturated fatty acids (MUFA), impairs lung cancer cell proliferation, survival and invasiveness, and dramatically reduces tumor formation in mice. In this report, we show that inhibition of SCD activity in human lung cancer cells with the small molecule SCD inhibitor CVT-11127 reduced lipid synthesis and impaired proliferation by blocking the progression of cell cycle through the G1/S boundary and by triggering programmed cell death. These alterations resulting from SCD blockade were fully reversed by either oleic (18:1n-9), palmitoleic acid (16:1n-7) or cis-vaccenic acid (18:1n-7) demonstrating that cis-MUFA are key molecules for cancer cell proliferation. Additionally, co-treatment of cells with CVT-11127 and CP-640186, a specific acetylCoA carboxylase (ACC) inhibitor, did not potentiate the growth inhibitory effect of these compounds, suggesting that inhibition of ACC or SCD1 affects a similar target critical for cell proliferation, likely MUFA, the common fatty acid product in the pathway. This hypothesis was further reinforced by the observation that exogenous oleic acid reverses the anti-growth effect of SCD and ACC inhibitors. Finally, exogenous oleic acid restored the globally decreased levels of cell lipids in cells undergoing a blockade of SCD activity, indicating that active lipid synthesis is required for the fatty acid-mediated restoration of proliferation in SCD1-inhibited cells. Altogether, these observations suggest that SCD1 controls cell cycle progression and apoptosis and, consequently, the overall rate of proliferation in cancer cells through MUFA-mediated activation of lipid synthesis
mTORC1 Inhibition via Rapamycin Promotes Triacylglycerol Lipolysis and Release of Free Fatty Acids in 3T3Γ’ L1 Adipocytes
Signaling by mTOR complex 1 (mTORC1) promotes anabolic cellular processes in response to growth factors, nutrients, and hormonal cues. Numerous clinical trials employing the mTORC1 inhibitor rapamycin (aka sirolimus) to immunoΓ’ suppress patients following organ transplantation have documented the development of hypertriglyceridemia and elevated serum free fatty acids (FFA). We therefore investigated the cellular role of mTORC1 in control of triacylglycerol (TAG) metabolism using cultured murine 3T3Γ’ L1 adipocytes. We found that treatment of adipocytes with rapamycin reduced insulinΓ’ stimulated TAG storage ~50%. To determine whether rapamycin reduces TAG storage by upregulating lipolytic rate, we treated adipocytes in the absence and presence of rapamycin and isoproterenol, a ΓΒ²2Γ’ adrenergic agonist that activates the cAMP/protein kinase A (PKA) pathway to promote lipolysis. We found that rapamycin augmented isoproterenolΓ’ induced lipolysis without altering cAMP levels. Rapamycin enhanced the isoproterenolΓ’ stimulated phosphorylation of hormone sensitive lipase (HSL) on SerΓ’ 563 (a PKA site), but had no effect on the phosphorylation of HSL S565 (an AMPK site). Additionally, rapamycin did not affect the isoproterenolΓ’ mediated phosphorylation of perilipin, a protein that coats the lipid droplet to initiate lipolysis upon phosphorylation by PKA. These data demonstrate that inhibition of mTORC1 signaling synergizes with the ΓΒ²Γ’ adrenergicΓ’ cAMP/PKA pathway to augment phosphorylation of HSL to promote hormoneΓ’ induced lipolysis. Moreover, they reveal a novel metabolic function for mTORC1; mTORC1 signaling suppresses lipolysis, thus augmenting TAG storage.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141428/1/lipd1089.pd
Tuberous Sclerosis Complex-1 Deficiency Attenuates Diet-Induced Hepatic Lipid Accumulation
Non-alcoholic fatty liver disease (NAFLD) is causally linked to type 2 diabetes, insulin resistance and dyslipidemia. In a normal liver, insulin suppresses gluconeogenesis and promotes lipogenesis. In type 2 diabetes, the liver exhibits selective insulin resistance by failing to inhibit hepatic glucose production while maintaining triglyceride synthesis. Evidence suggests that the insulin pathway bifurcates downstream of Akt to regulate these two processes. Specifically, mTORC1 has been implicated in lipogenesis, but its role on hepatic steatosis has not been examined. Here, we generated mice with hepatocyte-specific deletion of Tsc1 to study the effects of constitutive mTORC1 activation in the liver. These mice developed normally but displayed mild hepatomegaly and insulin resistance without obesity. Unexpectedly, the Tsc1-null livers showed minimal signs of steatosis even under high-fat diet condition. This βresistantβ phenotype was reversed by rapamycin and could be overcome by the expression of Myr-Akt. Moreover, rapamycin failed to reduce hepatic triglyceride levels in models of steatosis secondary to Pten ablation in hepatocytes or high-fat diet in wild-type mice. These observations suggest that mTORC1 is neither necessary nor sufficient for steatosis. Instead, Akt and mTORC1 have opposing effects on hepatic lipid accumulation such that mTORC1 protects against diet-induced steatosis. Specifically, mTORC1 activity induces a metabolic shift towards fat utilization and glucose production in the liver. These findings provide novel insights into the role of mTORC1 in hepatic lipid metabolism
Inhibition of StearoylCoA Desaturase-1 Inactivates Acetyl-CoA Carboxylase and Impairs Proliferation in Cancer Cells: Role of AMPK
Cancer cells activate the biosynthesis of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in order to sustain an increasing demand for phospholipids with appropriate acyl composition during cell replication. We have previously shown that a stable knockdown of stearoyl-CoA desaturase 1 (SCD1), the main Ξ9-desaturase that converts SFA into MUFA, in cancer cells decreases the rate of lipogenesis, reduces proliferation and in vitro invasiveness, and dramatically impairs tumor formation and growth. Here we report that pharmacological inhibition of SCD1 with a novel small molecule in cancer cells promoted the activation of AMP-activated kinase (AMPK) and the subsequent reduction of acetylCoA carboxylase activity, with a concomitant inhibition of glucose-mediated lipogenesis. The pharmacological inhibition of AMPK further decreased proliferation of SCD1-depleted cells, whereas AMPK activation restored proliferation to control levels. Addition of supraphysiological concentrations of glucose or pyruvate, the end product of glycolysis, did not reverse the low proliferation rate of SCD1-ablated cancer cells. Our data suggest that cancer cells require active SCD1 to control the rate of glucose-mediated lipogenesis, and that when SCD1 activity is impaired cells downregulate SFA synthesis via AMPK-mediated inactivation of acetyl-CoA carboxylase, thus preventing the harmful effects of SFA accumulation
Regulation of TFEB and V-ATPases by mTORC1
TORC1 is a key regulator of cell growth in response to nutrients and acts at the surface of the late endosome. This study identifies V-ATPase genes as transcriptional targets of TORC1 and implicates the transcription factor TFEB as an important mediator of TORC1-dependent gene expression and TORC1-regulated endocytosis
mTOR: from growth signal integration to cancer, diabetes and ageing
In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy
and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.National Institutes of Health (U.S.)Howard Hughes Medical InstituteWhitehead Institute for Biomedical ResearchJane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship)Human Frontier Science Program (Strasbourg, France
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