scholarly journals Nutrient-sensing mTOR-mediated pathway regulates leptin production in isolated rat adipocytes

2003 ◽  
Vol 284 (2) ◽  
pp. E322-E330 ◽  
Author(s):  
Cecilia Roh ◽  
Jianrong Han ◽  
Alexandros Tzatsos ◽  
Konstantin V. Kandror
Keyword(s):  
Endoplasmic Reticulum ◽  
Food Intake ◽  
Mammalian Target Of Rapamycin ◽  
Heavy Fraction ◽  
Nutrient Sensing ◽  
Rat Adipocytes ◽  
Cytosolic Extract ◽  
Adipose Cells ◽  
Isolated Rat

Leptin biosynthesis in adipose cells in vivo is increased by food intake and decreased by food deprivation. However, the mechanism that couples leptin production to food intake remains unknown. We found that addition of leucine to isolated rat adipocytes significantly increased leptin production by these cells, suggesting that postprandial leptin levels may be directly regulated by dietary leucine. The effect of leucine was inhibited by rapamycin and not by actinomycin D. Besides, leucine administration did not increase the amount of leptin mRNA in adipocytes. Therefore, we concluded that leucine activates leptin expression in adipose cells at the level of translation via a mammalian target of rapamycin (mTOR)-mediated pathway. Because leptin is a secreted protein, its biosynthesis is compartmentalized on the endoplasmic reticulum. To analyze mTOR signaling in this subcellular fraction, we separated adipose cells by centrifugation into a heavy membrane fraction that includes virtually all endoplasmic reticulum and the cytosolic extract. Phosphorylation of the major mTOR targets, the ribosomal protein S6 and the translational inhibitor 4E-binding protein (BP)/phosphorylated heat- and acid-stable protein (PHAS)-1, was stimulated by leucine in the cytosolic extract, whereas, in the heavy fraction, S6 was constitutively phosphorylated and leucine only induced phosphorylation of 4E-BP/PHAS-1. We also found that 60–70% of leptin mRNA was stably associated with the heavy fraction, and leucine administration did not change the ratio between compartmentalized and free cytoplasmic leptin mRNA. We suggest that, in adipose cells, a predominant part of leptin mRNA is compartmentalized on the endoplasmic reticulum, and leucine activates translation of these messages via the mTOR/4E-BP/PHAS-1-mediated signaling pathway.

scholarly journals The Mammalian Target of Rapamycin Complex 1 Regulates Leptin Biosynthesis in Adipocytes at the Level of Translation: The Role of the 5′-Untranslated Region in the Expression of Leptin Messenger Ribonucleic Acid

2008 ◽  
Vol 22 (10) ◽  
pp. 2260-2267 ◽  
Author(s):  
Partha Chakrabarti ◽  
Takatoshi Anno ◽  
Brendan D. Manning ◽  
Zhijun Luo ◽  
Konstantin V. Kandror
Keyword(s):  
Untranslated Region ◽  
Molecular Mechanisms ◽  
Mammalian Target Of Rapamycin ◽  
Dominant Negative ◽  
Target Of Rapamycin ◽  
Messenger Ribonucleic Acid ◽  
Leptin Expression ◽  
Adipose Cells

Abstract Leptin production by adipose cells in vivo is increased after feeding and decreased by food deprivation. However, molecular mechanisms that control leptin expression in response to food intake remain unknown. Here, we test the hypothesis that leptin expression in adipose cells is regulated by nutrient- and insulin-sensitive mammalian target of rapamycin complex 1 (mTORC1)-mediated pathway. The activity of mTORC1 in 3T3-L1 adipocytes was up-regulated by stable expression of either constitutively active Rheb or dominant-negative AMP-activated protein kinase. In both cases, expression of endogenous leptin was significantly elevated at the level of translation. To investigate the role of leptin 5′-untranslated region (UTR) in the regulation of protein expression, we created bicistronic reporter constructs with and without the 5′-UTR. We found that the presence of leptin 5′-UTR renders mRNA resistant to regulation by mTORC1. It appears, therefore, that mTORC1 controls translation of leptin mRNA via a novel mechanism that does not require the presence of either the 5′-terminal oligopyrimidine tract or the 5′-UTR.

scholarly journals Substrates of semicarbazide-sensitive amine oxidase co-operate with vanadate to stimulate tyrosine phosphorylation of insulin-receptor-substrate proteins, phosphoinositide 3-kinase activity and GLUT4 translocation in adipose cells

2000 ◽  
Vol 350 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Gemma ENRIQUE-TARANCÓN ◽  
Isabelle CASTAN ◽  
Nathalie MORIN ◽  
Luc MARTI ◽  
Anna ABELLA ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Glucose Transport ◽  
Kinase Activity ◽  
Amine Oxidase ◽  
Rat Adipocytes ◽  
Low Concentrations ◽  
Phosphoinositide 3 Kinase ◽  
Adipose Cells

It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H2O2 formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or β-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.

scholarly journals Drug library screen identifies inhibitors of toxic astrogliosis

2020 ◽  
Author(s):  
Ruturaj R Masvekar ◽  
Peter R Kosa ◽  
Christopher Barbour ◽  
Joshua L Milstein ◽  
Bibiana Bielekova
Keyword(s):  
Multiple Sclerosis ◽  
Endoplasmic Reticulum ◽  
Cerebrospinal Fluid ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Mammalian Target Of Rapamycin ◽  
Inhibitory Effect ◽  
Multiple Sclerosis Patients

Objective: Multiple sclerosis is a chronic neuroinflammatory disorder, in which activated immune cells directly or indirectly induce demyelination and axonal degradation. Inflammatory stimuli also change the phenotype of astrocytes, making them neurotoxic. The resulting toxic astrocyte phenotype has been observed in animal models of neuroinflammation and in multiple sclerosis lesions. Proteins secreted by toxic astrocytes are elevated in the cerebrospinal fluid of multiple sclerosis patients and reproducibly correlate with the rates of accumulation of neurological disability and brain atrophy. This suggests a pathogenic role for neurotoxic astrocytes in multiple sclerosis. Methods: Here, we applied a commercially available library of small molecules that are either Food and Drug Administration-approved or in clinical development to an in vitro model of toxic astrogliosis to identify drugs and signaling pathways that inhibit inflammatory transformation of astrocytes to a neurotoxic phenotype. Results: Inhibitors of three pathways related to the endoplasmic reticulum stress: 1) proteasome, 2) heat shock protein 90 and 3) mammalian target of rapamycin reproducibly decreased inflammation-induced conversion of astrocytes to toxic phenotype. Dantrolene, an anti-spasticity drug that inhibits calcium release through ryanodine receptors expressed in the endoplasmic reticulum of central nervous system cells, also exerted inhibitory effect at in vivo achievable concentrations. Finally, we established cerebrospinal fluid SERPINA3 as a relevant pharmacodynamic marker for inhibiting toxic astrocytes in clinical trials. Interpretation: Drug library screening provides mechanistic insight into the generation of toxic astrocytes and identifies candidates for immediate proof-of-principle clinical trial(s).

scholarly journals The protein phosphatases responsible for dephosphorylation of hormone-sensitive lipase in isolated rat adipocytes

1993 ◽  
Vol 295 (2) ◽  
pp. 531-535 ◽  
Author(s):  
S L Wood ◽  
N Emmison ◽  
A C Borthwick ◽  
S J Yeaman
Keyword(s):  
Adipose Tissue ◽  
Phosphatase Activity ◽  
Protein Phosphatases ◽  
Total Activity ◽  
Hormone Sensitive Lipase ◽  
Rat Adipocytes ◽  
Hormone Sensitive ◽  
Total Phosphatase Activity ◽  
Isolated Rat

The levels of the cytosolic serine/threonine protein phosphatases (PP) in rat adipocyte extracts have been determined, by using both reference substrates and hormone-sensitive lipase (HSL) as substrates. Adipocytes contain significant levels of both PP1 and 2A (1.6 and 2.0 m-units/ml of packed cells respectively), with lower levels of PP2C and virtually no PP2B activity. PP2A and 2C exhibit similar degrees of activity against HSL phosphorylated at site 1, together accounting for 92% of the total. In contrast, site 2 is dephosphorylated predominantly by PP2A (over 50% of total activity), whereas PP1 and PP2C contribute approx. 20% and 30% respectively to the total phosphatase activity against that site. Total phosphatase activity in the adipocyte extracts was 2-3-fold higher against site 2 than against site 1. The possible significance of these findings to the regulation of HSL activity in adipose tissue in vivo is discussed.

The effects of metabolic acidosis in vivo on insulin binding to isolated rat adipocytes

Metabolism ◽  
1982 ◽  
Vol 31 (6) ◽  
pp. 553-557 ◽  
Author(s):  
J. Whittaker ◽  
C. Cuthbert ◽  
V.A. Hammond ◽  
K.G.M.M. Alberti
Keyword(s):  
Metabolic Acidosis ◽  
Insulin Binding ◽  
Rat Adipocytes ◽  
Isolated Rat

scholarly journals In vivo and in vitro effect of p-chlorophenoxyisobutyrate on insulin binding and glucose transport in isolated rat adipocytes.

1985 ◽  
Vol 32 (6) ◽  
pp. 829-836
Author(s):  
NOBUAKI WATANABE ◽  
MASASHI KOBAYASHI ◽  
HIROSHI MAEGAWA ◽  
OSAMU ISHIBASHI ◽  
YASUMITSU TAKATA ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Insulin Binding ◽  
Rat Adipocytes ◽  
Vitro Effect ◽  
Isolated Rat

scholarly journals Role of Neuronal Energy Status in the Regulation of Adenosine 5′-Monophosphate-Activated Protein Kinase, Orexigenic Neuropeptides Expression, and Feeding Behavior

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 3-10 ◽  
Author(s):  
Kichoon Lee ◽  
Bing Li ◽  
Xiaochun Xi ◽  
Yeunsu Suh ◽  
Roy J. Martin
Keyword(s):  
Protein Kinase ◽  
Food Intake ◽  
Feeding Behavior ◽  
High Glucose ◽  
Ex Vivo ◽  
Nutrient Sensing ◽  
Central Administration ◽  
Energy Status ◽  
Low Glucose

Nutrient sensing in the hypothalamus is tightly related to food intake regulation. However, the mechanisms by which the nutrient-sensing cells of the brain translate this signal of energy need into feeding behavior via regulation of neuropeptide expression are not known. To address this issue, we investigated two neuronal cell lines expressing agouti-related protein (AgRP), ex vivo hypothalamic tissues, and in vivo whole animals. Maintaining cells in a low cellular ATP concentration generated by low glucose, 2-deoxyglucose (2-DG), ATP synthesis inhibitor, and 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside increased phosphorylation of AMP-activated protein kinase (AMPK) and increased AgRP expression, whereas maintaining cells in high ATP status by high glucose and pyruvate supplementation in 2-DG-treated cells decreased phosphorylation of AMPK and decreased AgRP expression. Overexpression of a dominant-inhibitory mutant of AMPK significantly decreased low-glucose- or 2-DG-induced AgRP expression. Furthermore, ex vivo hypothalamus culture in high glucose concentrations decreased both expression and phosphorylation of AMPK and expression of both AgRP and neuropeptide Y, whereas pyruvate supplementation suppressed a 2-DG-induced AgRP expression. Finally, our in vivo studies clearly show that central administration of pyruvate dramatically delayed 2-DG-induced food intake. These data indicate that modulation of ATP levels in neuronal cells triggers a cascade of events via AMPK that modulate feeding behavior to restore energy status of cells.

An Ultra-Structural Study of Human Melanoma in Vivo and Vitro

1976 ◽  
Vol 34 ◽  
pp. 258-259
Author(s):  
James R. Gaylor ◽  
Fredda Schafer ◽  
Robert E. Nordquist
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Protein Aggregation ◽  
Structural Study ◽  
Human Melanoma ◽  
Protein Matrix ◽  
Early Form ◽  
Endoplasmic Reticulum Protein ◽  
Mitochondrial Origin

Several theories on the origin of the melanosome exist. These include the Golgi origin theory, in which a tyrosinase-rich protein is "packaged" by the Golgi apparatus, thus forming the early form of the melanosome. A second theory postulates a mitochondrial origin of melanosomes. Its author contends that the melanosome is a modified mitochondria which acquires melanin during its development. A third theory states that a pre-melanosome is formed in the smooth or rough endoplasmic reticulum. Protein aggregation is suggested by one author as a possible source of the melanosome. This fourth theory postulates that the melanosome originates when the protein products of several genetic loci aggregate in the cytoplasm of the melanocyte. It is this protein matrix on which the melanin is deposited. It was with these theories in mind that this project was undertaken.

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