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Protein degradation in hepatocyte monolayers. Effects of glucagon, adenosine 3':5'-cyclic monophosphate and insulin.

ABSTRACT: 1. Hepatocytes were isolated by collagenase perfusion of livers from fed rats and established in stationary monolayer culture. 2. Degradation of intracellular protein was measured in these monolayers after labelling for 16h with [3H]leucine followed by a 3h chase period in medium containing 2mM-leucine. 3. Proteolysis in this system was stimulated by physiological concentrations of glucagon and also by added dibutyryl cyclic AMP. The effects of these two agents were not additive, which is consistent with the view that they act by the same mechanism. 4. A close correlation was found between intracellular cyclic AMP concentrations generated by glucagon and the degree of stimulation of proteolysis elicited by the hormone. 5. Insulin reduced glucagon-stimulated proteolysis, but not glucagon-elevated intracellular cyclic AMP concentrations. 6. The continual presence of either insulin or glucagon was necessary for the full expression of their effects on proteolysis. 7. In the presence of cycloheximide, proteolysis was normally responsive to glucagon but not to insulin. In contrast, proteolysis was not responsive to either hormone in the presence of ammonia, an agent that blocks the final lysosomal step of protein breakdown. 8. We propose that in hepatocyte monolayers glucagon may act via cyclic AMP to increase cellular autophagy and thus increase proteolysis, whereas insulin inhibits these processes independently of cyclic AMP.

SUBMITTER: Hopgood MF

PROVIDER: S-EPMC1161504 | biostudies-other | 1980 Jan

REPOSITORIES: biostudies-other

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Project description:BackgroundIntestinal smooth muscle cells (SMCs) undergo substantial morphological, phenotypic, and contractile changes during inflammatory bowel disease (IBD). SMCs act as a source and target for different inflammatory mediators, however their role in IBD pathogenesis is usually overlooked. Glucagon-like peptide-1 (GLP-1) is an incretin hormone reported to exert multiple anti-inflammatory effects in different tissues including the gastrointestinal tract through various mechanisms.AimThe aim of this research is to explore the effect of GLP-1 analog exendin-4 on the expression and secretion of inflammatory markers from mouse colon smooth muscle cells (CSMCs) after stimulation with lipopolysaccharide (LPS).Materials and methodsFreshly isolated CSMCs from male BALB/c mice were cultured in DMEM and treated with vehicle, LPS (1 μg/mL), LPS+exendin-4 (50 nM), or LPS+exendin-4 (100 nM) for 24 h. Expression of inflammatory cytokines was then evaluated by antibody array membrane.ResultsCSMCs showed basal expression of several cytokines which was enhanced with the induction of inflammation by LPS. However, exendin-4 (50 and 100 nM) significantly (p<0.05) reduced the expression of multiple cytokines including tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), T cell activation gene-3 (TCA-3), stromal cell-derived factor-1 (SDF-1), and macrophage colony stimulating factor (M-CSF). To confirm these results, expression of these cytokines was further assessed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction and similar results were also observed. Moreover, secretion of TNF-α and IL1-α into the conditioned media was significantly downregulated by exendin-4 when compared to LPS-treated cells. Furthermore, LPS increased NF-κB phosphorylation, while exendin-4 significantly reduced levels of NF-κB phosphorylation.ConclusionThese data indicate that GLP-1 analogs can exert significant anti-inflammatory effects on CSMCs and can potentially be used as an adjunct treatment for inflammatory bowel conditions.

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Protein degradation in hepatocyte monolayers. Effects of glucagon, adenosine 3':5'-cyclic monophosphate and insulin.

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