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The effects of glucagon and insulin on adenosine 3':5'-cyclic monophosphate concentrations in an organ culture of mature rat liver.

ABSTRACT: 1. A modified radioimmunoassay for cyclic AMP was developed from the method of Steiner et al. (1969). Cyclic [(3)H]AMP was used as the radioactive tracer. Free and antibody-bound nucleotides were separated by adsorption of protein to Millipore filters. The assay was used to measure amounts of cyclic AMP down to 0.1pmol in 50mul. 2. The effect of glucagon on cyclic AMP content in pieces of mature rat liver maintained for 6 days in organ culture was studied. 3. Cyclic AMP content in the tissue reached a maximum in 5-15min and then decreased. This may have been partly due to an inhibitor of glucagon action formed in the tissue. Small amounts of cyclic AMP were released into the incubation medium. 4. The maximal increase in cyclic AMP content produced by glucagon decreased over 6 days in culture. However, liver pieces cultured for 2 and 6 days were more sensitive to low concentrations of glucagon than were fresh liver pieces. Glucagon concentrations for half-maximal effects were approx. 1mum and 0.05mum for fresh liver and 2-day cultured liver respectively. 5. Insulin (3.5mum) lowered the cyclic AMP content by 30% in the presence of a submaximal glucagon concentration in liver cultured for 2 days. No effect of insulin was demonstrated on fresh liver pieces. 6. Insulin and glucagon were rapidly destroyed by fresh liver pieces.

SUBMITTER: Siddle K

PROVIDER: S-EPMC1177651 | biostudies-other | 1973 Apr

REPOSITORIES: biostudies-other

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Project description:Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein-coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and G? proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m-tolyl 5-chloro-2-[ethylsulfonyl] pyrimidine-4-carboxylate [SBI-115]), and (3) a combination of SBI-115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA-treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5-/- ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and G?s (but not G?i and G?q ) proteins was increased 2-fold to 3-fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ?40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5-/- ;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with G?s were increased ?2-fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ?30% in cystic cholangiocytes after treatment with SBI-115 alone and by ?50% when SBI-115 was combined with pasireotide. CONCLUSION:TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218).

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The effects of glucagon and insulin on adenosine 3':5'-cyclic monophosphate concentrations in an organ culture of mature rat liver.

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