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Fresh and cultured mouse islets differ in their response to nutrient stimulation.

ABSTRACT: Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be typical for mouse islets. This was also the response of cultured islets to the nutrient secretagogue alpha-ketoisocaproic acid, whereas the secretion of fresh islets increased similarly fast but remained strongly elevated. The responses of fresh and cultured islets to purely depolarizing stimuli (tolbutamide or KCl), however, were closely similar. Signs of apoptosis and necrosis were rare in both preparations. In cultured islets, the glucose-induced rise of the cytosolic Ca2+ concentration started from a lower value and was larger as was the increase of the ATP/ADP ratio. The prestimulatory level of mitochondrial reducing equivalents, expressed as the NAD(P)H/FAD fluorescence ratio, was lower in cultured islets, but increased more strongly than in fresh islets. When culture conditions were modified by replacing RPMI with Krebs-Ringer medium and FCS with BSA, the amount of released insulin varied widely, but the kinetics always showed a predominant first phase. In conclusion, the secretion kinetics of fresh mouse islets is more responsive to variations of nutrient stimulation than cultured islets. The more uniform kinetics of the latter may be caused by a different use of endogenous metabolites.

SUBMITTER: Morsi M

PROVIDER: S-EPMC7424343 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Fresh and cultured mouse islets differ in their response to nutrient stimulation.

Morsi Mai M Schulze Torben T Früh Eike E Brüning Dennis D Panten Uwe U Rustenbeck Ingo I

Endocrine connections 20200801 8

Observing different kinetics of nutrient-induced insulin secretion in fresh and cultured islets under the same condition we compared parameters of stimulus secretion coupling in freshly isolated and 22-h-cultured NMRI mouse islets. Stimulation of fresh islets with 30 mM glucose after perifusion without nutrient gave a continuously ascending secretion rate. In 22-h-cultured islets the same protocol produced a brisk first phase followed by a moderately elevated plateau, a pattern regarded to be ty ...[more]

PMID: 32688335

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Project description:BackgroundIn skeletal muscle, the nutrient-induced stimulation of protein synthesis requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Expression of the repressor of mTORC1 signaling, regulated in development and DNA damage 1 (REDD1), is elevated in muscle during various atrophic conditions and diminished under hypertrophic conditions. The question arises as to what extent REDD1 limits the nutrient-induced stimulation of protein synthesis.ObjectiveThe objective was to examine the role of REDD1 in limiting the response of muscle protein synthesis and mTORC1 signaling to a nutrient stimulus.MethodsWild type REDD1 gene (REDD1(+/+)) and disruption in the REDD1 gene (REDD1(-/-)) mice were feed deprived for 16 h and randomized to remain feed deprived or refed for 15 or 60 min. The tibialis anterior was then removed for analysis of protein synthesis and mTORC1 signaling.ResultsIn feed-deprived mice, protein synthesis and mTORC1 signaling were significantly lower in REDD1(+/+) than in REDD1(-/-) mice. Thirty minutes after the start of refeeding, protein synthesis in REDD1(+/+) mice was stimulated by 28%, reaching a value similar to that observed in feed-deprived REDD1(-/-) mice, and was accompanied by increased phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) by 81%, 167%, and 207%, respectively. In refed REDD1(-/-) mice, phosphorylation of mTOR (Ser2448), p70S6K1 (Thr389), and 4E-BP1 (Ser65) were significantly augmented above the values observed in refed REDD1(+/+) mice by 258%, 405%, and 401%, respectively, although protein synthesis was not coordinately increased. Seventy-five minutes after refeeding, REDD1 expression in REDD1(+/+) mice was reduced (?15% of feed-deprived REDD1(+/+) values), and protein synthesis and mTORC1 signaling were not different between refed REDD1(+/+) mice and REDD1(-/-) mice.ConclusionsThe results show that REDD1 expression limits protein synthesis in mouse skeletal muscle by inhibiting mTORC1 signaling during periods of feed deprivation and that a reduction in its expression is necessary for maximal stimulation of protein synthesis in response to refeeding.

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Fresh and cultured mouse islets differ in their response to nutrient stimulation.

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Fresh and cultured mouse islets differ in their response to nutrient stimulation.

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