Sirtuin 1 independent effects of resveratrol in INS-1E β-cells

Abstract

Resveratrol (Resv), a natural polyphenol, is suggested to have various health benefits including improved insulin sensitivity. Resv activates Sirtuin (Sirt1) in several species and tissues. Sirt1 is a protein deacetylase with an important role in ageing, metabolism and β-cell function. In insulinoma β-cells (INS-1E), Resv is previously shown to improve glucose-stimulated insulin secretion in a Sirt1-dependent mechanism and to protect against β-cell dedifferentiation in non-human primates, while inducing hypertrophy in myoblasts. Mammalian (mechanistic) Target of Rapamycin (mTOR), is a key regulator of cellular metabolism and regulates the cell size, β-cell survival and proliferation. In order to understand the interaction of Sirt1 and mTOR cascade activity with Resv-induced changes in the INS-1E cell line, we generated stable Sirt1-down-regulated INS-1E cells, and analysed Sirt1-dependent effects of Resv with respect to mTOR cascade activity. Sirt1-knockdown (KD) had a significant increase in cell size compared to negative-control (NEG CTR) cells. Resveratrol treatment increased cell size in both cell types in a dose-dependent manner at 24 h (Resv conc: 15-60 μM), and decreased the cell number (Resv conc: 30-60 μM). Cell area was increased in NEG CTR cells (Resv conc: 60 μM) at 24 h and KD cells at 48 h (Resv conc: 15-60 μM). Rapamycin, a specific mTOR inhibitor, counteracted the Resv-induced cell enlargement (both cell diameter and area). Furthermore, Sirt1-downregulation by itself did not affect the mTOR cascade activities as measured by Western blotting for total and phosphorylated Akt and mTOR. Rapamycin decreased the mTORC1 activity, while increasing the pAkt levels. Resveratrol did not interfere with the mTOR activity or with Sirt1 expression. Altogether, this work indicates that Sirt1 is a negative regulator of cell size. Moreover, the effect of Resv on cell size increase is mTOR-cascade dependent.

Lysine Acetylation Goes Global: From Epigenetics to Metabolism and Therapeutics

“…577,578 These polyphenols lack potency in sirtuin binding, have low retention times in humans, and likely have considerable off-target effects. 579,580 More recent high-throughput screening methodologies uncovered other SIRT1 activators, such as SRT1720, with interesting biological effects that lead to extended lifespan and improved health in mice and some efficacy against xenografted tumor growth models. 581-583…”

“…Polyphenolic compounds, namely the phytochemical resveratrol, were originally shown to activate sirtuin activity by enhancing cofactor and substrate binding via engagement of the SIRT1 N-terminus. , These polyphenols lack potency in sirtuin binding, have low retention times in humans, and likely have considerable off-target effects. , More recent high-throughput screening methodologies uncovered other SIRT1 activators, such as SRT1720, with interesting biological effects that lead to extended lifespan and improved health in mice and some efficacy against xenografted tumor growth models. − …”

Functional Mitochondria Are Important for the Effect of Resveratrol

“…Resveratrol (3,4′,5-trihydroxy- trans -stilbene) (Resv) is a naturally occurring polyphenol found in a high number of unrelated plant species including grapes, cranberries and peanuts, and also in a number of herbal remedies [ 2 ]. Resv shows cytostatic action, prolongs the cell cycle S phase [ 3 ], enlarges cell size [ 4 ], and there is a clear anticarcinogenic activity of Resv in animal experiments [ 5 , 6 ].…”

Physiological phenotyping of mammalian cell lines by enzymatic activity fingerprinting of key carbohydrate metabolic enzymes: a pilot and feasibility study

“…HaCaT [5], HEK293 [7], C2C12 [6] and HepG2 cells [8] were cultured in DMEM with 25 mM glucose, 10% FBS and P/S. ⁠INS-1E cells (a kind gift of Claes Wollheim [3, 9]) were cultured in RPMI 1640 with Glutamax supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (P/S), 10% heat-inactivated FBS, and 50 µM β-mercaptoethanol as previously described [11].…”