Fructose induces Hepatic Gluconeogenesis through a SIRT1-dependent mechanism

Paul Caton, Nanda Nayuni, Roger Corder

William Harvey Research Institute, London, UK

SIRT1, an NAD+ dependent protein deacetylase stimulates hepatic gluconeogenesis during fasting through deacetylation and induction of PGC1-alpha, an essential coactivator of gluconeogenic genes (Rodgers et al., 2005). Conversely, activators of SIRT1 are reported to have anti-diabetic actions and are under investigation as a treatment for obesity (Milne et al., 2007). Consumption of a fructose-rich diet leads to insulin resistance, induction of hepatic gluconeogenesis and hyperglycaemia. Recently SIRT1 induction in the hearts of fructose-fed rats has been described (Pillai et al., 2008), but the impact of fructose on hepatic SIRT1 has yet to be investigated. The aim of this study was to establish whether fructose induced hepatic SIRT1, leading to PGC1-alpha directed induction of gluconeogenesis

SIRT1 protein levels and activity were measured using western blot and fluorometric assay. To assess levels of gluconeogenesis, glucose production was measured using a colourimetric assay, and activity of phosphoenolpyruvate carboxykinase (PEPCK; gene code PCK1), the rate limiting enzyme of gluconeogenesis, was assayed using luminescence methodology based on the conversion of oxaloacetate to phosphoenolpyruvate. For PCK1 and PGC1-alpha measurements, total RNA was extracted using an RNA microprep kit (Stratagene, UK) and mRNA levels were measured using qRT-PCR. Statistical differences were determined by ANOVA. Results are means ± S.E.M from three experiments.

Incubation of rat hepatocytes (H4IIEC3 cell line) for 6 h with fructose (1 – 5 mM) increased SIRT1 protein (6.4 ± 1.6 fold; p<0.01) and activity (30 ± 1%; p<0.05) through increased NAD+/NADH ratio (63 ± 2.2%; p<0.05). Fructose increased mRNA levels for PGC1-alpha? (1.7 ± 0.8 fold; p<0.01), PCK1 mRNA (1.9 ± 0.3 fold; p<0.01) and enzyme activity (55 ± 1.2%; p<0.05) levels and elevated hepatocyte glucose production (10 ± 1 fold; p<0.001). Fructose-induced increases in all gluconeogenic parameters were abolished by the SIRT1 inhibitors nicotinamide (0.1 – 2 mM; p<0.05) and 6TCC (1 – 10 μM; p<0.05) (Napper et al., 2005). In comparison, SIRT1 activator 3 (20 – 60 μM) (Nayagam et al., 2006) increased glucose production (2.2 ± 0.5 fold; p<0.001), PEPCK activity (87 ± 17%; p<0.05) and mRNA levels for PCK1 (3.5 ± 0.6 fold; p<0.001) and PGC1-alpha (1.4 ± 0.5 fold; p<0.01). Similarly, SRT1720, another SIRT1 activating compound (Milne et al., 2007) also induced gluconeogenesis.

The results of this study indicate that induction of hepatic SIRT1 plays a key role in fructose induced changes in hepatic functions, including gluconeogenesis. This suggests that caution should be exercised with respect to the use of SIRT1 activators as therapies for T2DM and metabolic syndrome.

Rodgers J.T. et al., (2005) Nature, 434; 113-118

Milne J.C. et al., (2007)Nature, 450; 712-716

Pillai J.B. et al., (2008) Am. J. Physiol. Heart Circ. Physiol., 294, 1388-97

Napper A.D et al., (2005) J. Med. Chem., 48; 8045-54

Nayagam et al., (2006) Journal of Biomolecular Screening, 11, 959-967