Metabolic Engineering through Cofactor Manipulation and Its Effects on Metabolic Flux Redistribution in Escherichia coli

Abnormal elevation of hepatic gluconeogenesis is central to the onset of hyperglycaemia in patients with type 2 diabetes mellitus (T2DM). Metformin corrects hyperglycaemia through inhibition of gluconeogenesis, but its mechanism of action is yet to be fully described. SIRT1 and GCN5 (listed as KAT2A in the MGI Database) have recently been identified as regulators of gluconeogenic gene expression through modulation of levels and activity of the coactivators cAMP-response element binding protein-regulated transcription coactivator 2 (TORC2 or CRTC2 as listed in the MGI Database) and peroxisome proliferator-activated receptor-g coactivator-1a (PGC1a or PPARGC1A as listed in the MGI Database). We report that in db/db mice, metformin (250 mg/kg per day; 7 days) increases hepatic levels of GCN5 protein and mRNA compared with the untreated db/db mice, as well as increases levels of SIRT1 protein and activity relative to controls and untreated db/db mice. These changes were associated with reduced TORC2 protein level and decreased gene expression and activation of the PGC1a gene target phosphoenolpyruvate carboxykinase, and lower plasma glucose and insulin. Inhibition of SIRT1 partially blocked the effects of metformin on gluconeogenesis. SIRT1 was increased through an AMP-activated protein kinase-mediated increase in gene expression of nicotinamide phosphoribosyltransferase, the rate-limiting enzyme of the salvage pathway for NAD C . Moreover, levels of GCN5 were dramatically reduced in db/db mice compared with the controls. This indicates that loss of GCN5-mediated inhibition of gluconeogenesis appears to constitute a major mechanism for the onset of abnormally elevated hepatic glucose production in db/db mice. In conclusion, induction of GCN5 and SIRT1 potentially represents a critical mechanism of action of metformin. In addition, these data identify induction of hepatic GCN5 as a potential therapeutic strategy for treatment of T2DM.

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“…C /NADH ratio and ATP levels ATP levels and NAD C /NADH ratios were measured according to published methods (San et al 2002, Moynihan et al 2005.…”

Section: Nadmentioning

confidence: 99%

Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5

Caton¹,

Nayuni²,

Kieswich³

et al. 2010

Journal of Endocrinology

175

135

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“…It is conceivable that in coenzyme-dependent production systems, coenzyme availability and the proportion of coenzyme in the active form may play an important role in dictating the overall process yield. Hence, the manipulation of these coenzyme levels may be crucial in order to further increase production [75][76][77][78].…”

Section: Alterations In the Coenzyme Pools 374mentioning

confidence: 99%

Minimizing acetate formation in E. coli fermentations

Mey

Maeseneire

Soetaert

et al. 2007

J Ind Microbiol Biotechnol

199

144

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Escherichia coli remains the best established production organisms in industrial biotechnology. However, during aerobic fermentation runs at high growth rates, considerable amounts of acetate are accumulated as by-product. This by-product has negative effects on growth and protein production. Over the last 20 years, substantial research efforts have been spent to reduce acetate accumulation during aerobic growth of E. coli on glucose. From the onset it was clear that this quest should not be a simple nor uncomplicated one. Simple deletion of the acetate pathway, reduced the acetate accumulation, but instead other by-products were formed. This minireview gives a clear outline of these research efforts and the outcome of them, including bioprocess level approaches and genetic approaches. Recently, the latter seems to have some promising results

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“…This can be done by using a probe molecule that is bacterial cytoplasmic membrane-impermeable. We selected NAD + as the probe because NAD + is an important metabolite, and several analytic methods including HPLC (Sporty et al, 2008) and enzymatic cycling (San et al, 2002) have been established for precise measurement of NAD + level. As shown in Fig.…”

Section: Determining the V Ex Value By Using Nad + As The Probementioning

confidence: 99%

“…This solution was used to rupture cells and extract the intracellular NAD + (San et al, 2002;Wong and Whitesides, 1981). As shown in Fig.…”

Section: Determining the V In Valuementioning

confidence: 99%

“…NAD + was chosen as the probe because it can significantly affect the metabolic networks and has been routinely quantified during metabolic data collection (Roca et al, 2003;San et al, 2002). Furthermore, because prokaryotic cells are free of intracellular compartmentalization, the V in data can be readily used for the estimation of absolute concentrations of cellular components (Bennett et al, 2009;Rabinowitz, 2007).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

An accurate method for estimation of the intracellular aqueous volume of Escherichia coli cells

Wang¹,

Zhou²,

Ji³

et al. 2013

Journal of Microbiological Methods

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Metabolic Engineering through Cofactor Manipulation and Its Effects on Metabolic Flux Redistribution in Escherichia coli

Cited by 235 publications

References 23 publications

Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5

Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5

Minimizing acetate formation in E. coli fermentations

An accurate method for estimation of the intracellular aqueous volume of Escherichia coli cells

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