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H6PDH is a bifunctional enzyme that catalyses the first two steps of an ER-specific pentose phosphate pathway generating the cofactor, NADPH, for 11β-HSD1 promoting its oxo-reductase activity. H6PDH is a free-floating enzyme within the ER lumen whereas 11β-HSD1 is bound to the inner ER membrane

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(Odermatt et al. 2001). H6PDH regulates the direction of endogenous 11β-HSD1 enzyme activity within the ER/SR lumen (Lavery et al. 2008b).

H6PDH was initially purified from rabbit liver microsomes and subsequently the human H6PDH cDNA and gene were cloned and identified (Mason et al. 1999). Sequence analyses indicate that H6PDH is biochemically distinct from its cytosolic homolog, G6PDH (Hewitt et al. 2005; White et al. 2007). Similarities and differences between both enzymes are summarised in the Table 1-2 (Senesi et al. 2010).

Table 1-2 Comparison of G6PDH to H6PDH Characterisation

G6PDH

H6PDH

Chromosome location Xq28 1p36 Sequence length 515 AA 791 AA Catalysed reaction D-G6P + NADP+= D- glucono-1,5-lactone-6- phosphate + NADPH D-G6P + NAD(P)+ + H2O= 6- phospho-D-gluconate + NAD(P)H

Cofactors NADP+ NAD(P)+, deoxy NADP+

Substrates G6P G6P, hexose-6-phosphates,

glucose-6-sulfate, glucose Intracellular

location Cytosol, soluble ER, soluble

Interactions Homodimer or homotetramer Homodimer, association to _11β-HSD1 Involvement in pathology Chronic non-spherocytic hemolytic anemia Apparent cortisone reductase deficiency, skeletal myopathy

To investigate the role of H6PDH in regulating 11β-HSD1 activity and GC metabolism, H6PDH global knockout (H6PDHKO) mice were previously generated. Inactivation of murine H6PD gene changes the direction of 11β-HSD1, only dehydrogenase activity was detectable in a variety of H6PDHKO tissues.

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Additionally, urine analysis from H6PDHKO mice revealed the presence of mostly metabolites of 11-DHC. These findings confirm that H6PDH plays an essential role in the regulation of NADPH-dependent oxo-reductase activity of 11β-HSD1 (Lavery et al. 2006).

1.3.2.1 NADPH/NADP+ redox status in the SR

H6PDH is a housekeeping enzyme, and within the SR utilises G6P and NADP+ to generate reducing NADPH for several ER reductases including 11β-HSD1. H6PDH may act as a metabolic sensor by connecting intermediary metabolism to hormonal signalling (Banhegyi et al. 2009). Changes in an NADPH/NADP+ ratio in the SR might cause disruption in the thiol-disulfide redox leading to incorrect protein folding and activation of the unfolded protein response (UPR) pathway as a compensatory mechanism and consequently resulting in myopathy development (Rogoff et al. 2010). Previous findings show that cortisone reduction by 11β-HSD1 is driven by the luminal generation of NADPH by H6PDH and is independent of cytosolic NADPH resources (Csala et al. 2006). Therefore, H6PDH activity relies on the G6P transport across the ER membrane by G6PT (Zielinska et al. 2011).

1.3.2.2 NADH/NAD+ redox status in the cytosol

Interestingly, the cytosolic NADH/NAD+ redox is essential for initiation of muscle contraction as it triggers release of intracellular calcium ions from the SR by the calciumrelease channels or ryanodine receptors (RyR). Cytosolic NADH/NAD+ levels are regulated by GAPDH which is associated with the SR membrane. Therefore, changes in activity of this enzyme might lead to more significant changes of local

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NADH/NAD+ levels in close proximity to SR bound proteins (such as the RyR and SERCA) (Zima et al. 2003). The ratio of NADH/NAD+ varies as a response to changes in metabolism and indicates the metabolic state of a cell or tissue, for instance, during ischemia cytosolic NADH/NAD+ ratio increases up to 30 times (Park et al. 1998).

1.3.2.3 NAD+ metabolism

The role NAD+ fulfils in muscle energy signalling and homeostasis, and its ability to stimulate mitochondrial function, has recently risen to prominence. NAD+ is an enzyme co-factor central to metabolism and essential for life in all organisms and importantly, fundamental to skeletal muscle metabolism. It functions both as a co-enzyme for oxidoreductases and as a source of ADP-ribosyl groups for the sirtuins (SIRT) family of protein deactylases which can signal to increase mitochondrial metabolism and insulin sensitivity in skeletal muscle playing an important role in life span extension (Bieganowski and Brenner 2004; Tempel et al. 2007). Among seven members of the sirtuin family, SIRT1 and SIRT3 are of particular interest with regard to skeletal muscle metabolism following the discovery that they positively regulate the PGC-1α, a potent transcriptional co-activator of mitochondrial biogenesis. SIRT1 is involved in the process of glucose metabolism and insulin secretion. Down-regulation of SIRT1 protein levels under insulin-resistant conditions in C2C12 myotubes leads to insulin resistance (Sun et al. 2007) whereas SIRT3 is localised primarily in mitochondria and is associated with ageing by regulating mitochondrial function (Jing et al. 2011). As a substrate for SIRT1/3, NAD+ is rapidly consumed; thus, there is a necessity to

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generate new NAD+ or re-synthesise NAD+ from by-products of the NAD+- degradation reaction.

NAD+ can be de novo synthesised from tryptophan taken up from the diet or in the Preiss-Handler pathway by utilising the vitamin precursor of NAD+, nicotinic acid (Na) (Houtkooper et al. 2010). Na is generally acquired from the diet or from the hydrolysis of nicotinamide (NAM), whereas NAM is the breakdown product of NAD+. Both Na and NAM are first converted to their mononucleotide forms: nicotinic acid mononucleotide (NaMN) or nicotinamide mononucleotide (NMN), and then the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) produces the nicotinic acid adenine dinucleotide (NaAD+) or NAD+ respectively. Finally, NaAD+ is converted to NAD+ by the enzyme NAD+ synthetase (Khan et al. 2007). In cytosol, NAD+ can be phosphorylated by NAD+ kinase to NADP+ which then can be further reduced to NADH or NADPH in the pentose phosphate pathway (Figure 1-16) (Outten and Culotta 2003).

Figure 1-16 Schematic representation of NAD+ and NADP+ conversion

Because cellular NAD+ is consumed in numerous pathways, its replenishment is critical to preserving signalling competency and supporting NAD(P)(H)-dependent reactions.

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1.4 In vivo genetic manipulations of ER/SR genes

In document BANCO AGROPECUARIO - AGROBANCO (página 48-51)