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Diabetologia (1997) 40: S115–S117

 Springer-Verlag 1997

Cytosolic NADH/NAD + , free radicals, and vascular dysfunction in early diabetes mellitus

Y. Ido, C. Kilo, J. R. Williamson Washington University, St. Louis, Missouri, USA

To the extent that early vascular dysfunction and end- oxidized to coupled to reduction of NAD + stage diabetic vascular disease are mediated by com- to NADH by sorbitol dehydrogenase. In humans, in- mon pathogenetic factors (although early increases creased oxidation of non-esterified fatty acids (in per- in blood flow may not cause end-stage vascular dis- oxisomes in addition to mitochondria) and/or glucu- ease), elucidation of metabolic imbalances that medi- ronic acid pathway metabolites may be equally or ate the earliest manifestations of vascular dysfunction more important than oxidation of sorbitol. In con- may provide insights into the pathogenesis of vascu- trast to cytosolic reductive stress induced by in- lar complications of diabetes. The earliest detectable creased oxidation of substrates coupled to reduction evidence of vascular dysfunction induced by diabetes of NAD + to NADH, reductive stress induced by hy- is increased blood flow in retina, kidney, and periph- poxia is largely the consequence of impaired oxida- eral nerve. In non-diabetic humans and animals in- tion of NADH to NAD + by the mitochondrial elec- creased blood flow in these tissues is demonstrable tron transport chain. Thus, reductive stress arising in after only a few hours of acute hyperglycaemia in- the mitochondria due to hypoxia and that in the cyto- duced by intravenous infusion [1]; loss of en- sol due to increased substrate oxidation are mediated dothelial barrier function develops later. The earliest by independent mechanisms and are additive. Two to metabolic imbalance linked to increased blood flow four weeks after the onset of diabetes, when sciatic in these tissues in animal models of diabetes is cytoso- nerve blood flow is increased, metabolite couples lic reductive stress, i.e. an increased ratio of cytosolic that reflect mitochondrial NADH/NAD + do not free NADH/NAD + . This ‘hypoxia-like’ redox differ from normal rats. This finding indicates that cy- change is caused by increased oxidation of substrates tosolic reductive stress in these nerves (manifested by coupled to reduction of the cofactor NAD + to NADH (Fig.1). Candidate substrates include sorbi-

tol, non-esterified fatty acids, and glucuronic acid Hyperglycaemia pathway metabolites (UDP glucose, l-gulonate, and xylitol) [1]. Substrate oxid.NAD+ NADH In animal models of early diabetes the most impor- Sorbitol, NEFA, Glucuronates tant metabolic pathway contributing to cytosolic re- Reductive stress = NADH/NAD+ ductive stress appears to be increased flux of glucose via the sorbitol pathway [1–4]. In the first step of this GAP DH pathway glucose is reduced to sorbitol by re- Autox O – VEGF ductase. In the second step of the pathway sorbitol is NEG 2 snG3P DH Ca2+ DAG Corresponding author: Professor J.R. Williamson, Department NOS of Pathology, Box 8118, 660 S. Euclid Avenue, St. Louis, MO 63110, USA PKC Abbreviations: GAPDH, 3-phosphate dehy- + + drogenase; G3PDH, 3-phosphate dehydrogenase; Na K , PL A2 PG Vascular dysfunction DHAP, dihydroxyacetone phosphate; VEGF, vascular endo- ATPase thelial growth factor. Fig. 1. See text. S116 Y. Ido et al.: Cytosolic NADH/NAD + , free radicals, and vascular dysfunction in early diabetes mellitus increased lactate/pyruvate ratios which are in equilib- novo synthesis of diacylglycerol (which in turn acti- rium with NADH/NAD + via lactate dehydrogenase) vates protein C). Activation of protein kinase is not attributable to hypoxia; it is, instead, the conse- C has been implicated in mediating vascular dysfunc- quence of metabolic imbalances arising in the cytosol. tion in the retina, aorta, and kidney, since inhibitors The importance of the NADH/NAD + redox cou- of protein kinase C and/or of its sequelae prevent vas- ple in mitochondrial and cytosolic energy metabo- cular dysfunction in all three tissues in diabetic rats lism, and evidence that cytosolic free NADH/ [1, 4]. NAD + is modulated by mitochondrial NADH/ Reductive stress, whether induced by elevated glu- NAD + and by intra- and extracellular lactate and cose levels or by hypoxia, can promote free radical pyruvate (via lactate dehydrogenase and plasma production by several mechanisms including: 1)inhibi- membrane transporters of lactate and pyruvate), in- tion of xanthine dehydrogenase which will shift oxida- dicate that cytosolic free NADH/NAD + is a sensor tion of xanthine and hypoxanthineto xanthine oxidase of the (NADH/NAD + ) redox state of mitochondria, which yields superoxide; 2) autoxidation of NADH; 3) cytosol, and the extracellular milieu. It is unlikely to glycation of CuZn-superoxide dismutase by be a coincidence, therefore, that cytosolic NADH/ phosphates which inactivates the enzyme [1]. NAD + also appears to play an important role in reg- Several lines of evidence support a physiological ulating tissue blood flow in response to changes in role for superoxide in modulating numerous meta- tension, energy metabolism, and the extracel- bolic pathways, gene transcription, and biological lular lactate/pyruvate ratio. Reductive stress, regard- functions including increased blood flow in response less of the cause, is associated with increased blood to reductive stress [1]. We have hypothesized that flow. In addition to increased oxidation of glucose- the metabolic need for increased blood flow is sensed derived metabolites in diabetes, increased oxidation by reductive stress (regardless of the cause) which ini- of , electrochemical or mechanical work, hy- tiates a cascade of events that increase blood flow [1, perlactataemia, experimental galactosaemia, cyanide 4] as depicted in Figure 1. Reductive stress increases and poisoning, and fasting all cause superoxide levels which increase intracellular cal- reductive stress and are associated with increased cium and vascular endothelial growth factor blood flow in the affected tissue(s). (VEGF). Increased intracellular calcium activates The importance of cytosolic reductive stress is that constitutive synthase to produce small it impacts on the activity of many dehydrogenase en- amounts of nitric oxide which increases blood flow. zymes, several of which have been implicated in the This scenario is supported by evidence that: 1) pathogenesis of diabetic complications, that require VEGF expression is increased in cultured cells ex- NAD + or NADH as cofactors and are regulated by posed to elevated glucose levels; 2) increased blood NADH/NAD + . Several lines of evidence suggest flow in granulation tissue induced by elevated glu- that intracellular production of oxygen reactive spe- cose levels is prevented by polyclonal and mono- cies, glycation reactions, and activation of protein ki- clonal antibodies to VEGF. The role of each of the nase C are mediated in large part by a cascade of participants in this cascade is supported by evidence events initiated by the effects of cytosolic reductive that increased blood flow induced by elevated glu- stress on glyceraldehyde 3-phosphate dehydrogenase cose levels is also prevented by inhibitors of aldose (GAPDH) and glycerol 3-phosphate dehydrogenase reductase, sorbitol dehydrogenase, nitric oxide syn- (G3PDH) [1, 4]. thase, prostaglandin synthase, and superoxide dismu- Because of the equilibrium between NADH/ tase [1–4]. Increased blood flow in soleus muscle of NAD + and glyceraldehyde 3-phosphate/1,3-bisphos- rats induced by acute hyperglycaemia is also pre- phoglycerate established by GAPDH, an increased vented by superoxide dismutase. With increasing du- ratio of NADH/NAD + favours increased levels of ration of diabetes, increased production of superox- glyceraldehyde 3-phosphate which is in equilibrium ide by various mechanisms may scavenge nitric oxide with other trioses (dihydroxyacetone phosphate to the point that blood flow returns to normal or may [DHAP], and fructose 1,6-bisphosphate, referred to even be decreased. collectively as ‘triose phosphates’). Triose phosphates In conclusion, cytosolic reductive stress produced are highly reactive which undergo autoxida- by increased oxidation of glucose metabolites (via se- tion (with production of free radicals including super- lected metabolic pathways) coupled to reduction of oxide) resulting in non-enzymatic glycation and oxi- NAD + to NADH appears to play an important role dative damage to intracellular proteins, DNA, and in mediating early vascular dysfunction induced by membrane lipids (Fig.1). diabetes. With longer duration of diabetes, sustained As a result of the corresponding equilibrium be- reductive stress and associated increased production tween NADH/NAD + and glycerol 3-phosphate/ of free radicals and growth factors will lead to vascu- DHAP established by G3PDH, an increased ratio of lar sclerosis (and angiogenesis in selected tissues NADH/NAD + favours reduction of DHAP to glyc- such as the retina) resulting in obliterative end-stage erol 3-phosphate, the first step in one pathway for de vascular disease. Y. Ido et al.: Cytosolic NADH/NAD + , free radicals, and vascular dysfunction in early diabetes mellitus S117

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