Interactions Between Hyperglycemia and Hypoxia - Diabe...

Interactions Between Hyperglycemia and Hypoxia

Interactions Between Hyperglycemia and Hypoxia Interactions Between Hyperglycemia and Hypoxia
Received for publication February 27, 2004 and accepted in revised form July 27, 2004
Jens R. Nyengaard1, Yassuo Ido2, Charles Kilo3, and Joseph R. Williamson4
Diabetes
© 2004 by the American Diabetes Association, Inc.

Implications for Diabetic Retinopathy

1 Stereological Research and Electron Microscopical Laboratory, University of Aarhus, Aarhus C, Denmark
2 Departments of Medicine and Physiology, Diabetes & Metabolism Unit, Boston Medical Center, Boston University School of Medicine, Boston, Massachusetts
3 Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri
4 Department of Pathology, Washington University School of Medicine, St. Louis, Missouri

ABSTRACT

The primary aim of these experiments was to assess in vitro effects of hyperglycemia (30 mmol/l glucose) and hypoxia (PO2 = 36 torr) of 2-h duration, separately and in combination, on cytosolic and mitochondrial free NADH (NADHc and NADHm, respectively) in retinas from normal rats. NADH is the major carrier of electrons and protons that fuel ATP synthesis and several metabolic pathways linked to diabetic complications. Hyperglycemia and hypoxia increase free NADHc by different mechanisms that are additive. Hyperglycemia increases transfer of electrons and protons from sorbitol to NAD+c, reducing it to NADHc, but does not increase NADHm. Hypoxia increases NADHm by inhibiting its oxidation. Electrons and protons accumulating in NADHm restrain transfer of electrons and protons from NADHc to NAD+m via the malate-aspartate electron shuttle. Hyperglycemia and hypoxia also increase glycolysis by different mechanisms that are additive, and hyperglycemia increases ATP levels in hypoxic and in aerobic retinas. The additive effects of hyperglycemia and hypoxia on accumulation of electrons and protons in a common pool of free NADHc confirm the test hypothesis and the potential of a combination of these two risk factors to accelerate the onset and progression of diabetic retinopathy (and other complications of diabetes) by augmenting metabolic pathways fueled by free NADHc.

The importance of duration and severity of hyperglycemia in the development of diabetic retinopathy was clearly established by the Diabetes Control and Complications Trial (1); however, the mechanisms that mediate early and late retinal vascular dysfunction and structural changes remain unclear. Proliferative retinopathy is widely attributed to increased production of vascular endothelial growth factor evoked by hypoxia/ischemia caused by capillary closure and nonperfusion that develop relatively early after the onset of diabetes (2).

The possibility that hyperglycemia and hypoxia may interact via a common metabolic imbalance(s) to initiate and/or exacerbate complications of diabetes is suggested by the correspondence of several redox, metabolic, and pathophysiological changes evoked by either condition alone. Examples include an increased ratio of reduced to oxidized free cytosolic NADc (3–16), increased production of free radicals (3,17–24) and vascular endothelial growth factor (17,19,25–28), accumulation of triose phosphates (7,9,10,13–15), activation of protein kinase C (PKC) (22,28–30), decreased Na+/K+-ATPase activity (3,30–33), early increases in blood flow (3,33–35), and paradoxical protective effects of diabetes and brief periods of hypoxia/ischemia (i.e., ischemic preconditioning) that attenuate dysfunction/injury evoked by subsequent more severe hypoxia/ischemia (3,29,36–38).

An increase in free NADH/NAD+c appears to be the best candidate metabolic imbalance for mediating pathophysiological changes common to diabetes and hypoxia (3,38). This redox imbalance develops when electrons and protons are transferred to free oxidized NAD+c (reducing it to NADHc) faster than electrons and protons carried by NADHc are used for ATP synthesis in mitochondria by oxidative phosphorylation. Hypoxia increases free NADHc because lack of O2 impairs utilization of electrons and protons carried by mitochondrial NADHm for oxidative phosphorylation. The mass action effect of electrons and protons accumulating in NADHm restrains transfer of electrons and protons from free NADHc to NAD+m by the malate-aspartate electron shuttle; thus, electrons and protons accumulate in, and elevate, free NADHc.

In cells for which glucose uptake is insulin-independent, hyperglycemia augments glucose uptake and metabolism via the sorbitol pathway. In the second step of the pathway, sorbitol is oxidized to fructose by sorbitol dehydrogenase, which catalyzes transfer of a hydride ion (:H–) from sorbitol to free NAD+c (reducing it to NADHc) and removal of a second hydrogen atom that is released into solution as a proton (H+):


In such cells, increased electrons and protons carried by NADHc can fuel several metabolic pathways implicated in the pathogenesis of diabetic retinopathy (3,9,10,17,38).

We coined the term "hyperglycemic pseudohypoxia" to draw attention to numerous metabolic imbalances and pathophysiological changes common to hyperglycemia and hypoxia (3). The present studies were undertaken to determine whether increases in NADHc evoked by hyperglycemia and hypoxia are additive, which would have the potential to augment NADHc-fueled metabolic pathways implicated in the pathogenesis of diabetic retinopathy.

Our findings demonstrate that hyperglycemia and hypoxia increase NADHc and triose phosphate levels by different mechanisms that are additive, and inhibition of the sorbitol pathway prevents both of these increases evoked by hyperglycemia but not the increases evoked by hypoxia. They also demonstrate that hyperglycemia and hypoxia increase glycolysis (manifested by increased lactate production) by different mechanisms that are additive; however, the increases in glycolysis are independent of the sorbitol pathway.

Please visit the website to view the article in its entirety.