Research ReportExpression of hexokinase isoforms in the dorsal root ganglion of the adult rat and effect of experimental diabetes
Introduction
Diabetic sensory neuropathy in humans is associated with a spectrum of structural changes in peripheral nerve that includes axonal degeneration, microangiopathy, paranodal demyelination and loss of myelinated and unmyelinated fibers — the latter probably the result of a dying-back of distal axons (Malik et al., 2005, Yagihashi, 1997). In the streptozotocin (STZ)-diabetic rat and Bio-Breeding (BB) rat animal models of type I diabetes, similar structural abnormalities in peripheral nerve have been observed (Mizisin et al., 1999, Sima and Sugimoto, 1999, Yagihashi, 1997). The Diabetes Control and Complications Trial (DCCT) concluded that control of hyperglycemia is still the ideal means of preventing appearance of complications in diabetes, such as peripheral neuropathy (DCCT, 1990). However, such a goal remains an unrealized ideal and research continues to focus on biochemical transducers downstream from hyperglycemia that may directly induce neuropathic sensory nerve damage. Brownlee and colleagues have proposed that high cellular concentrations of glucose lead to mitochondrial dysfunction and the generation of damaging levels of reactive oxygen species (ROS) which then mediate cell degeneration (Nishikawa et al., 2000b). One component of such a theory presupposes that high glucose concentrations are converted by glycolysis into high levels of pyruvate that then enter the tricarboxylic acid cycle (TCA) within the mitochondrial matrix and provide high levels of reduced electron donors for electron transport. The inability of mitochondrial oxidative phosphorylation to efficiently deal with this high level of electron donation may lead to excessive superoxide production and oxidative stress (Nishikawa et al., 2000a, Nishikawa et al., 2000b).
Hexokinases serve as the gateway through which glucose enters glycolysis, by catalyzing the phosphorylation of glucose to yield glucose 6-phosphate (G-6-P), the initial and rate-limiting step of the glycolytic pathway (Wilson, 1995, Wilson, 2003). In mammals, four distinct hexokinase isozymes exist, designated types I, II, III and IV, with the latter commonly known as glucokinase (Wilson, 2003). The hexokinase I–III isozymes are 100 kDa molecules that display internal sequence repetition, and the N- and C-terminal halves have extensive sequence similarity to each other and to other members of the hexokinase family, in contrast, glucokinase has a molecular weight of approximately 50 kDa (Wilson, 1995). Based upon the kinetics of hexokinase isoform activities it is believed that hexokinase I functions primarily in a catabolic role, introducing glucose into glycolytic metabolism with the primary purpose of generating ATP. This is consistent with the ubiquitous expression of hexokinase I in tissues. In addition, hexokinase I is expressed at particularly high levels in the brain, which is virtually reliant on glycolytic metabolism of glucose to sustain a high rate of energy metabolism (Wilson, 2003). In contrast, hexokinase II is much more limited in its expression, primarily being found in insulin-sensitive tissues such as skeletal muscle and adipose tissue. Glucokinase has a relatively low affinity for glucose (Km = 5–15 mM), with the result that the rate of glucose phosphorylation is responsive to physiologically relevant changes in plasma glucose concentration. Glucokinase is expressed primarily in the liver and pancreatic β-cells (Roncero et al., 2000).
Hexokinase I and II have the ability to bind specifically and reversibly to hydrophobic sequences at the outer mitochondrial membrane (Muller et al., 1994, Polakis and Wilson, 1985). A majority of the hexokinase activity is found in the mitochondrial fraction from homogenates of brain from various species (Kabir and Wilson, 1993). It has been demonstrated that hexokinase II interferes with the ability of bax to bind to mitochondria and release cytochrome c, consequently inhibiting bax-induced mitochondrial dysfunction and cell death (Pastorino et al., 2002). The physiological significance of this association of hexokinase with mitochondria, the primary site for oxidative metabolism, is that bound hexokinase has ‘preferential’ access to an intramitochondrial compartment of ATP. Subsequently, the rate of glucose phosphorylation (i.e. the rate at which glucose is being introduced into glycolysis) is directly correlated with the rate of mitochondrial oxidative phosphorylation (de Cerqueira Cesar and Wilson, 1995). Thus, binding of hexokinase to brain mitochondria provides a physical basis for coordination of the initial step of glycolytic metabolism occurring in the mitochondria.
Even though hexokinase has such a central role in glucose metabolism there is little information on its expression or activity in peripheral sensory neurons. Additionally, the effect of experimental diabetes on such parameters has not been investigated and possible role in sensory neuropathy studied. Therefore, this study describes the expression, localization and activity of the hexokinase isoforms in dorsal root ganglia (DRG) and sciatic nerve of adult rats. Furthermore, the effect of streptozotocin (STZ)-induced diabetes on expression, localization and activity of hexokinase was investigated. A central finding is that hexokinase activity is actually reduced in DRG of STZ-diabetic rats and would suggest that glycolytic delivery of substrates to the TCA cycle is, in fact, diminished. Therefore, the results question the validity of theories proposing that high glucose concentrations induce mitochondrial dysfunction under diabetic conditions in sensory neuron perikarya.
Section snippets
Expression and activity of hexokinase isoforms in nervous tissues
A range of tissues, including brain, lumbar DRG and sciatic nerve, were isolated from adult rat and subjected to SDS–PAGE in order to determine the expression levels of the hexokinase isoforms I–IV (Fig. 1, Fig. 2A). Hexokinase I was expressed at high levels in brain, heart, kidney and lumbar DRG. There was also low expression in the sciatic nerve. Hexokinase II was expressed in muscle and heart (low level of expression that is not clear on blot). Hexokinase III was expressed at low levels in
Discussion
The results show that adult sensory neurons exhibit high levels of hexokinase I protein expression and enzyme activity. Hexokinase I expression was particularly high in small–medium neurons, but interestingly was very low or absent in large sensory neurons (N52 positive and mainly proprioceptive in terms of phenotype). There was clear evidence of positive staining for hexokinase I in satellite cells surrounding these large neurons. Immunostaining and biochemical analysis demonstrated that
Conclusion
In conclusion, this work shows that adult sensory neurons expression high levels of mitochondrially associated hexokinase I, however the pattern of expression is neuronal sub-type specific and also occurs in satellite cells. STZ-diabetes diminished hexokinase activity in lumbar DRG and, therefore, questions the hypothesis that high glucose concentrations drive production of high ROS levels by elevating electron donation into the electron transport chain.
Induction of diabetes
Male Wistar rats were made diabetic for 6 and 12 weeks by a single intraperitoneal injection of STZ (55 mg/kg, Sigma). The level of glucose in tail vein blood was measured using a glucose oxidase strip operated reflectance meter (Reflolux II BCL Boehringer Mannheim), and it was > 27 mmol/l for all STZ-injected rats. At death plasma glucose levels for all groups of diabetic rats were > 20 mmol/l (plasma glucose levels for control rats ranged from 10 to 11 mmol/l). Body weights for age-matched
Acknowledgments
The authors thank Dr John Wilson for gifts of antibodies to hexokinase. Dr. Zuocheng Wang was supported by a postdoctoral research fellowship from the St Boniface Hospital and Research Foundation. Dr. Natalie Gardiner was supported by a Research Council UK Postdoctoral Fellowship. This work was supported by grants from CIHR (grant #ROP-72893), NSERC (grant #311686-06) and the Manitoba Medical Services Foundation.
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