Elsevier

Metabolism

Volume 60, Issue 4, April 2011, Pages 550-556
Metabolism

Recurrent hypoglycemia increases hypothalamic glucose phosphorylation activity in rats

https://doi.org/10.1016/j.metabol.2010.05.009Get rights and content

Abstract

The mechanisms underpinning impaired defensive counterregulatory responses to hypoglycemia that develop in some people with diabetes who suffer recurrent episodes of hypoglycemia are unknown. Previous work examining whether this is a consequence of increased glucose delivery to the hypothalamus, postulated to be the major hypoglycemia-sensing region, has been inconclusive. Here, we hypothesized instead that increased hypothalamic glucose phosphorylation, the first committed intracellular step in glucose metabolism, might develop following exposure to hypoglycemia. We anticipated that this adaptation might tend to preserve glucose flux during hypoglycemia, thus reducing detection of a falling glucose. We first validated a model of recurrent hypoglycemia in chronically catheterized (right jugular vein) rats receiving daily injections of insulin. We confirmed that this model of recurrent insulin-induced hypoglycemia results in impaired counterregulation, with responses of the key counterregulatory hormone, epinephrine, being suppressed significantly and progressively from the first day to the fourth day of insulin-induced hypoglycemia. In another cohort, we investigated the changes in brain glucose phosphorylation activity over 4 days of recurrent insulin-induced hypoglycemia. In keeping with our hypothesis, we found that recurrent hypoglycemia markedly and significantly increased hypothalamic glucose phosphorylation activity in a day-dependent fashion, with day 4 values 2.8 ± 0.6-fold higher than day 1 (P < .05), whereas there was no change in glucose phosphorylation activity in brain stem and frontal cortex. These findings suggest that the hypothalamus may adapt to recurrent hypoglycemia by increasing glucose phosphorylation; and we speculate that this metabolic adaptation may contribute, at least partly, to hypoglycemia-induced counterregulatory failure.

Introduction

Intensified insulin therapy for diabetes is often limited in clinical practice by increased risk of hypoglycemia [1], [2], [3]. In health, blood glucose levels are tightly controlled within relatively narrow boundaries, with a falling blood glucose level being rapidly detected and a series of defensive counterregulatory responses triggered to prevent or limit hypoglycemia and restore euglycemia. Counterregulatory responses include the release of hormones such as glucagon and epinephrine and the generation of protective warning symptoms. Counterregulatory responses may become impaired after repeated exposure to hypoglycemia as experienced by diabetic patients undergoing intensive insulin therapy, placing them at significant risk of subsequent episodes of severe hypoglycemia—essentially a type of “stress desensitization” [1]. The mechanisms underlying the down-regulation of defensive counterregulatory responses following recurrent hypoglycemic exposure remain unclear.

To elicit counterregulatory responses, falling glucose levels need to be detected, probably in large part by sensors in specialized areas of the hypothalamus [4], [5], [6], [7], [8]. Although mechanisms used by specialized glucose sensors to detect changes in glucose availability are unclear, there is increasing evidence that these sensors may sense downstream products of glucose metabolism, for example, monitoring levels of adenosine triphosphate, analogous to the canonical pancreatic β-cell glucose-sensing mechanism [9], [10], [11].

Glucose metabolism in hypothalamus may therefore contribute to neuronal glucose sensing and potentially the control of hypoglycemia counterregulation. In keeping with this, several investigators have suggested that alterations in brain glucose uptake and/or metabolism may contribute to the impairment of hypoglycemia counterregulation following recurrent hypoglycemia [12], [13], [14]. In particular, recent human brain imaging studies have shown that recurrent hypoglycemia may selectively alter neuronal glucose utilization in hypothalamic [15] or thalamic [16] regions of the brain, although the biochemical adaptations underpinning these changes cannot easily be further defined in human studies.

Following glucose transport into neurons, the first committed step in glucose metabolism is phosphorylation, which is catalyzed by hexokinases (HK; EC 2.7.1.1), a group of related enzymes [17]. Given the potentially rate-limiting role of glucose phosphorylation in neuronal glucose sensing, we hypothesized that up-regulation of glucose phosphorylation activity may occur following recurrent hypoglycemia in parallel with impairment of glucose counterregulation. In this work, we aimed to examine whether brain glucose phosphorylation activity was up-regulated following recurrent hypoglycemia, reasoning that any adaptation related to glucose sensing (as opposed to a more general defensive adaptation to a decrease in glucose as the major brain fuel) would be restricted to hypothalamic and/or glucose-sensing areas rather than being a more global change in brain glucose phosphorylation. To address this issue directly, we used a rat model of impaired glucose counterregulation induced by exposing the animals to recurrent insulin-induced hypoglycemia and investigated glucose phosphorylation activity in brain protein preparations in vitro at a physiologic brain glucose concentration during hypoglycemia. Here, we demonstrate that recurrent hypoglycemia, with 4 sequential daily episodes of exposure to blood or plasma glucose concentration of less than 4 mmol/L [10], [11], significantly increased glucose phosphorylation activity in hypothalamus preparations.

Section snippets

Animals

We studied healthy adult male Sprague-Dawley rats (Charles River, UK) of approximately 300 g throughout. Rats were housed individually after surgery on a 12-hour on-off light-dark cycle. The procedures were approved in advance by both a local University and UK Home Office ethics review. All chemicals were from Sigma-Aldrich (Gillingham, UK) except where otherwise stated.

Surgical preparation

Under inhaled anesthetic, rats underwent survival surgery for placement of vascular catheters into the right jugular vein [18]

Study 1: validation of model of impaired counterregulation to hypoglycemia

In study 1, we investigated the pattern of impairment of epinephrine responses during recurrent hypoglycemia in another cohort of rats (Fig. 1A). Plasma epinephrine levels decreased significantly (P < .05) and day dependently with exposure to sequential hypoglycemia (Fig. 1A). Plasma glucagon levels also tended (P = not significant [NS]) to reduce following exposure to recurrent hypoglycemia (Fig. 1B). In summary, these data confirmed that epinephrine responses were suppressed over the course

Discussion

The mechanisms underlying impaired glucose counterregulation following repetitive hypoglycemia, as experienced by patients with diabetes undergoing aggressive insulin therapy, remain unclear. Several investigators have suggested that alterations in brain glucose metabolism may contribute to the impairment of glucose counterregulation following recurrent hypoglycemia [12], [13], [14], [15], [16]. Nonetheless, the precise nature of biochemical alterations in brain glucose metabolism that occurs

Acknowledgment

This work was supported by Juvenile Diabetes Research Foundation regular Grants 1-2003-78 and 1-2006-29 and Diabetes UK Grant RD05/003059 (to M.L.E.); the Diabetes Research Wellness Foundation (to M.A.O.); and the Cambridge Medical Research Council Centre for Study of Obesity and Related Disorders. L.K.H. was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01DK065171, American Diabetes Association, and the Wellcome Trust. PH was supported by the Sir Jules

References (30)

  • OomuraY. et al.

    Reciprocal activities of the ventromedial and lateral hypothalamic area of cats

    Science

    (1964)
  • KangL. et al.

    Glucokinase is a critical regulator of ventromedial hypothalamic neuronal glucosensing

    Diabetes

    (2006)
  • McCrimmonR.J. et al.

    Activation of ATP-sensitive K+ channels in the ventromedial hypothalamus amplifies counterregulatory hormone responses to hypoglycemia in normal and recurrently hypoglycemic rats

    Diabetes

    (2005)
  • EvansM.L. et al.

    Hypothalamic ATP-sensitive K + channels play a key role in sensing hypoglycemia and triggering counterregulatory epinephrine and glucagon responses

    Diabetes

    (2004)
  • LeiH. et al.

    Effect of chronic hypoglycemia on glucose concentration and glycogen content in rat brain: a localized 13C NMR study

    J Neurochem

    (2006)
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