OBJECTIVEAlthough intensive glycemic control achieved with insulin therapy increases the incidence of both moderate and severe hypoglycemia, clinical reports of cognitive impairment due to severe hypoglycemia have been highly variable. It was hypothesized that recurrent moderate hypoglycemia preconditions the brain and protects against damage caused by severe hypoglycemia.RESEARCH DESIGN AND METHODSNine-week-old male Sprague-Dawley rats were subjected to either 3 consecutive days of recurrent moderate (25–40 mg/dl) hypoglycemia (RH) or saline injections. On the fourth day, rats were subjected to a hyperinsulinemic (0.2 units · kg−1 · min−1) severe hypoglycemic (∼11 mg/dl) clamp for 60 or 90 min. Neuronal damage was subsequently assessed by hematoxylin-eosin and Fluoro-Jade B staining. The functional significance of severe hypoglycemia–induced brain damage was evaluated by motor and cognitive testing.RESULTSSevere hypoglycemia induced brain damage and striking deficits in spatial learning and memory. Rats subjected to recurrent moderate hypoglycemia had 62–74% less brain cell death and were protected from most of these cognitive disturbances.CONCLUSIONSAntecedent recurrent moderate hypoglycemia preconditioned the brain and markedly limited both the extent of severe hypoglycemia–induced neuronal damage and associated cognitive impairment. In conclusion, changes brought about by recurrent moderate hypoglycemia can be viewed, paradoxically, as providing a beneficial adaptive response in that there is mitigation against severe hypoglycemia–induced brain damage and cognitive dysfunction.

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose.

The cardiac subtype of the L-type voltage-sensitive Ca(2+) channel (VSCC) Cav1.2 (alpha(1C)) is the primary voltage-sensitive channel responsible for Ca(2+) influx into actively proliferating osteoblasts. This channel also serves as the major transducer of Ca(2+) signals in growth-phase osteoblasts in response to hormone treatment. In this study, we have demonstrated that 24-h treatment of MC3T3-E1 preosteoblasts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a coupling factor for bone resorption, coordinately downregulates Cav1.2 (alpha(1C)) and uniquely upregulates T-type channel Cav3.2 (alpha(1H)). No other voltage-sensitive channel alpha-subunit of the 10 that were surveyed was upregulated by 1,25(OH)(2)D(3). The shift from predominantly L-type to T-type channel expression has been demonstrated to occur at both mRNA and protein levels detected using quantitative PCR and immunohistochemistry with antibodies specific for each channel type. Functional and pharmacological studies using specific inhibitors have revealed that treatment with 1,25(OH)(2)D(3) also alters the Ca(2+) permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity. We conclude that the L-type channel is likely to support proliferation of osteoblast cells, whereas T-type channels are more likely to be involved in supporting differentiated functions after 1,25(OH)(2)D(3)-mediated reversal of remodeling has occurred. This latter observation is consistent with the unique expression of the T-type VSCC Cav3.2 (alpha(1H)) in terminally differentiated osteocytes as we recently reported.

Hypoglycemia is a common complication for insulin treated people with diabetes. Severe hypoglycemia, which occurs in the setting of excess or ill-timed insulin administration, has been shown to cause brain damage. Previous pre-clinical studies have shown that memantine (an Nmethyl-D-aspartate receptor antagonist) and erythropoietin can be neuroprotective in other models of brain injury. We hypothesized that these agents might also be neuroprotective in reponse to severe hypoglycemia-induced brain damage. To test this hypothesis, 9-week old, awake, male Sprague-Dawley rats underwent hyperinsulinemic (0.2 U.kg −1 .min −1 ) hypoglycemic clamps to induce severe hypoglycemia (blood glucose 10-15 mg/dl for 90 minutes). Animals were randomized into control (vehicle) or pharmacological treatments (memantine or erythropoietin). One week after severe hypoglycemia, neuronal damage was assessed by Fluoro-Jade B and hematoxylin and eosin staining of brain sections. Treatment with both memantine and erythropoietin significantly decreased severe hypoglycemia-induced neuronal damage in the cortex by 35% and 39%, respectively (both p<0.05 vs controls). These findings demonstrate that memantine and erythropoietin provide a protective effect against severe hypoglycemia-induced neuronal damage.

Although high dosages of insulin can cause hypoglycemia, several studies suggest that increased insulin action in the head may paradoxically protect against severe hypoglycemia by augmenting the sympathoadrenal response to hypoglycemia. We hypothesized that a direct infusion of insulin into the third ventricle and/or the mediobasal hypothalamus (MBH) would amplify the sympathoadrenal response to hypoglycemia. Nine week-old male rats had insulin (15 mU) or artificial cerebrospinal fluid (aCSF- control) infused bilaterally into the MBH or directly into the third ventricle. During the final two hours of the brain insulin or aCSF infusions, the counterregulatory response to either a hyperinsulinemic hypoglycemic (~50 mg/dl) clamp or to a 600 mg/kg IV bolus of 2-deoxy-glucose (2DG) was measured. 2DG was used to induce a glucoprivic response without peripheral insulin infusion. In response to insulin-induced hypoglycemia, epinephrine rose more than 60-fold, norepinephrine rose more than 4-fold, glucagon 8 fold, and corticosterone almost 2-fold, but these increments were not different in aCSF versus insulin treatment groups with either ICV or bilateratal MBH insulin protocols. ICV insulin infusion stimulated insulin signaling as noted by a 5-fold increase in AKT phosophorylation. In the absence of systemic insulin infusion, 2DG-induced glucopenia resulted in an equal counterregulatory response with brain aCSF and insulin infusions. Under the conditions studied, although insulin infusion acted to stimulate hypothalamic insulin signaling neither intrahypothalmic nor intracerebroventricular insulin infusion augmented the counterregulatory response to hypoglycemia nor to 2DG-induced glucoprivation. Therefore, it is proposed that the previously noted acute actions of insulin to augment the sympathoadrenal response to hypoglycemia are likely mediated via mechanisms exterior to the central nervous system.

Haywood SC, Bree AJ, Puente EC, Daphna-Iken D, Fisher SJ. Central but not systemic lipid infusion augments the counterregulatory response to hypoglycemia. Am J Physiol Endocrinol Metab 297: E50 -E56, 2009. First published May 5, 2009 doi:10.1152/ajpendo.90673.2008.-This study tests the hypothesis that lipids could act as an alternative fuel source in the brain during insulin-induced hypoglycemia. Male Sprague-Dawley rats were subjected to hyperinsulinemic (5 mU ⅐ kg Ϫ1 ⅐ min Ϫ1 ) hypoglycemic (ϳ50 mg/dl) clamps. In protocol 1, intralipid (IL), a fat emulsion, was infused intravenously to prevent the fall in free fatty acid levels that occurs in response to hyperinsulinemic hypoglycemia. Intravenous lipid infusion did not alter the counterregulatory responses to hypoglycemia. To test whether IL could have central effects in mediating the counterregulatory response to hypoglycemia, in protocol 2 the brains of precannulated rats were intracerebroventricularly (icv) infused with IL or artificial cerebrospinal fluid (aCSF) as control. Unexpectedly, the epinephrine and glucagon response to hypoglycemia was significantly augmented with icv IL infusion. To determine whether central IL infusion could restore defective counterregulation, in protocol 3 rats were made recurrently hypoglycemic (RH) for 3 days and on the 4th day underwent hyperinsulinemic hypoglycemic clamps with icv IL or aCSF infusion. RH rats had the expected impaired epinephrine response to hypoglycemia, and icv IL infusion again significantly augmented the epinephrine response in RH rats to normal. With regard to our experimental model of hypoglycemic counterregulation, we conclude that 1) systemic lipid infusion did not alter the counterregulatory response to hypoglycemia, 2) the icv infusion of lipids markedly increased CSF FFA levels and paradoxically augmented the epinephrine and glucagon responses, and 3) the blunted sympathoadrenal response in recurrently hypoglycemic rats was completely normalized with the icv lipid infusion. It is concluded that, in the setting of insulin-induced hypoglycemia, increased brain lipids can enhance the sympathoadrenal response.intralipid; intracerebroventricular HYPOGLYCEMIA CONTINUES TO BE THE RATE-LIMITING STEP for achieving blood sugar control for patients with diabetes (8). Because the brain is the primary glucose sensor (2), many efforts have been made toward finding alternate fuel sources that could maintain cognitive function and/or restore the defective counterregulatory response to hypoglycemia. Although the brain is critically dependent on glucose as a substrate, many recent studies have indicated that the brain is also sensitive to fatty acids (14,22). In particular, the intracerebroventricular (icv) infusion of fatty acids has been shown to regulate glucose homeostasis and feeding behavior (21, 23). The icv infusion of intralipid (IL; a lipid solution) was associated with a decreased sympathetic tone (6) that was shown to be mediated by the ␤-oxidation of the fatty acids in the brain (7). Analogous to gl...

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