Summary Background Disruption of circadian (daily) timekeeping enhances the risk of metabolic syndrome, obesity, and Type 2 diabetes. While clinical observations have suggested that insulin action is not constant throughout the 24 hour cycle, its magnitude and periodicity have not been assessed. Moreover, when circadian rhythmicity is absent or severely disrupted, it is not known whether insulin action will lock to the peak, nadir or mean of the normal periodicity of insulin action. Results We used hyperinsulinemic-euglycemic clamps to show a bona fide circadian rhythm of insulin action; mice are most resistant to insulin during their daily phase of relative inactivity. Moreover, clock-disrupted Bmal1-knockout mice are locked into the trough of insulin action and lack rhythmicity in insulin action and activity patterns. When rhythmicity is rescued in the Bmal1-knockout mice by expression of the paralogous gene Bmal2, insulin action and activity patterns are restored. When challenged with a high fat diet, arhythmic mice (either Bmal1-knockout mice or wild type mice made arhythmic by exposure to constant light) were obese prone. Adipose tissue explants obtained from high-fat fed mice have their own periodicity that was longer than animals on a chow fed diet. Conclusions This study provides rigorous documentation for a circadian rhythm of insulin action and demonstrates that disturbing the natural rhythmicity of insulin action will disrupt the rhythmic internal environment of insulin sensitive tissue, thereby predisposing the animals to insulin resistance and obesity.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[ 14 C]deoxyglucose to assess tissue-specific glucose uptake and [3-3 H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).

Jacobson, Lauren, Tasneem Ansari, and Owen P. McGuinness. Counterregulatory deficits occur within 24 h of a single hypoglycemic episode in conscious, unrestrained, chronically cannulated mice. Am J Physiol Endocrinol Metab 290: E678 -E684, 2006; doi: 10.1152/ajpendo.00383.2005.-Hypoglycemia-induced counterregulatory failure is a dangerous complication of insulin use in diabetes mellitus. Controlled hypoglycemia studies in gene knockout models, which require the use of mice, would aid in identifying causes of defective counterregulation. Because stress can influence counterregulatory hormones and glucose homeostasis, we developed glucose clamps with remote blood sampling in conscious, unrestrained mice. Male C57BL/6 mice implanted with indwelling carotid artery and jugular vein catheters were subjected to 2 h of hyperinsulinemic glucose clamps 24 h apart, with a 6-h fast before each clamp. On day 1, blood glucose was maintained (euglycemia, 178 Ϯ 4 mg/dl) or decreased to 62 Ϯ 1 mg/dl (hypoglycemia) by insulin (20 mU ⅐ kg Ϫ1 ⅐ min Ϫ1 ) and variable glucose infusion. Donor blood was continuously infused to replace blood sample volume. Baseline plasma epinephrine (32 Ϯ 8 pg/ml), corticosterone (16.1 Ϯ 1.8 g/dl), and glucagon (35 Ϯ 3 pg/ml) were unchanged during euglycemia but increased significantly during hypoglycemia, with a glycemic threshold of ϳ80 mg/dl. On day 2, all mice underwent a hypoglycemic clamp (blood glucose, 64 Ϯ 1 mg/dl). Compared with mice that were euglycemic on day 1, previously hypoglycemic mice had significantly higher glucose requirements and significantly lower plasma glucagon and corticosterone (n ϭ 6/group) on day 2. Epinephrine tended to decrease, although not significantly, in repeatedly hypoglycemic mice. Pre-and post-clamp insulin levels were similar between groups. We conclude that counterregulatory responses to acute and repeated hypoglycemia in unrestrained, chronically cannulated mice reproduce aspects of counterregulation in humans, and that repeated hypoglycemia in mice is a useful model of counterregulatory failure.hypoglycemia-associated autonomic failure; hypoglycemia unawareness; catecholamines; norepinephrine; glucocorticoids HYPOGLYCEMIA-ASSOCIATED COUNTERREGULATORY FAILURE is an increasingly common complication of intensive insulin therapy and poses new obstacles to glucose control in insulindependent diabetes. Counterregulatory failure is evident as decreases in either the absolute level or the threshold for activation of sympathoadrenal, glucocorticoid, and glucagon responses to hypoglycemia (3). Of these responses, the glucagon and sympathoadrenal responses are the most critical because of the rapidity with which they are activated and serve to increase blood glucose (4). Counterregulatory failure is not unique to individuals with type 1 diabetes and can be demonstrated experimentally in nondiabetic subjects after only one to two episodes of hypoglycemia (5, 6). However, because type 1 diabetes patients lack the ability to decrease insulin and often lose hypoglycemia-in...

Glucocorticoids have been implicated in hypoglycemia-induced autonomic failure but also contribute to normal counterregulation. To determine the influence of normal and hypoglycemia-induced levels of glucocorticoids on counterregulatory responses to acute and repeated hypoglycemia, we compared plasma catecholamines, corticosterone, glucagon, and glucose requirements in male wild-type (WT) and glucocorticoid-deficient, corticotropin-releasing hormone knockout (CRH KO) mice. Conscious, chronically cannulated, unrestrained WT and CRH KO mice underwent a euglycemic (Prior Eu) or hypoglycemic clamp (Prior Hypo) on day 1 followed by a hypoglycemic clamp on day 2 (blood glucose both days, 65 +/- 1 mg/dl). Baseline epinephrine and glucagon were similar, and norepinephrine was elevated, in CRH KO vs. WT mice. CRH KO corticosterone was almost undetectable (<1.5 microg/dl) and unresponsive to hypoglycemia. CRH KO glucose requirements were significantly higher during day 1 hypoglycemia despite epinephrine and glucagon responses that were comparable to or greater than those in WT. Hyperinsulinemic euglycemia did not increase hormones or glucose requirements above baseline. On day 2, Prior Hypo WT had significantly higher glucose requirements and significantly lower corticosterone and glucagon responses. Prior Hypo and Prior Eu CRH KO mice had similar day 2 glucose requirements. However, Prior Hypo CRH KO mice had significantly lower day 2 epinephrine and norepinephrine vs. Prior Eu CRH KO and tended to have lower glucagon than on day 1. We conclude that glucocorticoid insufficiency in CRH KO mice correlates with 1) impaired counterregulation during acute hypoglycemia and 2) complex effects after repeated hypoglycemia, neither preventing decreased hormone responses nor worsening glucose requirements.

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