Hypothalamic AMP-activated protein kinase activation with AICAR amplifies counterregulatory responses to hypoglycemia in a rodent model of type 1 diabetes

Animals.

Male diabetic BB/Wor rats (Biomedical Research Models, Worcester, MA) with established insulin-requiring diabetes (n = 30; 14–28 days disease duration) and male Sprague-Dawley rats (n = 24) were housed in the Yale Animal Resource Center on a 12:12-h day-night cycle, fed a standard pellet diet (22% protein, 5% fat, and 51% carbohydrate; cat. no. 2018; Harlan, Boston, MA) and maintained on once-daily PZI insulin (BCP Veterinary Pharmacy, Houston, TX). The animal care and experimental protocols were reviewed and approved by the Yale Animal Care and Use Committee.

Rodent surgery.

Ten days prior to each study the rats were anaesthetized with an intraperitoneal injection (1 ml/kg) of a mixture of xylazine (20 mg/ml AnaSed; Lloyd Laboratories, Shenandoah, IA) and ketamine (100 mg/ml Ketaset; Aveco, Fort Dodge, IA) in a ratio of 1:2 (vol:vol). The rats initially underwent vascular surgery for the implantation of chronic vascular catheters, as described previously (41). The catheters [PE-50 tubing with a tip made from Silastic laboratory tubing (0.51 mm ID)] are inserted via a neck incision into the internal jugular vein and carotid artery and extended to the level of the right atrium and aortic arch, respectively. They are then tunneled subcutaneously and externalized at the nape of the neck where the catheter ends are left free. Catheter patency is maintained by filling them with a heparin/polyvinylpyrrolidone solution. After catheter insertion, VMH (anterior-posterior, −2.6 mm; medial-lateral ± 3.8 mm; and dorsoventral, 8.3 mm; at an angle of 20 degrees) microinjection guide cannulas were inserted stereotaxically as described previously (4, 5). The coordinates chosen leave the guide cannula tip 1 mm from the VMH and minimizes tissue damage and gliosis in the area of interest. Previous studies have shown that microinjection to the VMH results in relatively little spreadout with the immediate microinjection site (5).

Study 1.

In this study, the effect of providing an additional pharmacological stimulus to AMPK in the VMH in chronically hyperglycemic or recurrently hypoglycemia diabetic BB rats was examined. Diabetic BB rats require insulin therapy to prevent ketosis and death. The diabetic rats in our facility are treated with once-daily PZI insulin (BCP Veterinary Pharmacy) injected subcutaneously at 1700 with doses based on body weight, tail vein glucose at 0900, and study protocol. Diabetic BB rats in the present study were divided into two groups; Chronic hyperglycemia (CH) and RH. For the CH group, insulin doses were adjusted pre- and postoperatively to avoid exposure to hypoglycemia and to maintain glucose levels in the moderate-to-high range. The average morning tail vein glucose throughout this prestudy phase for the CH group was 309 ± 14 mg/dl. RH diabetic rats in addition to basal PZI insulin replacement at 1700 received an IP 10 U/kg dose of human regular insulin (Eli Lilly, Indianapolis, IN) at 0900 on the 5 consecutive days prior to surgery. Postoperatively the rats were allowed a 5-day recovery period with moderate glucose control and then underwent a second 5-day period of recurrent once-daily hypoglycemia. During the hypoglycemia period, food was withheld for 3 h to allow for moderate sustained hypoglycemia. At the end of this period the rats were given free access to food. Average tail vein glucose readings for the 5-day preoperative, postoperative, and 5-day prestudy were, respectively, 39 ± 1, 304 ± 18, and 38 ± 2 mg/dl (the readings on the hypoglycemia study days were taken at the end of the 3-h period of hypoglycemia). On the clamp study day, the CH and RH diabetic BB rats were microinjected into the VMH with either AICAR or saline; thus four groups of rats were studied; 1) CH-VMH AICAR (n = 6; 0.1 μl/min for 5 min, total dose 16 ng); 2) CH-VMH control (n = 6; 0.1 μl/min for 5 min); 3) RH-VMH AICAR (n = 6; 0.1 μl/min for 5 min, total dose 16 ng); 4) RH-VMH control (n = 6; 0.1 μl/min for 5 min).

Study 2.

In this initial study, we sought to confirm the utility of AICAR (5-aminoimidazole-4-carboxamide; Sigma-Aldrich) as an agent for activating AMPK in the VMH. Concerns have arisen with regard to the selectivity of AICAR and, most importantly, the potential for activation of other AMP-regulated kinases and adenosine (15). We have recently shown that selectively downregulating expression levels of the α-catalytic subunit of AMPK through local delivery of an adeno-associated viral vector (AAV) containing a short hair pin RNA for the α-catalytic subunit reduced AMPK gene expression (AMPK-siRNA) in the VMH by ∼30–40% and significantly suppressed hormonal counterregulatory responses to hypoglycemia 7–10 days post-AAV microinjection (32). Thus we hypothesized that if AICAR was acting via AMPK, then the AICAR effect should be at least blunted following VMH AMPK downregulation. In this study, normal male Sprague-Dawley rats were microinjected with an AAV containing an AMPK-siRNA (n = 18), or a control RNA (n = 6) as described previously (32). These rats were allowed to recover from surgery and then 10-days later underwent a hyperinsulinemic hypoglycemic clamp study with VMH microinjection under three conditions: 1) control siRNA + VMH saline injection (n = 6), 2) AMPK-siRNA + VMH saline (n = 9), or 3) AMPK-siRNA + VMH AICAR (n = 9), (doses as above).

Hyperinsulinemic hypoglycemic clamp and VMH microinjection protocol.

Both normal Sprague-Dawley and diabetic BB rats underwent this protocol. All animals were fasted overnight. On the morning of the study the vascular catheters were opened and the animals were allowed to settle and recover from any stress of handling for at least 90 min. Thirty minutes prior to the hypoglycemia clamp study, 22-gauge microinjection needles, designed to extend 1 mm beyond the tip of the guide cannula (Plastics One, Roanoke, VA), were inserted through the guide cannula bilaterally into each VMH. The study rat was then microinjected over 5 min (0.1 μl/min) with either AICAR (5-aminoimidazole-4-carboxamide; Sigma-Aldrich; total dose, 16 ng) dissolved in 0.9% saline or 0.9% saline as a control, as described above. Microinjection was performed in vivo 30 min prior to initiating the hyperinsulinemic hypoglycemic clamp study using a CMA-102 infusion pump (CMA Microdialysis, North Chelmsford, MA). Following microinjection the needles were left in place for 5 min before being removed. Thereafter, a hyperinsulinemic hypoglycemic clamp technique as adapted for the rat (38) was used to provide a standardized hypoglycemic stimulus. At time 0, a 2-h 20 mU·kg⁻¹·min⁻¹ infusion of human regular insulin (Eli Lilly) was begun. The plasma glucose was allowed to fall to ∼2.8 mmol/l and was then maintained at this level for 120 min using a variable rate 20% dextrose infusion based on frequent (5–10 min) plasma glucose determinations. Plasma samples for insulin, c-peptide, epinephrine, and glucagon were obtained premicroinjection (t = −30 min), immediately before the clamp procedure (t = 0 min), and during hypoglycemia (t = 60 and 90 or 120 min). At the end of the study, the rats were euthanized and the probe position confirmed in all rats histologically. Only those rats with injection sites confirmed within the VMH (which represented ∼70% of studies) were included for analysis.

Analytical procedures.

Plasma levels of glucose were measured by the glucose oxidase method (Beckman, Fullerton, CA). Catecholamine analysis was performed by HPLC using electrochemical detection (ESA, Acton, MA); plasma-free insulin, c-peptide, and glucagon were measured by RIA (Linco, Millipore, Billerica, MA). All data are means ± SE, and were analyzed statistically using either a Student's t-test or repeated-measures ANOVA followed by post hoc testing to localize significant effects as indicated (Prism 4; Graphpad Software, San Diego, CA).

Study 1 (a): VMH AMPK activation in recurrently hypoglycemic diabetic BB rats.

Plasma glucose profiles (Fig. 1A) in the two RH animal groups were equivalent and did not differ significantly over the time course of the study [F = 0.3, P = ns (not significant)]. Glucose infusion rates (GIR), however, differed markedly between groups (Fig. 1B), with the RH-control rats requiring significantly more exogenous glucose to maintain the hypoglycemic plateau than RH-AICAR groups (overall F = 43.47, P < .001). Over the last 60 min of the hypoglycemic clamp the mean GIR was 20.2 ± 3.2 vs. 8.0 ± 2.7 mg·kg⁻¹·min⁻¹ for RH-control vs. RH-AICAR, respectively. These data show that RH-control rats required markedly more exogenous glucose to maintain the hypoglycemic control than RH-AICAR injected rats.

Plasma levels of insulin rose significantly in both groups during the hypoglycemia clamp studies to levels of 2,148 ± 546 and 2,405 ± 33 pmol/l for RH-control vs. RH-AICAR, respectively (P = ns). Plasma levels of c-peptide (normal fasting c-peptide in the rat ∼100 ± 15 pmol/l) were very low under basal (43 ± 7 vs. 42 ± 8 pmol/l, respectively) and hypoglycemic (34 ± 1 vs. 34 ± 2 pmol/l, respectively) conditions in these diabetic BB rats, and did not differ significantly between groups.

VMH microinjection of AICAR had no significant effect on basal levels (−30 to 0 min) of glucagon, epinephrine, and norepinephrine in either group, and all hormones rose in response to acute hypoglycemia (Fig. 2, A–C). However, distinct differences between groups were found in the counterregulatory hormonal response during the acute hyperinsulinemic hypoglycemic clamp study. VMH AICAR microinjection significantly amplified (∼180%) glucagon [means ± SE; area under the curve over time (AUC/t) of 144 ± 43 vs. 50 ± 11 ng·l⁻¹·min⁻¹; RH-AICAR vs. RH-control, respectively; P < 0.05] and epinephrine (∼300%) (4.27 ± 0.96 vs. 1.06 ± 0.26 nmol·l⁻¹·min⁻¹, respectively; P < 0.05), but not norepinephrine (3.07 ± 0.4 vs. 2.06 ± 0.25 nmol·l⁻¹·min⁻¹, respectively; P = 0.85) responses to the hypoglycemia challenge.

Study 1 (b): VMH AMPK activation in chronically hyperglycemic diabetic BB rats.

Plasma glucose profiles (Fig. 3A) in the animal groups were similar and did not differ significantly over the time course of the study (F = 1.3, P = ns). Mean ± SE plasma glucose during the hypoglycemic plateau (60–120 min) was 3.1 ± 0.1 vs. 3.3 ± 0.1 mmol/l for CH-control vs. CH-AICAR, respectively. Mean glucose infusion rates (GIR) required to maintain the hypoglycemia plateau (60–120 min) were slightly, but not significantly reduced in CH-AICAR vs. CH-control BB rats (1.0 ± 0.6 vs. 4.5 ± 2.0 mg·kg⁻¹·min⁻¹; P = 0.1; Fig. 3B).

Plasma levels of insulin rose significantly in both groups during the hypoglycemia clamp studies to levels of 2,481 ± 458 and 3,212 ± 268 pmol/l for CH-control vs. CH-AICAR, respectively (P = ns). Plasma levels of c-peptide were very low under basal (42 ± 6 vs. 28 ± 5 pmol/l, respectively) and hypoglycemic (37 ± 4 vs. 22 ± 1 pmol/l, respectively) conditions in these CH-control and CH-AICAR diabetic BB rats, and did not differ significantly between groups.

Microinjection had no effect on basal levels (−30 to 0 min) of glucagon, epinephrine, and norepinephrine in either CH group, and all hormones rose in response to acute hypoglycemia (Fig. 4 A–C). VMH AICAR microinjection significantly amplified glucagon (∼80%) (AUC/t 151 ± 22 vs. 85 ± 22 ng·l⁻¹·min⁻¹; CH-AICAR vs. CH-control, respectively; P < 0.05), but not epinephrine (3.25 ± 0.45 vs. 7.00 ± 1.1 nmol·l⁻¹·min⁻¹, respectively; P = 0.2) or norepinephrine (2.36 ± 0.9 vs. 2.00 ± 0.50 nmol·l⁻¹·min⁻¹, respectively; P = 0.07) responses to the hypoglycemia challenge.

Study 2: effect of VMH AICAR during hypoglycemia in normal rats following selective AMPKα downregulation.

To control for potential nonspecific effects of AICAR, initial studies were performed in normal Sprague-Dawley rats previously microinjected to the VMH with either a short-hairpin RNA designed to selectively downregulate AMPK (VMH AMPK-siRNA + VMH saline or VMH AMPK-siRNA + VMH AICAR groups) or a control short-hairpin RNA (VMH control-siRNA + VMH saline). Compared with the control siRNA study, and consistent with our previous work (32), VMH AMPK downregulation through local delivery of the AMPK siRNA led to a significant reduction in the glucagon (∼40%), epinephrine (∼30%), and norepinephrine (∼30%) responses to acute hypoglycemia (Fig. 5). This effect was not significantly reversed by microinjection of AICAR to the VMH (Fig. 5; P = ns for all hormones), a finding which supports the use of AICAR in subsequent studies in the BB rat as an activator of AMPK.