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Endocrinology, doi:10.1210/en.2005-1040
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Endocrinology Vol. 147, No. 4 1860-1870
Copyright © 2006 by The Endocrine Society

Effects of Insulin Treatment without and with Recurrent Hypoglycemia on Hypoglycemic Counterregulation and Adrenal Catecholamine-Synthesizing Enzymes in Diabetic Rats

Karen E. Inouye, Jessica T. Y. Yue, Owen Chan, Tony Kim, Eitan M. Akirav, Edward Park, Michael C. Riddell, Elena Burdett, Stephen G. Matthews and Mladen Vranic

Departments of Physiology (K.E.I., J.T.Y.Y., O.C., T.K., E.M.A., E.P., E.B., S.G.M., M.V.), Obstetrics and Gynecology (S.G.M.), and Medicine (M.V.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; and School of Kinesiology and Health Science (M.C.R.), York University, Toronto, Ontario, Canada M3J 1P3

Address all correspondence and requests for reprints to: Dr. Mladen Vranic, 1 King’s College Circle, Medical Sciences Building, Room 3358, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: mladen.vranic{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Untreated diabetic rats show impaired counterregulation against hypoglycemia. The blunted epinephrine responses are associated with reduced adrenomedullary tyrosine hydroxylase (TH) mRNA levels. Recurrent hypoglycemia further impairs epinephrine counterregulation and is also associated with reduced phenylethanolamine N-methyltransferase mRNA. This study investigated the adaptations underlying impaired counterregulation in insulin-treated diabetic rats, a more clinically relevant model. We studied the effects of insulin treatment on counterregulatory hormones and adrenal catecholamine-synthesizing enzymes and adaptations after recurrent hypoglycemia. Groups included: normal; diabetic, insulin-treated for 3 wk (DI); and insulin-treated diabetic exposed to seven episodes (over 4 d) of hyperinsulinemic-hypoglycemia (DI-hypo) or hyperinsulinemic-hyperglycemia (DI-hyper). DI-hyper rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. On d 5, rats from all groups were assessed for adrenal catecholamine-synthesizing enzyme levels or underwent hypoglycemic clamps to examine counterregulatory responses. Despite insulin treatment, fasting corticosterone levels remained increased, and corticosterone responses to hypoglycemia were impaired in DI rats. However, glucagon, epinephrine, norepinephrine, and ACTH counterregulatory defects were prevented. Recurrent hypoglycemia in DI-hypo rats blunted corticosterone but, surprisingly, not epinephrine responses. Norepinephrine and ACTH responses also were not impaired, whereas glucagon counterregulation was reduced due to repeated hyperinsulinemia. Insulin treatment prevented decreases in basal TH protein and increased PNMT and dopamine ß-hydroxylase protein. DI-hypo rats showed increases in TH, PNMT, and dopamine ß-hydroxylase. We conclude that insulin treatment of diabetic rats protects against most counterregulatory defects but not elevated fasting corticosterone and decreased corticosterone counterregulation. Protection against epinephrine defects, both without and with antecedent hypoglycemia, is associated with enhancement of adrenal catecholamine-synthesizing enzyme levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPOGLYCEMIA IS THE main acute complication of insulin-treated type 1 diabetes, and its occurrence is greatly increased with intensive insulin treatment (1, 2, 3). A major contributor to hypoglycemia is defective glucose counterregulation (4). In established type 1 diabetes, glucagon responses to hypoglycemia are absent (5, 6), and epinephrine responses are often impaired (6, 7, 8, 9, 10). Defects in cortisol and norepinephrine responses may also exist (8, 9, 10). Antecedent exposure to hypoglycemia is a primary contributor to defective counterregulation in diabetes (7, 8, 11, 12, 13). Antecedent hypoglycemia not only reduces neuroendocrine and autonomic responses to hypoglycemia (7, 11, 12, 13) but also increases glycemic thresholds for counterregulatory responses (7) and causes hypoglycemia unawareness (8, 11). Together, these defects increase susceptibility to further hypoglycemia (4).

Epinephrine responses are particularly sensitive to impairment by antecedent hypoglycemia. In nondiabetic (14, 15, 16, 17, 18, 19, 20, 21) and diabetic (7, 8, 11, 12, 22, 23, 24) subjects, responses are almost always diminished after hypoglycemia. In diabetes, defective epinephrine counterregulation in the presence of absent glucagon responses results in severely compromised counterregulation (25, 26). Thus, it is important to understand the mechanisms underlying defective epinephrine counterregulation. To investigate these mechanisms, we previously examined adaptations underlying impaired counterregulation in untreated streptozotocin (STZ)-diabetic rats (24, 27). In untreated 3-wk diabetic rats, blunted epinephrine responses were associated with decreased adrenomedullary levels of mRNA for tyrosine hydroxylase (TH) (27), the rate-limiting enzyme of catecholamine synthesis. These data suggested a possible contribution of decreased TH synthesis to the epinephrine defect. When diabetic rats were exposed to recurrent hypoglycemia, epinephrine responses were further reduced (24). This was associated with reduced mRNA for both TH and phenylethanolamine N-methyltransferase (PNMT) (27), the enzyme that converts norepinephrine to epinephrine. Thus, the further epinephrine defect after recurrent hypoglycemia may have been due to decreased synthesis of TH and PNMT.

It is not known whether the counterregulatory defects and changes in adrenal catecholamine-synthesizing enzymes that occur in untreated diabetic rats also occur in insulin-treated animals. Insulin-treated animals are a more clinically relevant model and thus may provide a better understanding of the counterregulatory defects in insulin-treated type 1 diabetic humans. The current study investigated the effects of insulin treatment of diabetic rats on: 1) counterregulatory responses and adrenal catecholamine-synthesizing enzymes and 2) adaptations in these parameters after recurrent hypoglycemia. Previously in untreated diabetic rats, due to insulin resistance, large doses of insulin were required to induce antecedent hypoglycemia (24). Here, insulin treatment of diabetic rats reduced insulin resistance, which allowed for the induction of antecedent hypoglycemia with a 10-fold lower insulin dose. The dose of insulin used during assessment of counterregulation with hypoglycemic clamps was also reduced by coinfusing phloridzin, a compound that decreases glucose levels by blocking renal glucose reabsorption (28). We report that insulin treatment prevented epinephrine and other counterregulatory defects. However, fasting corticosterone levels remained elevated, and corticosterone responses to hypoglycemia were mildly impaired. Antecedent hypoglycemia in insulin-treated diabetic rats reduced corticosterone responses to hypoglycemia. Surprisingly, epinephrine responses remained intact, indicating a protective effect of chronic insulin treatment on epinephrine counterregulation. The absence of epinephrine defects in diabetic rats without and with antecedent hypoglycemia was associated with an effect of insulin treatment to prevent decreases in adrenal TH and increase PNMT and dopamine ß-hydroxylase (DßH) levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Sprague Dawley rats (Charles River Laboratories, Quebec, Canada) initially weighing 275–300 g were studied. Rats were individually housed in a temperature- and light-controlled environment (12-h light, 12-h dark schedule) with free access to food (Rodent Laboratory Chow 5001, LabChows, Agribrands; Purina Canada, Woodstock, Ontario, Canada) and water. Groups included: 1) normal (n = 17), 2) insulin-treated diabetic control (DI; n = 15), 3) and insulin-treated diabetic exposed to recurrent hyperinsulinemic-hypoglycemia (DI-hypo; n = 18) or 4) recurrent hyperinsulinemic-hyperglycemia (DI-hyper, n = 17). All procedures were in accordance with Canadian Council on Animal Care standards and were approved by the Animal Care Committee of the University of Toronto.

Induction of diabetes and implantation of slow-release insulin implants
Diabetes was induced by ip injection of STZ (65 mg/kg, Sigma, St. Louis, MO) dissolved in saline. This dose produced diabetes with fed-state glucose levels ranging from 18–25 mM. Four days after induction of diabetes, rats were administered sc slow-release insulin implants containing bovine insulin and microrecrystallized palmitic acid (0.5–1 pellet; 1–2 U insulin/d; Linshin Canada, Inc., Toronto, Ontario, Canada). The purpose of insulin treatment was not to normalize glucose but to moderately lower glucose levels so as to reduce insulin resistance. The initial target glucose range was 15–20 mM (fed state). With this target, glucose levels decreased to 5–10 mM after surgery for implantation of catheters (see below). Normal rats received control implants consisting only of palmitic acid. The implants remained in place for the duration of the study.

Surgical implantation of carotid and jugular catheters
Fourteen days after induction of diabetes or 3 d before the 1st d of antecedent treatment, all rats were implanted with catheters into the left carotid artery and right jugular vein. Surgery was performed under general anesthesia (100 mg/kg ketamine chloride, MTC Pharmaceuticals, Cambridge, Ontario, Canada; 1 mg/kg acepromazine maleate, Wyeth-Ayerst Canada Inc., Montreal, Quebec, Canada; 1 mg/kg xylazine, Bayer Inc., Etobicoke, Ontario, Canada), as described previously (29).

Treatment protocol
On d 17 after induction of diabetes or 3 d after surgery, diabetic rats underwent either 4 d of recurrent hyperinsulinemic-hypoglycemia, recurrent hyperinsulinemic-hyperglycemia, or sham treatment. Normal rats underwent sham treatment.

At 0830 h on each treatment day, the rats’ catheters were extended outside the cages and connected to infusion (jugular) and sampling (carotid) syringes. The rats were allowed 2 h to recover from handling stress before blood was collected from the carotid catheter for measurement of basal blood glucose (Glucometer Elite blood glucose meter, Bayer Inc.; range, 1.1–33 mM) and plasma insulin and corticosterone levels. The treatments began after collection of the basal samples.

DI-hypo rats (n = 18).
DI-hypo rats underwent 3 d of two episodes per day of hyperinsulinemic-hypoglycemia, followed by a single episode on the morning of the 4th d. The single episode on d 4 allowed for insulin and counterregulatory hormones to return to basal levels before the rats underwent either hypoglycemic clamps or euthanasia on d 5. On each day, after collection of the 1030 h basal sample, insulin (100 U/cc Iletin II Regular Insulin Injection, Eli Lilly, Indianapolis, IN) was injected sc at a dose of approximately 0.2 U/100 g body weight to yield approximately 60 min of hypoglycemia at approximately 2.2 mM. This dose was 10-fold lower than what we used previously in diabetic rats not treated with insulin (24). We were able to use this lower dose because treatment with insulin implants reduced insulin resistance. Blood glucose was measured from carotid artery samples collected every 30 min over a 2.5-h period after insulin injection (0–150 min). After morning hypoglycemia, the animals were allowed to recover for 1 h before the afternoon episode was begun. Food was given to aid recovery. At 1400 h, an identical hypoglycemic episode was induced. On the 1st d of treatment, blood samples for plasma insulin and corticosterone were collected throughout the morning and afternoon. To prevent anemia, packed blood cells from the samples were resuspended in heparinized saline (10 U/ml) and reinfused into the rats. Throughout the treatment, 0.9% saline was infused via the jugular catheter to match the infusion of glucose in DI-hyper rats (see below).

DI-hyper rats (n = 17).
DI-hyper rats controlled for the insulin administered to DI-hypo rats and thus differentiated the effects of hyperinsulinemia per se from those of hypoglycemia. DI-hyper rats underwent identical treatment to DI-hypo rats but were maintained at basal hyperglycemic levels throughout treatment with an iv infusion of 40% dextrose (Abbott Laboratories Ltd., Montreal, Quebec, Canada). Hyperglycemia, rather than euglycemia, was maintained so that the effects of insulin, independent of its effects to normalize glucose, could be determined.

DI rats (n = 15).
DI rats underwent 4 d of sham treatment in which 0.9% saline was injected instead of insulin. DI rats underwent the same glucose measurement and blood sampling protocol as DI-hypo and DI-hyper rats. Saline (0.9%) was infused via the jugular catheter to match the infusion of glucose in DI-hyper rats.

Normal rats (n = 17).
Normal rats underwent the same sham treatment as DI rats.

After treatment on d 4, all rats were fasted from 1800 h before either undergoing a hypoglycemic glucose clamp [normal (n = 9), DI (n = 6), DI-hypo (n = 7), and DI-hyper (n = 8)] or being euthanized [normal (n = 8), DI (n = 9), DI-hypo (n = 11), and DI-hyper (n = 9)] on d 5. During fasting, diabetic rats were provided with 10% sucrose water to prevent hypoglycemia due to continuous insulin release from the implants. Normal rats received 10% sucrose to control for any potential effects of the sucrose on the parameters to be measured.

For rats to be euthanized without undergoing clamp experiments, carotid catheters were connected to sampling syringes at 0830 h. After 2 h of recovery from handling stress, carotid blood was collected for measurement of basal blood glucose and plasma ACTH, corticosterone, catecholamine, glucagon, and insulin levels. Plasma was stored at –20 C (or –80 C for catecholamines) until assayed. Rats were then euthanized by decapitation, and adrenal glands were removed and stored at –80 C for subsequent mRNA and protein analysis.

Hyperinsulinemic-hypoglycemic glucose clamp with phloridzin
Hyperinsulinemic-hypoglycemic clamps were performed in conscious unrestrained rats to determine counterregulatory responses to hypoglycemia. A scheme of the clamp protocol is shown in Fig. 1Go. At 0830 h, overnight fasted rats’ catheters were connected to infusion and sampling syringes. At 1030 h, blood was collected for measurement of plasma glucose, insulin, glucagon, ACTH, corticosterone, and catecholamines. A constant infusion of insulin (10 mU/kg·min; 100 U/cc Iletin II Regular Insulin Injection, Eli Lilly) and phloridzin (50 µg/kg·min; Sigma) was then begun. Phloridzin decreases plasma glucose levels by binding to SGLT1 sodium- and energy-dependent glucose cotransporters in the brush border of renal tubular cells to block renal glucose reabsorption (28). Previously, in untreated diabetic rats, large amounts of insulin (50 mU/kg·min) were required to induce hypoglycemia (2.5 mM) (24). Here, the infusion of phloridzin, in addition to insulin treatment, which reduced insulin resistance, allowed for the induction of hypoglycemia with a 5-fold lower insulin dose. Plasma glucose was maintained at a euglycemic target of 5.8 ± 0.2 mM with a variable infusion of 45% dextrose. Glucose infusion rates were based on measurements of plasma glucose determined every 5 min. At the onset of insulin and phloridzin infusion, a primed (4 µCi) 0.07 µCi/min infusion of HPLC-purified 3-[3H]-glucose (NEN Life Science Products, Inc., Boston, MA) was begun for measurement of glucose turnover. After at least 1 h of tracer equilibration, and once target euglycemia was maintained for 20 min (0–20 min), blood was collected for measurement of plasma insulin, glucagon, ACTH, corticosterone, catecholamines, and glucose turnover over a 20-min period (20–40 min). Plasma glucose was then dropped to a hypoglycemic target of 2.7 ± 0.2 mM and maintained in this range for 90 min (0–90 min). At the onset of hypoglycemia, the 3-[3H]-glucose infusion rate was reduced to 0.025–0.035 µCi/min to minimize the increase in specific activity during the transition to hypoglycemia. This ensured accurate measurement of glucose turnover (30). Blood samples for hormones and glucose turnover were collected only once the 2.7 ± 0.2 mM target was attained. Blood was centrifuged immediately for collection of plasma. Packed blood cells were resuspended in heparinized saline (10 U/ml) and infused into the rats to prevent volume depletion and anemia. At the end of the hypoglycemic clamp, animals were euthanized by decapitation. Adrenal glands were collected and stored at –80 C for mRNA and protein analysis.


Figure 1
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  		FIG. 1. Scheme of d 5 hyperinsulinemic hypoglycemic glucose clamp with phloridzin.

 
Adrenal sectioning and in situ hybridization
To determine the effects of the treatments and hypoglycemic clamps on adrenal catecholamine-synthesizing enzyme and glucocorticoid receptor (GR) mRNA, we performed in situ hybridization on the adrenals of all animals. Six to eight cryosections (12 µm) containing medulla and cortex were obtained from the left adrenal. The method of in situ hybridization has been described in detail previously (31). Briefly, 45-mer antisense oligonucleotide TH (bases 905–949), DßH (bases 1514–1558), PNMT (bases 265–309), and GR (bases 1321–1365) (Sigma Genosys, Sigma Aldrich Canada Ltd., Oakville, Ontario, Canada) probes were labeled with [35S]-deoxyadenosine 5'-({alpha}-thio)triphosphate (1300 Ci/mmol, NEN Life Science Products, DuPont Canada, Mississauga, Ontario, Canada) and applied to the tissues on each slide. Slides were incubated overnight at 42.5 C, washed in SSC, and dehydrated in 70 and 95% ethanol. They were then exposed (exposure time, GR, 3 wk; TH, DßH, and PNMT, 24 h) to autoradiographic film (Biomax MR, Eastman Kodak, Rochester, NY). The relative OD (ROD) of the signal on the film was quantified using a computerized image analysis system (Imaging Research, St. Catharines, Ontario, Canada).

Measurement of adrenal protein levels by Western blot
Protein levels of TH, DßH, and PNMT were measured in the right adrenal glands of all animals. Frozen adrenals were homogenized in radioimmunoprecipitation lysis buffer. Fifty micrograms of protein was resolved on a 10% SDS-polyacrylamide gel. Stacking gel (4%) was used for well formation. Protein was then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were soaked in blocking solution [5% powdered milk in PBS-Tween (PBS-T)] overnight. They were washed with PBS-T and incubated with primary antibody [rabbit anti-TH (1:20,000), goat anti-DßH (1:200), goat anti-PNMT (1:200), and goat antiactin (1:1,000), Santa Cruz Biotechnology Inc., Santa Cruz, CA] for 1 h. Each antibody was run separately. Membranes were then washed with PBS-T and incubated for 1 h with goat antirabbit (1:20,000) or bovine antigoat (1:10,000) antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.). Membranes were then washed for 35 min in PBS-T, immersed in chemiluminescence (PerkinElmer, Life Sciences, Boston, MA), and exposed to x-ray film (X-OMAT LS, Eastman Kodak). RODs of the bands were measured using a computerized image analysis system (Imaging Research). Levels of proteins of interest were expressed relative to actin levels.

Analytical methods
Plasma glucose during the hypoglycemic clamp was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Fullerton, CA) (32). 3-[3H]-glucose-specific activity was determined as previously described (scaled down for a smaller plasma volume of 50 µl) (30). Plasma insulin (Linco Research Inc., St. Charles, MO), glucagon (Diagnostics Products Corp., Los Angeles, CA), ACTH (ICN Biomedicals, Inc. Diagnostics Division, Costa Mesa, CA), and corticosterone (ICN Biomedicals Inc.) were measured by RIA. Plasma catecholamines were measured by the single isotope derivative radioenzymatic assay technique (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK).

Glucose turnover determinations
Data for specific activity and plasma glucose concentrations were smoothed using the optimized Optimal Segments program (33). Rates of glucose appearance and glucose use were calculated according to nonsteady-state equations (30, 34). Endogenous glucose production was calculated by subtracting the exogenous glucose infusion rate from the total rate of glucose appearance. Metabolic clearance rate of glucose (MCR) was calculated by dividing rate of glucose use by plasma glucose concentration.

Data analysis
Data are presented as mean ± SEM. Data were analyzed by one- or two-way ANOVA. Two-way ANOVA with a repeated measures design was used to analyze data from the antecedent treatments and glucose clamps. Significance was assumed at P < 0.05. Duncan’s post hoc test was performed if ANOVAs revealed P < 0.05. Statistical analysis was performed with Statistica software (Statsoft, Inc., Tulsa, OK).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weights and glucose levels
Body weights and fed-state blood glucose levels before and during antecedent treatment are summarized in Table 1Go. Body weights did not differ between groups before induction of diabetes (normal, 311 ± 2 g; DI, 307 ± 5 g; DI-hypo, 303 ± 3 g; DI-hyper, 305 ± 3 g) but were decreased (P < 0.05) after induction of diabetes. Insulin implant administration initially moderately decreased (P < 0.05) glucose levels in DI and DI-hypo rats. However, by d 1 of antecedent treatment (3 d after catheter implantation), glucose levels were near or at normal levels in all diabetic groups.


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  		TABLE 1. Body weights, blood glucose, plasma insulin, and plasma corticosterone levels in normal, DI, DI-hypo, or DI-hyper rats

 
Glucose, insulin, and corticosterone levels during antecedent treatment
Blood glucose levels on d 1 of treatment are shown in Fig. 2AGo. In DI-hypo rats, d 1 glucose levels were <2.7 mM for a total of 2.5 h. In normal rats, glucose levels were at 5.3 ± 0.1 mM. DI glucose levels were at 8.6 ± 0.2 mM (P < 0.05 vs. normal). DI-hyper rats had moderately elevated (10.8 ± 0.6 mM; P < 0.05) glucose levels compared with DI rats. This occurred because our main aim in DI-hyper rats was to prevent a drop in glucose after insulin injection. Glucose levels on the remaining treatment days did not differ from d 1, except morning basal glucose levels in DI-hypo rats, which paradoxically increased (P < 0.05) progressively from d 1–4 (Table 1Go). Fasting glucose levels did not differ (data not shown).


Figure 2
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  		FIG. 2. Blood glucose (A), plasma corticosterone (B), and plasma insulin (C) levels on d 1 of antecedent treatment in normal ({square}), DI ({blacksquare}), and DI-hypo ({diamond}) or DI-hyper ({diamondsuit}) rats. Insulin or saline was injected immediately after 0 and 210 min sampling. Values are expressed as mean ± SEM. *, P < 0.05 vs. normal. {dagger}, P < 0.05 vs. DI. {ddagger}, P < 0.05 vs. DI-hyper.

 
Plasma corticosterone levels during d 1 of treatment are shown in Fig. 2BGo. After morning insulin injection, DI-hypo rats displayed a more than 2-fold rise in corticosterone (P < 0.05 vs. all groups). Afternoon levels were lower (P < 0.05 vs. morning) but nonetheless remained elevated (P < 0.05) compared with all groups. DI corticosterone levels were mildly lower (P < 0.05) than in DI-hyper and normal rats. Basal fed-state corticosterone levels from d 1–4 did not differ, except on d 3, where they were lower (P < 0.05, by Student’s t test) in DI rats vs. the other groups (Table 1Go). Fasting corticosterone levels on d 5 were elevated (P < 0.05) by nearly 3-fold in DI and DI-hypo rats vs. normal and DI-hyper rats (normal, 148 ± 39; DI, 385 ± 90*; DI-hypo, 470 ± 94*; DI-hyper, 175 ± 47 nM; * refers to significance vs. normal and DI-hyper; P < 0.05 vs. normal and DI-hyper), whereas fasting ACTH levels did not differ (data not shown).

Insulin levels during the morning of d 1 of treatment are shown in Fig. 2CGo. DI-hypo and DI-hyper rats showed similar increases in insulin levels after insulin injection. Normal and DI insulin levels were similar and remained steady at 278 ± 42 and 261 ± 38 pM, respectively.

Glucose, insulin, and counterregulatory hormone levels during hyperinsulinemic-hypoglycemic glucose clamps
Plasma glucose levels during hyperinsulinemic-euglycemia were at similar steady-state levels for all groups [normal, 5.8 ± 0.2; DI, 6.0 ± 0.2; DI-hypo, 5.5 ± 0.2; DI-hyper, 5.8 ± 0.2 mM; P = not significant (NS)] and dropped to similar levels during the hypoglycemic period (normal, 2.8 ± 0.1; DI, 2.7 ± 0.2; DI-hypo, 2.6 ± 0.2; DI-hyper, 2.7 ± 0.1 mM; P = NS) (Fig. 3Go). Insulin levels during the clamp were at approximately 1800 pM and did not differ among the groups (data not shown). Glucagon responses to hypoglycemia did not differ between normal and DI rats, indicating that insulin treatment normalized glucagon counterregulation (Fig. 4AGo). However, in DI-hypo and DI-hyper rats, responses were blunted (P < 0.05 vs. normal), suggesting that repeated hyperinsulinemia suppressed glucagon counterregulation. DI rats also showed normalized epinephrine and norepinephrine responses (Fig. 4Go, B and C). Surprisingly, antecedent hypoglycemia in insulin-treated diabetic rats did not impair epinephrine counterregulation and, paradoxically, increased (P < 0.05 vs. all groups) norepinephrine responses. During the euglycemic phase of the clamp, hyperinsulinemia stimulated corticosterone release in normal, DI, and DI-hypo rats (P < 0.05) but not DI-hyper rats (Fig. 5AGo). Because hyperinsulinemia per se affected corticosterone release, and this effect differed among the groups, we assessed corticosterone responses to hypoglycemia by examining increments in corticosterone from the euglycemic to hypoglycemic period. Corticosterone responses were nearly absent in DI-hypo rats (P = NS vs. euglycemic period; P < 0.05 vs normal, DI-hyper), whereas levels increased (P < 0.05 vs. euglycemic period) in normal and DI-hyper rats (Fig. 5BGo). Thus, repeated hypoglycemia blunted corticosterone counterregulation. Although the responses of DI rats did not differ significantly from normal rats, they also did not increase significantly from euglycemia to hypoglycemia, indicating that some degree of impairment remained. In contrast to corticosterone, ACTH responses did not differ [e.g. normal rats, basal, 17 ± 2 pM; hyperinsulinemic-euglycemia, 44 ± 9 pM (P < 0.05 vs. basal); hypoglycemia, 117 ± 17 pM (P < 0.05 vs. hyperinsulinemic-euglycemia); remaining data not shown].


Figure 3
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  		FIG. 3. Plasma glucose levels during d 5 hyperinsulinemic hypoglycemic glucose clamps in normal ({square}), DI ({blacksquare}), and DI-hypo ({diamond}) or DI-hyper ({diamondsuit}) rats. Values are expressed as mean ± SEM.

 

Figure 4
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  		FIG. 4. Plasma glucagon (A), epinephrine (B), and norepinephrine (C) levels during d 5 hyperinsulinemic hypoglycemic clamps in normal ({square}), DI ({blacksquare}), and DI-hypo ({diamond}) or DI-hyper ({diamondsuit}) rats. Values are expressed as mean ± SEM. *, P < 0.05 vs. normal. {dagger}, P < 0.05 vs. normal, DI, DI-hyper.

 

Figure 5
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  		FIG. 5. Plasma corticosterone levels at baseline and during hyperinsulinemic-euglycemia (A) and incremental corticosterone responses from the euglycemic period to the hypoglycemic period (B) during d 5 hypoglycemic clamps in normal ({square}), DI ({blacksquare}), and DI-hypo ({diamond}) or DI-hyper ({diamondsuit}) rats. Values are expressed as mean ± SEM. *, P < 0.05 vs. normal. {dagger}, P < 0.05 vs. DI-hyper. {ddagger}, P < 0.05 vs. basal. §, P < 0.05 vs. euglycemic period.

 
Glucose turnover during hyperinsulinemic-hypoglycemic glucose clamps
Glucose turnover data are summarized in Table 2Go. Glucose production during hyperinsulinemic-euglycemia did not differ, indicating no differences in hepatic insulin sensitivity. In response to hypoglycemia, glucose production rose 2- to 3-fold (P < 0.05) to similar levels in all groups. Glucose use during the euglycemic period did not differ and decreased (P < 0.05) to similar levels in all groups during hypoglycemia. MCRs of glucose were similar and did not change throughout the clamp. The similar MCRs indicate that peripheral insulin sensitivity did not differ among the groups and did not change in response to hypoglycemia. Our data indicate that in the presence of constantly high insulin levels, hypoglycemia lowers glucose use. This contrasts with experiments where rat hindquarters were perfused with glucose in absence of insulin (35). In those experiments, low blood sugar did not affect glucose use because hypoglycemia increased MCR via increased muscle glucose transporter levels. We assume that the expression of glucose transporters during hyperinsulinemia is already at its maximum. Thus, glucose uptake depends on glycemia, rather than changes in muscle glucose transporter levels.


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  		TABLE 2. Glucose turnover during d 5 hyperinsulinemic hypoglycemic clamps in normal, DI, DI-hypo, or DI-hyper rats

 
Adrenal TH, DßH, PNMT, and GR mRNA and protein levels
In agreement with the lack of defects in epinephrine counterregulation, insulin treatment prevented reductions in basal adrenal TH mRNA and protein in all diabetic groups. Basal TH mRNA levels after the treatments did not differ (Fig. 6AGo), but surprisingly, basal TH protein levels were increased (P < 0.05) in DI-hypo vs. normal rats (Fig. 6BGo). In response to the hypoglycemic clamp, there were no changes in TH mRNA or protein in any of the groups. In DI and DI-hypo rats, basal DßH mRNA and protein levels and PNMT protein levels were also increased (P < 0.05 vs. normal; Figs. 7Go and 8Go). In response to the hypoglycemic clamp, there was an overall reduction (P < 0.05) in DßH mRNA levels in all groups. However, the decrease only reached statistical significance in DI-hyper rats. In DI and DI-hypo rats, DßH protein was also decreased (P < 0.05) in response to the hypoglycemic clamp. The hypoglycemic clamp induced marked 60% reductions (P < 0.01) in PNMT mRNA from baseline levels in normal and DI rats. In contrast, PNMT mRNA did not decrease in DI-hypo and DI-hyper rats, suggesting that recurrent hyperinsulinemia protected against the decreases in PNMT mRNA. Despite the marked decreases in PNMT mRNA in normal and DI rats, PNMT protein levels did not change.


Figure 6
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  		FIG. 6. Densitometric analysis of adrenomedullary TH mRNA (A) and protein levels (B) at baseline and after d 5 hyperinsulinemic hypoglycemic clamps in normal, DI, and DI-hypo or DI-hyper rats. Values are expressed as mean ± SEM ROD. *, P < 0.05 vs. normal at baseline. {dagger}, P < 0.05 vs. normal, DI, and DI-hyper after hypoglycemic clamp.

 

Figure 7
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  		FIG. 7. Computerized images of adrenomedullary DßH (A), densitometric analysis of DßH mRNA (B), and protein (C) levels, and representative Western blot bands for DßH and actin (D) at baseline and after d 5 hyperinsulinemic hypoglycemic clamps in normal, DI, and DI-hypo or DI-hyper rats. Values are expressed as mean ± SEM ROD. DßH protein levels are relative to actin levels. In A, the area representing adrenal cortex shows no signal. *, P < 0.05 vs. normal basal. {dagger}, P < 0.05 vs. DI-hyper basal. {ddagger}, P < 0.05 vs. basal.

 

Figure 8
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  		FIG. 8. Computerized images of adrenomedullary PNMT mRNA (A), densitometric analysis of PNMT mRNA (B), and protein (C) levels, and representative Western blot bands for PNMT and actin (D) at baseline and after d 5 hyperinsulinemic hypoglycemic clamps in normal, insulin-treated diabetic control, and DI-hypo or DI-hyper rats. Values are expressed as mean ± SEM ROD. PNMT protein levels are relative to actin levels. In A, the area representing adrenal cortex shows no signal. *, P < 0.05 vs. basal. {dagger}, P < 0.05 vs. normal. {ddagger}, P < 0.05 vs. DI-hyper.

 
In the adrenal cortex, basal GR mRNA levels did not differ. GR mRNA levels also did not change in response to the hypoglycemic clamp (data not shown). In the adrenal medulla, basal GR mRNA levels were increased (P < 0.05) in DI vs. DI-hyper rats (Fig. 9Go). At the end of the hypoglycemic clamp, medullary GR mRNA was decreased (P < 0.05) in normal, DI, and DI-hypo rats compared with basal levels. The decrease in GR mRNA in DI-hyper rats was significant by Student’s t test (DI-hyper basal vs. after clamp).


Figure 9
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  		FIG. 9. Densitometric analysis of adrenomedullary GR mRNA expression at baseline and after d 5 hyperinsulinemic hypoglycemic clamps in normal, DI, and DI-hypo or DI-hyper. Values are expressed as mean ± SEM ROD. *, P < 0.05 vs. DI-hyper basal. {dagger}, P < 0.05 vs. basal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we examined chronically insulin-treated diabetic rats to investigate the mechanisms underlying impaired hypoglycemic counterregulation. We examined the effects of insulin treatment on counterregulatory hormones and adrenal catecholamine-synthesizing enzymes and adaptations in these parameters after recurrent hypoglycemia. We report that insulin treatment of diabetic rats prevents most defects in hypoglycemic counterregulation but does not normalize fasting corticosterone levels and corticosterone responses to hypoglycemia. Recurrent hypoglycemia in insulin-treated rats impairs corticosterone responses but, surprisingly, not epinephrine responses. In both rats without and with recurrent hypoglycemia, the protective effect of insulin treatment on epinephrine counterregulation appears to be, at least in part, due to prevention of decreases in adrenal TH synthesis and enhancement of PNMT and DßH synthesis.

In untreated 3-wk STZ-diabetic rats, we have shown that fasting plasma ACTH levels tend to be increased, and corticosterone levels are more than 10-fold higher than in normal rats (24), indicating increased basal pituitary-adrenal activity. Here, insulin-treated diabetic rats displayed normal fed-state corticosterone levels. However, fasting corticosterone levels were nearly 3-fold higher than in normal rats, suggesting that pituitary-adrenal activity was only partially normalized. Although our STZ-diabetic rats differ in many ways from type 1 diabetic humans, it is interesting to note that in diabetic humans, insulin treatment also does not always normalize ACTH and corticosterone levels (36, 37, 38). Chronically elevated glucocorticoid levels are detrimental because they can have long-term damaging effects on metabolic function (39), as well as neuronal and cognitive function (40, 41, 42).

To assess counterregulatory responses, we performed hypoglycemic clamps using a combination of phloridzin and insulin. Phloridzin infusion combined with chronic insulin treatment, which reduced insulin resistance, enabled us to use a 5-fold lower insulin dose to induce hypoglycemia. This decreased plasma insulin during the clamp by 10-fold compared with clamps in untreated rats infused with insulin alone (1800 vs. 18,000 pM) (24). The lower insulin levels lessened possible effects of hyperinsulinemia on counterregulation. In untreated diabetic rats glucagon, epinephrine, norepinephrine, corticosterone, and glucose production responses to hypoglycemia are reduced compared with normal rats (24, 43). We now show that chronic fed-state insulin levels in diabetic rats fully normalize these responses, except those of corticosterone, which remain partially impaired. The normalized glucagon responses contrast with insulin-treated type 1 diabetic humans, where responses are usually impaired (5, 6). Recent data in STZ-diabetic rats and humans indicate that decreases in intraislet insulin are important for triggering glucagon release during hypoglycemia and that absence of this signal may underlie the loss of the glucagon response in diabetes (44, 45). Unlike humans with established diabetes, 3-wk STZ-diabetic rats have some residual insulin secretion (24). This may provide them with the capacity to secrete glucagon in response to hypoglycemia. Although insulin normally suppresses glucagon release (46), insulin treatment may restore other mechanisms that support glucagon secretion. For example, the improved responses may in part have been due to restored autonomic activity, which stimulates glucagon secretion (47) because insulin treatment normalized epinephrine and norepinephrine counterregulation.

Remarkably, recurrent hypoglycemia in insulin-treated rats blunted corticosterone responses but not epinephrine and other responses. The absence of an epinephrine defect was surprising, given that antecedent hypoglycemia consistently impairs epinephrine responses in nondiabetic (14, 15, 16, 17, 18, 19, 20, 21) and diabetic (7, 8, 11, 12, 22, 23, 24) humans and rats. Epinephrine responses were unaltered despite the fact that the rats were exposed to glucose levels of less than 3 mM for at least 2 h/d for 4 d. The reason for the intact epinephrine response is unclear, but it may have been due to a protective effect of chronic insulin treatment combined with repeated hyperinsulinemia because antecedent insulin enhances epinephrine responses to subsequent hypoglycemia (48, 49). Although chronic insulin treatment of diabetic rats allowed for the use of a lower insulin dose to induce antecedent hypoglycemia, insulin levels achieved during antecedent hypoglycemia were still in the supraphysiological range. It is possible that these high insulin levels contributed to the protective effect on epinephrine counterregulation. The intact epinephrine responses also occurred despite elevated plasma corticosterone levels during antecedent hypoglycemia. In humans and rats, antecedent exposure to elevated cortisol levels has been shown to reduce counterregulation to subsequent hypoglycemia (50, 51, 52). It should be noted, however, that in a study in nondiabetic rats, antecedent corticosterone did not affect counterregulation (21). Glucagon responses were also decreased after recurrent hypoglycemia; however, this may have been due to a suppressive effect of repeated hyperinsulinemia combined with chronic insulin treatment because DI-hyper rats, which also received antecedent insulin injections, showed similarly blunted glucagon responses. Interestingly, recurrent hypoglycemia led to enhanced norepinephrine responses. The reason for this effect is unclear, but it does not appear to be due to insulin administration because norepinephrine responses were normal in DI-hyper rats.

In untreated 3-wk diabetic rats, we have shown that impaired epinephrine counterregulation is associated with reduced basal adrenal levels of TH mRNA, suggesting that decreased TH synthesis may contribute to the epinephrine defect (24, 27). Here, insulin-treated diabetic control rats not only displayed normal TH mRNA and protein levels but also increases in DßH and PNMT protein. Thus, the restored epinephrine responses after insulin treatment may, at least in part, have been due to normalized TH synthesis and perhaps even increased DßH and PNMT synthesis. In insulin-treated rats exposed to recurrent hypoglycemia, PNMT and DßH levels remained elevated, and TH protein was increased. These data are consistent with the lack of a defect in epinephrine counterregulation in these animals. This contrasts with untreated diabetic rats exposed to repeated hypoglycemia, where the further defect in epinephrine counterregulation is associated with reduced PNMT mRNA (24, 27). In response to the hypoglycemic clamp, PNMT mRNA decreased markedly in normal and insulin-treated diabetic control rats but, surprisingly, was unchanged in diabetic rats exposed to recurrent hypoglycemia or hyperglycemia. The similar reductions in PNMT mRNA in normal and diabetic rats indicate that hypoglycemia has similar effects on PNMT mRNA in both the nondiabetic and diabetic states. Conversely, the lack of reductions in PNMT mRNA in DI-hypo and DI-hyper rats suggests that insulin treatment with repeated hyperinsulinemia protects against hypoglycemia-induced decreases in PNMT mRNA. Despite the marked reductions in PNMT mRNA in normal and diabetic control rats, there were no reductions in PNMT protein. The hypoglycemic phase of the glucose clamp lasted 90 min. It is possible that changes in PNMT protein take longer to occur. Hypoglycemia had a mild effect to decrease DßH mRNA levels in all groups and reduced protein levels in diabetic control and DI-hypo rats. Reduced stimulation by corticosterone (53, 54) may have contributed to the decreases in DßH mRNA because adrenal medulla GR mRNA decreased in all groups in response to the hypoglycemic clamp. Unlike PNMT and DßH, neither TH mRNA nor protein levels changed. Our data show that insulin treatment of diabetic rats increases or at least protects against decreases in adrenal catecholamine-synthesizing enzymes. These findings are consistent with data showing an effect of acute hyperinsulinemic-euglycemia to increase adrenal TH and PNMT mRNA (55). We suggest that insulin’s stimulatory effect on catecholamine-synthesizing enzymes may contribute to the protective effect of insulin treatment on epinephrine counterregulation.

In summary, we have shown that insulin treatment of STZ-diabetic rats prevents defects in counterregulatory responses to hypoglycemia and, surprisingly, protects against impairment of epinephrine counterregulation after antecedent hypoglycemia. The restored epinephrine responses in rats without and with antecedent hypoglycemia are associated with an effect of insulin treatment to prevent decreases in, or increase, adrenal TH, PNMT, and DßH. On the other hand, insulin treatment does not prevent defects in HPA function because basal corticosterone levels remain elevated and corticosterone responses to hypoglycemia remain reduced in insulin-treated diabetic rats. Insulin also does not protect against defective corticosterone counterregulation after antecedent hypoglycemia. We conclude that chronic insulin treatment of diabetic rats prevents most counterregulatory defects but not defects in HPA function. The protective effect on epinephrine counterregulation may be due to restored or enhanced adrenal catecholamine-synthesizing capacity. Although it is not known whether alterations in adrenal catecholamine synthesis contribute to defective epinephrine counterregulation after antecedent hypoglycemia in type 1 diabetic humans, our data raise the possibility that targeting of adrenal catecholamine synthetic capacity might be a means of improving hypoglycemic counterregulation in type 1 diabetes.


   Footnotes
 
This work was supported by grants from the Canadian Institutes of Health Research and the Juvenile Diabetes Foundation International (to M.V. and S.G.M). Graduate students K.I. and O.C. were supported by scholarships from the University of Toronto’s Department of Physiology, Canadian Institutes of Health Research, and by Novo-Nordisk studentships from the Banting and Best Diabetes Centre in Toronto.

K.E.I., J.T.Y.Y., O.C., T.K., E.M.A., E.P., M.C.R., E.B., S.G.M., and M.V. have nothing to declare.

First Published Online January 5, 2006

Abbreviations: DßH, Dopamine ß-hydroxylase; DI, insulin-treated diabetic control; DI-hyper, insulin-treated diabetic exposed to recurrent hyperinsulinemic-hyperglycemia; DI-hypo, insulin-treated diabetic exposed to recurrent hyperinsulinemic-hypoglycemia; GR, glucocorticoid receptor; MCR, metabolic clearance rate of glucose; NS, not significant; PBS-T, PBS-Tween; PNMT, phenylethanolamine N-methyltransferase; ROD, relative OD; STZ, streptozotocin; TH, tyrosine hydroxylase.

Received August 15, 2005.

Accepted for publication December 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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