Metabolic effects of prazosin on skeletal muscle insulin resistance in glucocorticoid-treated male rats

Abstract

High-dose glucocorticoids (GC) induce skeletal muscle atrophy, insulin resistance, and reduced muscle capillarization. Identification of treatments to prevent or reverse capillary rarefaction and metabolic deterioration caused by prolonged elevations in GCs would be therapeutically beneficial. Chronic administration of prazosin, an α₁-adrenergic antagonist, increases skeletal muscle capillarization in healthy rodents and, recently, in a rodent model of elevated GCs and hyperglycemia. The purpose of this study was to determine whether prazosin administration would improve glucose tolerance and insulin sensitivity, through prazosin-mediated sparing of capillary rarefaction, in this rodent model of increased GC exposure. Prazosin was provided in drinking water (50 mg/l) to GC-treated or control rats (400 mg implants of either corticosterone or a wax pellet) for 7 or 14 days (n = 5–14/group). Whole body measures of glucose metabolism were correlated with skeletal muscle capillarization (C:F) at 7 and 14 days in the four groups of rats. Individual C:F was found to be predictive of insulin sensitivity (r² = 0.4781), but not of glucose tolerance (r² = 0.1601) and compared with water only, prazosin treatment decreased insulin values during oral glucose challenge by approximately one-third in corticosterone (Cort)-treated animals. Cort treatment, regardless of duration, induced significant glycolytic skeletal muscle atrophy (P < 0.05), decreased IRS-1 protein content (P < 0.05), and caused elevations in FOXO1 protein expression (P < 0.05), which were unaffected with prazosin administration. In summary, it appears that α₁-adrenergic antagonism improves Cort-induced skeletal muscle vascular impairments and reduces insulin secretion during an oral glucose tolerance test, but is unable to improve the negative alterations directly affecting the myocyte, including muscle size and muscle signaling protein expression.

glucocorticoids (GCs) are naturally occurring stress hormones, which are frequently prescribed for a number of immune and inflammatory conditions (39) and in combination with cancer treatments (52). Acute high doses or chronic usage of GCs has been shown to induce skeletal muscle atrophy (30), insulin resistance (8, 41), capillary rarefaction (43), nonalcoholic fatty liver disease (12), and the development of Type 2 diabetes mellitus (T2DM) (23, 38). GCs also have various effects on vascular function, inhibiting inflammation and cell proliferation (43), preventing endothelium-dependent vasodilatation (50) and altering vasoconstrictor responses (49).

The profound metabolic deterioration caused by high GCs may be related, at least in part, to changes in the vascular network. Indeed, a key characteristic of both insulin resistance and T2DM is an inability for the peripheral vascular networks to sufficiently perfuse skeletal muscle during periods of elevated metabolic demand (27), ultimately leading to poor muscle oxygenation (18, 47) and likely decreased hormone and nutrient provision (24, 26). The relationship between insulin resistance within the skeletal muscle and microvascular perfusion continues to suggest an important role for the microvasculature as a target for insulin action, and that when impaired, insulin's vasodilatory action within the microvasculature can significantly affect glucose metabolism (24).

New capillary growth within the skeletal muscle occurs via existing capillaries through sprouting or nonsprouting angiogenesis (15). Exercise training and mechanical loading triggers sprouting angiogenesis, while vasodilation through α-adrenergic antagonism promotes nonsprouting angiogenesis (16). The α₁-adrenergic antagonist prazosin has been used in numerous rodent studies to selectively increase skeletal muscle capillary-to-fiber ratio (C:F) (13, 54). Very recent work from our laboratory has established that 14 days of prazosin treatment is sufficient to prevent capillary rarefaction associated with elevated GCs through the maintenance of multiple aspects of shear stress signaling (28). Additionally, there is evidence that α₁-adrenergic blockade can enhance hepatic function (14, 32), insulin sensitivity, and the lipid profile in hypertensive and non-insulin-dependent diabetes mellitus patients (20, 29), in addition to decreasing blood pressure (22). Furthermore, individuals with T2DM, when compared with healthy lean and obese controls, have elevated resting sympathetic activation (21) and demonstrate an augmented plasma glucose response after a noradrenaline load, primarily due to elevated hepatic glucose output, suggesting that these individuals could be more responsive to sympathetic stimulation (7).

Recent work has established that the administration of prazosin to healthy rats results in enhanced insulin sensitivity (2), while in aging adults at risk for T2DM development, augmented skeletal muscle capillarization due to aerobic exercise training caused improved insulin sensitivity, which persisted even after 2 wk of detraining (36). To examine the relationship between improved microvasculature and insulin sensitivity in a model of skeletal muscle insulin resistance, we used the α₁-adrenergic antagonist prazosin to increase skeletal muscle capillarization and measured markers of glucose metabolism and skeletal muscle atrophy. We hypothesized that the increased angiogenesis due to α₁-adrenergic antagonism would increase peripheral tissue perfusion, enhancing insulin sensitivity and skeletal muscle glucose uptake.

METHODS

This study was carried out in accordance with the recommendations of the Canadian Council for Animal Care guidelines and was approved by the York University Animal Care Committee (2013–5).

Animals.

Male Sprague-Dawley rats (Charles River Laboratories, initial mass of 225–250 g, 6 wk postweaned, n = 74) were individually housed (12:12-h light-dark cycle with lights on at 0700 and lights off at 1900) after 1 wk of acclimatization to room temperature (22–23°C) and humidity (50–60%) controlled facilities. After 7 days of acclimation, animals were randomly divided into one of four groups: corticosterone-treated or wax-treated (controls), with either regular drinking water (Cort-water and Control-water), or drinking water containing prazosin hydrochloride (P7791, 50 mg/l; Sigma Aldrich Canada) (Cort-prazosin and Control-prazosin). Animals were then randomly allocated to complete either 7 (n = 20) or 14 days (n = 54) of water-prazosin treatment. All animals were fed a standard rodent chow diet (14% fat, 54% carbohydrate, 32% fat; 3.0 calories/g) ad libitum.

Pellet surgeries.

On day 0, each rat received a subcutaneous implantation of either corticosterone (Cort; the major GC found in rodents) pellets (4 × 100 mg, cat. no. C2505; Sigma-Aldrich, Canada) or wax pellets (Control), as previously described (44). Briefly, purified corticosterone was carefully melted in a small stainless-steel spoon over a low gas flame and then poured into a 6-mm diameter hole drilled in a paraffin block to create a pellet. Once the pellet was fully solidified, it was removed from the mold and trimmed to the correct weight (~100 mg) and sterilized using ultraviolet light. Immediately following surgery, rats were placed into standard sterile cages with ad libitum access to chow and water. Two days after Cort or wax pellet implantation, prazosin was administered to those groups allocated to receive it, a timeline of the experimental protocol is shown in Fig. 1.

Fig. 1.

Schematic of experimental design. On day 0, before pellet implantation, all animals had saphenous vein measurement of pretreatment corticosterone (Cort) concentrations followed by pellet implantation of either wax or Cort pellets, with prazosin or vehicle treatment started on day 2. Saphenous vein measurement of plasma Cort took place on days 7 and 14, post-pellet implantation. An oral glucose tolerance test (OGTT) was administered on day 8 after an overnight fast to examine the effects of 1 wk of concurrent Cort and prazosin treatment, these animals were euthanized on day 9 (endpoint 1). The second round of animals were administered their OGTT on day 12 while an insulin tolerance test (ITT) was given to a subgroup of animals in each experimental condition on day 14. These animals did not have plasma corticosterone measured to ensure ideal ITT testing conditions. All second-round animals were euthanized on day 16 (endpoint 2).

Blood sampling.

On day 0, 7, or 14, at ~0800, blood samples were collected from each fed animal via saphenous vein (~50 μl) for the determination of plasma corticosterone concentrations. For this, an experienced person manually handled the rat while another performed the blood draw. One hind limb was exposed and a small area of hair on the lateral upper hind limb was shaven with an electric razor and wiped with a thin layer of petroleum jelly. Once the saphenous vein was located, a sterile 25-gauge needle was used to puncture the vein, and whole blood was collected in lithium–heparin-coated Microvette capillary tubes (cat. no. 16.443.100; Sarstedt, Montreal, QC, Canada) and centrifuged at ~9,500 g (Eppendorf Mini-Spin Plus, Brinkman Instruments, Westbury, NY) for 5 min, transferred into polyethylene tubes, and stored at −80°C until further analysis of corticosterone concentrations using a radioimmunoassay kit (cat. no. 07120102; MP Biomedicals, Solon, OH). This type of blood collection is known to cause a minor/moderate stress response, which may confound some of our results, as such, throughout the duration of the protocol, all rats were minimally handled each day, to become accustomed to being held.

Oral glucose tolerance and insulin tolerance tests.

Animals were fasted overnight (16 h) on days 7 and 11 and were administered an oral glucose tolerance test (OGTT; 1.5 g/kg body mass) on the following day, days 8 and 12, respectively, as previously described (44). Each animal’s fasting blood glucose (time 0, min) was measured via saphenous vein bleed from the opposite leg used for the prior day's corticosterone collection (~50 μl) with a sterile needle (25 gauge) and glucometer (Bayer One Touch meter, Contour, Tarrytown, New York). Plasma was also collected from saphenous vein bleed (see above for this procedure) for subsequent analysis of insulin concentrations via ELISA (ELISA kit, cat. no. 90060; Crystal Chem, Downer's Grove, IL). Blood glucose concentration was also measured from saphenous vein at 5, 15, 30, 60, 90, and 120 min after oral glucose gavage. In the four groups completing 14 days of the protocol, blood was collected to measure plasma insulin at 15, 30, 60 and 120 min after oral gavage. Insulin concentrations were not measured in the OGTT in the 7-day treatment group for technical reasons. Nonesterified fatty acid (NEFA) and glucagon concentrations were measured from overnight fasted plasma collections, collected before OGTT, at time 0, min (NEFA kit, cat. no. 999-34691; Wako Diagnostics, Richmond, VA; Glucagon ELISA, cat. no. 10-1281-01, Mercodia, Uppsala, Sweden). Glucose and insulin area under the curves (AUCs) were measured relative to each animals individual fasting glucose or insulin level, respectively. Homeostatic model assessment for β-cells (HOMA-β) and insulin resistance (HOMA-IR), as previously reported by Beaudry et al. and others (3, 44, 51), was calculated on the basis of the following equations: HOMA-β = 20 × insulin (μU/l)/glucose (mM) − 3.5, HOMA-IR = [insulin (μU/ml) × glucose (mmol/l)]/22.5. An intraperitoneal insulin tolerance test (ITT) was administered on day 14 to a subset of animals in each experimental group (n = 24); these animals did not have a saphenous vein bleed for corticosterone determination. The ITT (0.75 U/kg body mass) was administered following a partial overnight fast (15 g), as previously described (4, 44). Briefly, ~5 µl of blood was collected from the tail vein (time 0, min) immediately before the administration of the intraperitoneal injection of insulin, to measure glucose concentrations with a glucometer. Further tail vein blood glucose measurements were collected at 5, 10, 20, and 30 min after insulin injection.

Euthanization.

Animals were anesthetized through inhalation of isoflurane gas (2–5%). All animals were euthanized after the skeletal muscles and liver were removed, weighed, and immediately frozen and stored at −80°C. One subset of animals (n = 24) had the extensor digitorum longus (EDL) removed; then these animals were administered an intraperitoneal injection of insulin (3 U/kg body mass), and 20 min after insulin injection, they had the other EDL removed. This protocol allows for the assessment of insulin stimulation within the skeletal muscle.

Histology.

Skeletal muscle (tibialis anterior, TA) and liver tissue from euthanized animals were embedded in tissue-freezing medium, frozen in liquid nitrogen, and cryosectioned (10 µm thick). The TA was stained for fiber type, succinate dehydrogenase (SDH) activity, and C:F, while the liver was stained with Oil Red O (ORO) for neutral lipid content, as previously described (4), and hematoxylin and eosin was used to perform structural assessments.

Fiber type and SDH analysis.

To identify skeletal muscle fiber type, a metachromatic myosin ATPase stain was performed on cross sections of the TA muscle using a modified protocol (33). Sections were preincubated in an acidic buffer (pH = 4.25) to differentially inhibit myosin ATPases within the different fiber types. In this protocol, type I fibers appear dark blue, type IIa appear very light blue, and type IIb and IIx are not apparent from each other and are, thus, classified as IIb/x. These fibers appear bluish-purple. A representative image of the TA for each group was acquired for analysis. SDH activity was then assessed using histochemical analysis and expressed as total optical density. SDH activity was assessed with Adobe Photoshop CS6, converted to grayscale and reported as the average optical density (two ×10 images were averaged per animal). The grayscale is evaluated on a range of completely black (set as 0 U) to white (set at 255 U). All images were acquired with a Nikon Eclipse 90i microscope and Q-Imaging MicroPublisher with Q-Capture software at ×10 magnification.

Capillary-to-fiber analysis.

TA capillary-to-fiber measurements, as reported by Mandel et al. (28), were used in the current study to correlate with factors related to insulin sensitivity and glucose uptake.

ORO staining.

Liver sections were fixed with 3.7% formaldehyde for 1 h at room temperature, while an ORO solution composed of 0.5 g ORO powder (Sigma-Aldrich, Oakville, ON, Canada) and 100 ml of 60% triethyl phosphate (Sigma-Aldrich) was mixed and filtered. Following fixation in 3.7% formaldehyde, slides were immersed in filtered ORO solution for 30 min at room temperature. Slides immediately underwent five washes with ddH₂O, were allowed to dry for 10 min, and were sealed with Permount (Sigma-Aldrich). Liver images were acquired at ×20 magnification using a Nikon Eclipse 90i microscope (Nikon, Canada) and Q-imaging MicroPublisher 3.3 RTV camera with Q-capture Software.

Hematoxylin-and-eosin staining.

Liver sections were hydrated with ethanol, washed with running tap water, then stained with hematoxylin (Sigma-Aldrich), differentiated (0.1% HCl, 0.1% NH₄OH), and stained with eosin. After staining was complete, the sections were dehydrated, allowed to dry, and then sealed with Permount (Sigma-Aldrich). Liver images were acquired at ×20 and ×10 magnifications, respectively, using a Nikon Eclipse 90i microscope (Nikon, Canada) and Q-imaging MicroPublisher 3.3 RTV camera with Q-capture Software.

Western blot analysis.

We quantified protein expression for IRS-1, FOXO1, and phosphor-Akt (pAkt) Ser⁴⁷³ to assess the insulin signaling and atrophic pathways in the skeletal muscle, and Glucose-6-phosphatase (G6Pase) to assess the gluconeogenic pathway in the liver. Western blot analysis was carried out according to previously published work (4). In brief, 20–40 μg of protein lysate from the TA or the liver was run on a 10% (FOXO1, G6Pase, pAkt Ser⁴⁷³) or 6% (total IRS-1) SDS-PAGE gel, and proteins were transferred to a PVDF membrane (Bio-Rad, Mississauga, ON, Canada). Membranes were blocked in 5% powdered milk and Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h. Membranes were then incubated overnight at 4°C with their respective primary antibodies (FOXO1, 1:1,000, cat. no. 2880; Cell Signaling, Beverly, MA; G6Pase 1:1,000, cat. no. sc-25840; Santa Cruz Biotechnology, Santa Cruz, CA; total IRS-1, 1:1,000, cat. no. 2382; Cell Signaling; pAkt Ser⁵⁷⁴, 1:1,000, cat. no. 4060, Cell Signaling). The following morning, the membranes were washed with TBST and incubated with anti-mouse (1:10,000, cat. no. ab6789; Abcam, Toronto, ON, Canada) or anti-rabbit (1:10,000, cat. no. ab6721; Abcam) secondary antibodies for 1 h at room temperature. Membranes were then washed and imaged. Images were detected on a Kodak In vivo FX Pro imager and molecular imaging software (Carestream Image MI SE, version S.0.2.3.0; Rochester, New York) was used to quantify protein content. GAPDH, β-actin, or total Akt (GAPDH, 1:10,000, ab9484, Abcam; β-actin, cat. no. 47778, 1:5,000, Santa Cruz; total Akt, cat. no. 9272, 1:1,000; Cell Signaling) were used as loading controls.

Statistical analysis.

All data were analyzed using an appropriate one- or two-way ANOVA with a criterion of P < 0.05. A Shapiro-Wilks test was performed to analyze symmetry of the data. All significant differences for both one- and two-way ANOVA testing of parametric data were evaluated using a Bonferroni post hoc test (GraphPad Prism v. 6.03). All data are means ± SE. A Pearson parametric correlation was performed using individual 14-day AUC, C:F, and fasted values (GraphPad Prism v. 6.03; GraphPad Software, San Diego, CA).

RESULTS

Body composition, muscle and liver mass.

A significant reduction in body mass and relative muscle mass of the TA was observed; however, an increase in relative liver mass was observed after exogenous Cort treatment at both time points (P < 0.05, Table 1), consistent with the known influence of Cort on body mass, muscle atrophy, and liver mass (41, 44). Prazosin did not modify body mass, relative muscle mass, or relative liver mass at either time point (Table 1).

Table 1.

Animal characteristics

	Control-Water	Control-Prazosin	Cort-Water	Cort-Prazosin
	7 Days
Body mass, g	361.7 ± 10	357.2 ± 7.7	260.7 ± 5.5#	251.3 ± 2.3#
TA relative mass, g/kg body mass	1.77 ± 0.06	1.85 ± 0.04	1.71 ± 0.05	1.71 ± 0.08
EDL relative mass, g/kg body mass	0.5 ± 0.02	0.52 ± 0.01	0.52 ± 0.02	0.51 ± 0.01
Soleus relative mass, g/kg body mass	0.41 ± 0.01	0.38 ± 0.02	0.49 ± 0.01#	0.50 ± 0.02#
Liver relative mass, g/kg body mass	40.22 ± 2.0	38.62 ± 1.7	56.42 ± 2.3*	54.45 ± 0.7*
	14 Days
Body mass, g	383.6 ± 7.7	383.6 ± 9.3	249.5 ± 5.6#	289.4 ± 12.3#
TA relative mass, g/kg body mass	1.83 ± 0.03	1.87 ± 0.04	1.64 ± 0.08#	1.62 ± 0.06#
EDL relative mass, g/kg body mass	0.47 ± 0.01	0.50 ± 0.01	0.50 ± 0.02	0.46 ± 0.01
Soleus relative mass, g/kg body mass	054 ± 0.06	0.56 ± 0.07	0.57 ± 0.02	0.49 ± 0.01
Liver relative mass, g/kg body mass	36.3 ± 0.9	35.5 ± 1.4	53.8 ± 2.3*	47.7 ± 1.5*

All values are expressed as means ± SE. All analysis was completed by two-way ANOVA with Bonferroni post hoc analysis, where P < 0.05; n = 5 for 7 days and n = 11–14 for 14 days. #Significantly different compared with the respective Control Group. *Main effect of corticosterone (Cort) treatment.

Plasma Cort, fasting NEFAs, glucose, insulin, and glucagon concentrations.

Cort levels measured at 0800 on days 7 and 14 were elevated in both Cort-treated groups (P < 0.05, Table 2), as expected and previously observed by our laboratory (5). There was no effect of prazosin on Cort concentrations within either the Control or Cort-treated Groups. Fasted NEFA levels were similar in all groups at both day 7 (P < 0.05, Table 2) and day 14 (P < 0.05, Table 2).

Table 2.

Plasma and metabolic effects of corticosterone and prazosin cotreatment

	Control-Water	Control-Prazosin	Cort-Water	Cort-Prazosin
	7 Days
Plasma corticosterone
Day 0, ng/ml	44.3 ± 17.6	61 ± 46.1	28 ± 17.5	37.6 ± 14.7
Day 7, ng/ml	17.5 ± 1.8	18.9 ± 7.1	583.1 ± 51.3*	485.5 ± 93.3*
Capillary-to-fiber ratio	1.9 ± 0.1	2.0 ± 0.1	1.7 ± 0.2*	1.7 ± 0.1*
Fasted glucose, mmol/l	4.8 ± 0.1	5.2 ± 0.2	6.56 ± 0.26*	6.6 ± 0.8*
Fasted insulin, ng/ml	0.39 ± 0.2	0.29 ± 0.2	5.3 ± 0.6*	3.7 ± 0.4*#
HOMA-IR	1.6 ± 1.3	1.9 ± 1.2	44.7 ± 5.5*	36.3 ± 6.9*
HOMA-β	222.6 ± 131.4	112 ± 74	1009.9 ± 94.9*	1136.8 ± 396*
	14 Days
Plasma corticosterone
Day 0, ng/ml (n = 7–9)	39.8 ± 25.1	47.3 ± 25.2	64.5 ± 20.6	51.6 ± 19.5
Day 14, ng/ml (n = 7–9)	16.2 ± 6.5	9.6 ± 0.6	296.3 ± 34.5*	284.7 ± 34.2*
Capillary-to-fiber ratio (n = 7–9)	2.1 ± 0.1	2.3 ± 0.03†	1.6 ± 0.1*	2.0 ± 0.1*†
NEFAs, mmol/l (n = 7–9)	0.67 ± 0.1	0.66 ± 0.1	0.79 ± 0.1	0.65 ± 0.1
Fasted glucose, mmol/l (n = 11–14)	5.3 ± 0.2	5.6 ± 0.2	6.8 ± 0.4*	7.3 ± 0.5*
Fasted insulin, ng/ml (n = 11–14)	1.2 ± 0.2	1.8 ± 0.3	5.2 ± 0.7*	4.6 ± 0.5*
Fasted glucagon, pmol/l (n = 6)	8.2 ± 1.0	13.3 ± 1.7	13.3 ± 0.8	15.6 ± 1.2
HOMA-IR (n = 11–14)	7.8 ± 1.2	12.7 ± 2.5	45.3 ± 7.0*	42.7 ± 5.0*
HOMA-β (n = 11–14)	4440.0 ± 99.9	548.6 ± 136.5	1407.2 ± 463.3**	976.5 ± 245.6

All values are expressed as means ± SE. All analysis was completed by two-way ANOVA with Bonferroni post hoc analysis, where P < 0.05; n = 5 for 7 days. †Main effect of prazosin consumption. #Mean was statistically significant from control-prazosin. *Main effect of Cort treatment. **Significantly different from controls.

Fasting blood glucose values were significantly elevated before the oral glucose challenge after both 8 (P < 0.05, Table 2) and 12 days (P < 0.05, Table 2) of Cort treatment. No effect of prazosin to alter the fasting blood glucose concentration was observed in either Cort or Control Groups. Fasting insulin values were approximately four-fold higher after Cort-treatment compared with placebo-treated groups regardless of treatment duration (P < 0.05, Table 2). Prazosin treatment of only 7 days caused a slight reduction in fasted insulin concentrations in the Cort-prazosin animals compared with the Cort-water animals (3.7 ± 0.4 vs. 5.3 ± 0.6 ng/ml, P < 0.05, Table 2); however, this reduction was not maintained after 14 days of prazosin treatment (3.8 ± 0.6 vs. 3.7 ± 0.2, Table 2). Fasted glucagon values were not statistically different between groups following 12 days of cotreatment (Table 2).

Oral glucose tolerance test and insulin tolerance test.

Cort, regardless of the duration of treatment, resulted in elevated fasting blood glucose values and deterioration in glucose tolerance when compared with the Control Groups (P < 0.05, Fig. 2, A and B). Seven and 14 days of prazosin treatment did not improve glucose tolerance significantly, although the AUC appeared to be slightly reduced with 14 days of prazosin exposure, and values at the 120-min time point were normalized with prazosin treatment (P < 0.05, Fig. 2, A′, B, and B′). Plasma insulin concentrations were highest in the Cort water-treated animals before and throughout the oral challenge (P < 0.05, Fig. 2C). Interestingly, prazosin administration lowered the insulin AUC during oral glucose gavage at the 2-wk time point, primarily by restoring the insulin levels back to baseline by the 30-min time point (P < 0.05, Fig. 2C′). The Cort-water animals had the highest blood glucose values over the 30-min insulin tolerance test (P < 0.05, Fig. 2, D and D′). HOMA-IR was used to determine the level of fasting insulin resistance, with both 7 and 14 days of Cort treatment causing significant insulin resistance when compared with both control groups, with prazosin not having any significant effect in either group (P < 0.001, Table 2). HOMA-β was used to calculate basal β-cell function, where higher HOMA-β scores indicate greater β-cell function. Cort treatment, after 7 days, caused ~4- to 5-fold increase in HOMA-β compared with controls, with prazosin having no significant effect (P < 0.001, Table 2). Two weeks of Cort-water treatment was also associated with higher HOMA-β score, while the Cort-prazosin animals had an attenuated rise in HOMA-β (P < 0.05, Table 2), suggesting that prazosin administration was able to release the demand on β-cell function.

Fig. 2.

Glucose and insulin responses to an oral glucose tolerance test and exogenous insulin treatment after 8 (A and A′), 12 (B, B′, C, and C′) and 14 days (D and D′) of corticosterone and prazosin treatment. An OGTT was administered after 8 and 12 days of Cort treatment. The Cort-treated animals given water were highly glucose intolerant by 8 days of treatment, (P < 0.05); n = 5. The impaired glucose tolerance observed following 12 days of Cort treatment was improved with concurrent prazosin consumption (P < 0.05); n = 11–14. Insulin levels throughout the OGTT were elevated following Cort treatment and were improved 30 min into OGTT, with prazosin consumption (P < 0.05); n = 11–14. An ITT was administered to a subgroup of animals following 14 days of cotreatment and was calculated as the percent change in blood glucose concentration over time (D and D′). Prazosin treatment significantly ameliorated the insulin insensitivity caused by Cort treatment (P < 0.05); n = 5 or 6. Bars that do not share similar letters denote statistical significance (P < 0.05), using a two-way ANOVA with Bonferroni post hoc test (A′, B′, C,′ and D′). #Significantly different from all other groups (P < 0.05). *Main effect of Cort-treatment (P < 0.05). **Significantly different from Control groups (P < 0.05) using a one-way ANOVA with Bonferroni post hoc test at each individual time point. All values are expressed as means ± SE.

Capillary-to-fiber ratio vs. glucose tolerance and insulin sensitivity.

The TA C:F analysis was originally reported in Mandel et al. (28); a series of representative images (Fig. 3A) and reference TA C:F values (Table 2) were reported here to allow for correlation analysis. To determine whether glucose tolerance and insulin sensitivity are associated with TA C:F in the 14-day treatment groups, individual glucose AUC, fasted insulin, and insulin AUC values from each animal were plotted, and a Pearson correlation was calculated for each variable (Fig. 3, B, C, D). Tibialis anterior C:F was found to be moderately predictive of insulin sensitivity (r² = 0.5783, P < 0.05, Fig. 3C; r² = 0.4781, P < 0.05, Fig. 3D) but not of glucose tolerance (r² = 0.1601, Fig. 3B).

Fig. 3.

Capillary-to-fiber ratio (C:F) is moderately predictive of insulin sensitivity, as measured by fasted insulin levels and insulin area under the curve (AUC) following oral glucose challenge, but not glucose tolerance, as measured by glucose AUC. Representative images depicting C:F within the tibialis anterior (TA) muscle (A) after 14 days of cotreatment. Individual C:F, glucose AUC (B), fasted insulin (C), and insulin AUC (D) values after 14 days of concurrent Cort and prazosin treatment were plotted, and a Pearson parametric correlation was calculated. C:F was found to have no relationship with glucose tolerance (r² = 0.1601) but was moderately predictive of insulin sensitivity (r² = 0.5783, P < 0.05; r² = 0.4781, P < 0.05); n = 7–9. All values are individual.

Muscle cross-sectional area.

The TA muscle of representative animals from each treatment group was stained for cross-sectional area (CSA). CSA, indicating individual muscle fiber size, was lowest in type IIb/x fibers of Cort-treated animals after both 7 and 14 days (Fig. 4, A–D, P < 0.01). The Control-prazosin animals had the largest muscle fibers across all three fiber types after 7 days (P < 0.05), but this difference was not sustained by 14 days of treatment.

Fig. 4.

Cross-sectional area (CSA) in I, IIa, and IIb/x fiber types of the tibialis anterior (TA) after 7 (A and B) and 14 days (C and D) of concurrent prazosin and corticosterone treatment. Cort treatment, regardless of duration, resulted in significant atrophy of the IIb/x fibers (P < 0.01). The control-prazosin animals had the largest muscle fibers across all three fiber types after 9 days (P < 0.05), which was not sustained after 14 days of cotreatment; n = 5. #Significantly different from all other groups (P < 0.05). *Significantly different from control groups (P < 0.01). **Significantly different from control-prazosin (P < 0.05) using a two-way ANOVA with Bonferroni post hoc analysis. All values are expressed as means ± SE.

FOXO1, IRS-1, and insulin-stimulated Akt phosphorylation at Ser⁴⁷³ protein content.

Cort treatment is known to stimulate muscle atrophy and impair insulin signaling; therefore, FOXO1, IRS-1, and insulin-stimulated phosphorylated Akt at serine-473 (pSer⁴⁷³) protein concentrations were analyzed since these are considered critical signaling molecules in these two pathways. After 7 days of Cort treatment, there was a significant increase in FOXO1 protein content and an approximately twofold decrease in total IRS-1 (Fig. 5, A and B, P < 0.05). These changes were sustained after 14 days, regardless of prazosin treatment (Fig. 5, C and D, P < 0.05). Within both Control Groups (water and prazosin), there was an effect of insulin to stimulate pSer⁴⁷³ phosphorylation (Fig. 5, E and G, P < 0.01). Within the Cort-water animals, pAkt Ser⁴⁷³ was unaffected by insulin stimulation; however, this effect was blunted in Cort-water animals. Moreover, there was a trend (P = 0.065) for increased insulin-stimulated phosphorylation with prazosin administration in Cort-treated animals (Fig. 5, F and G).

Fig. 5.

Effect of prazosin and corticosterone on total FOXO1 (A and C) and IRS-1 (B and D) protein content TA after 7 (A and B) and 14 (C and D) days of cotreatment, and insulin-stimulated phosphorylated Akt at serine-473 (pSer⁴⁷³) (E) protein content in the extensor digitorum longus (EDL) after 14 days of cotreatment. Regardless of protocol duration, Cort treatment caused increased FOXO1 content and decreased total IRS-1 content, impairments that were unaffected by prazosin administration; n = 5. Insulin stimulation increased pSer⁴⁷³ within both Control groups, which was blunted with Cort treatment, and tended to be improved with prazosin. Bars that do not share similar letters, denote statistical significance (P < 0.05). **Main effect of insulin (P < 0.05), using a two-way ANOVA with Bonferroni post hoc analysis. All values are expressed as means ± SE.

Succinate dehydrogenase content.

The TA muscle of representative animals from each treatment group was then stained for SDH. SDH staining, which correlates with muscle oxidative capacity (42), was not different among all treatment groups after both 7 and 14 days (Fig. 6, A–D).

Fig. 6.

Representative cross sections of the TA depicting succinate dehydrogenase (SDH) content after 7 (A and B) and 14 days (C and D) of concurrent prazosin and corticosterone (Cort) treatment. Muscle oxidative capacity was unaffected by either Cort or prazosin treatment at either time point; n = 5 or 6. A two-way ANOVA was used with Bonferroni post hoc analysis. All values are expressed as means ± SE.

Liver histology and enzymatic content.

Hematoxylin-and-eosin staining of liver sections was used to visually examine possible structural damage in all treatment groups after both 7 (Fig. 7A) and 14 (Fig. 7B) days. Nuclei are visualized as purple (dark) and the cytosol is pink (light). Cort treatment appeared to cause damage to the liver, seen as vacuoles or holes in the cytosol, at both 7 and 14 day end points, and 14 days of prazosin consumption seemed to have a small visual beneficial effect in reducing the number of vacuoles. Liver sections after 14 days of treatment were also stained for ORO, to examine lipid accumulation (Fig. 7C). Lipid staining was visibly more pronounced following Cort treatment, with no apparent effect of prazosin administration. G6Pase protein content was measured to assess the gluconeogenic pathway in the 14-day treatment groups (Fig. 7D). Cort treatment increased G6Pase content (P < 0.05) in comparison to both control groups, with no obvious effect of prazosin treatment in either controls or Cort-treated groups.

Fig. 7.

Representative images of the liver depicting hematoxylin and eosin staining after 7 (A) and 14 (B) days, Oil Red O staining of the liver (C), and protein content of the gluconeogenic enzyme glucose-6-phosphatase (G-6-Pase) (D) after 16 days of cotreatment. Cort treatment of both 7 and 14 days caused vacuole deposition in the liver, which appeared to be slightly improved with 2 weeks of prazosin consumption. There was no effect of prazosin to improve the fat accumulation in the liver after 16 days of Cort treatment. Two weeks of Cort treatment caused a significant elevation in liver G-6-Pase protein content, which was unaffected by prazosin consumption; n = 5–9. Bars that do not share similar letters, denote statistical significance (P < 0.05) using a two-way ANOVA with Bonferroni post hoc analysis. All values are expressed as means ± SE.

DISCUSSION

This study demonstrates, for the first time, that prazosin improves skeletal muscle capillarity and enhances whole body insulin sensitivity and glucose tolerance in GC-treated rats. However, in spite of improvements in muscle capillarization and insulin sensitivity with prazosin treatment, GC-induced muscle atrophy still is observed in this animal model. These findings suggest that promoting muscle capillarization through prazosin treatment significantly improves, but does not fully normalize, the metabolic Cushing’s-like phenotype in this particular animal model.

Elevations in GCs for a 2-wk period in male rats negatively impact ectopic fat deposition, glucose tolerance, insulin sensitivity, and skeletal muscle growth and capillary number (5, 12, 43, 44). Elevated GCs directly modulate angiogenesis via GC receptors in the endothelial cells (45) and reduce capillary growth indirectly through inhibition of the primary regulating growth factor vascular endothelial growth factor (19, 34). Recently, we have established that regular exercise improves the metabolic profile and skeletal muscle phenotype in male Sprague-Dawley rats exposed to prolonged corticosterone treatment (4). Prazosin is an established method to increase skeletal muscle angiogenesis, through interference with peripheral sympathetic function, selectively blocking postsynaptic α-adrenoceptors (17), without any of the other metabolic and morphological adaptations that can occur with exercise training. Blockade of the α-adrenergic pathway with prazosin leads to angiogenesis and enhanced capillary flow in skeletal muscle in healthy rats, a phenomenon that is linked to improvement in whole body insulin sensitivity without apparent changes in skeletal muscle insulin signaling per se (2). We have also established that prazosin administration is able to prevent the capillary rarefaction associated with pathophysiological elevations in GCs, through maintenance of multiple aspects in shear stress signaling (28). It is thought that the dominant proangiogenic effect of prazosin results from the vasodilation of skeletal muscle arterioles, which chronically increases shear stress at the level of the capillary (13, 16). The lack of angiogenic response in the Cort-prazosin rats suggests that shear stress was not increased sufficiently with prazosin treatment. This could result from GC-induced expression of other vasoconstrictors. Further investigations are required to define the modulatory influence of GC treatment on skeletal muscle blood flow. While muscle capillarization was improved by prazosin treatment, as was whole body insulin sensitivity and glucose tolerance to some degree, it is important to note that skeletal muscle size was unaffected by prazosin treatment. These findings suggest that an increase in skeletal muscle capillarization may improve glucose tolerance directly, perhaps via increased nutrient disposal and insulin delivery, but this does not fully restore the muscle growth and development.

Two weeks of Cort treatment causes insulin resistance and glucose intolerance in male Sprague-Dawley rats (3). Here, we show that this metabolic response can occur as early as 7 days of Cort treatment and that this phenotype is improved, albeit not completely, within ~2 wk of prazosin treatment. It may be that a more prolonged treatment of prazosin could have improved glucose metabolism further. Early work examining the effect of prazosin in human hypertensive subjects found that 12 wk of prazosin consumption was associated with improved carbohydrate metabolism, specifically improving both glucose uptake and insulin sensitivity (35). A number of studies have since been completed further verifying the beneficial effect that prazosin can have on glucose tolerance and insulin sensitivity in human subjects (29, 48). The relationship between insulin sensitivity and muscle capillarization was recently explored, also using prazosin to alter skeletal muscle angiogenesis, in sedentary, nondiabetic, male rats (2). The authors found that after 21 days of prazosin treatment, there was an increase in capillarization that was accompanied by improved insulin sensitivity and glucose disposal, but with no obvious effect on the major insulin-signaling proteins within the muscle itself. The authors attributed the improvement in insulin sensitivity to improved muscle glucose diffusion due to enhanced surface area for nutrient exchange. In line with these observations, we also saw improvements in muscle capillarization, reduced insulin secretion throughout the OGTT, and enhanced insulin sensitivity/glucose tolerance after only 14 days of prazosin administration.

It is worth noting that fasted insulin and glucose values were still elevated in the Cort-treated animals that were given prazosin (Fig. 2). The elevations in glucose and insulin with Cort treatment in the fasted state may be related, in part, to hyperglucagonemia (37). Interestingly, α-adrenergic receptor activation has been shown to increase murine glucagon levels (46), a phenomenon that might contribute to hyperglycemia. As such, we considered the possibility that prazosin, an α-adrenergic receptor antagonist, might lower circulating glucagon levels, which, in turn, could help explain the improved glucose control in the Cort-prazosin group. However, we found no significant differences in fasted glucagon concentrations between groups.

To further examine the link between muscle capillarization and insulin sensitivity, we analyzed individual capillary-to-fiber and insulin and glucose AUC values and found that the individual capillary-to-fiber ratio was predictive of insulin sensitivity but not of glucose tolerance. The lack of significant improvement to glucose tolerance could be due to the fact that Cort treatment caused such pronounced skeletal muscle atrophy and hepatic insulin resistance that the modest improvements in skeletal muscle vasculature were unable to overcome these impairments.

The alterations in glucose tolerance observed in the Cort-treated animals appear to be mediated by activation of the atrophic pathway identified by significantly decreased skeletal muscle CSA, specifically within the glycolytic (IIb/x) fibers and elevations in total FOXO1 protein content, an atrophic protein with a glucocorticoid response element (GRE). We also observed an obvious deficit in IRS-1 total protein content, which was evident after just 7 days of Cort treatment, in addition to impaired insulin-stimulated pSer⁴⁷³ activation following 14 days of Cort treatment. These results parallel the general findings of the effects of elevated Cort on the skeletal muscle machinery (9, 40, 41). While we know prazosin was able to improve the capillary rarefaction observed after Cort treatment and increase insulin sensitivity, the Cort-treated animals were still glucose intolerant, insulin resistant, and were physically smaller than their placebo counterparts. In general, GCs decrease absolute body mass but increase relative fat mass, a result that is especially pronounced in young growing animals (44) or in human adolescence (1). The loss in total mass is usually attributed to reduced skeletal muscle mass, but it can also represent reductions in body length or height. While we did not expect α₁-adrenoceptor antagonism to completely reverse these symptoms of GC excess, it was hypothesized that increasing the skeletal muscle capillary supply could, in fact, enhance nutrient and hormone provision, possibly attenuating some growth impairments within the muscle itself.

We hypothesized that pharmacologically increasing the capillarization of insulin-resistant rodent skeletal muscle could subsequently impact its oxidative capacity. After basic quantification of SDH staining within the TA, neither 7 nor 14 days of prazosin administration caused augmentation to SDH staining intensity within either the control or Cort-treated groups. These results suggest that the capillary supply to skeletal muscle can be enhanced independently of the metabolic profile, oxidative capacity, and fiber size, an interesting finding also observed in the skeletal muscle of young men (6). As such, it is likely that regular exercise, which can increase both muscle capillarization and oxidative capacity, as well as improve skeletal muscle insulin sensitivity, might be a preferred means to improve metabolic function in this particular animal model of sustained hypercorticosteronemia.

The promoter of the G6Pase gene also has a GRE, which suggests initiation of gene transcription by activated glucocorticoid receptors, stimulating an increased rate of gluconeogenesis (53). Our results show increased G6Pase protein content in the liver of Cort-treated rats, suggesting elevated gluconeogenesis, which was unaffected by prazosin administration. Very early research determined that the glycogenolytic and gluconeogenic actions of epinephrine and norepinephrine in the rat liver were primarily mediated through an α-adrenergic, cAMP-independent mechanism (11) and that after adrenalectomy, there was a reduction in α-adrenergic action in rat livers. Additionally, hepatic triglyceride secretion rate was reduced in prazosin-treated rats (10), which correlated with reduced plasma triglyceride concentrations. This points to a direct effect of α-adrenergic blockade on liver triglyceride secretion rate. Additionally, antagonism of the α-adrenergic pathway leads to increased availability of lipoprotein lipase, which also can decrease plasma triglycerides and triglyceride-rich lipoproteins (31). These are possible mechanisms for the improved lipid profile observed in multiple chronic α-adrenergic blockade studies, and also a reason why we measured NEFAs and lipids in the liver. Although there was no effect of Cort to increase NEFA concentrations, there was also no effect of prazosin to lower NEFA concentrations in either the control or Cort groups. Histochemical analysis of the liver sh ows slight improvement in structural integrity but no effect on neutral lipid deposition after 14 days of prazosin administration in the Cort-treated rats. There was a fair amount of variability involved in the ORO analysis, as some Cort-treated rats given prazosin did not improve to the extent as others, a result that was additionally observed with both glucose tolerance and insulin sensitivity assessments. This is not uncommon with our model of elevated GCs, as some rats tend to respond to the Cort pellets more dramatically than others. This points to a possible enhanced therapeutic action of prazosin if administered for a longer period of time, as here in this study, we only examined either 7 or 14 days of treatment. Interestingly, 7 days of Cort produced minimal visible defects in the liver. Furthermore, 14 days of Cort treatment caused elevations in fasted glucose values, regardless of prazosin administration, which also points to the conclusion that 2 wk of α₁-adrenoceptor antagonism was unable to provide major functional improvements to the liver of Cort-treated rodents.

Perspectives and Significance

Currently, there is a suggested association between skeletal muscle insulin resistance, microvascular perfusion, and overall capillarity. The microvasculature is a major target for insulin action, and when impaired, insulin's vasodilatory action within the microvasculature can directly affect glucose metabolism. The present findings confirm the association between reduced skeletal muscle capillary content and insulin resistance in a rodent model of exogenous corticosterone treatment. Furthermore, the administration of prazosin enhanced skeletal muscle capillary content though α₁-adrenergic blockade, bypassing the metabolic and morphological adaptations normally associated with exercise training or caloric restriction, resulting in a corresponding enhancement in insulin sensitivity. Overall, these findings highlight the significant and influential role that the vasculature plays in regulating glucose metabolism in health and disease and suggests that the augmentation of skeletal muscle capillarization is of major therapeutic potential for glucocorticoid-induced insulin resistance and hyperglycemia.

GRANTS

This work was funded by the Natural Science and Engineering Research Council of Canada Discovery Grant to T. L. Haas and M. C. Riddell. E. C. Dunford is a recipient of the Natural Science and Engineering Research Council of Canada Doctoral Scholarship.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.C.D., E.R.M., and S.M. performed experiments; E.C.D., E.R.M., S.M., and M.C.R. analyzed data; E.C.D., E.R.M., and M.C.R. interpreted results of experiments; E.C.D. prepared figures; E.C.D. drafted manuscript; E.C.D., E.R.M., S.M., T.L.H., and M.C.R. edited and revised manuscript; E.C.D., E.R.M., S.M., T.L.H., and M.C.R. approved final version of manuscript.