Glucocorticoid Ablation Restores Glycemic and Thermogenic Parameters in Obesity

Abstract

Glucocorticoid ablation following adrenalectomy resulted in restoration of the impaired non-shivering thermogenesis and impaired glucose tolerance and insulin resistance in obese LA/Ntul//-cp rats. This is a congenic rat strain where the only difference between the lean and obese phenotypes was the presence of the epigenetic expression of obesity in an NIDDM-free animal model. Groups of young adult obese animals were adrenalectomized, followed by thermogenesis and glycemic assessment thereafter. In an additional subgroup, animals were administered insulin daily in an attempt to maintain the insulin resistance state. Adrenalectomy resulted in a complete restoration of normal resting and norepinephrine stimulated thermogenesis and an amelioration of the glycemic parameters of insulin resistance.

1. Introduction

The development of overweight and obese conditions is commonly associated with insulin resistance in man and animals along with multiple associated pathophysiologic comorbidities. Among the comorbidities, the insulin resistance of obesity and overweight conditions is strongly associated with numerous common pathophysiologic sequelae including but not limited to non-insulin dependent diabetes (NIDDM), hypertension (HTN), stroke, renal complications, certain cancers, musculoskeletal, and cardiovascular disorders to cite just a few of the possible complications that may develop over time. The prevalence of obesity and overweight conditions is increasing at an alarming rate in Westernized populations, with the onset now beginning at earlier ages than in past generations [1, 2, 3].

The LA/Ntul//-cp rat model is a unique congenic animal model of epigenetic obesity. The initial expression of the obese stigmata typically begins by 5 to 6 weeks of age, where the affected offspring begin to express the stigmata, including an altered gait and outward more rounded appearance [4, 5]. The only obvious genetic difference between the lean and obese phenotypes is the autosomal recessive expression of the obesity (-cp) trait in one quarter of the offspring of heterozygous breeding pairs. Therefore all other inherited characteristics being equal, this rat strain is an excellent animal model to investigate the metabolic processes contributing to the development of an obese state and its pathophysiologic complications [4, 6]. The obese phenotype typically develop hyperinsulinemia and insulin resistance, elevations in plasma amylin, and disordered glycemic response to carbohydrate loading by early adulthood in both sexes, but have been found to remain free of diabetes of wither type throughout most or all of their projected lifespan [4, 5, 7, 8]. Dysregulation of glucocorticoid actions have also been found in obesity and overweight conditions, where they provide further impairments in the regulation of glucose uptake and glycemic status, and contribute to the magnitude of glucose intolerance and impaired thermogenesis, which like other aspects of glucose oxidation is dependent on efficient glucose uptake in brown adipose tissue for expression of BAT-mediated elements of nonshivering thermogenesis [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. The closely related SHR/Ntul//-cp strain share the same genetic trait for obesity (the -cp trait) but unlike the LA/Ntul//-cp strain, the SHR/Ntul//-cp strain also develops symptoms of moderate to severe NIDDM in both sexes by early adulthood. In both strains the obesity trait was obtained from the Koletsky rat and purified via multiple rounds of back-crossing to attain the congenic status, whereby the only known apparent surviving trait from the Koletsky origin was the evidence of the expression of the autosomal recessive obesity (-cp) trait [10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. To date, the obese of both -cp strains have not been observed to reproduce due to unknown endocrinologic or other factors, similar to that which has been observed in other autosomal recessive obese rodent strains although the histologic presence of spermatozoa in obese LA/Ntul//-cp rats has been reported [20]. In addition, the obese phenotype of the LA/Ntul//-cp rats also exhibit an impaired thermogenic response to caloric overnutrition induced by the cafeteria diet and other thermogenic regimens, and which has been observed to persist throughout most if not all of the projected lifespan in the obese phenotype of this stain [4, 6, 8, 14, 15, 16].

While the specific biochemical mechanisms that contribute to the impaired capacity for not shivering thermogenesis and caloric expenditure in the obese phenotype remain unclear, the thermogenic defect is presumed to contribute to their enhanced caloric efficiency and contribute at least in part to their propensity for excess fat accretion and storage in adipose tissue depots. Dysregulation of glucocorticoid actions involving the insulin-dependent GLUT4 transporters and insulin insensitivity in addition to impaired thyroidal and sympathetic actions have been reported in the obese of several strains, and likely contribute a role in this animal model as well [9, 10, 11, 12, 20, 21, 22, 23]. Regardless of the energy mechanisms involved, the LA/Ntul//-cp rat strain ranks among the longest surviving obese rat strains known due to the longevity-prone Lister/Albany/NIH background, with the lean phenotype often surviving to 4 years or more and the obese littermates to 2.5 to 3 years under standard laboratory conditions [16, 17, 18, 19]. The current investigation was designed to further elucidate the mechanisms of carbohydrate intolerance and its association with impaired nonshivering thermogenesis, and to determine if the metabolic derangements were associated with the expression of the obese trait in this rat strain. Rats were studied from the earliest visible onset of the expression of the obese trait throughout postweaning development and early adulthood. Rats typically become of reproductive age in the lean phenotype by early adulthood, but reproductive activity has not been observed in the obese phenotype. Thes physiologic developments occur at an age at which the onset of metabolic evidence of insulin resistance in the obese phenotype will have developed [20]. Michaelis et al. have demonstrated that the obese phenotype of this strain demonstrate little if any glycosuria throughout much of their normal lifespan despite the carbohydrate source in the diet being fed [7, 24].

In human populations, familial syndromes of obesity and NIDDM often occur together, suggesting a possible genetic predisposition in their development, and consistent with an implication of a receptor defect in insulin actions, including possible dysregulation of the GLUT4 transporter system. However, the magnitude of the expression of the obesity and NIDDM parameters may differ in their molecular mechanisms in the two disorders. The -cp trait for obesity was originally derived from the Koletsky rat, and bred into the NIH colony of the SHR rat, followed by 12 or more cycles of backcrossing, whereby the resulting obese offspring exhibit NIDDM by early adulthood. The metabolic basis for the development of NIDDM has been presumed to occur due to a combination of a receptor and postreceptor linked activity in carbohydrate metabolism implicating the insulin receptor. In contrast, when the same -cp trait was bred into the LA/N strain, the obese offspring developed moderate insulin resistance but not NIDDM, suggesting that the impairment in insulin action in that substrain survived as a receptor mediated effect only [6, 7, 17, 19, 25]. In the current investigation, the experimental design was developed in an attempt to further elucidate key parameters resulting in the epigenetic expression of obesity, to include the potential impact of the counterregulatory effects of adrenal glucocorticoid hormones on thermogenic and glycemic parameters.

2. Methods and materials

The rats for this investigation were obtained from Drexel University at 6 weeks of age. Rats were designated as lean or pre-obese on the basis of visible observation of emerging obese stigmataThe animals consisted of normally reared lean and obese male animals as biological littermates (1 lean plus 1 obese, n = 6 rats/treatment group). The preobese rats were selected based on physical characteristics, including typical indicators of changes in gait, stance, and palpable subcutaneous fat combined with lower resting VO2 measures than occurred in their lean littermates [6]. The animals that were selected from the above criterion were placed in shoebox cages lined with one inch of fresh pine shavings, changed periodically 3 times a week, and housed under standard laboratory conditions from 6 until 12 weeks of age. Room temperatures were maintained at 22–24°C with controlled humidity of 50 to 60% relative humidity. Ll animals received free access to Purina Chow #5012 and house water throughout, beginning at weaning (21 days of age) and continuously throughout the remainder of the study. Purina chow formula # 5012 manufacturers certificate of analysis reported an energy density of 3.34 kcal/gram. The detailed composition lacked refined carbohydrates and was indicative of a low glycemic index. When animals were 9 to 12 weeks of age during the final 3 weeks of the study they were offered the highly palatable cafeteria (Café) diet thereafter in addition to the Purina chow diet. The Café regimen consisted ofa rotation of four tasty items each day out of 20 total Café food items. The quantities of all foods consumed were carefully recorded after correction for spillage and uneaten residuals. The twenty different Café foods that were provided in the daily master menu included at least one reliable nutritive protein source.

The daily caloric Intakes were determined periodically during the 6-week study. Each animal was placed in an individual metabolic cage for 3 days, during which time all total food that had been consumed was tabulated, measured by weight, corrected for spillage, and resulting caloric intake during the final 2 days, while the data obtained during the first day discarded, thus enabling the individually housed animals to acclimate to the change in environment of the metabolic cages. The Purina chow was ground in a homogenizer for ease in determing the accuracy of measurement throughout the duration of the study. The groups of animals were also offered the highly palatable cafeteria (Café) diet in addition to the Purina chow during weeks 9 through 12. The Café diet included numerous typical ‘finger foods’ such as snack items that included Oreo and peanut butter cookies, assorted sweets and sugary confections, hot dogs, white bread, popcorn, etc., and totals consumed corrected to account for wastage and uneaten residues each day. The net caloric intake was determined from the manufacturer’s published nutritional information for each individual food item consumed. Thus, the caloric intake reported includes the total Purina Chow plus the Café food items consumed after correction for spillage and uneaten remnants.

Determination of the Resting Metabolic Rates (RMR). Measurement of the RMR of lean and obese rats were determined in the fasted state. Measures of RMR were obtained with a Collins Small Animal Spirometer apparatus (WE Collins, Braintree MA, USA) fitted with a locally manufactured closed circuit 1 cubic foot volume watertight Plexiglas animal chamber [6, 8, 26, 27, 28]. All measures of VO2 were conducted at thermal neutrality (30°C) by immersing the sealed canister in a constant temperature water bath, allowed to equilibrate for up to 15 min, and the temperature was kept constant by immersing the animal chamber in the temperature controlled circulating water bath. The data was corrected for conditions of local altitude and relative humidity, and expressed per kg of body weight ^0.75 power to correct for potential differences in body composition and surface area [6, 8, 26, 27, 28]. Typically, with this procedure, animals were observed to explore their new surroundings for 2 to 3 min upon initial placement in the submersible chamber, after which time they were observed to be resting comfortably during equilibration of the system and for the remainder of their occupation and measurement of RMR in the chamber. Reliable measures were typically obtained within 15 min of observation and continued for an additional 15 min segment. Animals were familiarized with the procedure prior to obtaining measure of record to minimize the effects of stress or training issues on the final results. Significant differences in body weights and this metabolizing tissues were observed in the two phenotypes due to the drastic differences in actual body composition due to the obesity [6].

Adrenalectomy. A partial (hemi-) or complete adrenalectomy (Hemi-ADX/ADX) was performed in groups (n = 6 rats/group) of pre-obese rats as described by Marchington et al. In this procedure rats were subjected to pentobarbital anesthesia (5.5 mg/kg BW, intraperitoneally) when 6 weeks of age [29]. Briefly, the surgical procedure consisted of a single 1 to 1.5 cm mid-posterior incision, and the adrenals located by palpation and inspection followed by surgical removal in toto. The animals warmed on an electronic heating pad during and after the surgical procedure to prevent anesthesia-induced post-surgical hypothermia. Animals were given a 0.9% NaCl solution supplement ad libitum in addition to house water following adrenal surgery. An additional group received a hemi-adrenalectomy and were treated exactly the same as the obese and bilateral adrenalectomy groups were treated. A final subgroup (n = 4) of adrenalectomized rats received a daily Lente insulin injection, starting at 1 U / day and ramping up to a max of 8 u/day on the final day of the study in an attempt to continue the magnitude of insulin resistance in the absence of the adrenal secretions. Animals were closely monitored daily, and were observed to survive the surgical procedure without difficulty. During the final dissection, the completeness of the adrenalectomy procedure was determined by inspection to confirm the presence of one or no remaining adrenal tissue remants. All groups had 6 rats / group at the onset, but in the Obese+ADX + Ins group only 4 rats remained at 12 weeks of age, as some animals inadvertently succumbed due to apparent hypoglycemia during fasting and thus their data was not included in the final data computations.

Measures of glycemic status. Measures of glycemic status included a) an oral glucose tolerance (OGT), plasma insulin (I), glucose (G) and the computing the I:G ratio of rats. In addition, the area under the OGT curve (AUC) was computed as previously described [7, 30]. The measures of oral glucose tolerance (OGT) were performed in 8-h fasted animals as outlined by Tulp et al. [30] The glucose challenge consisted a measured quantity based on body weight of a 50% glucose solution (25 mg glucose/kg BW) administered slowly via gavage over a 1-min duration in fasting animals via oral gavage. Periodic bloods were obtained for measurement of blood glucose concentration from the tail vein in heparinized capillary tubes initially and at the 30-, 60-, 90 and 120-min post gavage. The bloods were subjected to plasma glucose determination with a glucose oxidase method, and recorded as mg/dl [30]. The AUC for the glucose curve from 0 to 120 min was calculated as described previously [30]. Upon sacrifice, truncal blood were collected for measures of fasting glucose and insulin. Insulin concentrations were determined via solid phase radioimmunoassay. Data were analyzed via standard descriptive statistics, ANOVA, and Students unpaired t tests [31]. This study was reviewed and approved by Institutional IACUC (Institutional Animal Care and Use Committee prior to the investigation.

3. Results

The initial and final body weights of rats are depicted in Figure 1 at each age studied. These data show that the body weights of all groups were similar upon assignment to the respective treatment groups. In contrast, at 12 weeks of age, the obese and adrenalectomized obese rats were observed to be significantly greater than those of their unoperated lean littermates. Since all groups had the same genetic background the same diets and were housed under identical laboratory conditions with the exception of the autosomal recessive expression of the -cp trait, the differences in final body weights and weight gain following ADX are considered significant.

Figure 1.

Body weights of rats at 6, 9 and 12 weeks of age. Data are mean ± 1 SEM, n-4-6 rats/treatment group. P = < 0.05 obese vs. lean at each age studied. All groups were observed to gain weight with age (p = <0.05, students T test for paired comparisons).

When adrenalectomized obese rats were 9 weeks of age midpoint and prior to the addition of the Café diet however, their body weights were still similar to those of their lean littermates. Upon introduction of the Café diet regimen however, the obese adrenalectomized rats exhibited significant hyperphagia. The hyperphagia was followed by additional weight gain that was equal to or greater than their unoperated obese littermates by the culmination of the study at 12 weeks of age. The final data point for bode weights is indicated in Figures 2–4 in Figure 5, the mean weight gain between 6 and 9 weeks of age is depicted and indicates that weight gain in both lean and obese-ADX rats was similar, averaging 70 grams per rat. However as noted, when the obese+adx group consumed the Café regimen, their weight gain during the final 3 weeks of the study significantly exceeded that of their lean littermates by a factor of 2.75. This rate of weight gain was similar to a 2.7-fold greater rate of weight gain in the unoperated obese group. Thus, those rats attained a final rate of weight gain that measured equal to or greater than that which was observed among the other experimental groups.

Figure 2.

Orl Glucose tolerance Test in lean and obese rats at 6 weeks of age. Data are mean ± 1 SEM. * = P = <0.05 (Lean Vs Obese) at 60 and 90 min. P = n.s. at baseline, 30 and 120 min. *Comparisons by unpaired Student’s ‘t’ test.

Figure 3.

The effect of ADX on glycemic responses at 9 weeks of age. The data are mean ± 1 SEM, n = 6 rats/group. The plasma glucose and postprandial absorptive phase of obese rats were significantly greater than the other groups at +30 and + 60 min (p = <0.05; students unpaired t test). WOA = weeks of age.

Figure 4.

Effect of ADX and diet on glycemic excursions at 12 weeks of age. Data are mean ± 1 SEM, n = 6 rats/group. The glycemic responses of the obese rats were significantly greater than the other groups at +30 and + 60 min (p = <0.05; students unpaired t test). WOA = weeks of age. OGT obtained after 3 weeks of the Café diet regimen. Note the difference in the vertical axis in Figure 4 vs. Figures 2 or 3 (0 to 300 in 4 vs. 0 to 250 mg/dl in 2 and 3).

Figure 5.

Weight gain of rats at 6, 9 and 12 weeks of age. Data are mean ± 1 SEM, 4–6 rats/group. P = <0.05 (lean vs. obese, Student’s unpaired t test) at each age studied. In addition, all groups gained weight between 6 and 12 weeks of age (p = < 0.05, students paired comparisons t test).

The measures of Oral Glucose tolerance are depicted in Figures 2–4 and the and the glucose area under the glucose curves (the AUCglucose) is depicted in Figure 6. These data are consistent with a prolonged glycemic response in the obese phenotype that also included a greater magnitude of glycemic excursion at the highest glucose concentrations observed than occurred in their similarly treated lean littermates. These observations are suggestive of a prolonged absorptive phase in the obese phenotype. When 9 weeks of age, the glycemic responses and the AUCglc in the obese+ADX rats were now similar to their unoperated lean littermates, while only the untreated obese remained elevated. At 12 weeks of age, as depicted in Figure 4, the glycemic excursions in the lean and adrenalectomized obese rats remained of similar magnitude. The unoperated obese continued to demonstrate a deranged glycemic response at 12 weeks of age, consistent with a prolonged absorptive phase, but glycosuria was absent, as was diagnostic criterion indicative of NIDDM.

Figure 6.

Mean glucose AUC from 4-point OGT at 12 weeks of age. Data are the mean ± 1 SEM, N = 4–6 RATS/GROUP. Bars computed from of plasma glucose values depicted in Figure 4 above. Obese, obese + hemi-ADX and obese + ADX+ insulin is similar (n.s) while glucose AUC of lean and obese + ADX are similar. Presurgical AUCglc at 6 weeks of age from Figure 2 was 264 vs. obese 312. Numerical values above each bar above are the mean AUC at 12 weeks of age.

The data reflecting ADX of obese rats at 12 weeks of age is shown in Figure 4 above. The glycemic responses in the unoperated lean and in the obese+ADX groups are similar, reflecting a normalization of the glycemic responses in the obese animals following the ADX procedure. In contrast, the glycemic responses in the unoperated obese animals continue to indicate modest glucose dysregulation, with peak glucose concentrations exceeding 200 mg/dl at the 30-min data point but returning to pre-glucose administration plasma concentrations at the 120-min time point in all groups including the unoperated obese, Café fed animals.

The final glycemic parameters including plasma glucose, plasma insulin, and the Inulin to glucose ratio as an estimate of insulin resistance are shown in Table 1 and indicate that fasting plasma glucose concentrations were similar in all groups, with a trend toward elevations in plasma glucose in the Obese+ADX + Insulin group. The greater value in the insulin groups is reflective of their non-fasting state, as fasting in that group resulted in premature hypoglycemic deaths of some animals. Plasma insulin concentrations were markedly elevated in the obese phenotype but were similar to those of lean animals following complete adrenalectomy. In contrast, hemi-adrenalectomy resulted in plasma insulin concentrations that were similar to unoperated obese rats, while administration of exogenous insulin resulted in yet still greater plasma insulin concentrations and insulin to glucose ratios, indicative of insulin resistance in those animals.

Group	n	Glucose	Insulin	I:G ratio
Lean	6	144.0 ± 9.0^a	253 ± 20^a	1.43 ± 0.16^a
Obese	6	145.5 ± 15.7^a	874 ± 30^b	6.01 ± 1.40^b
Obese Hemi-ADX	4	141.0 ± 27.0^a	1060 ± 41^b	7.52 ± 1.5^b
Obese-ADX	6	126.3 ± 4.9^a	299.5 ± 71.5^a	2.40 ± 1.08^a
Obese ADx + INS	3	223.0 ± 21.0^b	1941 ± 110^c#	8.70 ± 1.8^b#
ANOVA		ns^#	p = <0.05	p = <0.05

Table 1.

Glycemic parameters in lean and obese LA/Ntul//-cp rats.

Effects of ADX and diet on glycemic parameters of lean and obese rats at 12 weeks of age. All data are reported as the mean ± 1 SEM (n = 6 rats/group). Truncal bloods were collected in heparin at 12 weeks of age. Rats in a semi-fasted (2–4 h) state. Different subscript letters denote distinct statistical subgroups via Student-Neuman-Keuls application for identification of significance in different subgroups. ^# = non-fasting.

The mean AUCglc of the different groups at 12 weeks of age is shown in Figure 6, in addition to the numerical mean above each bar. Note that the AUCglu of the unoperated lean and the obese+ADX AUCglc are virtually identical (261.2 vs. 257.25, respectively), indicating the ADX procedure resulted in a virtual normalization in the glycemic responses. In contrast, the final AUCglc in the obese, Obese+hemiADX and the Obese+ADX + Insulin ate similar, (315.9, 320.1 and 334.0 respectively), and consistent with insulin resistance of similar magnitude in the 3 groups.

The effects of adrenalectomy on parameters of adiposity at 12 weeks of age are shown in Table 2 and indicate that the combined fat pad mass and percent adiposity of the obese animals was markedly greater than was observed in their lean littermates. Adrenalectomy resulted in a partial normalization in fat accretion, while fat pad mass and percent adiposity of the hemi-adrenalectomized rats were similar to those of unoperated obese rats. When adrenalectomized rats were administered supraphysiologic doses of insulin however, fat pad mass and the percent adiposity exceeded that of the unoperated obese littermates. The insulin administration resulted in a return of parameters of insulin resistance, and combined with the greater overall caloric intake, resulted in yet greater fat accretion as predicted. The caloric efficiency in column 4 of the data table is consistent with a greater caloric efficiency in the obese, obese-hemi adrenalectomy and insulinized animals.

Group	n	AT Mass, g.	% Adiposity	Net CE_6-9W	CE 9-12W
Lean	6	5.54 ± 0.11^a	1.87 ± 0.04e-2^a	0.08 ± 0.01^a	0.05 ± 0.01^a
Obese	6	21.18 ± 2.10^c	4.74 ± 0.47e-2^c	0.09 ± 0.01^b	0.06 ± 0.01^b
Obese Hemi-ADX	4	22.04 ± 1.90^c	4.85 ± 0.25e-2^c	0.10 ± 0.01^b	0.06 ± 0.00^b
Obese-ADX	6	12.81 ± 2.30^b	3.02 ± 0.54e-2^b	0.08 ± 0.01^a	0.05 ± 0.01^a
Obese ADx + INS	4	28.2 ± 2.50^c	5.29 ± 0.25e-2^c	0.10 ± 0.011^b	0.06 ± 0.01^b
ANOVA		ns	p = <0.05	p = <0.05	p = <0.05

Table 2.

Effects of adrenalectomy on adipose parameters in lean and obese LA/Ntul//-cp rats.

Data are mean ± 1 SEM, n = 4–6 rats/group. The mass of adipose tissue was computed as the sum of the Dorsal, Retroperitoneal and Epididymal fat pad depots as dissected at 12 weeks of age; The percent adiposity data in column two reflects the combined mass of the three depots expressed as g / g. BW x 100. CE = caloric efficiency from 6 to 9 weeks and 9 to 12 weeks of study. The groups with different superscripts indicate statistically different subgroups at p = < 0.05 via Student Neuman Keuls analysis.

The levels of relative adiposity of the 5 groups are indicated in Table 2. The grams of the 3 fat pad depots are presented in the far-left column and the percent adiposity in the adjacent column. Comparing the relative adiposity with the Insulin to glucose ratio as an indicator of insulin resistance in Table 1, the relative adiposity corresponds with the magnitude of insulin resistance in each group, where the Obese+ADX is similar to the similarly fed unoperated lean controls (Figure 7), while all other treatment groups remain similarly elevated by comparison. The net caloric intake during weeks 6 to 9 is reflected in column 3 of Table 2 and indicates that caloric intake in the obese+ADX is similar to unoperated lean animals, while the caloric intake of all other groups was similar. The caloric efficiency indicated in the far-right column corresponds with the caloric intake data, consistent with the magnitude of insulin resistance and adiposity observed.

Figure 7.

Association between percent adiposity and insulin to glucose ratio. Data are mean ± 1 SEM, N = 4–6 rats/group.

Rhus, the results of this investigation indicate that bot unoperated obese and obese-adrenalectomized developing a greater magnitude of adiposity than occurred in their lean littermates, indicative a = of a phenotype effect on weight gain and the expression of obesity. The significant differences in weight gain between the phenotypes occurs despite their having the same genetic origins and being reared under the same environment and dietary conditions. The coexistence of insulin resistance is evident in the obese phenotype, as reflected by greeter plasma insulin concentrations in addition to an exaggerated insulin to glucose ratio in the obese. The obese phenotype exhibited hyperphagia and an improved efficiency of weight gain following hemi-adrenalectomy or adrenalectomy plus insulin replacement in an attempt to maintain a state of insulin resistance in the absence of adrenal secretions. In contrast, adrenalectomized obese animals exhibited glycemic and thermogenic parameters that were similar to those of their unoperated lean littermates, consistent with the impression that the counterregulatory effects between insulin and adrenal glucocorticoid hormones are key contributory factors in the insulin resistance and in parameters reflecting insulin sensitivity.

The Interscapular Brown adipose tissue (IBAT) depots were excised in their entirety after sacrifice and are depicted in Figure 8. The data indicates that the IBAT mass of obese rats and the proportion of IBAT mass to body weight were significantly greater in the obese phenotype. Neither partial or complete adrenalectomy was effective in restoring iBAT mass to normal, likely because increases in the IBAT mass associate with hyperphagia beginning soon after postweaning development, via processes of hypertrophy and hyperplasia [4, 13]. The IBAT to BW ratio was however only partially corrected since the final body weights of the adrenalectomized animals were intermediate between those of the lean and obese phenotype while the IBAT mass was similar n both groups.

Figure 8.

Brown adipose tissue mass of lean and obese rats at 12 weeks of age. Data are mean ± 1 SEM, n = 4–6 rats/group. P = <0.05 obese vs. lean for IBAT mass and IBAT: Body weight (Students unpaired t test). WOA = weeks of age.

The effect of ADX on RMR is depicted in Figure 9. Measures on RMR in the lean, obese, and obese-adrenalectomized rats are depicted in Figure 9, and indicate that in lean animals, the RMR increased with age, in contrast, the RMR of obese rats decreased with advancing age. The RMR of the adrenalectomized obese rats however, attained levels similar to those of unoperated lean littermates by 12 weeks of age, reflective of the improvements in insulin sensitivity in those animals.

Figure 9.

The effect of adrenalectomy on resting VO2 in LA/Ntul//-cp rats. Data are expressed as the mean ± 1 SEM (n = 6 rats/treatment group). Significance was determined with a p = < 0.05 via students unpaired t test comparing lean vs. obese, and obese+ADX vs. obese. Final VO2 of adrenalectomized obese rats at 12 weeks of age was similar to lean littermates (p = n.s.).

Measures of RMR at in all 5 treatment groups are depicted in Figure 10 and indicate that RMR of the lean animals remained similar at all 3 ages studies. In contrast, the RMR of the obese rats were lower at the onset at 6 weeks and increased only modestly with age and dietary treatment. The effects of partial and complete adrenalectomy were similar at 6 and 9 weeks of age, and increased when fed the cafeteria diet supplement at 12 weeks of age only in fully adrenalectomized animals. RMR increased in magnitude in the insulinized adrenalectomized obese animals to levels similar to obese adrenalectomized at the 12-week time point, after 3 weeks of the cafeteria diet supplement, during which time they also gained excessive weight and greater fat accretion.

Figure 10.

Effect of treatment groups on RMR at 12 weeks of age. Data are mean ± 1 SEM, n = 4–6 rats/group except group obese + ADX + INS, where n = 4 due to fasting induced premature deaths in 2 animals.

The effects of norepinephrine administration on VO2 in the various treatment groups is depicted in Figure 11. The data shown are the increase in VO2 over the RM, taken 15 min after administering the NE (100 μg/kg BW, s.c. in the interscapular region). In lean rats, the NE induced increase was greater at each age studied. In contrast, in the obese phenotype, NE had only a modest effect, initially about one half of the response of lean animals, and which response decreased at each subsequent time point. Adrenalectomy in obese animal restored the NE response to that of their lean littermates, while partial adrenalectomy and adrenalectomy followed by inulin treatment were ineffective in restoring the thermic response to NE.

Figure 11.

Effect of treatment groups on NE increase in RMR at 12 weeks of age. Data are mean ± 1 SEM, n = 6 rats/group except group obese + ADX + INS, n = 4 due to fasting induced premature deaths of 2 animals. 0 = < 0.05 (lean vs. obese, obese hemi ADX and obese ADX + INS; p = n.s. (lean vs. obese ADX at 9 and 12 WOA; WOA = weeks of age.

4. Discussion

The contributions of glucocorticoid actions on insulin-stimulated formation and cytoplasmic translocation of GLUT4 transporters has been well documented [9, 32, 33, 34]. When the regulation of the transporters become disrupted due to glucocorticoids or other factors, the typical physiologic response is an increase in insulin secretion in an apparent attempt to overcome the glucose transporter impediment. The cascade of metabolic events which follow contributes to the progressive development of insulin resistance along with its pathophysiologic sequelae. In humans and likely rats as well, as the level of excess glucocorticoid stimulation continues, the elevations in cortisol contribute to preadipocyte differentiation and additional lipid accretion. Thus, pathophysiologic influences on adipose tissue depots may further exaggerate the impact of hyperinsulinemia and insulin resistance which may extend to additional tissues including skeletal muscle, brain and others that are also dependent on molecular glucose for oxidative metabolism [32, 33, 34]. Therefore, pharmacologic or surgical ablation of adrenal glucocorticoid inhibitory activity would seem to be an effective strategy to restore GLUT4 transporter functions, permit the restoration of endoplasmic reticulum synthesis and intracellular translocation to the plasma membranes, improve cellular glucose uptake and oxidation, and thus diminish the overall magnitude of insulin resistance in the various tissues of the obese phenotype of this and other genetically obese rat strains. Insulin is known to impact multiple aspects of energy metabolism and storage, including lipogenesis, activation of preadipocytes, and substrate oxidative processes, all of which may serve as primary or secondary factors that contribute to excess energy storage in adipocytes and other tissues, including hepatic fatty acid and glycogen synthesis and storage [35, 36, 37].^.

Evidence of the onset of insulin resistance were already apparent in the obese phenotype by 6 weeks of age, as demonstrated by the delayed glycemic excursions during the oral glucose tolerance challenge. Moreover, the disordered glycemic responses became more exaggerated by 12 weeks of age in the same obese animals, consistent with a progression of the insulin resistance state. In contrast, the magnitude of disordered glycemic responses following bilateral adrenalectomy became normalized to those of their lean littermates by 12 weeks of age, consistent with a restoration of normal glycemic parameters following adrenal ablation. However, when a subgroup of the obese adrenalectomized animals were subjected to the hyperphagia inducing Café diet regimen plus daily insulin injections in an attempt to re-establish insulin resistance in the absence of adrenal glucocorticoid secretions, adiposity progressed at an accelerated rate and with a similar magnitude of insulin resistance. These observations are consistent with adrenal mediated contributions to the process leading to insulin resistance and adiposity.

Insulin resistance also is known to impede the metabolic process of non-shivering thermogenesis in brown adipose tissue [38, 39, 40, 41, 42]. Insulin resistance also contributes to dysregulation of lipid metabolism in both brown and white adipose tissues, where it impacts on activity of lipoprotein lipase, essential for the mobilization and release of stored triglyceride [10, 11, 12, 13, 14]. Brown adipose tissue is recognized as a major contributor to the process of non-shivering thermogenesis, and has been attributed to play a role in energy balance during periods of over- and under nutrition [38, 39, 40, 41, 42]. Brown adipose tissue mass increases during early prepubucent life via the processes of hyperplasia, and via hypertrophy of brown adipocytes beyond adolescence and during periods of metabolically- or pharmacologically-mediated decreases in thermogenic activity [38, 39, 42, 43]. Thus, the presence of insulin resistance has been shown to impair cellular glucose uptake in isolated brown adipocytes, and to generate deceases in resting and pharmacologically-stimulated alterations in thermogenic activity in isolated cells and in vivo responses to nutritional and pharmacologic simuli [10, 11, 12, 43]. In other studies, the presence of chronic insulin resistance was associated with the ‘unbowning’ of brown adipose tissue, as the numerous small lipid locules normally active during thermogenic activity accumulated additional lipid doing their decreased periods of thermogenic activity, giving way to a less intense brown coloration of the tissue [41]. In the present study, ablation of glucocorticoid influences via bilateral adrenalectomy resulted in a recovery of normal thermogenic responses by 12 weeks of age, consistent with adrenal glucocorticoid-induced contributions to insulin resistance and impaired nonshivering thermogenesis, in vivo.

Insulin resistance impedes the process of non-shivering thermogenesis, in addition to contributing to dysregulation of lipid metabolism in both brown and white adipose tissues [10, 11, 12, 13, 14]. Brown adipose tissue is recognized as a major contributor to the process of non-shivering thermogenesis, and to play a role in energy balance during periods of over- and under nutrition [38, 39, 40, 41, 42]. Brown adipose tissue mass increases via the processes of hyperplasia during early life, and via hypertrophy of brown adipocytes beyond adolescence, and during periods of metabolically- or pharmacologically-mediated decreases in thermogenic activity [38, 39, 42, 43]. The presence of insulin resistance impaired cellular glucose uptake in isolated brown adipocytes, and in deceases in resting and pharmacologically-stimulated alterations in thermogenic activity [10, 11, 12, 43]. In other studies, the presence of chronic insulin resistance was associated with the ‘unbowning’ of brown adipose tissue, as the numerous small lipid locules normally active during thermogenic activity accumulated additional lipid doing their decreased periods of thermogenic activity, giving way to a less intense brown coloration of the tissue [41]. In addition, the thermic increases following the Café diet regimen are manifested by a combination of short-term neurosympathetic and longer-term thyroidal actions, both of which hormonal systems interact with the lipogenic activities that modulate energy metabolism and expenditure [36]. Thus, the roles of insulin and glucocorticoid hormones cannot be viewed independently with respect to parameters of the integrated complex regulation of energy balance in man and animals.

Acknowledgments

The author thanks the University of Science Arts and Technology, Montserrat for the provision of institutional resources to develop this manuscript.