In vivo tissue specific modulation of rat insulin receptor gene expression in an experimental model of mineralocorticoid excess

Molecular and Cellular Biochemistry 185: 177–182, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.

177

In vivo tissue specific modulation of rat insulin receptor gene expression in an experimental model of mineralocorticoid excess Javier Campión,1 Vicente Lahera,2 Victoria Cachofeiro,2 Begoña Maestro,1 Norma Dávila,3 María del Carmen Carranza1 and Consuelo Calle1 1

Deptartamento de Bioquímica y Biología Molecular; 2Deptartamento de Fisiología, Facultad de Medicina, Universidad Complutense; 3Servicio de Endocrinología, Hospital C.P.H., Madrid, Spain

Abstract Insulin receptor (IR) gene expression at the mRNA level was investigated in hindlimb skeletal muscle, epididymal adipose tissue and in the liver of rats exposed to prolonged in vivo administration of deoxycorticosterone acetate (DOCA). Following treatment, plasma insulin levels were reduced while glucose levels increased compared to values in control rats. DOCA-treated animals showed an increase in blood pressure and a reduction in body weight. This treatment also induced hypokalemia and decreased plasma protein levels. Sodium levels were unaffected. Moreover, no differences in DNA and protein content or in the indicator of cell size (protein/DNA) were observed in the skeletal muscle or adipose tissue of animals. In contrast, there was a clear increase in the protein and DNA contents of the liver with no change in the indicator of cell size. Northern blot assays revealed 2 major IR mRNA species of approximately 9.5 and 7.5 Kb in the 3 tissues from control animals. DOCA treatment induced no change in the levels of either RNA species in skeletal muscle. However, a decrease of approximately 22% was detected in the levels of both species in adipose tissue whereas the liver showed an increase of 64%. These results provide the first evidence for an in vivo tissue-specific modulation of IR mRNA levels under experimental conditions of mineralocorticoid excess. (Mol Cell Biochem 185: 177–182, 1998) Key words: insulin receptor gene expression, mineralocorticoid-treated rats

Introduction Mineralocorticoid hormones increase Na+ re-absorption and promote K+ and H+ secretion in classical mineralocorticoid target tissues such as the kidney [1]. More recent observations suggest additional activities of mineralocorticoids in cells which are not directly related to electrolyte and water homeostasis i.e. differentiation in 3T3-L1 adipocytes [2] and immune processes in human mononuclear cells [3]. Mineralocorticoids may also modulate insulin secretion and action in patients with primary hyperaldosteronism [4, 5]. Thus, impaired glucose tolerance and decreased insulin levels have been observed in patients with this syndrome [5, 6]. In addition, a reduction in the number of insulin receptors (IR)s

and affinity has been also detected in the adipose tissue of a patient with primary hyperaldosteronism [7]. The mineralocorticoid receptor (MR) mediates mineralocorticoid action in target cells and may be described as a class I nuclear receptor [8]. This class also includes receptors for glucocorticoids (GR)s, progesterone, androgens and estrogens. These ligand-dependent transcription factors modulate gene expression by binding to specific DNA sequences [9]. Earlier in vitro and in vivo studies of our laboratory and others have reported regulation of IR gene expression by glucocorticoids [10–13], progestins [14] and androgens [15]. In addition, it has recently been observed by the present authors that mineralocorticoids are able to modulate IR gene expression in vitro [16].

Address for offprints: C. Calle, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain

178 Therefore, the aim of the present investigation was to demonstrate an in vivo tissue-specific modulation of IR gene expression by mineralocorticoids, analyzing IR mRNA levels in hindlimb skeletal muscle, epididymal adipose tissue and liver of rats in which the mineralocorticoid concentration had been increased by prolonged treatment with deoxycorticosterone, the main mineralocorticoid in the rat. The experimental animals were also subjected to other minor manipulations to reproduce conditions of primary hyperaldosteronism in humans.

Materials and methods

blot assays, RNA samples (40 µ g skeletal muscle, 30 µg adipose tissue and 50 µg liver) were denatured, electrophoresed in 1.1% agarose-formaldehyde gels and blotted onto nylon membranes (Hybond N, Amersham). Ethidium bromide staining of 28 S and 18 S ribosomal RNAs was routinely checked before blotting as a control of sample loading, and after blotting as a control of RNA transfer. RNA blots were prehybridized, hybridized with excess [32P]-labeled probe (the 0.98-Kb rat IR specific EcoRI fragment of the p16 clone, gift from Prof. Goldstein), washed under stringent conditions and finally autoradiographed [22]. The autoradiographs were scanned with a laser densitometer (Molecular Dynamics) and the readings normalized with the respective amounts of 28 S rRNA, as revealed by ethidium bromide.

Animals Male Wistar rats (n=14) weighing approximately 210 g at the onset, maintained on ad libitum rat chow, tap water and a 12 h dark/light cycle were employed for the study. All animals were pre-uninephrectomized under thiopental anesthesia. Seven days after surgery, some of the animals (n = 8) were administered deoxycorticosterone acetate (DOCA) (Sigma) by subcutaneous injection (20 mg/Kg body wt) twice a week for 4 weeks (DOCA-treated rats). These animals were maintained on 4% saline ad libitum instead of tap water. The remaining animals (n=6) received only the vehicle (oil) and tap water to drink. These formed the control group. Systolic blood pressure was estimated using a tail-cuff plethysmograph (Narco Bio-Systems), as described previously [17]. At the end of the experimental period, animals were sacrificed by decapitation without anesthesia. Trunk blood samples were collected for plasma measurements. The hindlimb skeletal muscle, epididymal adipose tissue and liver were immediately removed and individually packed and frozen in liquid nitrogen for nucleic acid and protein determination.

Analytical methods Plasma insulin levels were determined by radioimmunoassay using rat insulin as standard [18]. Glucose levels in plasma were estimated using a commercially available glucoseoxidase technique. Plasma sodium and potassium levels were measured by flame photometry. Protein levels of the homogenates of individual tissues were determined by the method of Bradford [19]. Total DNA content was estimated using a spectrofluorimetric method [20]. RNA blot assays Total RNA was extracted from individual tissues according to the method of Chomczynski and Sacchi [21]. For Northern

Statistical analysis Unless otherwise indicated, data are expressed as the mean ± S.E.M. Statistical comparisons (Student’s t-test) were performed using a computer program (Sigma Plot version 1.02, Jandel Scientific). Differences were considered statistically significant when p < 0.05.

Results and discussion After treatment for 4 weeks with DOCA, the plasma insulin levels of the animals were found to be significantly reduced whereas glucose levels showed an increase (Table 1). In addition, blood pressure was seen to increase while the body weights of the animals suffered a reduction (Table 1). Prolonged DOCA treatment also induced hypokalemia and a decrease in plasma protein as shown in Table 1. DOCAtreated animals showed normal plasma sodium concentrations. This experimental model of mineralocorticoid excess was designed to reproduce conditions of primary hyperaldosteronism in humans. In both the model and pathological condition, the association of hyperglycemia, hypoinsulinemia, hypokalemia and hypertension is clear [5–7]. These alterations were initially induced by the DOCA treatment. The elevation of blood pressure was favoured by the increase in salt intake and the reduction of renal mass [23]. A virtual glucose intolerance accompanied by impaired insulin release was observed in the DOCA-treated animals of the present study. However, Dai et al. have reported [24, 25] that prolonged DOCA treatment lowers insulin plasma levels in rats but glucose levels and glucose tolerance remain unaffected. This difference in results may be attributable to: strain variation (Wistar vs. Sprague-Dawley rats), variations in the experimental conditions of the animals (fed vs. fasting; uninephrectomized vs. unnephrectomized) and/or to the presence or no of hypokalemia induced by the DOCA

179 Table 1. Plasma glucose, insulin, sodium, potassium and protein levels, body weight and blood pressure of DOCA-treated rats in comparison to control rats

Plasma glucose (mg/dl) Plasma insulin (ng/ml) Plasma sodium (mEq/l) Plasma potassium (mEq/l) Plasma protein (g/dl) Body Weight (g) Blood presure (mm Hg)

Control rats (n = 6)

DOCA-treated rats (n = 8)

135

150 ± 4*

±5

0.56 ± 0.05 160

±1

0.34 ± 0.04** 158

±1

7.7 ± 0.2

5.1 ± 0.3**

5.27 ± 0.18

4.63 ± 0.16*

356

±2

331

± 6*

120

±4

184

± 9**

Values are means ± S.E.M. for the number of animals indicated in parentheses, **p < 0.01, *p < 0.05 vs. respective control values.

treatment. With regard to the latter, it is known that potassium deficiency can induce decreased insulin release leading to an impairment of glucose tolerance [5, 26]. The results shown in Table 2 indicate that were no differences in the total DNA and protein contents and in the indicator of cell size (protein/DNA) in hindlimb skeletal muscle or epididymal adipose tissue of DOCA-treated animals in comparison to that of controls. In contrast, a clear Table 2. Tissue characteristics of hindlimb skeletal muscle, epididymal adipose tissue and liver of DOCA-treated rats compared to those of control rats

Hindlimb muscle DNA (mg/g) Protein (mg/g) Prot/DNA (mg/mg) RNA (mg/g)

Control rats (n=6)

DOCA-treated rats (n=6)

0.49 ± 0.05 128 ± 11 265 ± 10 0.42 ± 0.02

0.49 ± 0.03 122 ± 4 261 ± 26 0.43 ± 0.05

Epididymal adipose tissue DNA (mg/g) Protein (mg/g) Prot/DNA (mg/mg) RNA (mg/g) Liver DNA (mg/g) Protein (mg/g) Prot/DNA (mg/mg) RNA (mg/g)

0.21 ± 12.6 ± 71.3 ± 0.12 ±

1.8 132 73.4 2.9

± ± ± ±

0.06 1.8 7 0.01

0.1 5 5 0.1

0.25 ± 17.2 ± 79.8 ± 0.15 ±

2.4 155 64.3 3.8

± ± ± ±

0.05 2.3 10 0.02

0.1** 5** 1 0.1**

Values are means ± S.E.M. for the number of animals indicated in parentheses, **p < 0.01 vs. respective control values.

increase in both protein and DNA content, with no change in the indicator of cell size, was seen in the liver of DOCAtreated rats (Table 2). The increments in protein and DNA contents of livers may be attributable to hypertrophy of the organ but, given that the protein/DNA ratio remained unaltered, this seems unlikely. Another explanation could be that treatment with DOCA causes hyperplasia of the liver although indirect data suggest that mineralocorticoids are not mitogenic in liver cells [27]. An alternative possibility is that DOCA treatment causes a reduction in liver weight as a consequence of a retardation in the growth of the animals [26]. This last explanation seems to be supported by the fact that DOCA treatment caused a reduction in body weights (Table 1). Northern blot assays of the 3 tissues obtained from control animals, revealed 2 major IR mRNA species of approximately 9.5 and 7.5 Kb in size (Figs 1–3). The relative amounts of the 2 IR mRNA species, measured as the 9.5 Kb/ 7.5 kb ratio, were: 0.52 ± 0.03 in skeletal muscle, 1.04 ± 0.07 in adipose tissue and 0.96 ± 0.07 in the liver. These results are in agreement with those previously reported by the present authors [11, 16] and others [28–30] using similar rat tissues. Since there is only one IR gene, the variation in transcript length has been related to the existence of alternative polyadenylation sites [31]. Prolonged treatment with DOCA did not affect IR mRNA species in skeletal muscle (Fig. 1). However, DOCA treatment induced a decrease of approximately 22% in the levels of both IR mRNA species in epididymal adipose tissue (27% corresponding to the 9.5 Kb and 18% to the 7.5 Kb species) (Fig. 2). In view of the fact that DOCA treatment did not alter the total RNA content per gram of tissue (Table 2), IR mRNA levels in both hindlimb skeletal muscle and epididymal adipose tissue were expressed per unit of RNA (Figs 1 and 2). In the liver, treatment with DOCA produced an increase of approximately 64% in the levels of both IR mRNA species (66% corresponding to the 9.5 Kb and 62% to the 7.5 Kb species) expressed per unit of RNA (Fig. 3). However, given that the treatment augmented the total RNA content of this organ (Table 2), the results were also expressed with respect to DNA and protein. As shown in Fig. 3, regardless of the form of expression, IR mRNA levels in the livers of DOCAtreated animals increased by 62–88% in comparison to control animals. This indicates that after prolonged treatment with DOCA, there is a tissue-specific modulation of the levels of IR mRNA. The different responses observed in the tissues could be related to the presence or absence of specific MRs which are able to mediate the process. The expression of MR mRNA has been detected in the developing muscle of mouse fetus [32] but not in adult rat muscle [33]. This lack of MRs could be related to the present unaffected IR mRNA levels in the

180

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B

Fig. 1. Insulin receptor (IR) mRNA levels in hindlimb skeletal muscle of DOCA-treated rats compared to control rats. (A) Autoradiograph of a representative Northern blot experiment in hindlimb skeletal muscle of 2 control rats and 2 DOCA-treated rats. The sizes of the 2 major IR mRNA species are shown in the margin. Also indicated are the respective amounts of 28 S rRNA revealed by ethidium bromide. (B) Densitometric readings of 3 independent Northern blot experiments. IR mRNA species were quantified separately, normalized with the respective 28 S rRNA values and expressed per unit of RNA as a percentage of the values obtained in control rats (mean ± S.E.M.).

Fig. 2. Insulin receptor (IR) mRNA levels in epididymal adipose tissue of DOCA-treated rats compared to control rats. (A) Autoradiograph of a representative Northern blot experiment in epididymal adipose tissue of 2 control rats and 2 DOCA-treated rats. The sizes of the 2 major IR mRNA species are shown in the margin. Also indicated are the respective amounts of 28 S rRNA revealed by ethidium bromide. (B) Densitometric readings of 3 independent Northern blot experiments. IR mRNA species were quantified separately, normalized with the respective 28 S rRNA values and expressed per unit of RNA as a percentage of the values obtained in control rats (mean ± S.E.M. *p < 0.05).

skeletal muscle of DOCA-treated rats, suggesting a minor role for the biological action of mineralocorticoids in this type of tissue. The presence of MRs in isolated rat adipocytes [34] and 3T3-L1 adipose cells [2] has been clearly demonstrated. Presumably, these MRs would mediate the decrease in IR gene expression observed in the adipose tissue of DOCAtreated rats. The decrease in IR mRNA levels could, at least in part, account for a possible reduced insulin sensitivity of the adipose tissue of these animals. This assumption is in line

with the decrease in number and affinity of IRs observed in the adipocytes of a patient with primary hyperaldosteronism [7]. Several authors have reported a lack of [33] or very low expression of MR mRNA in the liver [35, 36] suggesting a minor physiological role for MRs in this organ. However, GRs are highly expressed in liver [36]. These ligand-activated GRs are capable of inducing an increase in IR mRNA levels in the livers of rats treated with dexamethasone [11, 28]. More recently, Bamberger et al. [37] have clearly demonstrated

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B

Finally, the present results do not indicate whether the DOCA-induced tissue-specific modulation of IR mRNA levels in the rat is due to transcriptional or post-transcriptional regulation, or to both. Although specific mineralocorticoid response elements have not been identified in the IR gene promoter, previous studies have reported the presence of up to 5 putative glucocorticoid response elements in this promoter [41]. The implication of these sequences in the DOCAinduced tissue-specific regulation of IR mRNA in the rat, remains to be investigated. The present findings indicate that DOCA-treated rats may be an appropriate model for the investigation of the chronic effects of mineralocorticoids in Man.

Acknowledgements We are most grateful to Dr. B. J. Goldstein, Director of the Endocrinology Division, Jefferson Medical College, Philadelphia, for providing the rat IR cDNA probe. This work was financed by research funds from the Universidad Complutense de Madrid, Grant: 4830 and the Fondo de Investigaciones Sanitarias de la Seguridad Social, Grants: 94/0276 and 97/ 1218 .

References Fig. 3. Insulin receptor (IR) mRNA levels in the liver of DOCA-treated rats compared to control rats. (A) Autoradiograph of a representative Northern blot experiment in the liver of 2 control rats and 2 DOCA-treated rats. The sizes of the 2 major IR mRNA species are shown in the margin. Also indicated are the respective amounts of 28 S rRNA revealed by ethidium bromide. (B) Densitometric readings of 3 independent Northern blot experiments. IR mRNA species were quantified separately, normalized with the respective 28 S rRNA values and expressed (per unit of RNA, per unit of DNA and per unit of protein) as a percentage of the values obtained for control rats (mean ± S.E.M. *p < 0.05).

expression of MR mRNA in human liver. The possible mediation of a mechanism of MR/GR interaction [9, 37, 38] in the DOCA-induced increase in hepatic IR mRNA levels, requires further investigation. Irrespective of the mechanism which mediates the process, the functional significance of the increase in hepatic IR mRNA may be attributed to enhanced insulin sensitivity in this organ. In this respect, no insulin binding and/ or postbinding studies have been performed using livers from DOCA-treated rats or patients with primary hyperaldosteronism that could support this hypothesis. However, data reported by Dai et al. [24, 25] suggest that DOCA may play a role in glucose metabolism leading to an improvement in insulin sensitivity. In addition, several investigations have demonstrated increased insulin sensitivity in certain patients with primary aldosteronism [39, 40].

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