Streptozotocin-induced diabetes has differential effects ... - Springer Link

Diabetologia (1998) 41: 660±665 Ó Springer-Verlag 1998

Streptozotocin-induced diabetes has differential effects on atrial natriuretic peptide synthesis in the rat atrium and ventricle: a study by solution-hybridization-RNase protection assay S. Q. Wu1, C. Y. Kwan2, F. Tang Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China 1 Department of Physiology, Beijing Medical University, Beijing, China 2 Department of Biomedical Science, Mc Master University, Canada

Summary In rats with streptozotocin (STZ)-diabetes for 2 or 4 weeks, the atrial natriuretic peptide (ANP) concentrations in the atria decreased whilst that of ANP in the plasma and ventricles increased. ANP concentrations in the hypothalamus and in the brainstem did not change in either 2- or 4-week diabetic rats. Atrial ANP content was partly restored by insulin replacement in 4-week diabetic rats. Plasma ANP concentrations and ventricular ANP contents were reversed to normal by insulin treatment in both 2- and 4-week diabetic rats. Solution-hybridization-RNaseprotection assay showed a significant increase in the preproANP mRNA expression in the ventricles but

not in the atria. These results indicated that the STZdiabetes increased the synthesis of ANP in the ventricles and consequently its release from the ventricles. The synthesis of ANP in the atria did not change as judged from the preproANP mRNA expression but the release of ANP from the atria might also be increased for ANP content decreased in the atria. The reason for the difference in the response of atrial and ventricular preproANP concentrations to STZ-diabetes is not known. [Diabetologia (1998) 41: 660±665]

Atrial natriuretic peptide (ANP), a 28 amino acid peptide hormone, is mainly synthesized by the atria and heart ventricles and is released in response to acute and chronic extracellular volume expansion. ANP facilitates the excretion of water and sodium and has a vasodilator effect on the blood vessels [1, 2]. The release, storage and synthesis of ANP in the heart are intimately linked to changes in intravascular volume and blood pressure [3]. Diabetes mellitus may lead to abnormalities in fluid and electrolyte balance and consequently affect blood volume and blood pressure [4]. Alterations in

renin-angiotensin-aldosterone system and arginine vasopression (AVP) secretion, the two well-known mechanisms of controlling extracellular fluid volume homeostasis, have been observed both in humans with diabetes mellitus and in rats treated with alloxan [5]. Atrial natriuretic peptide also plays an important role in body fluid and electrolyte balances and in regulating blood pressure [1, 2]. Plasma ANP concentrations have been reported to be either increased [6] or normal [7] in Type-I diabetic patients compared with normal subjects. Plasma ANP concentrations in chronic STZ-diabetic rats have also been shown to increase [8, 9, 10, 11] though unaltered plasma ANP concentrations have been reported [12, 13]. It is still not clear whether the increased plasma ANP concentrations are due to increase in cardiac ANP secretion. Therefore, this study was to examine ANP content in the brain, the atria and the ventricles of the heart and the plasma. ANP mRNA expression in the atria and the ventricles were also quantified using solution-hybridization-RNase protection assay.

Received: 29 October 1997 and in revised form: 29 January 1998 Corresponding author: Professor F. Tang, Department of Physiology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China Abbreviations: ANP, Atrial natriuretic peptide; STZ, streptozotocin; AVP, arginine vasopression; ANOVA, analysis of variance.

Keywords Atrial natriuretic peptide, rat, solution-hybridization-RNase protection assay, streptozotocin.

S. Q. Wu et al.: Diabetes on cardiac ANP synthesis

Materials and methods Male Sprague-Dawley rats weighing 250±300 g were used. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg body weight), whereas the control animals received the buffered vehicle (0.1 mol/l citrate, pH 4.5). Induction of diabetes was confirmed 1 week after the injection of STZ, by the presence of polyuria and polydipsia in the rats and by testing urine glucose. Later it was further confirmed by serum glucose measurement. Half of the diabetic rats (insulin replacement group) were subcutaneously injected with insulin in the afternoon (4 u/day) 1 week after STZ injection. Control and diabetes rats received saline (NaCl 154 mmol/l) injection only. The rats were killed in the morning by decapitation 2 weeks or 4 weeks after the injection of STZ. The hypothalamus, brainstem, atria and heart ventricles were quickly excised, frozen on dry ice and stored at Ÿ 70 C. The trunk blood was collected into prechilled plastic test tubes containing 100 ml 7.2 % EDTA. The blood samples were centrifuged for 20 min (2000 ” g ), and the plasma was aspirated, snap-frozen and stored at Ÿ 70 C until extraction. Measurement of serum glucose. Serum glucose levels were measured by the glucose-oxidase method. Extraction of ANP from tissues and plasma. The left and right atria, and the 2 ventricles were not separated. Both sides were extracted together. Tissues were homogenized in 2N acetic acid and boiled for 10 min. An aliquot of 50 ml of homogenate was aspirated for protein assay. The remaining homogenate was centrifuged for 20 min at 17 000 ” g at 4 C. The supernatant was lyophilized and stored at Ÿ 20 C until assay. For plasma ANP extraction, an aliquot of 2 ml of plasma was acidified with 1 % TFA and centrifuged for 20 min at 17 000 ” g at 4 C. The supernatant was applied to Sep-column which had been pretreated with 1 ml acetonitrile-1 % TFA (3:2 by volume) and 9 ml 1 % TFA. The peptide was slowly eluted with 3 ml 60 % acetonitrile in 1 % TFA. The eluent was evaporated to dryness in a speed-vac concentrator. ANP radioimmunoassay. ANP was measured using a specific radioimmunoassay. Duplicate samples of ANP standards (0±500 pg/100 ml) and the extracted samples were incubated for 18 h at 4 C with 100 ml of ANP antiserum (1/10,000) (a gift from Dr. T. Yandle, Christchurch, New Zealand) and 100 ml of 125 I-labelled ANP (8000 cpm). Separation of antibody-bound from free 125I-ANP was achieved using activated charcoal. Protein measurements. We boiled 50 ml of homogenate or standard (bovine serum albumin) with 1 N NaOH for 10 min and mixed 50 ml of the boiled samples with 2.5 ml of protein assay reagent (Bio-Rad, Hercules, CA, USA). After 10 min of incubation at room temperature, samples were measured spectrophotometrically at 595 nm. Extraction of tissue total RNA. Total RNA was extracted using Trizol reagent (Gibco-brl, Gaithersburg, MD, USA). About 50±100 mg of the atria and the ventricles were homogenized in 1 ml Trizol reagent using a polytron. The homogenate was incubated at room temperature for 5 min. We added 0.2 ml of chloroform, the contents were mixed by shaking the tubes vigorously for 15 s and then incubated at room temperature for 3 min. The samples were centrifuged for 15 min at 10,000 ” g at 4 C. The aqueous phase was collected and mixed with an equal volume of isopropanol. The RNA was precipitated at room temperature for 10 min and centrifuged for 10 min at 10,000 ” g at 4 C. The RNA pellet was washed with 1 ml of

661 70 % ethanol and was centrifuged. The pellet was air dried and dissolved in TE buffer (pH 7.5) and stored at Ÿ 70 C until assay. Solution-hybridization-RNase-protection assay. This assay is similar to the one used for preproenkephalin mRNA [14]. Plasmid preproANP cDNA and b-actin cDNA (both kindly provided by Dr. D. J. Autelitano, Baker Medical Research Institute, Prahran, Australia) were transformed into E. coli JM 109. After harvest, these plasmid DNAs were linearized with restriction enzymes (preproANP: Eco RI for synthesis of probe and Sal I for standard; b-actin: Eco RI for probe and Hind III for standard). The standard RNA and the riboprobes were synthesized using polymerases (preproANP: T7 polymerase for probe and SP6 polymerase for standard; b-actin: SP6 polymerase for probe and T7 polymerase for standard) and reagents available in a kit obtained from Promega (Madison, Wis. USA). The sizes of the riboprobes for preproANP and b-actin were 755 and 387 nucleotides, respectively. The appropriate amount of RNA samples/standards were hybridized with 32p-riboprobe (100,000 cpm) in hybridization buffer (80 % formamide, 40 mmol/l PIPE, 400 mmol/l NaCl and 1 mmol/l EDTA) at 45 C overnight. Ventricular total RNA samples (10 mg) were co-hybridized with preproANP and b-actin riboprobes. For the atrial samples, 1 mg of total RNA was used for preproANP mRNA assay and 10 mg was used for b-actin mRNA assay. The unhybridized RNAs were digested at 37 C for 45 min with RNase A and RNase T1 (Sigma St. Louis, MO, USA). The enzymes were digested by proteinase K and 1 mg of yeast RNA was added as carrier. After purification by phenol-chloroform extraction and ethanol precipitation, hybrids of different sizes were separated on a 4 % polyacrylamide gel. The position of the hybrids in the gel was visualized by exposure to a X-ray film overnight at Ÿ 70 C in a cassette with intensifying screens. The hybrid bands on the gel were cut out and counted by a scintillation counter (Tricarb 2000 AC; Packard Meriden, CT, USA). Standard curves were drawn by plotting the radioactivity against the standards in picograms. The values of preproANP and bactin mRNA in the samples were read off from the standard curves. The atrial and ventricular preproANP mRNA contents were expressed as pg mRNA/pg b-actin mRNA. The values of preproANP mRNA and b-actin mRNA were not corrected to their native lengths. Statistical analysis. All results are means ± SE. The data were analysed using one-way analysis of variances (ANOVA) and multiple comparison procedure was performed using Tukey's test with p less than 0.05 as the levels of significance.

Results Body weights and serum glucose levels (Table 1). The initial body weight of the three groups were not different. However, 2 and 4 weeks after the injection of STZ, the body weight of each diabetic rat was significantly lower than that of control and insulin replacement rats. Serum glucose concentrations of each 2 and 4-week diabetic rat was about 6 times higher than that of control and 3 times higher than insulin replacement rats. The higher plasma glucose concentrations in the insulin replacement group was probably due to the time lapse between insulin injection and killing.

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S. Q. Wu et al.: Diabetes on cardiac ANP synthesis

Table 1. The effect of STZ-induced diabetes in rats on: body weights (g) and serum glocose levels (mg/100 ml) Control (11) Diabetes (10)

Insulin replacement (9)

Body weight 2 week 4 week

468 ± 13 430 ± 6

386 ± 19* 271 ± 10**

383 ± 18* 381 ± 13*

Serum glucose 2 week 4 week

112 ± 5 117 ± 5

623 ± 30** 632 ± 24**

257 ± 23* 359 ± 53*

ANP contents (pg/mg protein) in the hypothalamus and the brainstem

Hypothalamus 2 week 4 week Brainstem 2 week 4 week

Control (11)

Diabetes (10) Insulin replacement (9)

275 ± 18 284 ± 18

236 ± 15 276 ± 10

255 ± 16 283 ± 16

38 ± 4 33 ± 2

35 ± 2 36 ± 4

41 ± 3 33 ± 4

Fig. 1. Effect of STZ-induced diabetes on plasma ANP in the rats. * p < 0.05 ** p < 0.01 compared with control; n = 11, 10, 9 for control, diabetic and insulin replacement groups, respectively

PreproANP mRNA levels (pg/pg b-actin mRNA) in the atrium and the ventricle

Atrium 2 week 4 week Ventricle 2 week 4 week * **

Control (6)

Diabetes (6)

Insulin replacement (6)

896 ± 140 941 ± 210

621 ± 158 525 ± 114

555 ± 130 758 ± 267

1.62 ± 0.17 1.07 ± 0.31

5.36 ± 0.73** 2.92 ± 0.55**

2.37 ± 0.39 2.06 ± 0.19

p < 0.05 compared with control p < 0.01 compared with control

Fig. 2. Effect of STZ-induced diabetes on ANP in the atria. * p < 0.05 ** p < 0.01 compared with control; n = 11, 10, 9 for control, diabetic and insulin replacement groups, respectively

ANP content in the hypothalamus and the brainstem (Table 1). There was no significant change in these parameters. Plasma ANP levels increased in the diabetic rats (Fig. 1). Plasma ANP concentrations increased in diabetic rats (134 ± 16.6 pg/ml) compared with control (79.7 ± 7.6 pg/ml) and insulin replacement rats (94.3 ± 13.3 pg/ml) 2 weeks after the injection of STZ. There was also a significant increase in plasma ANP concentrations in 4-week diabetic rats (control: 57.2 ± 14.9 pg/ml; diabetes: 124 ± 23.1 pg/ml; insulin replacement: 53 ± 9.5 pg/ml). ANP content in the atria decreased in the diabetic rats (Fig. 2). ANP content in the atria is the highest in all the tissues tested. ANP content in the atria decreased in the diabetic rats (218 ± 33.2 ng/mg protein) compared with control (318.1 ± 25.2 ng/mg protein) two weeks after the injection of STZ. ANP content in 4-week diabetic rats also decreased (control: 328.6 ± 37.6; diabetes: 205.6 ± 32.3; insulin replacement: 247.0 ± 35.5 ng/mg protein).

Fig. 3. Effect of STZ-induced diabetes on ANP in the ventricles. * p < 0.05 ** p < 0.01 compared with control; n = 11, 10, 9 for control, diabetic and insulin replacement groups, respectively

ANP content in the heart ventricles increased in the diabetic rats (Fig. 3). Ventricular ANP content was much lower than that in the atria. But in contrast to the atria, ANP content in the ventricle in the diabetic rats increased significantly in both 2-week (152 ± 27.7 pg/mg protein) and 4-week (125 ± 19.0 pg/mg protein) diabetic rats compared with con-

S. Q. Wu et al.: Diabetes on cardiac ANP synthesis

Fig. 4. Effect of STZ-induced diabetes on preproANP mRNA in the atria. X-ray film of polyacrylamide gel electrophoresis of RNA hybrids of preproANP mRNA and 32P-labelled preproANP riboprobe after overnight exposure. Upper panel: preproANP standards and samples from 2 week experiment. Lower panel: samples from 4 week experiment. Lane 1±5: 0, 10, 50, 100 and 500 pg; C lanes with samples from control rats, D lanes from diabetic rats, I lanes from insulin replacement rats

trol and insulin replacement rats (2 week control: 72.4 ± 9.0; 2 week insulin: 54.3 ± 4.9; 4 week control: 60.8 ± 3.7; 4 week insulin: 61.0 ± 5.5). ANP mRNA levels in the atria (Fig. 4, Fig. 5, Table 1). ANP mRNA expression in the atria did not change in either 2- or 4-week diabetic rats. Fig. 5. Effect of STZ-induced diabetes on b-actin mRNA in the atria. X-ray film of polyacrylamide gel electrophoresis of RNA hybrids of b-actin mRNA and 32P-labeled b-actin riboprobe after overnight exposure. Upper panel: b-actin standards and samples from 2 week experiment. Lower panel: samples from 4 week experiment. Lane 1±5: 0, 5, 10, 50 and 100 pg of b-actin standards. C lanes with samples from control rats, D lanes from diabetic rats, I lanes from insulin replacement rats

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ANP mRNA in the ventricles (Fig. 6 and Table 1). ANP mRNA expression in the heart ventricles were much lower than that in the atria. However there was a significant increase in the ANP mRNA expression (in terms of pg b-actin mRNA) in rats with diabetes for 2 weeks (5.36 ± 0.73 pg/pg b-actin mRNA) and for 4 weeks (2.92 ± 0.55 pg/pg b-actin mRNA) compared with control and insulin replacement rats (2-week control: 1.62 ± 0.17; 2-week insulin: 2.37 ± 0.39; 4-week control: 1.07 ± 0.31; 4-week insulin: 2.06 ± 0.19).

Discussion In the present study we found that plasma ANP concentrations increased in both 2- and 4-week STZ-induced diabetic rats. These results are similar to those of Ortola et al. [9], who found elevated plasma ANP concentrations in moderately hyperglycaemic rats studied 2 weeks after induction of diabetes. Plasma ANP returned to normal when the rats were rendered normoglycaemic with insulin. It is pertinent to note that blood glucose in the insulin replacement rats were slightly above that in the controls in spite of daily treatment by long acting insulin [15]. Al-

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Fig. 6. Effect of STZ-induced diabetes on preproANP mRNA and b-actin mRNA in the heart ventricles. X-ray film of polyacrylamide gel electrophoresis of RNA hybrids of preproANP and b-actin mRNAs co-hybridized with 32P-labelled preproANP and b-actin riboprobes after overnight exposure. The upper bands are the preproANP mRNA hybrids while the lower bands are the b-actin mRNA hybrids. Upper panel: Standards and samples from the 4 week experiment. Lower panel: Samples from the 2 week experiment. Lane P is the riboprobes; lane 1 to 5 are the standards (for preproANP mRNA: 0, 10, 50, 100, 500 pg; for b-actin mRNA: 0, 5, 10, 50, 100 pg). C lanes with RNA samples from control rats, D lanes from diabetic rats, I lanes from insulin replacement rats

though an increase of blood glucose may increase plasma volume and thus also ANP secretion and plasma ANP concentrations, the finding of a normal plasma ANP concentration in rats on insulin replacement (Fig. 1) suggests adequate glucose control. Thus, we did not find a higher concentration of plasma ANP in 4-week STZ-diabetic rats compared with 2-week diabetic rats. The increases over the control at both time intervals were 100 %, similar to that reported in 10-week STZ-diabetic rats [10]. Such increases in plasma ANP concentrations may down-regulate receptors in both the kidney and the endothelium [16]. Although the significance of an increase in plasma ANP concentration in response to hyperglycaemic hyperosmotic volume expansion is easy to understand, the underlying mechanism is by no means clear. Our data showed that ANP content in the atria decreased significantly in both 2- and 4-week diabetic rats, consistent with the finding of Todd et al. [11] of a decreased atrial granularity in rats with diabetes. The decrease of atrial ANP content could be due to the increase of release or degradation, or the decrease of synthesis in the atria. Jin et al [8] observed a lowering of ANP mRNA expression in 11-week diabetic rats

S. Q. Wu et al.: Diabetes on cardiac ANP synthesis

using dot blot analysis. Our solution-hybridizationRNase protection assay results indicated that there was no statistically significant change of ANP mRNA expression in the atria in either 2- or 4-week diabetic rats though the mean values are slightly lower than that of control rats. It is generally accepted that a change in mRNA content is paralleled by peptide synthesis and that atrial ANP content appears to be more dependent on ANP release than on biosynthesis [3]. A decrease in atrial ANP content together with a lack of significant change in preproANP mRNA expression in 2- and 4-week diabetic rats would indicate an increase in ANP release precedes a change in synthesis in the early stage of STZ-induced diabetes. On the other hand, though the atria have the highest ANP peptide content and the highest ANP mRNA expression in all the tissues, the contribution of the heart ventricles to the plasma ANP should not be ignored, especially in the pathophysiological state, as shown in cardiomyopathic hamsters with heart failure [17]. Thus, we feel that both the atria and the ventricles may contribute considerably to increased plasma ANP concentrations in STZ-induced diabetic rats. This is supported by a concomitant increase in both ANP peptide and preproANP mRNA expression in the ventricle of diabetic rats. Given the evidence that the heart ventricles have a different secretion pattern through which ANP is released without being stored in granules [3, 18, 19], our data strongly indicate that the elevation in plasma ANP concentrations is at least partially due to the increased synthesis and release of ANP from the ventricles. The reason for the difference in the responses of atrial and ventricle preproANP mRNA expression to STZ-induced diabetes is not known. A similar differential RNA synthesis between the atrium and the ventricle has been noted (this time a decrease in the ventricle) in the vasopressin deficient Brattleboro rats [20]. Stretch is believed to be a common haemodynamic stimulus for synthesis and release of ANP within the heart [3]. Both atrial and ventricular ANP secretion are increased by increasing blood volume [21, 22, 23]. Increased blood pressure (afterload) can also increase the secretion of ANP from the heart [24, 25] and much greater change of ANP secretion in the ventricle than the atrium has been observed in spontaneously hypertensive rats (SHR) [26, 27]. One would expect to find an increase in both atrial and ventricular ANP synthesis in response to distension and stretch. Perhaps the atrial cells are more susceptible than ventricular cells to the adverse effects of the lack of insulin or hyperglycaemia with the result that atrial synthesis of ANP is impaired. Another possibility is the effect of glucocorticosteroids which are elevated in STZ rats [15] on ventricular release of ANP. This mediated glucocorticosteroid effect is greater on the ventricle than on the atrium [28]. If this is the

S. Q. Wu et al.: Diabetes on cardiac ANP synthesis

case, the ventricle can be an important source of plasma ANP in the diabetic state. Further experiments especially those involving the use of cell cultures are needed to confirm this hypothesis. Acknowledgments. This study was supported by a grant from the Committee on Research and Conference Grant (CRCG). We are grateful to Dr. Tim Yandle (Christchurch, New Zealand) for the gift of hANP anti-serum and to Dr. Dominic Autelitano (Prahran, Australia) for the gifts of preproANP cDNA and b-actin cDNA used in this study.

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