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Epidermal growth factor in serum, urine, submandibular glands and kidneys of diabetic mice
Life Sciences, Vol. 47, pp. 679-685 Printed in the U.S.A.
EPIDERMAL
Pergamon Pre
G R O W T H F A C T O R IN SERUM, URINE, S U B M A N D I B U L A R GLANDS A N D K I D N E Y S OF D I A B E T I C M I C E D.L.Hwang
and A . L e v - R a n
D e p a r t m e n t of Diabetes, E n d o c r i n o l o g y and M e t a b o l i s m City of H o p e N a t i o n a l M e d i c a l C e n t e r Duarte, California, U S A (Received in final form June 18, 1990) Summary L e v e l s of e p i d e r m a l g r o w t h factor (EGF) in serum w e r e s i g n i f i c a n t l y d e c r e a s e d in s t r e p t o z o t o c i n ( S T Z ) - d i a b e t i c m i c e (446+168 pg/ml a f t e r 1 w e e k and 423_+52 a f t e r 4 w e e k s vs 766_+162 pg/ml in controls, P.002 and <.001. respectively) and in g e n e t i c a l l y d i a b e t i c ob/ob m i c e (455+285 vs 962-+453 pg/ml in nond i a b e t i c ob/+ controls, P.043). The u r i n a r y e x c r e t i o n of EGF was s i g n i f i c a n t l y i n c r e a s e d in STZ m i c e (104-+53 vs 51_+23 ng/h, P.013) but u n c h a n g e d in ob/ob mice (33-+9 vs 45_+16 ng/h, P.134). However, w h e n e x p r e s s e d per mg c r e a t i n i n e it was d e c r e a s e d in b o t h cases: in STZ m i c e to 680_+250 n g / m g at i w e e k and 684_+211 at 4 weeks vs 1250_+303 n g / m g in c o n t r o l s (P<.Ol) ; and in the ob/ob m i c e to 552+117 vs 1237_+300 n g / m g in ob/+ c o n t r o l s (P<.01). EGF c o n t e n t of the s u b m a n d i b u l a r g l a n d s of STZ m i c e r e m a i n e d u n c h a n g e d at 1 w e e k (13.1_+2.9 vs 11.0_+1.8 ~g/mg protein, P.170) but d r o p p e d by 4 weeks (4.7_+1.2 ~g/mg, P<.001); in the o b / o b m i c e it was less than 20% that of c o n t r o l s (2.1-+0.8 vs 12.2_+3.6 ~ g / m g protein). In kidneys, the EGF c o n t e n t was not a l t e r e d in e i t h e r ob/ob (524_+50 vs 571+33 p g / m g protein) or STZ m i c e (652-+183 vs 665_+80 pg/mg). The p r e p r o E G F m R N A level in STZt r e a t e d m i c e was r e d u c e d a f t e r 4 w e e k s in s u b m a n d i b u l a r g l a n d s but not in kidneys. The results s h o w that d i a b e t e s affects EGF production, utilization a n d / o r e x c r e t i o n in m i c e and that k i d n e y s are s p a r e d from s u p p r e s s i o n of EGF s y n t h e s i s that is p r o n o u n c e d in the s u b m a n d i b u l a r glands. One of the e a r l i e s t e x p r e s s i o n s of b o t h c l i n i c a l and e x p e r i m e n t a l d i a b e t e s is renal h y p e r t r o p h y and h y p e r p e r f u s i o n (see ref. 1 for r e c e n t r e v i e w ). In rats, this p r o c e s s is e v i d e n t a l r e a d y a f t e r 3 days and c o n t i n u e s for several weeks (2). The p r e c i s e m e c h a n i s m for these c h a n g e s is unknown. Since e p i d e r m a l g r o w t h factor (EGF) exerts m u l t i p l e e f f e c t s (including mitogenesis) on k i d n e y s (see r e v i e w 3), we d e c i d e d to a s s a y its d y n a m i c s in m i c e w i t h g e n e t i c and e x p e r i m e n t a l diabetes. EGF in m i c e i s - p r o d u c e d m a i n l y in the s u b m a n d i b u l a r g l a n d s (4), w h i c h d e l i v e r it to the c i r c u l a t i o n (5). U r i n a r y EGF is m o s t p r o b a b l y not a p l a s m a u l t r a f i l t r a t e but o r i g i n a t e s 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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in the k i d n e y s (6,7). Therefore, our h y p o t h e s i s was that both p r o d u c t i o n and e x c r e t i o n of EGF by the k i d n e y s in d i a b e t e s w o u l d d e m o n s t r a t e c h a n g e s d i f f e r e n t from t h o s e o b s e r v e d in other organs. Several organs of d i a b e t i c r o d e n t s show sharply d e c r e a s e d EGF r e c e p t o r n u m b e r s and k i n a s e activity. This was r e v e r s e d by insulin t r e a t m e n t (8-13). It s e e m e d logical to study if t h e r e w e r e c h a n g e s in EGF content. In this report we m e a s u r e d the EGF c o n t e n t s of serum, urine, s u b m a n d i b u l a r glands and k i d n e y s as well as p r e p r o E G F m R N A e x p r e s s i o n in the s u b m a n d i b u l a r glands and k i d n e y s of m i c e w i t h S T Z - i n d u c e d h y p o i n s u l i n e m i c d i a b e t e s and of ob/ob m i c e w i t h g e n e t i c h y p e r i n s u l i n e m i c disease. Materials
and M e t h o d s
Two m o n t h old m a l e N I H / S w i s s m i c e w e r e o b t a i n e d from S i m o n s e n Lab (Gilroy, CA). D i a b e t e s was induced by two c o n s e c u t i v e d a i l y i.v. i n j e c t i o n of s t r e p t o z o t o c i n , i00 mg/kg each. R a n d o m b l o o d g l u c o s e d e t e r m i n e d 5-7 days later was >450 mg% in the d i a b e t i c a n i m a l s as c o m p a r e d to 115±19 in n o n d i a b e t i c s . In our p r e l i m i n a r y o b s e r v a t i o n we n o t i c e d that the size of s u b m a n d i b u l a r g l a n d s in m i c e one m o n t h after STZ t r e a t m e n t was m u c h s m a l l e r t h a n in the controls. Therefore, two g r o u p s of STZ mice, one and four w e e k s after the STZ a d m i n i s t r a t i o n , w e r e used. T h e i r serum g l u c o s e levels w e r e c o r r e s p o n d i n g l y 564±185 and 691±55 mg/dl, and u r i n e g l u c o s e 8.5±2.4 and 10.7±0.9%. G e n e t i c a l l y obese 2 m o n t h old m a l e s h y p e r g l y c e m i c and h y p e r i n s u l i n e m i c C 5 7 B L / 6 J ob/ob m i c e and t h e i r lean ob/+ c o n t r o l s w e r e p u r c h a s e d from J a c k s o n L a b o r a t o r i e s (Bar Harbor, ME). The serum g l u c o s e was 426±55 m g / d l in ob/ob m i c e vs 226±39 mg/dl in ob/+, the urine g l u c o s e in ob/ob m i c e was 9.7±2.1%. The a n i m a l s w e r e m a i n t a i n e d a c c o r d i n g to the NIH G u i d e for the Care and Use of L a b o r a t o r y Animals. M o s t studies w e r e done in the n o n - f a s t i n g state in the morning, u r i n e was c o l l e c t e d in the m e t a b o l i c cages and removed a f t e r each s p o n t a n e o u s u r i n a t i o n (to p r e v e n t drying); its t i m i n g was recorded. All c o n t r o l animals w e r e a g e - m a t c h e d to the d i a b e t i c mice. M o u s e EGF and rabbit a n t i - E G F a n t i s e r u m l ~ e r e p u r c h a s e d from C o l l a b o r a t i v e R e s e a r c h (Bedford, MA). [ I]-EGF was p r e p a r e d by the C h l o r a m i n e - T m e t h o d (14) to a s p e c i f i c activity 150-200 mCi/mg. The EGF r a d i o i m m u n o a s s a y was done as d e s c r i b e d (15) w i t h slight m o d i f i c a t i o n (13). G l u c o s e w a s d e t e r m i n e d with a B e c k m a n A n a l y s e r II. S u b m a n d i b u l a r g l a n d s and k i d n e y s were h o m o g e n i z e d w i t h 5 v o l u m e s (w/v) of s o l u t i o n c o n t a i n i n g 50 mM Tris-HCl, pH 8.0, I00 m M NaCI, 1 m M EDTA, 0.5% NP40 and 0.5% S o d i u m d e o x y c h o l a t e , and c e n t r i f u g e d at I0,000 g for 15 min. The r e s u l t i n g s u p e r n a t a n t w a s u s e d for p r o t e i n d e t e r m i n a t i o n (Bio-Rad p r o t e i n assay kit) and for EGF r a d i o i m m u n o a s s a y . EGF c o n c e n t r a t i o n in the s u b m a n d i b u l a r g l a n d s and the k i d n e y s was e x p r e s s e d p e r mg of e x t r a c t a b l e p r o t e i n and per organ. S t a t i s t i c a l e v a l u a t i o n of the results was done by the p a r a m e t r i c t-test. For the d e t e r m i n a t i o n of the p r e p r o E G F m R N A expression, total R N A was i s o l a t e d from s u b m a n d i b u l a r g l a n d s and kidneys the g u a n i d i n e / c e s i u m c h l o r i d e m e t h o d (16). R N A was s e p a r a t e d
by by
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e l e c t r o p h o r e s i s in a 1% a g a r o s e gel c o n t a i n i n g 6.6% f o r m a l d e h y d e and b l o t t e d (Northern blot) onto n y l o n m e m b r a n e (ICN R a d i o c h e m i c a l s , Irvine, CA). The cDNA p r o b e for mouse preproEGF pmEGF-26FI2 (17) was o b t a i n e d from A m e r i c a n Type C~ture C o l l e c t i o n (Rockville,MD). The p r o b e was l a b e l ~ d with [ P] by n i c k - t r a n s l a t i o n to a s p e c i f i c a c t i v i t y 2-3xi0 cpm/~g. The n y l o n m e m b r a n e was p r e h y b r i d i z e d at 42°C for 1 h o u r with b u f f e r c o n t a i n i n g 50% formamide, 5 x D e n h a r d t ' s solution, 5 x SSC (0.75M NaCl, 0 . 0 7 5 M s o d i u m citrate), 50 m M s o d i u m p h o s p h a t e (pH 6.5), 0.1% SDS, 200 ~g/ml calf thymus DNA and i00 ~ / m l y e a s t tRNA. The m e m b r a n e was then h y b r i d i z e d w i t h the [ ~ P ] l a b e l e d p r o b e (2x107 cpm in i0 ml) at 42°C for 48 hours with the same b u f f e r but u s i n g 1 x D e n h a r d t ' s s o l u t i o n and 20 m M sodium p h o s p h a t e pH 6.5. The m e m b r a n e was w a s h e d 4 times for i0 min. e a c h w i t h 2 x S S C - 0 . 1 % SDS at r o o m t e m p e r a t u r e f o l l o w e d by 2 w a s h i n g s 15 min. each w i t h 0.2 x S S C - 0 . 1 % SDS at 45°C. The air d r i e d m e m b r a n e was e x p o s e d to K o d a k XRP-5 x - r a y film at - 7 0 ° C w i t h an i n t e n s i f y i n g screen for 3-24 hours. Results The m a i n r e s u l t s are p r e s e n t e d in Figure i. In STZ and o b / o b m i c e EGF level in serum was s i g n i f i c a n t l y d e c r e a s e d as c o m p a r e d to a g e - m a t c h e d controls. U r i n a r y EGF (ng/h) showed s l i g h t d e c r e a s e s in the ob/ob and s i g n i f i c a n t i n c r e a s e s in STZ mice. However, w h e n e x p r e s s e d t r a d i t i o n a l l y p e r mg creatinine, it s h o w e d a s i g n i f i c a n t d e c r e a s e in b o t h d i a b e t i c g r o u p s since in b o t h g r o u p s e x c r e t i o n of c r e a t i n i n e was s i g n i f i c a n t l y higher t h a n in c o n t r o l s (in STZ mice, to 3.87±1.48 vs 0.96±0.28 mg/day, P<.001; in ob/ob mice, to 1 . 4 5 ± 0 . 2 6 vs 0.99±0.18 mg/day, P<.01). The c o n c e n t r a t i o n of EGF in s u b m a n d i b u l a r g l a n d s was s h a r p l y d e c r e a s e d in ob/ob mice. In STZ m i c e a s i m i l a r d r o p was e v i d e n t at 4 weeks but not at one w e e k after STZ. T h i s p h e n o m e n o n was r e f l e c t e d in the total EGF c o n t e n t of the glands: 32 ~g in ob/ob vs 225 #g in ob/+ mice; 351 ~g at 1 w e e k and 88 ~g at 4 weeks in STZ m i c e vs 289 ~g in controls. E G F c o n c e n t r a t i o n in the kidneys e x p r e s s e d in pg EGF per mg p r o t e i n r e m a i n e d u n c h a n g e d in d i a b e t i c mice (Fig.l), and so was the total EGF c o n t e n t per organ (39-41 ng for two kidneys). The d e c r e a s e of p r e p r o E G F m R N A level in the s u b m a n d i b u l a r g l a n d s of d i a b e t i c m i c e (Fig.2) p a r a l l e l e d the d e c r e a s e of EGF p r o t e i n in the same organ. P r e p r o p E G F m R N A in the k i d n e y s r e m a i n e d u n c h a n g e d (Fig.2) c o r r e s p o n d i n g to the stable renal EGF content. Discussion Our o b s e r v a t i o n s c l e a r l y s h o w that b o t h h y p e r i n s u l i n e m i c (ob/ob) and h y p o i n s u l i n e m i c (STZ) d i a b e t e s are a c c o m p a n i e d by s i g n i f i c a n t c h a n g e s in EGF s y n t h e s i s or secretion. Serum EGF level was d e c r e a s e d in all d i a b e t i c mice, even t h o u g h the s u b m a n d i b u l a r gland EGF c o n t e n t in STZ m i c e was d e c r e a s e d only a f t e r 4 w e e k s but not after 1 w e e k of diabetes. These data c o r r e s p o n d to those r e c e n t l y r e p o r t e d (18). E i t h e r EGF d e l i v e r y to the c i r c u l a t i o n s u f f e r s e a r l i e r than EGF s y n t h e s i s in the s u b m a n d i b u l a r gland, or a n o t h e r source of EGF d e l i v e r y is a f f e c t e d by d i a b e t e s b e f o r e the s u b m a n d i b u l a r gland. In the
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Fig. 1 EGF in serum, urine, s u b m a n d i b u l a r g l a n d s and k i d n e y s of nondiabetic (control, ob/+), s t r e p t o z o t o c i n - d i a b e t i c (A) and ob/ob diabetic (B) mice.
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FIG.2 A u t o r a d i o g r a p h of the N o r t h e r n b l o t of total RNA i s o l a t e d from s u b m a n d i b u l a r g l a n d s ~ 0 #g/lane) and k i d n e y s (20 ~g/lane) hybridized with P - l a b e l e d p r e p r o E G F DNA probe.
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first case we w o u l d expect r e l a t i v e l y h i g h EGF e n r i c h m e n t of the g l a n d s at 1 w e e k b u t it w a s not so, and EGF c o n t e n t c o r r e s p o n d e d to the level of m R N A (18, and p r e s e n t study). S i n c e ob/ob m i c e s u f f e r from c h r o n i c disease, the finding of d e c r e a s e d EGF c o n t e n t in t h e i r s u b m a n d i b u l a r g l a n d s was to be expected. It is i n t e r e s t i n g to note that 2 w e e k s of insulin t r e a t m e n t (started at 1 w e e k of diabetes) r e s t o r e d s u b m a n d i b u l a r g l a n d EGF c o n t e n t of STZ m i c e but did not i n c r e a s e it to a normal p l a s m a EGF level (18). U r i n a r y e x c r e t i o n of EGF (ng/h) w a s i n c r e a s e d in early d i a b e t i c STZ m i c e and u n c h a n g e d in the c h r o n i c d i a b e t i c ob/ob mice. T h i s can c o r r e s p o n d to the o b s e r v a t i o n s m a d e in rats that renal c e l l u l a r h y p e r t r o p h y and h y p e r p l a s i a in e x p e r i m e n t a l d i a b e t e s slowly t a p e r off (2). U r i n a r y e x c r e t i o n of EGF was s h a r p l y d e c r e a s e d in b o t h types of diabetes. A note of caution s h o u l d be added at this point. In humans, it is c u s t o m a r y to e x p r e s s EGF e x c r e t i o n p e r mg creatinine. This is j u s t i f i e d by the great s t a b i l i t y of c r e a t i n i n e excretion. In mice, however, as far as we know, diurnal s t a b i l i t y of c r e a t i n i n e e x c r e t i o n was n e v e r shown. G l o m e r u l a r f i l t r a t i o n in m i c e per body w e i g h t is e n o r m o u s even w h e n c o m p a r e d to rats. We found that in d i a b e t e s c r e a t i n i n e e x c r e t i o n was m u c h increased. Creatinine e x c r e t i o n r e f l e c t s g l o m e r u l a r f i l t r a t i o n and EGF e x c r e t i o n r e f l e c t s distal t u b u l a r function. Therefore, w h e n g l o m e r u l a r f i l t r a t i o n is not c o n s t a n t (like in mice), EGF e x c r e t i o n should be d e t e r m i n e d in t i m e d collection, a l t h o u g h this is m u c h more c u m b e r s o m e and t i m e - c o n s u m i n g . S t r e p t o z o t o c i n d i a b e t e s c a u s e d no c h a n g e s in the p r e p r o E G F mRNA, nor in the EGF c o n t e n t of k i d n e y s but in a s i g n i f i c a n t i n c r e a s e of u r i n a r y EGF excretion. I n c r e a s e d e x c r e t i o n requires a b s e n c e of s i g n i f i c a n t t u b u l a r r e a b s o r p t i o n , w h i c h is indeed the case w i t h EGF (19). We s u g g e s t t h a t in early d i a b e t e s with s i g n i f i c a n t h y p e r f i l t r a t i o n or in m o r e severe d i a b e t e s w i t h h i g h o s m o t i c d i u r e s i s some u r i n a r y EGF o r i g i n a t e s in plasma. T h i s m e c h a n i s m was shown in rats (20) and m i c e (D.L.Hwang, u n p u b l i s h e d o b s e r v a t i o n s ) , and d i u r e s i s was m u c h h i g h e r in STZ t h a n in ob/ob m i c e (correspondingly, 0.47 vs 0.ii m l / h in c o n t r o l s in STZ m i c e and 0.06 vs 0.04 m l / h in c o n t r o l s in ob/ob mice). We did not d e t e r m i n e p r e p r o E G F m R N A in the k i d n e y s of o b / o b m i c e but r e c e n t l y it w a s r e p o r t e d that in g e n e t i c a l l y d i a b e t i c d b / d b m i c e it was normal (18). The o b s e r v a t i o n s show that d i a b e t e s d i f f e r e n t i a l l y affects two m a i n organs k n o w n to p r o d u c e EGF ( s u b m a n d i b u l a r glands and kidneys) and results in a l t e r e d EGF b l o o d and u r i n e levels. T h e y also p o i n t to a p o s s i b l e d i f f e r e n c e in this r e s p e c t b e t w e e n s h o r t - t e r m and l o n g - t e r m a n d / o r m o r e or less severe disease. In conclusion, d e c r e a s e d EGF s y n t h e s i s a n d / o r s e c r e a t i o n in the s u b m a n d i b u l a r g l a n d s of d i a b e t i c m i c e c o n t r i b u t e d to the d e c r e a s e d EGF levels in the blood. EGF s y n t h e s i s in the kidneys was not a f f e c t e d by d i a b e t i c conditions. I n c r e a s e d u r i n a r y EGF e x c r e a t i o n in STZ m i c e was due to the i n c r e a s e d o s m o t i c diuresis, d e c r e a s e d t u b u l a r r e a b s o r p t i o n , and a p o r t i o n of the u r i n a r y EGF o r i g i n a t e d in blood.
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Acknowledc[ments This w o r k was s u p p o r t e d in part by a N I H C a n c e r C e n t e r S u p p o r t G r a n t (CA 33572) and the F o u n d e r s for D i a b e t i c Research. The t e c h n i c a l a s s i s t a n c e of Y-C Tay and C-R Chen appreciated.
is
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6. 7. 8. 9. i0. ii.
12. 13. 14. 15. 16. 17. 18. 19. 20.
A.S. REDDI, and R.A. C A M E R I N I - D A V A L O S , Arch. Intern. Med. 150:31-43 (1990) K. S E Y E R - H A N S E N , K i d n e y Int. 2_/3:643-646 (1983) D.A. FISHER, E.C. SAL£DO, and L. BARAJAS, Ann. Rev. Physiol. 5_!1:67-80 (1989) S. COHEN, J . B i o l . C h e m . 237 1555-1562 (1962) J. PERHEENTUPA, J. LAKSHMANAN, S.B. HOATH, U. BERI, H. KIM, T. MACASO, and D.A. FISHER, Am.J. Physiol. 248 E 3 9 1 - E 3 9 6 (1985) J. PERHEENTUPA, J. LAKSHMANAN, and D.A. FISHER, A c t a Endocrinol. 108, 428-432 (1985) P.S. OLSEN, E. NEXt, S.S. POULSEN, H.F. HANSEN, and P. KIRKEGAARD, Regulat. Peptides. I_O0 37-45 (1984) M. KORC, L.M. MATRISIAN, R. NAKAMURA, and B.E. MAGUN, Life Sci. 3_55 2 0 4 9 - 2 0 5 5 (1984) A. LEV-RAN, D.L. HWANG, and G. BARSEGHIAN, Biochem. Biophys. Res. Comm. 137 258-262 (1986) M. KASHIMATA, M. HIRAMATSU, and N. MINAMI, Biochem. Biophys. A c t a 923 496-500 (1987) P.J. BLACKSHEAR, D.J. STAMP, E.A. KENNINGTON, J.S. TUTTLE, D.N. ORTH, K.L. THOMPSON, M.C. HUANG, and M.R. ROSNER, J.Biol. Chem. 262 12356-12364 (1987) M. OKAMOTO, C.R. KAHN, R. MARON, and M.F. WHITE, Am. J. Physiol. 254 E 4 2 9 - E 4 3 4 (1988) D.L. HWANG, A. LEV-RAN, Y.C. TAY, C.R. CHEN, and N. DEV, Life Sci. 44 407-416 (1989) J.R. GAVIN, J. ROTH, P. JEN, and P. FREYCHET, Proc. Natl. Acad. Sci. U S A 6_99 747-751 (1972) S.B. HOATH, J. LAKSHMANAN, S.M. SCOTT, and D.A. FISHER, E n d o c r i n o l o g y 112 308-314 (1983) T. MANIATIS, E.F. FRITSCH, and J. SAMBROOK, M o l e c u l a r cloning. Cold S p r i n g s H a r b o r t Lab., N.Y. (1982) A. GRAY, T.J. DULL, and A. ULLRICH, N a t u r e 303 722-725 (1983) S. KASAYAMA, Y. OHBA, and T. OKA, Proc. Natl. Acad. Sci. USA 86:7644-7648 (1989) S. NIELSEN, E. NEXO, and E.I. CHRISTENSEN, Am. J. Physiol. 256 E55-E63 (1989) D.C. KIM, Y. SUGIYAMA, T. FUWA, S. SAKAMOTO, T. IGA, and M. HANANO,Biochem. Pharmacol. 3_88:241-249 (1989)
EPIDERMAL
Pergamon Pre
G R O W T H F A C T O R IN SERUM, URINE, S U B M A N D I B U L A R GLANDS A N D K I D N E Y S OF D I A B E T I C M I C E D.L.Hwang
and A . L e v - R a n
D e p a r t m e n t of Diabetes, E n d o c r i n o l o g y and M e t a b o l i s m City of H o p e N a t i o n a l M e d i c a l C e n t e r Duarte, California, U S A (Received in final form June 18, 1990) Summary L e v e l s of e p i d e r m a l g r o w t h factor (EGF) in serum w e r e s i g n i f i c a n t l y d e c r e a s e d in s t r e p t o z o t o c i n ( S T Z ) - d i a b e t i c m i c e (446+168 pg/ml a f t e r 1 w e e k and 423_+52 a f t e r 4 w e e k s vs 766_+162 pg/ml in controls, P.002 and <.001. respectively) and in g e n e t i c a l l y d i a b e t i c ob/ob m i c e (455+285 vs 962-+453 pg/ml in nond i a b e t i c ob/+ controls, P.043). The u r i n a r y e x c r e t i o n of EGF was s i g n i f i c a n t l y i n c r e a s e d in STZ m i c e (104-+53 vs 51_+23 ng/h, P.013) but u n c h a n g e d in ob/ob mice (33-+9 vs 45_+16 ng/h, P.134). However, w h e n e x p r e s s e d per mg c r e a t i n i n e it was d e c r e a s e d in b o t h cases: in STZ m i c e to 680_+250 n g / m g at i w e e k and 684_+211 at 4 weeks vs 1250_+303 n g / m g in c o n t r o l s (P<.Ol) ; and in the ob/ob m i c e to 552+117 vs 1237_+300 n g / m g in ob/+ c o n t r o l s (P<.01). EGF c o n t e n t of the s u b m a n d i b u l a r g l a n d s of STZ m i c e r e m a i n e d u n c h a n g e d at 1 w e e k (13.1_+2.9 vs 11.0_+1.8 ~g/mg protein, P.170) but d r o p p e d by 4 weeks (4.7_+1.2 ~g/mg, P<.001); in the o b / o b m i c e it was less than 20% that of c o n t r o l s (2.1-+0.8 vs 12.2_+3.6 ~ g / m g protein). In kidneys, the EGF c o n t e n t was not a l t e r e d in e i t h e r ob/ob (524_+50 vs 571+33 p g / m g protein) or STZ m i c e (652-+183 vs 665_+80 pg/mg). The p r e p r o E G F m R N A level in STZt r e a t e d m i c e was r e d u c e d a f t e r 4 w e e k s in s u b m a n d i b u l a r g l a n d s but not in kidneys. The results s h o w that d i a b e t e s affects EGF production, utilization a n d / o r e x c r e t i o n in m i c e and that k i d n e y s are s p a r e d from s u p p r e s s i o n of EGF s y n t h e s i s that is p r o n o u n c e d in the s u b m a n d i b u l a r glands. One of the e a r l i e s t e x p r e s s i o n s of b o t h c l i n i c a l and e x p e r i m e n t a l d i a b e t e s is renal h y p e r t r o p h y and h y p e r p e r f u s i o n (see ref. 1 for r e c e n t r e v i e w ). In rats, this p r o c e s s is e v i d e n t a l r e a d y a f t e r 3 days and c o n t i n u e s for several weeks (2). The p r e c i s e m e c h a n i s m for these c h a n g e s is unknown. Since e p i d e r m a l g r o w t h factor (EGF) exerts m u l t i p l e e f f e c t s (including mitogenesis) on k i d n e y s (see r e v i e w 3), we d e c i d e d to a s s a y its d y n a m i c s in m i c e w i t h g e n e t i c and e x p e r i m e n t a l diabetes. EGF in m i c e i s - p r o d u c e d m a i n l y in the s u b m a n d i b u l a r g l a n d s (4), w h i c h d e l i v e r it to the c i r c u l a t i o n (5). U r i n a r y EGF is m o s t p r o b a b l y not a p l a s m a u l t r a f i l t r a t e but o r i g i n a t e s 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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in the k i d n e y s (6,7). Therefore, our h y p o t h e s i s was that both p r o d u c t i o n and e x c r e t i o n of EGF by the k i d n e y s in d i a b e t e s w o u l d d e m o n s t r a t e c h a n g e s d i f f e r e n t from t h o s e o b s e r v e d in other organs. Several organs of d i a b e t i c r o d e n t s show sharply d e c r e a s e d EGF r e c e p t o r n u m b e r s and k i n a s e activity. This was r e v e r s e d by insulin t r e a t m e n t (8-13). It s e e m e d logical to study if t h e r e w e r e c h a n g e s in EGF content. In this report we m e a s u r e d the EGF c o n t e n t s of serum, urine, s u b m a n d i b u l a r glands and k i d n e y s as well as p r e p r o E G F m R N A e x p r e s s i o n in the s u b m a n d i b u l a r glands and k i d n e y s of m i c e w i t h S T Z - i n d u c e d h y p o i n s u l i n e m i c d i a b e t e s and of ob/ob m i c e w i t h g e n e t i c h y p e r i n s u l i n e m i c disease. Materials
and M e t h o d s
Two m o n t h old m a l e N I H / S w i s s m i c e w e r e o b t a i n e d from S i m o n s e n Lab (Gilroy, CA). D i a b e t e s was induced by two c o n s e c u t i v e d a i l y i.v. i n j e c t i o n of s t r e p t o z o t o c i n , i00 mg/kg each. R a n d o m b l o o d g l u c o s e d e t e r m i n e d 5-7 days later was >450 mg% in the d i a b e t i c a n i m a l s as c o m p a r e d to 115±19 in n o n d i a b e t i c s . In our p r e l i m i n a r y o b s e r v a t i o n we n o t i c e d that the size of s u b m a n d i b u l a r g l a n d s in m i c e one m o n t h after STZ t r e a t m e n t was m u c h s m a l l e r t h a n in the controls. Therefore, two g r o u p s of STZ mice, one and four w e e k s after the STZ a d m i n i s t r a t i o n , w e r e used. T h e i r serum g l u c o s e levels w e r e c o r r e s p o n d i n g l y 564±185 and 691±55 mg/dl, and u r i n e g l u c o s e 8.5±2.4 and 10.7±0.9%. G e n e t i c a l l y obese 2 m o n t h old m a l e s h y p e r g l y c e m i c and h y p e r i n s u l i n e m i c C 5 7 B L / 6 J ob/ob m i c e and t h e i r lean ob/+ c o n t r o l s w e r e p u r c h a s e d from J a c k s o n L a b o r a t o r i e s (Bar Harbor, ME). The serum g l u c o s e was 426±55 m g / d l in ob/ob m i c e vs 226±39 mg/dl in ob/+, the urine g l u c o s e in ob/ob m i c e was 9.7±2.1%. The a n i m a l s w e r e m a i n t a i n e d a c c o r d i n g to the NIH G u i d e for the Care and Use of L a b o r a t o r y Animals. M o s t studies w e r e done in the n o n - f a s t i n g state in the morning, u r i n e was c o l l e c t e d in the m e t a b o l i c cages and removed a f t e r each s p o n t a n e o u s u r i n a t i o n (to p r e v e n t drying); its t i m i n g was recorded. All c o n t r o l animals w e r e a g e - m a t c h e d to the d i a b e t i c mice. M o u s e EGF and rabbit a n t i - E G F a n t i s e r u m l ~ e r e p u r c h a s e d from C o l l a b o r a t i v e R e s e a r c h (Bedford, MA). [ I]-EGF was p r e p a r e d by the C h l o r a m i n e - T m e t h o d (14) to a s p e c i f i c activity 150-200 mCi/mg. The EGF r a d i o i m m u n o a s s a y was done as d e s c r i b e d (15) w i t h slight m o d i f i c a t i o n (13). G l u c o s e w a s d e t e r m i n e d with a B e c k m a n A n a l y s e r II. S u b m a n d i b u l a r g l a n d s and k i d n e y s were h o m o g e n i z e d w i t h 5 v o l u m e s (w/v) of s o l u t i o n c o n t a i n i n g 50 mM Tris-HCl, pH 8.0, I00 m M NaCI, 1 m M EDTA, 0.5% NP40 and 0.5% S o d i u m d e o x y c h o l a t e , and c e n t r i f u g e d at I0,000 g for 15 min. The r e s u l t i n g s u p e r n a t a n t w a s u s e d for p r o t e i n d e t e r m i n a t i o n (Bio-Rad p r o t e i n assay kit) and for EGF r a d i o i m m u n o a s s a y . EGF c o n c e n t r a t i o n in the s u b m a n d i b u l a r g l a n d s and the k i d n e y s was e x p r e s s e d p e r mg of e x t r a c t a b l e p r o t e i n and per organ. S t a t i s t i c a l e v a l u a t i o n of the results was done by the p a r a m e t r i c t-test. For the d e t e r m i n a t i o n of the p r e p r o E G F m R N A expression, total R N A was i s o l a t e d from s u b m a n d i b u l a r g l a n d s and kidneys the g u a n i d i n e / c e s i u m c h l o r i d e m e t h o d (16). R N A was s e p a r a t e d
by by
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e l e c t r o p h o r e s i s in a 1% a g a r o s e gel c o n t a i n i n g 6.6% f o r m a l d e h y d e and b l o t t e d (Northern blot) onto n y l o n m e m b r a n e (ICN R a d i o c h e m i c a l s , Irvine, CA). The cDNA p r o b e for mouse preproEGF pmEGF-26FI2 (17) was o b t a i n e d from A m e r i c a n Type C~ture C o l l e c t i o n (Rockville,MD). The p r o b e was l a b e l ~ d with [ P] by n i c k - t r a n s l a t i o n to a s p e c i f i c a c t i v i t y 2-3xi0 cpm/~g. The n y l o n m e m b r a n e was p r e h y b r i d i z e d at 42°C for 1 h o u r with b u f f e r c o n t a i n i n g 50% formamide, 5 x D e n h a r d t ' s solution, 5 x SSC (0.75M NaCl, 0 . 0 7 5 M s o d i u m citrate), 50 m M s o d i u m p h o s p h a t e (pH 6.5), 0.1% SDS, 200 ~g/ml calf thymus DNA and i00 ~ / m l y e a s t tRNA. The m e m b r a n e was then h y b r i d i z e d w i t h the [ ~ P ] l a b e l e d p r o b e (2x107 cpm in i0 ml) at 42°C for 48 hours with the same b u f f e r but u s i n g 1 x D e n h a r d t ' s s o l u t i o n and 20 m M sodium p h o s p h a t e pH 6.5. The m e m b r a n e was w a s h e d 4 times for i0 min. e a c h w i t h 2 x S S C - 0 . 1 % SDS at r o o m t e m p e r a t u r e f o l l o w e d by 2 w a s h i n g s 15 min. each w i t h 0.2 x S S C - 0 . 1 % SDS at 45°C. The air d r i e d m e m b r a n e was e x p o s e d to K o d a k XRP-5 x - r a y film at - 7 0 ° C w i t h an i n t e n s i f y i n g screen for 3-24 hours. Results The m a i n r e s u l t s are p r e s e n t e d in Figure i. In STZ and o b / o b m i c e EGF level in serum was s i g n i f i c a n t l y d e c r e a s e d as c o m p a r e d to a g e - m a t c h e d controls. U r i n a r y EGF (ng/h) showed s l i g h t d e c r e a s e s in the ob/ob and s i g n i f i c a n t i n c r e a s e s in STZ mice. However, w h e n e x p r e s s e d t r a d i t i o n a l l y p e r mg creatinine, it s h o w e d a s i g n i f i c a n t d e c r e a s e in b o t h d i a b e t i c g r o u p s since in b o t h g r o u p s e x c r e t i o n of c r e a t i n i n e was s i g n i f i c a n t l y higher t h a n in c o n t r o l s (in STZ mice, to 3.87±1.48 vs 0.96±0.28 mg/day, P<.001; in ob/ob mice, to 1 . 4 5 ± 0 . 2 6 vs 0.99±0.18 mg/day, P<.01). The c o n c e n t r a t i o n of EGF in s u b m a n d i b u l a r g l a n d s was s h a r p l y d e c r e a s e d in ob/ob mice. In STZ m i c e a s i m i l a r d r o p was e v i d e n t at 4 weeks but not at one w e e k after STZ. T h i s p h e n o m e n o n was r e f l e c t e d in the total EGF c o n t e n t of the glands: 32 ~g in ob/ob vs 225 #g in ob/+ mice; 351 ~g at 1 w e e k and 88 ~g at 4 weeks in STZ m i c e vs 289 ~g in controls. E G F c o n c e n t r a t i o n in the kidneys e x p r e s s e d in pg EGF per mg p r o t e i n r e m a i n e d u n c h a n g e d in d i a b e t i c mice (Fig.l), and so was the total EGF c o n t e n t per organ (39-41 ng for two kidneys). The d e c r e a s e of p r e p r o E G F m R N A level in the s u b m a n d i b u l a r g l a n d s of d i a b e t i c m i c e (Fig.2) p a r a l l e l e d the d e c r e a s e of EGF p r o t e i n in the same organ. P r e p r o p E G F m R N A in the k i d n e y s r e m a i n e d u n c h a n g e d (Fig.2) c o r r e s p o n d i n g to the stable renal EGF content. Discussion Our o b s e r v a t i o n s c l e a r l y s h o w that b o t h h y p e r i n s u l i n e m i c (ob/ob) and h y p o i n s u l i n e m i c (STZ) d i a b e t e s are a c c o m p a n i e d by s i g n i f i c a n t c h a n g e s in EGF s y n t h e s i s or secretion. Serum EGF level was d e c r e a s e d in all d i a b e t i c mice, even t h o u g h the s u b m a n d i b u l a r gland EGF c o n t e n t in STZ m i c e was d e c r e a s e d only a f t e r 4 w e e k s but not after 1 w e e k of diabetes. These data c o r r e s p o n d to those r e c e n t l y r e p o r t e d (18). E i t h e r EGF d e l i v e r y to the c i r c u l a t i o n s u f f e r s e a r l i e r than EGF s y n t h e s i s in the s u b m a n d i b u l a r gland, or a n o t h e r source of EGF d e l i v e r y is a f f e c t e d by d i a b e t e s b e f o r e the s u b m a n d i b u l a r gland. In the
682
Epidermal Growth Factor in Diabetic Mice
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Fig. 1 EGF in serum, urine, s u b m a n d i b u l a r g l a n d s and k i d n e y s of nondiabetic (control, ob/+), s t r e p t o z o t o c i n - d i a b e t i c (A) and ob/ob diabetic (B) mice.
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Glonds
Kb
-9.5 -7.5 4.4 -2.4
FIG.2 A u t o r a d i o g r a p h of the N o r t h e r n b l o t of total RNA i s o l a t e d from s u b m a n d i b u l a r g l a n d s ~ 0 #g/lane) and k i d n e y s (20 ~g/lane) hybridized with P - l a b e l e d p r e p r o E G F DNA probe.
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Epidermal Growth Factor in Diabetic Mice
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first case we w o u l d expect r e l a t i v e l y h i g h EGF e n r i c h m e n t of the g l a n d s at 1 w e e k b u t it w a s not so, and EGF c o n t e n t c o r r e s p o n d e d to the level of m R N A (18, and p r e s e n t study). S i n c e ob/ob m i c e s u f f e r from c h r o n i c disease, the finding of d e c r e a s e d EGF c o n t e n t in t h e i r s u b m a n d i b u l a r g l a n d s was to be expected. It is i n t e r e s t i n g to note that 2 w e e k s of insulin t r e a t m e n t (started at 1 w e e k of diabetes) r e s t o r e d s u b m a n d i b u l a r g l a n d EGF c o n t e n t of STZ m i c e but did not i n c r e a s e it to a normal p l a s m a EGF level (18). U r i n a r y e x c r e t i o n of EGF (ng/h) w a s i n c r e a s e d in early d i a b e t i c STZ m i c e and u n c h a n g e d in the c h r o n i c d i a b e t i c ob/ob mice. T h i s can c o r r e s p o n d to the o b s e r v a t i o n s m a d e in rats that renal c e l l u l a r h y p e r t r o p h y and h y p e r p l a s i a in e x p e r i m e n t a l d i a b e t e s slowly t a p e r off (2). U r i n a r y e x c r e t i o n of EGF was s h a r p l y d e c r e a s e d in b o t h types of diabetes. A note of caution s h o u l d be added at this point. In humans, it is c u s t o m a r y to e x p r e s s EGF e x c r e t i o n p e r mg creatinine. This is j u s t i f i e d by the great s t a b i l i t y of c r e a t i n i n e excretion. In mice, however, as far as we know, diurnal s t a b i l i t y of c r e a t i n i n e e x c r e t i o n was n e v e r shown. G l o m e r u l a r f i l t r a t i o n in m i c e per body w e i g h t is e n o r m o u s even w h e n c o m p a r e d to rats. We found that in d i a b e t e s c r e a t i n i n e e x c r e t i o n was m u c h increased. Creatinine e x c r e t i o n r e f l e c t s g l o m e r u l a r f i l t r a t i o n and EGF e x c r e t i o n r e f l e c t s distal t u b u l a r function. Therefore, w h e n g l o m e r u l a r f i l t r a t i o n is not c o n s t a n t (like in mice), EGF e x c r e t i o n should be d e t e r m i n e d in t i m e d collection, a l t h o u g h this is m u c h more c u m b e r s o m e and t i m e - c o n s u m i n g . S t r e p t o z o t o c i n d i a b e t e s c a u s e d no c h a n g e s in the p r e p r o E G F mRNA, nor in the EGF c o n t e n t of k i d n e y s but in a s i g n i f i c a n t i n c r e a s e of u r i n a r y EGF excretion. I n c r e a s e d e x c r e t i o n requires a b s e n c e of s i g n i f i c a n t t u b u l a r r e a b s o r p t i o n , w h i c h is indeed the case w i t h EGF (19). We s u g g e s t t h a t in early d i a b e t e s with s i g n i f i c a n t h y p e r f i l t r a t i o n or in m o r e severe d i a b e t e s w i t h h i g h o s m o t i c d i u r e s i s some u r i n a r y EGF o r i g i n a t e s in plasma. T h i s m e c h a n i s m was shown in rats (20) and m i c e (D.L.Hwang, u n p u b l i s h e d o b s e r v a t i o n s ) , and d i u r e s i s was m u c h h i g h e r in STZ t h a n in ob/ob m i c e (correspondingly, 0.47 vs 0.ii m l / h in c o n t r o l s in STZ m i c e and 0.06 vs 0.04 m l / h in c o n t r o l s in ob/ob mice). We did not d e t e r m i n e p r e p r o E G F m R N A in the k i d n e y s of o b / o b m i c e but r e c e n t l y it w a s r e p o r t e d that in g e n e t i c a l l y d i a b e t i c d b / d b m i c e it was normal (18). The o b s e r v a t i o n s show that d i a b e t e s d i f f e r e n t i a l l y affects two m a i n organs k n o w n to p r o d u c e EGF ( s u b m a n d i b u l a r glands and kidneys) and results in a l t e r e d EGF b l o o d and u r i n e levels. T h e y also p o i n t to a p o s s i b l e d i f f e r e n c e in this r e s p e c t b e t w e e n s h o r t - t e r m and l o n g - t e r m a n d / o r m o r e or less severe disease. In conclusion, d e c r e a s e d EGF s y n t h e s i s a n d / o r s e c r e a t i o n in the s u b m a n d i b u l a r g l a n d s of d i a b e t i c m i c e c o n t r i b u t e d to the d e c r e a s e d EGF levels in the blood. EGF s y n t h e s i s in the kidneys was not a f f e c t e d by d i a b e t i c conditions. I n c r e a s e d u r i n a r y EGF e x c r e a t i o n in STZ m i c e was due to the i n c r e a s e d o s m o t i c diuresis, d e c r e a s e d t u b u l a r r e a b s o r p t i o n , and a p o r t i o n of the u r i n a r y EGF o r i g i n a t e d in blood.
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Epidermal Growth Factor in Diabetic Mice
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Acknowledc[ments This w o r k was s u p p o r t e d in part by a N I H C a n c e r C e n t e r S u p p o r t G r a n t (CA 33572) and the F o u n d e r s for D i a b e t i c Research. The t e c h n i c a l a s s i s t a n c e of Y-C Tay and C-R Chen appreciated.
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