Diabetic syndrome in sand rats

Diabetologia, Apr 1967

A. A. Like, E. Miki

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Diabetic syndrome in sand rats

Diabetic Syndrome in Sand Rats* Elliott P. Joslin Research 0 1 Laboratory 0 1 Boston 0 1 Mass. 0 1 0 Key-words: Spontaneous Diabetes , Sand rat, Psammorays obesus, Pancreas. Ultrastructure, Beta cells, Alpha cells, Protein synthesis, Insulin in plasma, Insulin in pancreas, Obesity, Zqutrition and diabetes, Diet and diabetes 1 Medicine, J:Iarvard Medical School , The of Pathology Summary. UltrastucturM and light microscopic studies of pancreatic islets in normal and diabetic sand rats are reported. Following the institution of a synthetic chow diet, beta cell degranulation and enhanced protein synthesis were observed. With the appearance of diabetes, glycogen infiltration occurred, with displacement of cellular organelles and eventual cytoplasmic degeneration and liquefaction. These alterations were correlated with blood glucose and serum immunoreactive insulin levels. -The sand rats are unique in that they are not able to cope with the increased caloric load of synthetic chow. They respond by marked insulin production; an increase that usually maintains the animal free of ketosis, occasionally returns them to a euglyeemic state, and may rarely be terminated by beta ceil degeneration and necrosis with fatal ketoacidosis. - Syndrome diabdtique ehez le rat du sable (Psammomys obesus). I V . Altdrations morphologiques du tissu insulaire. t~dsum~. ~ o u s avons 6tudid avec les microscopes optique et 61ectronique les riots de Langerhans du pancr6as de rats des sables n o r m a u x et diabdtiques. Lorsqu'ils sont maintenus ~ un r6gime normal de laboratoire, on observe chez ces animaux une ddgranulation des cellules fl et les signes d ' u n e synth~se protdique augment6e. D~s q u ' a p p a r a l t le diab~te, on volt a p p a r a l t r e une infiltration glycogdnique avec d6placement des organelles cellulaires et, plus tard, une d6g6ndrescence eyptoplasmique avee liqu6faetion. L ' a p p a r i t i o n de ces anomalies morphologiques a 6t~ raise en corrdlation avec les alt6rations du glucose sanguin et de l'insuline immunor6active sdrique. Ce qui est remarquable chez le r a t des sables, c'est qu'il ne semble pas 6ire ~ m6me de s'adapter ~ l'apport calorique plus coneentr6 de la nourriture de laboratoire. II r6agit par une surproduction insulinique, surproduction qni, en g~n6ral, dvite la c6tose. Parfois, eerie surproduction suffit ramener le sucre sanguin ~ la normale. Bans des eas plus rares, la stimulation de la s6erdtion insulinique se termine par une d6g~n~rescence et une n6crose des cellules fl avee cdto-acidose mortelle. Zusammenfassung. Es wird fiber elektronen- und lichtmikroskopisehe Untersuchungen an Pankreas-Inseln normaler und diabetischer S a n d r a t t e n berichtet. Nach Verabreichm~g einer synthetischen K e k s - D i a t wurden eine Degranulation der fi-Zellen und Zeichen einer vermehrten Proteinsynthese beobachtet. Gleiehzeitig m i t dem Auftreten yon Diabetes erfolgte Glykogeninfiltration, begleitet yon einer Verdrangung der Zellorganellen und gelegentlicher Degeneration und Verfliissigung des Cytoplasmas. Diese Veranderungen wurden mit Blutzuckerund i m m u n r e a k t i v e m Serum~Insulinspiegel in ZusammerLhang gebracht. -- S a n d r a t t e n sind einzigartig in der I-Iinsicht, dab sie nicht in der Lage sind, das vermehrte Xalorienangebot der synthetischen Diat auf normale A r t zu bewaltigen. Sie reagieren mit Mehrproduktion yon Insulin. Die gesteigerte Insulinausschfittung halt das Tier gewStmlich frei yon Ketose, bringt den Blutzucker gelegentlich auf normale W e r t e zuriick, endet aber in seltenen Fallen mit einer Degeneration und lX!ekrose der fi-Zellen m i t anschlieBender fataler Ketoacidose. I n t h e course of a s y s t e m a t i c s t u d y of t h e d i a b e t i c s y n d r o m e in t h e E g y p t i a n s a n d r a t , Psammomys obesus, a n i m a l s were i m p o r t e d from t h e U n i t e d A r a b R e p u b l i c (U.A.I~) d u r i n g t h e s u m m e r of 1964 a n d 1965. Those r e c e i v e d in 1964 were o b s e r v e d t o develop a f u l m i n a t i n g , a c u t e k e t o t i c d i a b e t i c s y n d r o m e when P u r i n a l a b o r a t o r y chow was g i v e n in place of a diet of m i x e d fresh v e g e t a b l e s (MIKI et al., 1966). The a n i m a l s i m p o r t e d in 1965, a n d t h e i r A m e r i c a n - b o r n offspring, c o n s u m e d less l a b o r a t o r y chow a n d develo p e d a m i l d e r form of t h e disease (MIKI et al., 1967) r e s e m b l i n g m o r e closely t h e s y n d r o m e o r i g i n a l l y r e p o r t e d b y SC~MII)T-~IELSV.~ (1964 ) a n d t I A c ~ L (1965). M a t e r i a l for m o r p h o l o g i c e x a m i n a t i o n was o b t a i n e d from t h e a n i m a l s s t u d i e d physiologically, a n d a d d i t i o n a l specimens were J u l y - A u g u s t , 1965. o b t a i n e d in t h e U . A . R . d u r i n g This r e p o r t gives details of t h e l i g h t microscopic a n d u l t r a s t r u c t u r a l a p p e a r a n c e of t h e p a n c r e a t i c islets of t h e n o r m a l s a n d rat, a n d t h e changes o b s e r v e d a f t e r i n s t i t u t i o n of a s y n t h e t i c chow diet, w i t h a n d w i t h o n t t h e a p p e a r a n c e of d i a b e t e s mellitus. Since physiologic studies were also p e r f o r m e d on t h e s e a n i m a l s , s t r u c t u r a l - f u n c t i o n a l c o r r e l a t i o n s w e r e o f t e n p o s s i b l e . Material and Methods P o r t i o n s of, or t h e e n t i r e p a n c r e a s of 75 s a n d r a t s w e r e o b t a i n e d for m o r p h o l o g i c s t u d y . T h e a n i m a l s w e r e d i v i d e d i n t o 5 g r o u p s . i n g d i a b e t i c s y n d r o m e . T h i s g r o u p of r a t s w a s m a i n t a i n e d e x c l u s i v e l y o n P u r i n a l a b o r a t o r y c h o w . 1 3. 18 a n i m a l s , i n c l u d i n g t h o s e i m p o r t e d i n t o t h e U . S . i n 1965, a n d t h e i r A m e r i c a n - b o r n offspring. T h e s e r a t s c o n s u m e d P u r i n a l a b o r a t o r y c h o w or 01d G u i l f o r d b r e e d i n g p e l l e t s ~ less a v i d l y a n d d e v e l o p e d a m i l d e r f o r m of d i a b e t e s . Key for figures' lettering 1. 12 a n i m a l s k i l l e d i m m e d i a t e l y a f t e r or w i t h i n o n e d a y of c a p t u r e i n t h e U . A . R . w i t h f o o d i n t a k e t h e r e f o r e l i m i t e d e x c l u s i v e l y t o t h a t f o u n d i n t h e v i c i n i t y of t h e i r b u r r o w s (Salicornia fruticosa or Suaeda fruticosa). 2. 10 m e m b e r s of t h e g r o u p i m p o r t e d t o t h e U . S . i n 1964, s o m e of w h o m d e v e l o p e d t h e a c u t e f u l m i n a t 4 . 2 3 r a t s , all A m e r i c a n - b o r n o f f s p r i n g of t h e g r o u p i m p o r t e d i n 1965. T h e y w e r e f e d a m i x e d d i e t of l a b o r a t o r y c h o w a n d f r e s h v e g e t a b l e s a n d w e r e u t i l i z e d t o e v a l u a t e t h e e f f e c t of age o n t h e r e s p o n s i v e n e s s of 1 P u r i n a l a b o r a t o r y c h o w : St. Louis, Missouri. E m e r y Morse C o m p a n y : Guilford, C o n n e c t i c u t . the epididymal fat p a d to exogenous insulin (DE F ~ o ~ z o et al., 1967). 5. 15 animals, which were imported in 196~ and 1965, maintained exclusively on a diet of mixed fresh vegetables and served as controls for the physiologic studies cited above (MIKIet al., 1966, 1967). For electron microscopy, small fragments of pancreas were fixed at 0--5~ in 1.33~o osmium tetroxide, buffered with S-oollidine (0.07 M) with added sucrose (0.122 M) and calcinm chloride (0.005 M). Final p t I was 7.4 to 7.6. Tissue was also fixed at room temperature in 3 % glutaraldehyde buffered with sodium For light microscopy, tissue fragments were fixed in ]~ouin's solution, embedded in paraffin and stained with hematoxylin and eosin and a modified Gomori aldehyde fuchsin m e t h o d (W~aRE~ and LECoM~TE, 1952) . For the demonstration of glycogen, tissue was fixed in alcoholic formalin and paraffin sections were stained with PAS with and without prior digestion with Diastase. oaeodylate (0.1 M) p H 7.4 to 7.6, and a mixture of paraformaldehyde (4%) and glutaraldehyde (5%) buffered with sodium cacodylate (0.07 M) p H 7.1 to 7.2 (KAI~I~OVSKu 1965) . Calcium chloride (0.005 M) was added to both aldehyde fixatives. After aldehyde fixation, tissues were washed in buffer for variable periods of time and subsequently postfixed at 0 - - 5 ~ in osmium tetroxide (prepared as detailed above). All A.A. LncE and E. MINI: Diabetic Syndrome in Sand l~ats tissues were rapidly dehydrated in a graded series of ethanol solutions and embedded in a mixture of E p o n 812 (LvF~, 1961). Sections cut at 1 micron were Animals, diets, methods of glucose and insulin analysis are all described in the accompanying manuscripts (MIKI et al., 1967; DE FRo~zo et al., 1967) . Fig, 7. l'ancreatie islet of euglycemie sand rat, fed chow for 4 days. ~Blood glucose 84 rag/100 hal, serum Ii~I 240 /~U/ml. :Beta cell degrannia~ion is s~riking. Aldehyde fuehsin stain. 260 Fig. 8. Islet from same animal illustrated in Fig. 7. Cytoplasmic granules are virtually absent. :Enhanced staining intensity is due to increased cellular content of ribosomes. Tolnidine blue stain. 640 stained with 1~o toluidine-blue-O in 1~o borax for light microscopic identification of theislets. An occasional islet was stained with PAS (CA~D~O and STEINEI%, 1965) . Thin sections were cut with a diamond knife, using a Porter-Blum ServM1 MT-1 ultramicrotome, m o u n t e d on 100 or 150 mesh copper grids, without supporting membranes, double stained with aqueous or acetone solutions of uranyl acetate followed b y lead (Kmt{movs~:y, i961) and examined with a n 1%CA EMU-3 G microscope. Results Normal islets of Langerhans. The pancreatic islets of the sand rats killed immediately or shortly after capture in the U. A.R. and with no change in diet, are identical in appearance to the islets of animals born either in the U.A. 1%. or the U.S. and fed exclusively a mixture of fresh vegetables. None of the animals killed in the U. A. R. were diabetic. Their blood glucose varied from 35 to 127 rag/100 ml, and the serum Ii%I from 0 to 45/~U/ml. Only one animal among the vege~ig. 9. D e g r a n u l a t e d islet of euglycemie s a n d r a t f e d l a b o r a t o r y c h o w for 4 days. I)eripheral alpha cells r e t a i n their granules. B e t a cells occupy the r e m a i n d e r o f the m i c r o g r a p h a n d contain few cytoplasmic granules arrows w i t h w h i t e head). Mitochondria are a b u n d a n t . O s m i u m tctroxide. A p p r o x i m a t e l y 4300 ~ig. 10. ( P a g e 151) Several b e t a cells f r o m a n a n i m a l w i t h diabetes of b r i e f duration. Blood glucose 326 rag/100 mI, s e r u m I I ~ I > 1500 ttU/ml. Cytoplasmic granules are v i r t u a l l y absent, a n d g r a n u l a r endoplasmie r e t i e u l u m is substantially increased. Dilated s m o o t h surfaced tubules a n d vesicles are p r o m i n e n t (?AR). The latter are s o m e t i m e s continuous w i t h coated vesicles (*arrow). Small arrows indicate the n u m e r o u s coated vesicles, perhaps a n indication of enhanced p r o t e i n (insulin) t r a n s p o r t f r o m the cell. Glutaraldchyde. A p p r o x i m a t e l y 21000 A.A. LIKE and E. lVIIKI:Diabetic Syndrome in Sand l~ats to distinguish the alpha and beta cells on the basis of the staining intensity of the granules (Fig. 2). With the added resolution and magnification of the electron microscope, the beta and alpha cells were more readily identified and differentiated. No other cell types were closely resembled those of the l a b o r a t o r y albino r a t in distribution, n u m b e r and size. The aldehyde-fuchsin stain revealed the numerous well granulated, centrally located beta cells to be surrounded b y a rim of alpha cells (Fig. 1). When 1 micron sections of tissue fixed and embedded for electron microscopy were studied after staining with toluidine blue, the cytoplasmic granules of the islet cells were readily visualized without special stains, and it was sometimes possible observed. The beta cells, which usually occupy the central portion of the islet, were commonly separated from the exocrine tissue b y the more peripheral alpha cells. The a b u n d a n t beta granules of the normal animal were round, and enclosed within loosely fitting s m o o t h - m e m b r a n e d sacs. After p r i m a r y fixation in osmium tetroxide, the beta granules were of uniform density and size (Fig. 3). After aldehyde fixation, beta granules were more numerous, and more varied in A.A. LIKE and E. MI~t: Diabetic Syndrome in Sand Rats size and staining intensity (Fig. 4, 5). Mitochondria were relatively numerous, dispersed throughout the cell and appeared as slender structures with transverse cristae mitochondriales. The granular endoplasmic reticulum was not prominent in the well-granulated electron-dense secretory granules, which were conrained within more tightly fitting sacs (Fig. 4 and 18). The uniform size and staining qualities of the alpha granules did not appreciably v a r y with the fixative utilized. Fig. 12-15. Pancreatic islets of diabetic sand rats Fig. 12. Beta cell degranulation and vacuolization is pronounced. Vacuoles appeal' empty in this prepara tion. Aldehyde fuchsin stain. 260 Fig. 13. Most "vacuoles" are filled with PAS stainable material (arrows) in sections of Epon embedded tissue. Several "empty" vacuoles (V) are visible. PAS stain. 640 cell, and varied inversely with the number of cytoplasmic secretory granules (Fig. 5). The inconspicuous Golgi complex rarely contained secretory granules (Fig. 6) but was otherwise typical in appearance with cisternae, vacuoles, smooth vesicles and coated vesicles. Glycogen was not observed in normal islet cells. The alpha cells were distinguished b y their peripheral location and more specifically b y their more Islets of Langerhans after chow feeding. With the institution of a synthetic chow diet (Purina or Old Guilford pellets) the beta cells in susceptible animals eventually became relatively degranulated. This was particularly apparent in those animals offered synthetic chow prior to weaning. A l t h o u g h observed in the presence of euglycemia (Fig. 7 and 8), degranulation was usually accompanied b y increased levels of A.A. LIKE and E. MIKI: Diabetic Syndrome in Sand l~ats serum immunoreactive insulin (IRI). I n the presence of resting hyperglycemia, beta cell degranulation was more pronounced and the associated serum I R I levels even greater. The degranulated islets of the chow-fed, euglyeemie and hyperglycemic rats appeared considuration in Figs. 10 and 11. The peripheral alpha cells were without alterations. The more centrM b e t a cells were virtually devoid of secretory granules, contained increased numbers of mitochondria (Fig. 9), a corresponding increase in granular endoplasmic derably larger than those of the vegetable-fed animals. Quantitative m e a s u r e m e n t s of islet volume, however, were not performed. T h e duration of c h o w feeding required for degranulation of the b e t a cells was extremely variable and ranged from 4 days to 4 months. The electron microscopic appearance of beta cell degranulation in euglycemic sand rats is illustrated in Fig. 9, and in a sand r a t with diabetes of short reticulum (Fig. i0, ii) a n d m o r e prominent Golgi structures. Present in the vicinity of the cell m e m branes were m a n y m o r e coated vesicles a n d a significant n u m b e r of s m o o t h (agranular) m e m b r a n e d tubules (Fig. I0). Islets of Langerhans in diabetic animals. I n the presence of more pronounced resting hyperglycemia, glycogen infiltration was almost always present. B y A.A. LIKE and E. MIKI: Diabetic Syndrome in Sand Rats light microscopic examination, cytoplasmic vacuolization was observed and a t t r i b u t e d to PAS-stainable, with a non-structured material t h a t stained strongly with PAS (Fig. 13). Alcoholic formalin fixation a n d diastase-digestible material within greatly enlarged beta cells. The " e m p t y " vacuoles observed in paraffin sections (Fig. 12) were usually filled in E p o n sections paraffin embedding permitted diastase digestion prior to PAS staining, and therefore proved the presence of glycogen in the beta cell vacuoles (Fig. 14 a n d 15). A.A. LIKE a n d E. MII4I: D i a b e t i c S y n d r o m e in S a n d R a t s VoI. 3, No. 2, 1967 A.A. LIKE and E. I~_IKI: Diabetic Syndrome in Sand Rats The electron microscopic appearance of degranulated, glycogen-filled beta cells is illustrated in Figs. 16--21. The peripheral alpha cells were unaltered and retained their usuM complement of seeretory granules these cells contained increased quantities of granular endoplasmic reticulum, enlarged Golgi structures, and numerous enlarged mitochondria. The morphologie evidence of enhanced protein synthesis was most (Fig. 16--18). The degranulated beta cells were distorted b y the intraeellular accumulation of glycogen, which displaced and compressed cytoplasmic organelles (Fig. 17--21). The cytoplasmic remnant of obvious in those beta cells with smaller accumulations of glycogen (Fig. 18). Nuclei were never observed with glycogen accumulations. In one sand rat with sustained hyperglycemia A.A. LiKE a n d E . M n ~ : D i a b e t i c S y n d r o m e in Sand R a t s Diabetologia, "Vol. 8 A.A. LIKE and E. Mix:I: Diabetic Syndrome in Sand Rats Diabetotogia (600 mg/100 ml) and a disproportionally low serum I g I (51 /~U/ml), evidence of beta cell degeneration was noted b y light (Fig. 22 and 23) and electron microsnumber of degenerating beta cells (Fig. 25, 26) and small numbers of inflammatory cells (Fig. 27). The evidence of degeneration and possible necrosis was copy (Fig. 24--27). True vacuoles first appeared within the loci of glycogen accumulation (Fig. 24). Together with the increasing number and size of these "true vacuoles" (i.e., not glycogen filled) there appeared a limited to the single sand rat obtained for electron microscopic study in which elevated levels of blood glucose were not associated with the expected markedly elevated levels of serum I R I (MIKI et al., 1967). A.A. LIKE and E. 1VIIKI:Diabetic Syndrome in Sand Rats Discussion The light microscopic and ultrastructural appearance of the pancreatic islets in the normal sand rat cannot be distinguished from t h a t of the albino laboratory rat (LACY, 1961). A third granular cell was not observed in the sand rat (CA~AmA, 1963) ; and although alpha granules varied somewhat in diameter, these differences occurred within the same cell and hence no basis for subdividing the alpha cells into secondary groups was present (CaI~A~A e t a l . , 1965) . Beta cells were more adequately preserved after aldehyde fixation with retention of a greater number of beta granules and the occasional presence of secretory granules within the Golgi apparatus. In general, the amount of granular endoplasmie reticulum was inversely proportional to the number of secretory granules. When susceptible sand rats were fed a diet of synthetic chow, instead of mixed fresh vegetables or their native plants, increased levels of serum Ii~I and beta cell degranulation became manifest, at times in the absence of hyperglycemia. UltrastructurM evidence of enhanced protein synthesis was invariably present. Evidence suggestive of increased release of protein from the cell included the prominently enlarged Golgi complex, and the increased number of coated vesicles and agranular tubules, possibly of Golgi origin, in the vicinity of the cell membranes. Evidence of "emiocytosis" (LACY, 1961) was not present. With the appearance of hyperglycemia, protein synthesis was further increased and the beta cells revealed the presence of variable quantities of glycogen limited to the cytoplasm. The glycogen was not related to any particular cell organelle, but appeared to displace and compress all of the cytoplasmic structures. The extent of the glycogen infiltration varied considerably from cell to cell, perhaps accounting for the maintenance of elevated levels of serum I1%I in these animals. The ultrastructural appearance of the glycogen in the beta cells of the sand rat differs from the classical (R]~v~L, 1960) in t h a t the granules are less electron dense, regardless of the fixative utilized. T h a t the granular infiltrate is indeed glycogen was proven b y the diastase digestibility of comparable infiltrates, as visualized b y light microscopy. The explanation for the different ultrastructural appearance is not presently available. The appearance of liquefaction and true vacuolization was apparently limited to the areas of most concentrated glycogen infiltration. Concomitant alterations include cytoplasmic degeneration, nuclear irregularity with decreased size and increased electron density ( ?.pyknosis), and presumably cell death. The presence of these alterations, in the only diabetic animal without evidence of increased I g I in whom tissue was obtained for electron microscopic study, provides the b~sis, albeit slim, for suggesting t h a t these changes m a y Mso have been present in those animals t h a t developed the fulminating ketotic diabetic syndrome. 11" Diabetologia l~ig. 24. Cytoplasmic r e m n a n t s o f beta cells a t upper left a n d lower right are flattened against cell membranes b y accumulated glycogen. Vacuole formation within glycogen " s t u f f e d " cells parallels other degenerative changes. Alpha cell a n d capillary are normal. Paraformaldehyde-glutaraldehyde. Approximately 13700 A.A. LIxE and E. Mzxz: Diabetic Syndrome in Sand R a t s A.A, L i n e and E. MIni: Diabetic Syndrome in Sand R a t s Acknowledgment. The authors acknowledge the skillful technical assistance of Miss BA~BAn~ BEStir. AI~TEuI~ A. LIICE M.D. Elliott P. Joslin ~esearch Laboratory H a r v a r d Medical School 170 Pilgrim l~oad Boston, M~ssachusetts, 02215 U . S . A . CA~AlWTA ,F. : Electron microscopic description of a third cell type in the islets of the rat pancreas . Amer. J. Anat . 12 , 53 - 64 ( 1963 ). -- B . L . MUNGE ~ and P . E . L ~ c u The ultr~structural basis for the identification of cell types in the pancreatic islets. I. Guinea pig . Z. Zellforsch . 67 , 533 - 546 ( 1965 ). C~DNO , S.S. , and J . W . STEI ~E~: I m p r o v e m e n t of staining technics for thin sections of epoxy-embedded tissue . Amer. J. clin. Path . 43 , 1 - 8 ( 1965 ). DEFtCo~zo, I~ ., E. MI~:I and J. S~I~INKI~: Diabetic syndrome in sand rats. I I I . Observations on adipose tissue and liver in the non-diabetic stage . Diabetologia , 3 , 140 - 142 ( 1967 ). HAOKE~ , D.B. , L. F R O ~ A N , E. Mr~A% I L E . L~ ,novI~z, K. SCHMIDT-NIELSEN and T.D. KINNEY : Effect of diet on tlae glucose tolerance and plasma insulin levels of the sand rat (Psammomys Obesus) . Diabetes 15 , 105 - 114 ( 1966 ). -- K. SCmVXID ~-NmLSESr,t t . B . II-IAI~r~Sand E. MIKA ~: Diabetes mellitus in the sand rat, Psammomys obesus . Pathologic studies. Lab . Invest. 14 , 200 - 207 ( 1965 ). HAI >tEs, H.B., D.B. HAC ~Ea~ and ] K :. SCItlWIDT-:N'IELSEN: E x p e r i m e n t a l diabetes mellitus induced by diet in the sand rat . Amer. J. Physiol. 21)8 , 297 - 300 ( 1965 ). I~2a~ NOVSKY , M . J . : A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy . J. Cell. Biology 27 , 137A ( 1965 ). -- Simple methods for "Staining with lead" at high pIK in electron microscopy . J. biophys, biochem. Cytol . 11 , 729 - 732 ( 1961 ). LACY , P . E . : Electron microscopy of the normal islets of Langerhans . Diabetes 6 , 498 - 507 ( 1957 ), -- A . F . C~ DEZ~ and W. D. WILSON : Electron microscopy of the rat pancreas . Effects of glucagon administration. Diabetes 8 , 36 - 44 ( 1959 ). LUFT , J . t t . : I m p r o v e m e n t s in epoxy resin embedding methods . J. biophys, bioehem. Cytol . 9 , 409 - 414 ( 1961 ). 3 /[IKI, E. , A.A. LIKE , J . S . SO ]~LD~ER , J. S ~ EIN~d~ and G.F. CA]~ ILL ,Jr. : Acute ketotic-type diabetic syndrome in sand rats (Psammomys obesus) with special reference to the pancreas . Metabolism 15 , 149 - 160 ( 1966 ). -- -- J. STnI ~KE and J. S. SO ~ LD~ER : Diabetic syndrome in sand rats. II. Variability and association with diet . Diabetologia , 3 , 135 - 139 ( 1967 ). ]~]~v]~L, J . P . , L. NAPOLI~ A~O and D . W . FAWCE ~ T: Identification of glycogen in electron mierographs of thin tissue sections . J. biophys, biochem. Cytol . 8 , 575 - 589 ( 1960 ). SC~MII)T-N]: ELSEN , K. , H . B . HAINES and D . B . HACKEI ~: Diabetes me]litus in the sand rat induced by standard laboratory diets . Science 143 , 689 - 690 ( 1964 ). W ~ E N , S. and LECo~PwE P. : The pathology of diabetes mellitus . Philadelphia, Lea and Febige, 3rd Edition . p. 325 , 1952

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A. A. Like, E. Miki. Diabetic syndrome in sand rats, Diabetologia, 1967, 143-166, DOI: 10.1007/BF01222192