Studies of hereditary-obese mice (obob) after implantation of pancreatic islets in Millipore filter capsules

Diabetologia, Jun 1970

Summary Obese mice were implanted with Millipore diffusion chambers containing islets isolated from pancreas of normal littermates. Controls in this study were either obese implanted with encapsulated obese islets and empty chambers or non-obese implanted with encapsulated obese islets, non-obese islets and empty chambers. — Regular checks were made on all mice for weight gains and glucose levels. In addition, samples of blood were pooled from each group for immunoreactive insulin determinations. All mice, except one-half the experimental group, were sacrificed at 45 days andpost-mortem examinations performed. The capsules containing non-obese islets were removed from the remaining obese mice and weight gains followed for an additional 45 days. These mice were then sacrificed and determinations made for glucose and insulin levels. — The results showed that weight gain was stabilized, glucose and insulin levels in obese mice were reduced within 14 days after implanting Millipore diffusion chambers containing non-obese pancreatic islets. Weight gain resumed immediately and elevated glucose and insulin levels were found 45 days after removing Millipore diffusion chamber. — These results led to the following conclusions: 1. The spontaneous obesity which develops in the hereditary obese mouse results from a missing or defective pancreatic islet factor or factors. — 2. This factor originates from the islets of Langerhans and is capable of passing through 0.45 μ pores of the Millipore membrane. — 3. This factor's presence is necessary for normal glucose and lipid metabolism and insulin sensitivity.

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Studies of hereditary-obese mice (obob) after implantation of pancreatic islets in Millipore filter capsules

Studies of Hereditary-Obese Mice (obob) after Implantation of Pancreatic Islets in Millipore Filter Capsules R . L . STI~ 0 0 D e p a r t m e n t of Biology, The American University , Washington D. C., U S A , USA Summary. Obese mice were implanted with Millipore diffusion chambers containing islets isolated from pancreas of normal littermates. Controls in this study were either obese implanted with encapsulated obese islets and e m p t y chambers or non-obese implanted with encapsulated obese islets, non-obese islets and e m p t y chambers. -- l~egular cheeks were made on all mice for weight gains and glucose levels. I n addition, samples of blood were pooled from each group for immunoreactive insulin determinations. All mice, except one-half the experimental group, were sacrificed at 45 days and post-mortem exami~ nations performed. The capsules containing non-obese islets were removed from the remaining obese mice and weight gains followed for an additionaI 45 days. These mice were then sacrificed and determinations made for glucose and insulin levels. -- The results showed t h a t weight gain was stabilized, glucose and insulin levels in obese mice were reduced within 14 days after implanting Millipore diffusion chambers containing non-obese pancreatic islets. Weight gain resumed immediately and elevated glucose and insulin !evels wore found 45 days after removing Millipore diffusion chamber. -- These resuits led to the following conclusions: 1. The spontaneous obesity which develops in the hereditary obese mouse results from a missing or defective pancreatic isIet factor or factors. -- 2. This factor originates from the islets of Langerhans and is capable of passing through 0.45 ~ pores of the Millipore membrane. -- 3. This factor's presence is necessary for normal glucose and Iipid metabolism and insulin sensitivity. Etude de la souris obob aprgs implantation d'~lots pancrdatiques dans des capsules Millipore Rgsumd. Des ehambres ~ diffusion Millipore contenant des dots isolds de paner4as d ' a n i m a u x n o r m a u x out dt6 implant4es ~ des souris obbses. Dens cette 6rude, los anim a u x t4moins 4taient soit des animaux ob@ses implantds avec des ilots eneapsulds d ' a n i m a u x oh@sea ou avec des chambres vides, soit des animaux non-ob@ses implant4s avee des ilots oneapsul4s d ' a n i m a u x ob~ses, non ob@ses et avee des chambres rides. Des contr61es r4guliers ont 4t4 effectu4s chez tous les animaux quant au gain de poids et aux glyc4mies. De plus, des mesures de 'insuline immunor4active ont 4t4 effectugs sur des 4chantillons de sang provenant de plusiem-s animamx de cheque groupe. -Toutes les souris, ~ I'exception de la moiti4 du groupe exp4rimental, out 4t4 sacrifi4es au 45e jour de l'exp4rienee et des examens post-mortem ont dr4 effectuds. Los capsules contenant des ilots d ' a n i m a u x non-obbses out 4t4 retir4 des souris ob~ses restantes et le gain de poids a 4t4 suivi pour une entre pdriode de 45 jours. Apr@s sacrifice de cos souris, des mesures de glyc4mies et des t a u x d'insuline out dt4 effectuges. -- Los r4sultats de eette exp4rience out montr4 que le gain de poids s'4tait stabilis4, que los glycgmies et los t a u x d'insuline plasmatique des souris ob@ses s'dtaient r4duits au tours des 14 jours suivant l'implantation des ehambres Millipores contenant des ilots paner6atiques des animaux non-ob~ses. Le gain de poids reprenait imm6diatement et lea glyegmies et les t a u x d'insuline plasmatiques 41evds ont 4t4 trouv4s 45 jours aprbs I'ablation des chambres de diffusion Millipore. -- Les r4sultats de cette dtude nous e m i n e n t aux conclusions suivantes: 1. L'ob4sit6 spontange qui se dgveloppe chez la souris ayant une obgsit4 h4r6ditaire r4sulte d'une terence relative ou absolue d ' u n ou de plusieurs facteurs insulaires pancrgatiques. 2. Co facteur est originaire des ilots de Langerhans et il est capable de traverser los pores de 0.45 ~ des filtres Millipore. 3. La prgsence de ce faeteur est ngcessaire pour maintenir aussi bien une glyc4mie normale qu'uno sensibilit4 ~ l'insuline et un mdtabolisme lipidique normaux. Untersuchung der obob M a u s nach Implantation yon Langerhans'schen Inseln in Milliporekammern Zusammenfassung. Isolierte Langerhans'sche Inseln normalgewichtiger Geschwister wurden in Millipore-Kapseln in die PeritonealhShle yon obob Mgusen implantiert. Als Kontrollen dienten obob M/iuse, denen yon obob erhaltene Inseln odor leere Milliporekammern implantiert wurden und normale Tiere, die entweder Inseln yon obob, yon Kontrolltieren odor leere K a m m e r n trugen. I n regelm/il?igen Abstiinden wurden K6rpergewicht und Blutzuckerkonzentration kontrolliert. Augerdem wurden bei dieser Gelegenheit Blutproben allot Tiere zur radioimmunologisehen Bestimmung der Insulinkonzentration gepoolt. Nach 45 Tagen wurden, mit Ausnahme der H/ilfte der mit Normalinseln implantierten obob M/~use, s/imtliche Tiere getStet. Bei den iiberlebenden obob M/iusen wurden die von Normaltieren erhaltenen Langerhans'schen Inseln samt Millipore-t~apseln entfernt und die Beobachtung weitere 45 Tage fortgefiihrt. Es zeigte sieh, dal3 die Implantation normaler Inseln in obob M/iusen innerhalb yon 14 Tagen zu einer Stabilisierung des K6rpergewiehts a n d zu einem Abfall der Blutzueker- und Insulinkonzentrationen ffihrte. Nach Entfernung dot Kapseln erfolgte ein erneuter Gewichtsanstieg und 45 Tage nach dieser letzten Operation waren Blutzueker und Insulinkonzentrationen wieder erh6ht. -- Die Resultate lessen folgende Schliisse zu: 1. Des Spontanauftretcn des obes-hyperglykiimischen Syndroms der obob Ms ist auf den abolsuten oder roleriven Mangel eines in den Langerhans'sehen Inseln enthaltenen Faktors zuriickzufiihren. 2. Diese Substanz diffundiert dutch die 0.45 g weiten Porch der MilliporelVIembran. 3. Dieser F a k t o r gew/ihrleistet die Aufrechterhaltung eines normalen GIueose- a n d Fettstoffwechsels sowie einer normalen Insulinempfmdlichkeit. Key-words: Spontaneous diabetes, mutation obob, diabetes in mice, obesity in mice, implantation, transplantation, millipore filters, pancreas, insulin. On l o n g t e r m diet restriction, t h e obese-hyperglyof t h e blood will be reduced, b u t n o t to the n o r m a l cemic mouse becomes sensitive to insulin, t h e islets decrease in size a n d n u m b e r of cells a n d life span is l e n g t h e n e d (Lane a n d Dickie, 1958) . T h e glucose level level o b s e r v e d in non-obese mice. The obese-hyperglycemic mouse, on l i m i t e d food intake, still appears obese e v e n w h e n w e i g h t is c o m p a r a b l e t o controls (Mayer, 1960) . Alloxan t r e a t m e n t of obese mice produces a transient lowering of blood sugar levels and causes an increase in granulation of the beta cells, but produces a degranulation in normal sibling (Solomon and Mayer, 1962). Adipose tissue from obese mice shown h y p e r t r o p h y and hyperplasia of i n d i v i d u a l fag cells (Hausberger, 1959). I t was observed t h a t adipose isografts, made between obese and normal littermates, take on the characteristics of the host and not the donor. This would appear to indicate t h a t the metabolic defect found in obese adipose tissue is dependent upon some extra-adipose factor and not inherent in the fat cells. IIausberger (1958) demonstrated t h a t parabiosis of the hereditary-obese mouse with a non-obese litterm a t e suppressed, and in some cases, prevented weight gains beyond pre-union levels. After separation, all obese parabionts gained weight rapidly, indicating t h a t some factor which can be t r a n s m i t t e d b y successful parabiosis is missing in the hereditary-obese mouse. The fact t h a t subtotal and partial p a n c r e a t e e t o m y in the obese mouse reduces insulin levels, but fails to reduce obesity and weight gains (Mayer, et al., 1953) leads to the hypothesis t h a t the pancreas m a y be involved in the production of the lipostatic factor. Further evidence for this theory was obtained when islets implants from normal littermates were transferred to obese mice (Strautz, 1968) . The blood sugar levels, plasma insulin concentration, and weight gains were reduced in these mice, while no effect was seen in non-obese mice which received obese islets. The absence of such an islet factor in fat mice has been postulated b y several authors (Hausberger, 1958; Mayer and Thomas, 1967; Strautz, 1968) , although to date, no one has conclusive proof for its humeral characteristics or origin. The following study was designed to reexamine this problem b y repeating an earlier experiment (Strautz, 1968) on islet implants. Materials and Methods Obese and non-obese mice of the inbred strain C57/ B L / 6 J were weaned, separated according to sex at five to six weeks and separated into groups as indicated in Table 1 a. Blood was taken, at seven weeks, via the tail vein from each animal for glucose and insulin determination, after which each received intraperitoneal implants of sealed mlllipore chambers conraining either: a) 200--250 (approximately) isolated islets from the pancreas of non-obese mice, b) 200--250 (approximately) isolated islets from the pancreas of obese mice, or c) no tissue (empty chambers). The obese mice with islets from non-obese were the experimental group and the mice of all other groups regarded as controls. The weight was checked daily for 45 days and blood withdrawn for glucose and insulin levels at 7, 14, 30 and 45 days post-implantation for each animal. Blood glucose levels were determined for each animal, insulin levels on pooled blood samples from each group. Source and care of mice. All mice used in this study originated from a single breeding pair, heterozygous for the obese gene (-~/ob) and obtained from the Jackson Labs, Bar Harbor, Maine, 1965. The litters were weaned at a b o u t 5 weeks of age and separated according to sex and Jlater according to phenotype. Mice homozygous for tJhe obese gene (ob/ob) could be distinguished about this time b y the excess accumulation of abdominal fat. The heterozygous (4z/ob) and homozygous (-~ / + ) non-obese cannot be differentiated. All animals were housed in plastic lab cages a t relatively constant t e m p e r a t u r e (24~ The mice had free access to food, in the form of Purina lab pellets and t a p water. Greens were given once a week and a special vitamin supplement added to the drinking water every other week (Preterit-Roche Pharmaceuticals). Technique of islet isolation. Pancreatic islet isola tion was performed in obese and non-obese donors b y following, with a few modifications, the procedures described b y K o s t i a n o v s k y and L a c y (1966) for the rat. All operations were performed with the aid of 10 magnification of a binocular dissecting microscope. Animals at seven weeks of age were anesthetized with N e m b u t a l (Abbott Laboratories) and secured to an operating board in a supine position. The skin in the abdominal area was cleaned with 70~o alcohol. All instruments were sterilized b y immersion in a tincture of zephiran chloride. A mid-line incision was made through the skin, muscle, and peritoneal lining extending a p p r o x i m a t e l y 3 - - 4 centimeters caudal to the sternum. The skin was retracted and the intestines m o v e d to expose the diffuse pancreatic tissue. The duodenum was clamped at the pyloric sphincter and below the level of the pancreatic ducts entrance. The upper clamp also occluded the bile duct. The acinar tissue of the pancreas was disrupted b y injecting a p p r o x i m a t e l y 5 ml of H a n k ' s solution into the duodenum. This method was used since, in the mouse, the exoerine pancreas drains directly into the duodenum through several pancreatic ducts. The b o d y and tail of the pancreas were removed, trimmed, and washed in two changes of t I a n k ' s solution. The pancreas was then placed in a freshly prepared solution of 1~o collagenase (Nutritional Biochemical Corporation, Cleveland, Ohio) and cut into small pieces with scissors. The tissue was stirred in a closed weighing bottle using a magnetic stirrer and incubated 20 min at 37~C. After incubation of the pancreas with collagenase, the mixture was diluted with 15 to 25 ml of H a n k ' s solution in a conical, graduated centrifuge tube and allowed to settle for one minute. The intact islets would settle to the b o t t o m of the cylinder during this interval. The supernatant was removed with a syringe and needle and discarded. The sediment containing the islets was resuspended in t I a n k ' s solution and allowed to settle for 30 sec. This procedure was repeated for a total of eight times using cold H a n k ' s solution Diabetologic~ for the last four suspensions. The sediment remaining was diluted with H a n k ' s solution, transferred t o a glass dish surrounded b y an ice bath, and examined with a dissecting microscope. The unstained islets could be recognized easily under a dissecting microscope when viewed aginst a black background. T h e y appeared as free, round or ovoid structures with a greyish-white to brownish-red color. A small glass loop, which was slightly larger t h a n the diameter of the islets, was used to transfer individual islets into another dish containing cold H a n k ' s solution. A p p r o x i m a t e l y 100--120 islets could be transferred within a period of 30 min. The final transfer of islets into the diffusion chambers was accomplished b y using a 0.5 ml syringe. Diffusion chambers. To retain the islet cells, diffusion chambers were constructed of nylon reinforced Millipore m e m b r a n e s with a 0.45 ~ pore rating and a thickness of 100 ~ ~= 10 ~. Squares of slightly less t h a n one cm. were cut and the edges of two pieces were approximated. Three sides were sealed using a thin film of MF cement (formulation No. 2). The assembled chambers were sterilized b y formalin v a p o r for several hours and t h e n filled with a p p r o x i m a t e l y 200--250 isolated pancreatic islets from either obese or nonobese mice. The remaining side of the chambers were sealed with MF cement and k e p t in sterile H a n k ' s solution for not more t h a n 10 min before implanting. Method of implanting. Within a few hours following the initial blood glucose and plasma insulin determinations, obese and non-obese mice were anesthetized with N e m b u t a l and secured, in a supine position, to an operating board. After cleaning the abdominal area with 70% alcohol, a mid-line incision was made just large enough to a d m i t the diffusion chamber. The abdominal muscles and fascia were separated and the chambers inserted among the intestinal coils. The wound was closed using a single autoclip (ClayAdams), which was removed at 5 days post-operation. The animals were allowed to recover under a warming lamp for a few hours. Weight and blood glucose determination. All animals were k e p t for 45 days and weighed, on a dietary balance, daily at a p p r o x i m a t e l y the same time of day. Blood was t a k e n at m i d - d a y from the tail at 0, 7, 14, 30, and 45 days. A p p r o x i m a t e l y 0.15 ml was used for b o t h glucose and insulin levels. The blood glucose concentration of all animals was determined b y a micro-modification of the Somogyi-Nelson method. Duplicate determinations were done on 0.05 ml of whole blood and group averages compared using a s t a n d a r d "T" test. Immunoassay for insulin. The insulin level was determined b y an i m m u n o a s s a y which consisted of a double a n t i b o d y system (Morgan and Lazarow, 1962) . The theory behind this m e t h o d of determination is based on the observation t h a t l a I tagged insulin (Abbott Laboratories, Oak Ridge, Tennessee) can be bound in vitro with anti-insulin serum obtained from immunized guinea pigs (AIS-GP, obtained from Penrex, Kankakee, I l l . ) to form a soluble complex. This complex is then quantitatively precipitated b y antiguinea pig serum a n t i b o d y obtained from immunized rabbits (AGPS-R obtained from Fisher Scientific, Silver Spring, Md.). W h e n l a I insulin is used as a tracer, the a m o u n t of radioisotope present in the precipitate is a function of the concentration of unlabeled insulin present in the reaction mixture. The difference in the percent of l a I tagged insulin precipitated, when varying amounts of unlabeled insulin were added, forms the basis of the quantitative assay. A mouse insulin preparation for immunoassay has not yet been described and the mouse insulin values stated are approximations expressed in beef equivalents. Histological examination. A t 45 days, all mice except one-half the experiment group were sacrificed and the endogenous pancreas and Millipore filter chamber, with contents, preserved in Bouin's fixative. Histological preparation were made and stained b y the Azan technique (gomeis, 1948). Supplementary studies. The remaining animals in the experimental group of obese mice were anesthetized and the diffusion chamber, containing non-obese islets, removed and prepared as above. The obese mice were then followed b y daily weighings for an additional 45 days, after which blood glucose and insulin levels were determined, the animal sacrificed, and its pancreas preserved for histological observation. Results The results summarized in Tables 1--3 are based on 88 animals t h a t survived the initial implantation of Millipore diffusion chambers. E a c h figure (except insulin levels) represents the arithmetic m e a n of group samples  standard error. The body weight for the obese and lean mice are given in Table 1 a for 45 days following implantation of the Millipore chambers. No significant difference in weight exists, at a n y interval, between the groups of non-obese mice containing encapsulated obese islets, non-obese islets or e m p t y chambers ( P = >0.05). A p p a r e n t l y the animals own islet tissues m a i n t a i n a balanced carbohydrate-lipid metabolism for normal weight gains. W h e n comparing groups of obese mice with encapsulated obese islets and obese mice with e m p t y diffusion chambers it was observed t h a t there was a significant and uniform weight gain. The hypothetical factor necessary to prevent this gain in weight is lacking in the islets of the obese mice. The obese mice with non-obese encapsulated islets, however, show a significant reduction in weight gain after 7 days (P=0.003), when compared to other obese groups. This weight gain in obese with non-obese islets parallels the minimal weight increase of all non-obese groups. As was true in a previous experiment (Strautz, 1968) , the prior accumulation of abdominal fat rerosined, even though there was a stabilization in weight gains. 45 days post-implantation, four obese mice from the group containing encapsulated non-obese islets, were anesthetized with ~qembutal and restrained onto an operation board. The abdominal walls, of the obese mice were opened surgically, as described in material and methods and the Millipore diffusion chamber removed from its' vascular connective tissue bed in the host's abdominal cavity. The capsules were prepared for histological examination as described previously. The surgical wounds of these obese mice, were closed, using wound clips and the mice placed under a warming lamp until they had recovered. As shown in table l b, the weight gains returned almost immediately after removal of the diffusion chamber and continued to rise up to d a y 90 (45 days after removal of chamber) the elevated glucose levels lead to glucosuria, symptoms pointing to a similarity between the obese mouse and the m a t u r i t y onset diabetes in man. The hyperglycemia, noted in the obese mouse with non-obese islets was reduced at d a y 14 and remained significantly lower through d a y 45 ( P ~ - < 0 . 0 0 l ) . At d a y 30 through day 45 it compared statistically with levels shown in non-obese groups (P---->0.25). The glucose concentration in obese mice after the normal islets were removed did not, however, approach the level observed in other obese groups at 45 days. The importance of this fact is noted in the similar responses in weight gains and insulin levels (see below) after the encapsulated normal islets had been removed from the obese mice. Immunoreactive insulin was determined in obese and lean mice at various intervals, as is shown in Average weight in grams -- days following diffusion chamber implantation 29.170.5 31.0~:0.5 30.5q:0.5 20.8~:0.4 20.0 zF0.6 21.1 ~:0.4 30.0~:0.5 34.6 :F0.5 33.0:F0.8 21.7T0.5 19.7T0.6 21.8T0.5 14 29.8~:0.5 38.6~:0.9 37.7~:0.7 22.5:~0.5 21.1 ~:0.5 22.4:F0.7 28 31.3~:0.8 43.3:F1.0 42.5:~0.9 23.4:F0.6 22.9:~0.5 22.9~:0.6 45 33.6:F0.9 50.9 :]: 1.3 50.4:[:1.2 25.0~:0.6 24.9:F0.6 24.6:F0.6 ~o increase 13.9 39.1 39.5 16.8 19.7 14.2 Group Average weight in grams -- days following removal of chamber at which time the animals were sacrificed and blood t a k e n for glucose and insulin level determinations. The total weight gain was still lower a t 90 days then the other two obese groups (those with islets and e m p t y chambers) a t 45 days. These obese control groups had 39.1~ and 39.5% increase in weight during the 45 days following implantation, whereas the obese group (with non-obese islets) gained only 23.5% after the chambers had been removed. The increase in weight between 45 and 90 days (after removal of diffusion chambers) was seen as additional accumulation of abdominal fat. Blood glucose levels both before and after islet implantation are shown in Table 2. The levels were similar in all non-obese groups t h r o u g h o u t the experim e n t ( P = >0.2), but rose with each determination in obese mice containing obese islets and e m p t y chambers. These results agree with previous published findings (Wrenshall, et al., 1955) in showing higher glucose levels in m a t u r e obese mice. After the fourth m o n t h Diabetologia a m o u n t sufficient to cause convulsions in normal nonobese mice. The blood glucose concentration had fallen b y 43% after one hour, as compared to levels in these same mice before the insulin injection. This quantity of insulin has been shown to produce an elevation in an already high blood sugar in obese mice (Mayer et al., i951). I n this case the minimal reduction of hyperglycemia, in obese mice shown to be insulin resistant, is probably masked b y the concomitant rise initiated, during the stress of handling and injection. Post-mortem examinations revealed t h a t the diffusion chambers had become enclosed in a well vascularized fibrous connective tissue envelop. There was no evidence of disruption of the cell retaining Millipore Discussion Factors responsible for preventing excessive weight, hyperglycemia and hyperinsulinism in normal mice can be transmitted to obese mice b y intraperitoneal implants of pancreatic islets taken from non-obese littermates, as shown in previous experiments (Strautz, 1968) . Results of the present work summarized in Table 1 a, clearly show t h a t obese mice implanted with nonobese islets in Millipore diffusion chambers, did not have the weight gains recorded for obese mice with empty chambers. At the same time glucose and insulin levels of the blood were reduced to limits observed in non-obese mice (Tables 2 and 3). This evidence suga p ~ 0.06 at each interval. b p ~ 0.2 at all intervals. c 45 days after removal of chamber from 4 mice chambers and all contained granulated cells. Staining revealed cell constituents within the capsules differing from normal islets. The predominant cell type observed, was large (about 20 ~) and contained purple staining granules. There was little increase in the fibrous elements around groups of cells and an 0eeasional pancreatic duct could be identified among the other components. No morphologic or pathologic changes could be seen in the endogenous pancreatic tissue taken from non-obese mice. Obese mice did, however, reveal hypert r o p h y and hyperplasia of the islets, as seen after the onset of this syndrome in previous studies. No differences in the size of islets was noted between obese groups but an occasional mitotic figure was seen in obese mice with implanted obese islets or e m p t y diffusion chambers. gests t h a t some component of the non-obese pancreas produces and releases the factors capable of influencing fat deposition, probably through it's control on glucose metabolism. If this observation is analogous to the normal process of fat deposition, then the influence m a y be mediated either by direct action on the peripheral tissue as postulated b y Mayer and Thomas (1967), or indirectly b y its control of insulin response to blood glucose loads as suggested b y Paulsen, et al. (1968). The data presented in Table 1a also implies t h a t substances originating in the non-obese pancreatic islets are capable of passing through 0.45 ~ pores of the Millipore membrane and influencing weight gains in the obese mice. Post-mortem gross and microscopic examinations of Millipore chambers from obese mice gave no evidence of disruption or loss of ability to retain cells. The results presented here show t h a t diffusable factors, elaborated b y non-obese pancreatic islets play a major role in reversing, and probably preventLug, conditions responsible for the obese state in mice. The pancreatic source of this factor was suspected after the observation t h a t islets in obese mice were deficient in silver positive alpha (al) cells (ttellman, 1961). Although no specific function has been attributed to these cells, evidence from other work suggest t h a t the a I cell is the source of a third pancreatic factor (Epple, 1965; Hellman and Lernmark, 1969) . The present study lends support to the existence of another hormone elaborated b y islet cells, although there is no direct evidence, to indicate which cells are involved. Evidence t h a t the primary defect in obese mice is not caused b y hypersecretion of beta (insulin) or a 2 cells (glueagon) is provided b y experiments in which non-obese mice received islets from obese animals. I n such experiments the weight gains of normal mice were not affected b y the presence of obese islets (Table 1a). The levels of glucose and insulin in nonobese with obese islets, remained low, throughout the study, as compared to other non-obese mice (Tables 2 and 3). The experiments described in this thesis do not support the suggestion of Clark, et al. (1956), t h a t the development of obesity in the hereditary obese mouse is caused b y an excess secretion of a "hyperglycemiaglycogenolytic" factor from the pancreas of these animals. The experiments in Clark's study, which indicated t h a t the pancreas of obese mice released abnormal quantities of a glucagon-like substance, involved substance, involved the injection of obese pancreatic extract into other obese animals. The pancreatic extract caused the hyperglycemia to increase; a reaction identical to t h a t seen when insulin is injected into these obese-hyperglycemic mice. Injection of this same extract into non-obese mice lowered the blood sugar; an anticipated reaction, since earlier workers observed elevated levels of pancreatic insulin in the obese mouse (Wrenshall, et al., 1955) . If in fact the pancreas of obese mice release a hyperglycemic factor, then transplants of these islets into normal mice should elevate the blood sugar. As seen in Table 2, no such elevation occurred at a n y interval, even though the islets in diffusion chambers remained viable. I t is probable t h a t glucagon plays a less important role in mice than man, since injections of this hormone, into well-fed and fasted non-obese mice, cause an insignificant rise in blood glucose (Shull and Mayer, 1956) . There is, then, no reliable evidence to support the theory t h a t the obese syndrome could be caused b y a hyperglycemic factor. Chronic studies might well show t h a t glucagon could lower weight gains b y decreasing food intake in the obese. In this case the error would be in a high setting for glucose response in the hypothalamie satiety centers. I t would, therefore, seem probable t h a t the obese state in mice ensues from the lack of some factor. Evidence in support of this theory is provided b y Hausberger's parabiosis experiment (1958). Hausberger found t h a t the weight gains of hereditary obese mice, in parabiotic union with a non-obese littermate, were suppressed until the two mice were separated, when all obese parabionts gained weight rapidly. Both m y earlier experiment and the present work support this thesis, t h a t a factor is missing in the obese mouse. Moreover, the s t u d y described in this paper would imply the origin of this factor is the cells of the nonobese pancreatic islets, l~emoval of the capsule, conraining non-obese islets, could be compared to separation of Hausberger's parabionts. As seen in Table 1 b, weight gains resumed immediately in the obese after removal of the encapsulated non-obese islets containing the factor responsible for suppression of fat deposition. Obese mice did not, however, lose weight b u t retained excess abdominal fat, deposited before implantation of Islets. This observation was a]so noted in previous experiments (Strautz, 1968) . I t is possible t h a t additional factors are responsible for the obese state and these substances originate from a source other t h a n the pancreas. The factor described in the present study can only regulate the balance between lipogenesis and lipolysis, having no effect on the volume of fat mass which is probably also genetically determined. There is indirect evidence to suggest t h a t the prim a r y action of this factor is on lipid metabolism. The first recognizable symptoms in weaning obese mice are insulin tolerance and excess fat deposition, each identified several weeks before hyperglycemia and hyperinsulinism are apparent (Westman, 1968) . T h a t the imbalance in fat metabolism precedes the other characteristics associated with this obesity in mice gives support to Mayer's (1967) proposal t h a t this factor is directly involved in lipogenesis in adipose tissue. He suggests t h a t this factor represses glycerokinase activi t y in adipose and other extrahepatic tissues. This enzyme allows the cells in other tissue to reutilize glycerol, released b y lipolysis. In the absence of glyeerokinase activity, adipose tissue must depend upon glycerol phosphate, prov~'Lded b y glucose catabolism, to resynthesize fats. Lipogenesis, in adipose tissue, is normally controlled b y the levels of glycerol phosphate and indirectly of glucose. Although the rate of conversion from glucose to glycerol phosphate is reduced (Christophe et al., 1961 ), the rate of glycerol incorporated into glycerol phosphate in adipose tissue is elevated (Fried and Antopol, 1960) . Simultaneous elevation of glycerokinase activity is noted in adipose tissue of obese mice (Locchaya et al., 1963). The lack of some pancreatic islet factor, which normally suppresses glycerokinase activity in extrahepatic tissue, could lead to increased concentrations of glycerol phosphate and at the same time, decreased glucose uptake. As a result, insulin peripheral antagonism develops with a secondary hyperplasia and hyperand Lernmark, I969) is compatible with t h e results of t h e p r e s e n t s t u d y , b u t e x a c t l y h o w t h i s a c t i o n is m e d i a t e d , b e y o n d its d i r e c t effect on inhib i t i o n of insulin e l a b o r a t i o n is difficult t o d e t e r m i n e w i t h t h e p r e s e n t knowledge. 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R. L. Strautz. Studies of hereditary-obese mice (obob) after implantation of pancreatic islets in Millipore filter capsules, Diabetologia, 1970, 306-312, DOI: 10.1007/BF01212243