Carbohydrate impairment and insulin secretory abnormalities among Macaca mulatta from Cayo Santiago.код для вставкиСкачать
American Journal of Primatology 11:147-162 (1986) Carbohydrate Impairment and Insulin Secretory Abnormalities Among Macaca mulatta From Cay0 Santiago CHARLES F. HOWARD JR.', MAlT J. KESSLER', AND SUSAN SCHWARTZ2 'Division o Metabolic and Immune Diseases, Oregon Regional Primate Research Center, Beaverton, Caribbean Primate Research Center, University of Puerto Rico School of Medicine, Sabana Seca L Rhesus macaques (Macaca mulatta) from Cay0 Santiago were examined for evidence of carbohydrate intolerance indicative of potential development of non-insulin-dependent diabetes mellitus (NIDDM). Monkeys 6 to > 20 years from natal Groups J, M, and P, a n AGED Group (all >20 years), and unrelated monkeys removed from the island in association with other groups (CAYO), were examined with intravenous glucose tolerance tests (iv-GTT). Morphometric measurements were made on all tested monkeys. Impairments included fasting hyperglycemia ( > 115 mg/dl), impaired glucose clearance (K < 2.0%/min), fasting hyperinsulinemia (> 150 pU/ml) or hypoinsulinemia (<20 pU/ml), and insulin secretory abnormalities (>500 pU/ ml or < 75 pU/ml). Natal groups J and M had 31%with impairments, group P had 0% the AGED group had 45%, and the CAYO group had 33%. Impaired glucose clearance was usually attributable to a reduced insulin response. Impairments correlated significantly (p < 0.05) to body weight and a modified Quetelet index, but not to sex, acute stress, or islet cell antibodies; the relationships to age could not be delineated in this survey. Impairments in monkeys are indicative of various stages in the asynchronous development of carbohydrate intolerance leading to NIDDM. Key words: non-insulin-dependent diabetes mellitus (NIDDM), insulin secretion, genetics, Modified Quetelet Index, morphometry, islet cell antibody INTRODUCTION Spontaneous diabetes mellitus has been reported to occur in several species of nonhuman primates [Howard, 1983; Howard, 19841. The primate disease has usually been similar to that classed in human beings as non-insulin-dependent diabetes mellitus (NIDDM). In humans, NIDDM is associated with hyperglycemia and glucose intolerance, usually obesity, and often aberrations in insulin secretion or utilization [Ganda & Soeldner, 1977; Luft et al, 1981; Fajans, 1981; Defronzo et al, 19831. There are no consistent lesions in the islets of Langerhans. Although genetic factors play a major role in the development of NIDDM, the disease is aggravated by environmental factors. NIDDM cannot be readily induced in laboratory animals. In Received October 16, 1985; revision accepted May 5, 1986. Address reprint requests to Dr. Charles Howard, Jr., Oregon Regional Primate Research Center, 505 N.W. 185th Ave., Beaverton, OR 97006. 0 1986 Alan R. Liss, Inc. 148 / Howard, Kessler, and Schwartz contrast, a n insulin-deficient state, closely analogous to insulin-dependent diabetes mellitus (IDDM) most commonly found in younger human beings, can be produced in numerous laboratory animal species by pancreatic beta cell ablation techniques, eg, surgical removal of the pancreas or use of diabetogenic drugs [Howard, 1983; Howard, 19841. Much of the published work on diabetes in nonhuman primates consists of case reports on individual animals [Sokoloverova, 1960; Valerio et al, 1969; Digiacomo et al, 1971; Kirk et al, 1972; Jones, 1974; Leathers & Schedewie, 1980; Rosenblum et al, 1981; Howard, 19831. When investigators have sought NIDDM in primates, their studies have often focused on the associations of diabetes, obesity, and aging. Hamilton and co-workers [Maller & Hamilton, 1968; Hamilton, 1972; Hamilton et al, 1972; Hamilton & Ciaccia, 19781 found that some rhesus macaques caged for many years became obese; a few developed metabolic changes associated with diabetes. Hypothalamic lesions [Ranson et al, 1938; Hamilton & Brobeck, 19631 have also been used to induce overfeeding, obesity, and diabetes, but lack of predictability in metabolic developments after lesion placement has prevented this experimental induction technique from being more widely adopted. Efforts to overfeed macaques [Hansen, 1979; Hansen & Jen, 19791 or surveys of primate groups [Walike et al, 19771 for possible carbohydrate impairment have produced results of limited value. Although obesity occurs in several primate species, it is not necessarily accompanied by carbohydate impairment or diabetes [Kemnitz, 19841. To be most useful for studies on NIDDM, the disease should develop spontaneously in the animals; the causes should encompass genetic and environmental vectors analogous to those of their human counterparts; there should be information available on genetic relationships; and it should be possible to recognize the potential for future development of NIDDM in the animals early enough to permit longitudinal studies on the etiologies and progression of the disease. The rhesus macaques maintained on Cay0 Santiago fulfill many of these criteria. Some anecdotal evidence suggested the occurrence of diabetes among the freeranging rhesus macaques maintained on Cay0 Santiago, Puerto Rico. Loy  observed “One of the dead females suffered from diabetes mellitus, which may have been exacerbated by the food shortage”; after observing the diabetic female eat two lizards, he wondered “. . . whether this female’s diabetes was connected with her consumption of animal protein.” Sade et a1  commented that a “. . . genealogy with two diabetic females showed a n average pattern of early infant mortality.” Both reports apparently referred to females EK and JJ in group F, a group that later fissioned to yield groups F, M, P, and 0. None of the reported cases was definitively diagnosed or confirmed. Spontaneous obesity has also been reported in the Cay0 Santiago population [Rawlins & Kessler, 1982; Kemnitz, 19841. Because of these observations and because the matrilines are known as far back as six generations, these macaques seemed ideal for evaluation of carbohydrate impairment and insulin secretory abnormalities. MATERIALS AND METHODS Subjects Rhesus macaques from India were introduced to the island of Cay0 Santiago in 1938. The colony has been used for a variety of behavioral and biomedical studies [Rawlins, 1979; Rawlins et al, 1984; Rawlins & Kessler, 19861. Animals have been removed periodically from the island, but no new stock has been added except through births. Figure 1, which illustrates relationships among the various natal groups, modifies and extends information contained in a n earlier report [Sade et al, 19761. Several groups were culled from the island in the early 1970s. By 1976, the Glucose Intolerance in Macaca mulatta I 149 0 Fig. 1. Interrelationships among natal groups on Cay0 Santiago between 1954 and 1986. Remnants of groups A, C, E, H, and K were removed after troop size had been significantly decimated through selective culling of individual monkeys. Intact social groups J,0, M, and P were removed in 1984 and 1985. Group 0 was sold and was unavailable for testing. Solitary males are not included in this illustration. population had become organized into five naturally formed social groups (groups F, I, J, L, and M consisting of 17 matrilines) and a small band of peripheral males. Group 0 formed in 1977 and group P in 1984 as a result of the fission of group F along matrilineal lines [Rawlins, 19791. Detailed life histories were kept on each monkey through a daily census [Rawlins & Kessler, 19861. Data on natality, morbidity, mortality, and group affiliation were recorded by a team of experienced observers. Paternity was not known. Each troop comprised one to four matrilines with a variable number of associated adult males. The matrilines consisted of a n adult female, her adult female offspring, and their juveniles and infants. Females remained with the natal group for life or until fission occurred. Adult males were often nonnatal members of the group, since most adolescent males disperse from their natal troop at puberty [Rawlins & Kessler, 19821. The animals on Cay0 Santiago were provisioned daily with 0.23 kg per monkey of commercial, high-protein (24-26%) monkey diet (Agway, Inc., Syracuse, NY; Allied Mills, Inc., Chicago, IL;Ralston Purina Co., St. Louis, MO). They also foraged on the tropical vegetation and were geophagic [Sultana & Marriott, 19821. Water was available ad libitum from a n automatic water collection and distribution system. The macaques received no disease prophylaxis, but moribund animals were permanently removed from the island for treatment or euthanasia [Rawlins & Kessler, 19821. Groups J and M were removed from Cay0 Santiago in January and February of 1984, and group P was removed in January of 1985; all were housed at the Caribbean 150 / Howard, Kessler, and Schwartz Primate Research Center (CPRC) Sabana Seca Field Station near San Juan. Monkeys were caged individually, in outdoor group enclosures (7.6 m2), or in corrals (0.25-2 ha). Thirty-six mature monkeys captured as part of groups J, M, or P belonged to other natal groups. Data on these monkeys, all males, were included as a miscellaneous group designated CAYO. In addition to the monkeys from Cay0 Santiago, other macaques maintained as part of a study on aging (>20 years) at Sabana Seca were also examined (AGED group). Monkeys weighed from 6 to 12 kg. Only those at least 6 years old were examined; some were more than 20 years of age. Data in this report are on 93 females and 57 males; the preponderance of females reflects the availability of defined matrilines. Thirty-two females were pregnant during a t least one test. Pregnancy could be detected by palpation at 30 days gestational age. By 80 days, gestational age could be determined within +5%, as verified later by birth records. Lactation or the presence of a n infant was usually recorded along with other pertinent information at the time of the iv-G'M7. Examination of birthldeath records also provided information about whether a female would still have been nursing. Tests and Assays Intravenous glucose tolerance test. Monkeys were caged individually overnight; food was removed by 1600, and the macaques were sedated the following morning between 0800 and 1030 h r with a n intramuscular dose of 12 mg of ketamine-HC1 (Vetalar@,Parke-Davis, Morris Plains, NJ) per kg of body weight. Sedation was maintained with additional ketamine as necessary. For a n intravenous glucose tolerance test (iv-GTT), a 7-ml blood sample was drawn (zero time sample); 3 ml was allowed to clot, centrifuged, and serum was collected and frozen. The remaining 4 ml was added to a tube containing 10 U of heparin and 0.02 ml of 1 M bemamidine-HCI for collection of plasma. After withdrawal of the zero-time sample, 0.45 g of glucosekg of body weight (1 ml of D50W glucose solutionkg of body weight) was injected, and 3-ml blood samples were withdrawn at 10, 20, 30, 45, and 60 min. Serum was analyzed for glucose (Beckman glucose analyzer) and immunoreactive insulin (IRI) (AmershadSearle radioimmunoassay kit). A K-value (percent glucose clearance/min) was calculated from the glucose levels in the samples removed from 10 min onward until the glucose concentration was within 10% of the fasting level; thus, the K values represent only the time of metabolic glucose clearance. A AIRI value was calculated as the increment of insulin secreted during the first 10 min (insulin level at 10 min minus that at zero rnin). The AIRI has been established as a sensitive indicator of the acute insulin response [Howard & Fang, 19841. Plasma was analyzed for glucagon with Unger 04A antibody, a n antibody that gives results identical to those obtained with the earlier 30K antibody [Howard & Van Bueren, 19811. Islet Cell Antibody (ICA) Assay. Neonatal Papio anubis pancreas was fixed in Bouin's solution, embedded in paraffin, and sections were cut and processed through organic solvents and rehydration. After a n overnight incubation with Macaca mulatta plasma at dilutions of 1:4 and 1132,sections were washed, and reactions of antibodies with islet cell antigens were visualized by the immunoperoxidase technique using diaminobenzidine [Erlandsen et al, 1975; Howard & Fang, 19841. The reaction was scored as negative or minimal (0to 1) or positive (2 to 3);only scores of 2 or 3 are reported [Howard & Fang, 19841. Morphometric measurements. Measurements were taken for a Quetelet index modified for monkeys (MQD at the time of the iv-GTT [Rawlins et al, 1984; Walker et al, 19841. Crown-rump lengths (vertex to ischial callousities) were measured with Vernier anthropometric sliding calipers accurate to 1.0 mm; the macaques were in Glucose Intolerance in Macaca mulatta I 151 left lateral recumbency. Scales accurate to 0.1 kg were used to weigh animals. The MQI was calculated a s [body weight (kg)/crown-rump length (ern)]' x 1000. Subcutaneous, abdominal skin folds were measured with skin fold calipers (Cambridge Scientific Instruments); measurements were made approximately 1 cm above the navel. RESULTS Intravenous Glucose Tolerance Tests Although fasting hyperglycemia a t > 140 mg/dl is definitive for a diagnosis of diabetes in human beings, levels of 115 mg/dl or greater in humans [Brunzell et al, 19761 as well as in monkeys [Howard, 19831 are considered evidence of fasting glucose abnormalities. K values of >2.0%/min are characteristic of the nondiabetic state. Values of 1.0 to 2.0%/min indicate impaired glucose tolerance (IGT); < l.O%/ min occurred in overt diabetes. Limits of normality for insulin measurements were established from the data on these macaques. All of the values for fasting IRI and of AIRI were averaged, even though some values were obviously excessive. After a mean and standard deviation were computed, those values exceeding the mean by more than 3 SD were discarded. Values above this limit would constitute only 0.26% of the total sample frequency; values included four AIRI values of > 800 pU/ml and three fasting IRI of >400 pU/ml. The second calculated mean for AIRI was established at 216.1 pU/ml with a SD of 141.6 and a SEM of 13.7; the upper limits of normality for AIRI were set at the mean f 2 SD, ie, a AIRI value of >500 pU/ml was considered as abnormally great. A similar procedure followed for the fasting IRI values gave a second mean of 61.5 pU/ml with a SD of 42.9 and a SEM of 4.0. Fasting IRI values greater than mean + 2 SD, ie, > 150 pU/ml, were established as abnormal. Fasting hypoinsulinemia has been previously established at < 20 pU/ml [Howard, 19781. Minimal insulin secretion is also abnormal; a AIRI of <75 pU/ml was established as indicating impairment (AIRI mean minus 1SD). Means and limits of glucose and insulin values measured during a n iv-G'M' were established. Fourteen nonimpaired males and 14 nonpregnant, nonimpaired females were selected (every third monkey on the lists), and means for K and for glucose and insulin values at each sampling time during the iv-GTT were calculated. Results were similar whether K values were averaged or whether K was computed from the mean of glucose values a t 10, 30, and 45 min; inclusion of the 60-min sample gave erroneous results since the glucose concentration was close to baseline by 45 min. The average K for females was 3.91 f 0.32%/min, significantly greater (p < 0.05) than the average K of 3.04 0.24%/min for males. Fasting insulin levels were essentially the same in males and females (57.2 f 9.4 vs. 55.1 f 6.5), but the AIRI of 220.3 i 18.1 in females was greater than the AIRI of 152.4 & 13.1 in males (p < 0.05). There was a slight difference in secretory patterns; insulin peaked at 10 min in females and then decreased, whereas the insulin in males rose slightly more by 20 min before diminution. These average glucose and insulin values measured during a n iv-G'M' are used in Figure 2 to indicate limits of normality. The vertical shaded area represents the mean f 1 SD. Since males and females were not identical, the appropriate control is plotted for each sex. Representative iv-G'M' results are plotted in Figure 2 to convey some of the more common patterns. Figure 2A (OT Female) shows results from a diabetic monkey with hyperglycemia, impaired glucose clearance, and virtually no insulin response. In Figure 2B (Z1 Female), insulin secretion was minimal, but glucose clearance was only slightly impaired. Figure 2C (738 Male) illustrates impaired glucose clearance and insulin secretion; fasting levels of insulin were slightly elevated, but insulin response was minimal. Examples of fasting and secre- + K = 5.32 A5Female AIR1 = 28 K=17O .OOTE C 738 Male 4oo- - 300 - P g 2 8 0 200- joo- n- 4oo- 800- AIR1 - 66 r=,w -E -- F 565 Female AIR1 = 506 800- K=081 600 - 600- - ? L E 2 400- 200- 0- 0- 0- tory hyperinsulinemia are shown in Figures 2D (748 Male) and 2E (A5 Female). Figure 2F (565 Female) illustrates an uncommon pattern in which there was impaired glucose clearance accompanied by fasting hyperinsulinemia, but with a gradual diminution in insulin levels. Table I summarizes the different combinations of impairments by sex and pregnancy status. Five males and two females had impaired glucose clearance with no evidence of insulin impairments, although most had fasting insulin values and AIR1 well below the mean. Fifteen monkeys had decreased glucose clearance associated with minimal fasting insulin and/or AIRI; only one of these was hyperglycemic. The largest group included 23 monkeys with fasting insulin values or AIRI above the established limits, but with no evidence of changes in fasting glucose or glucose clearance. Glucose Intolerance in Macaca mulatta t 153 TABLE 1. Distribution of Different Impairments by Sex and Pregnancy Status* Possible impairmentsa Fasting A IRI > FBG <K X X X X X lRI < > > < X X X Males 5 2 2 1 X X X X X X X - 1 1 1 18 23 6 3 1 4 X X Totals 3 - 2 2 2 2 1 8 1 4 1 - - X X Females Non-PG PG - *IRI, immunoreactive insulin; PG, pregnant. aImpairments: <K, glucose clearance of <2%/min; > FBG, fasted blood glucose of > 115 mg/dl; >AIRI, insulin secretory increment of > 500 pYml; < AIRI, insulin secretory increment of < 75 pU/ml; >fasting IRI, fasting insulin concentrations of > 150 pU/ml; c fasting IRI, fasting insulin concentrations of < 20 pU/ml. TABLE 11. Distribution of Impairments by Group, Sex, and Pregnancy Status* Females Group N J 6 5 0 4 24 39 M P Aged Cay0 Totals Males I 2 2 0 2 12 18 (%I) N (25) (28) (0) (33) (33) (32) 25 4 5 7 0 41 Nonpregnant I (%I) 13 4 0 6 0 23 (34) (50) (0) (46) (0) (36) N 6 11 6 0 0 23 Pregnant I (%I) 2 3 0 1 0 6 (25) (21) (0) (100) (0) (21) Group totals 54 29 11 20 36 150 (%I) (31) (31) (0) (45) (33) (31) *N, nonimpaired I, impaired, which include: glucose clearance of < 2%/min;fasting glucose levels of > 115 mg/dl; increased or decreased insulin secretory response ( >500 or < 75 pU/ml);increased or decreased fasting insulin levels (> 150 or < 20 pU/ml);PG, pregnant; (%I), percentage of monkeys with some form of impairment. Table I1 contains a summary of results within the different groups, separated by sex and by pregnancy status. Impairments include any abnormalities in fasting levels of glucose or insulin, insulin secretory response, and/or glucose clearance. The percentages of the monkeys with impairments were virtually the same in natal groups J and M and in the CAY0 males. However, the females of group P lacked any impairments. The AGED group had a slightly greater percentage of those with impairments, especially in females, but it was not statistically significant. Average glucagon concentrations were 290 f 74 pg/ml for impaired and 188 f 21 pg/ml for nonimpaired monkeys (p > 0.05). Examination of selected impaired vs. nonimpaired groups or subgroups produced no significant differences among mean glucagon values. However, glucagon levels did correlate with the ICA scores. Glucagon levels in monkeys with ICA scores of 0 averaged 163 f 10 pg/ml, whereas 154 / Howard, Kessler, and Schwartz TABLE 111. Comparisons of Insulin Secretory and Fasting Values Among Normal Males, and Pregnant and Nonpregnant Females* Males Females Nonpregnant Nonlactating Lactating Pregnant No. AIR1 (pU/ml) Fasting IRI (pU/ml) 39 143.7 k 9.3" 56.0 k 5.9 41 24 16 23 200.0 f 16.2" 227.8 f 20.1" 216.9 f 30.4" 182.3 f 19.5 48.0 f 3.8 51.6 f 4.9 43.4 6.2 64.6 8.7 *Values are the mean Ifr SEM AIRI is the insulin secretory increment, and fasting IRI is the fasting insulin concentrations. "Values differ by p < 0.01 for females vs. males. monkeys with ICA scores of 2 or 3 had glucagon levels of 240 & 23 pg/ml (p < 0.01). The prevalence of ICA averaged 30% among both nonimpaired and impaired monkeys. Sequential Comparisons The effects of stress on carbohydrate clearance were assessed in order to differentiate between inherent carbohydrate impairment and changes related to new caging conditions, handling, or sedation. Of the monkeys in group M tested in January 1984, soon after removal from Cay0 Santiago, 11were tested again in May 1984. Data were analyzed both by comparing means with the Student's t-test and by analyzing changes in individual monkeys with the Wilcoxon signed rank test. Neither the decrease in mean K value from 4.0 f 0.5 to 3.2 f 0.4%/min nor the increase in AIRI from 148 f 29 to 165 f 46 pU/ml were significant. Changes in individual monkeys were not significant. Comparison by Age and Sex All monkeys were > 6 years. The average age of both impaired and nonimpaired monkeys in groups J and M was about 11 years. Although the 45% incidence of impairment in the AGED group was greater than in most other groupings, age differences were not significant. Sex had no effect on the prevalence of the impairments; 23 of 64 nonpregnant females (36%) and 18 of 39 males (32%) had impairments. There were no apparent differences when specific types of impairments were compared. Effects of Pregnancy Results from pregnant and nonpregnant monkeys are tabulated separately in Tables I and I1 in order to examine for possible effects of pregnancy on the various impairments. The 21% incidence of impairments in pregnant monkeys is less than the 36% in nonpregnant monkeys, but not significantly. Table I11 contains further comparisons of fasting and secretory insulin values by sex, and by pregnancy and lactation status for nonimpaired monkeys. The average AIRI values for all females were greater than the average AIRI values for males; results are consistent with the group of monkeys selected for analysis of iv-GlT results. Values for nonpregnant females were different when results were compared to those of the males. Fasting IRI values did not differ significantly among various groups of monkeys. Testing individual monkeys when pregnant and again when they are nonpregnant is the only way to establish whether impairments in pregnant females are due Glucose Intolerance in Macaca mulatta I 155 TABLE IV. Effects of Pregnancy on Metabolic Values* Nonpregnant Monkey No. 514 989 268 K (%/mid 6.8 4.1 2.4 Pregnant (mg/dl) AIRI (pU/ml) Fasting IRI (pU/ml) K (%/mid FBG (mg/dl) AIR1 (pU/ml) Fasting IRI (pU/ml) 46 66 64 254 137 38 149 28 26 4.1 7.0 0.6 57 46 151 519 746 87 90 137 166 FBG * K , glucose clearance; FBG, fasted blood glucose; AIRI, insulin secretory increment; fasting IRI, fasting insulin concentrations. TABLE V. Comparisons of Morphometric Measurements Among Macaca mulatta With and Without Metahalic Imoairments* Modified Quetelet Index Nonimpaired Impaired Males Femalesb 32.0 20.8 1.5' + 1.1" * 39.4 2.9" 26.8 + 2.4" Skinfold (cm) Weight (kg) Nonimpaired Impaired Nonimpaired Impaired 9.5 f 0.2" 7.2 0.2" 11.0 f 0.4" 8.2 0.3" 6.0 f 0.6 5.9 0.7 6.8 f 0.9 8.0 + 1.4 + *Values are the mean SEM. "Valuesbetween nonimpaired and impaired monkeys are significantly different by p < 0.01. 'Nonpregnant females. to inherent metabolic defects or to the pregnancy. Three such monkeys are presented in Table IV and are not included in other tabulations. Numbers 514 and 989 from group M had greater AIRI when pregnant than when nonpregnant; changes were sufficient to place them in the impaired classification when pregnant. Number 268 was the subject of an earlier report [Kessler et al, 19851. More recent testing showed her to have normal fasting glucose and glucose clearance, but insulin secretion remained impaired and fasting IRI was quite low. In addition to the three monkeys listed in Table IV, four other monkeys tested while pregnant and again when nonpregnant are included in the data in Tables I, 11, and III; three of these tested normal under all conditions and one had impairments in iv-G?T during and after pregnancy. Morphometric Comparisons Morphometric measurements for males and nonpregnant females, both lactating and nonlactating, are presented in Table V. The average values for the MQI and weights were significantly greater for impaired than for nonimpaired monkeys. Impaired males averaged 1.5 kg more and impaired females 1.0 kg more than their nonimpaired counterparts (p < 0.01). Skinfold thicknesses of impaired monkeys were also greater than in nonimpaired monkeys, but not significantly. Genealogy Figure 3 shows abbreviated matrilines for the monkeys tested in groups J, M, and P. Monkeys tested are presented in boldface type; other monkeys are included to delineate matrilines. Asterisks indicate the presence of impairment, whether of fasting glucose, glucose clearance, insulin secretion, or fasting insulin levels. Im- 156 / Howard, Kessler, and Schwartz [GlOUpJ] 073-1-4 (00-001 JJ I-d (00-00) 'OT-I(65- ) 092-1-6 !00-001 SO09 I-d IW-00) Ui-f-d (61-83) 418-m (71- I 6251(14- 1 xo-1-a( 6 3 . ~ 1 24-1-6 167-841 '303md (69-84) 458-1-6 171-84) 824.1 (74. ) 940.1R8- 1 563.1-6 (73-84) '685-1-4 (75.85) 8K-I-d 166-82) 662-Id (I3-81) A114IIP ) 8034 (I4. I 447-1-4 ! I t -791 682-1 (75- 1 TN-I-d (82.77) 9K.M (€4441 561-m(73784-I(16 '327-l(10 I '924-rn(78- 1 1 I 486-1-6 (72-841 946-1 (787E-m-d (68-85) I 107-rn(71. ) 522 l(72- ) 751-1(76- ) 656-1(7& ) 899-1 (78- ) E w a (56-79) *P84 (66- ) 780-1(76- ) 972-m(78- ) 022-1-6 (54-00) W-14 (MI-84) JI-I-d 161-00) Jz-1-a (65 oa) 518-1172- ) 734-1 ( 7 6 ) '832.1(77. ) *937-1(78- ) 6J-I-d (68-80) '514-1(72- ) 8571-6 (77-84) 640m(75- ) 293-1(69 ) 793-1(77. ) '989-1179- ) 517-1-6 (72- 1 740-1(76- ) 939-Id (18.81) 994-1 ( 7 9 ) '571-1-6 (73- ) 7%-1(77- ) 076.1-6 171-00) KO-I-d (00-001 VL-1(63- ) '6461(75- ) 831-m(77- ) ZM-I-d 164.00) '611-m(74- ) WX-I(M- ) 53&m(73- ) Kl-f-d (65-85) 476-f (71- ) 700-f (75- ) 861-f (77- ) 984-f (79- ) 789-f (76- ) N2-f (66- ) 41 1-f-d (71-82) 649-f (75- ) A73-f (79- ) 854-f (77- ) 979-f (79- ) 840-f (77- ) Fig. 3. Genealogy of groups J, M, and P. The code numbersiletters of the monkeys tested are in boldface type; the asterisk indicates the presence of impairment. Monkey numbers in light type were not tested, but have been included in the chart to allow delineation of familial relationships. The year of birth and of death, where known, are in parentheses. f, female; m, male; d, dead. pairments appeared frequently throughout both groups J and M, and several matrilines had some increased prevalence. Two of three offspring of WF in group J had impairments. Only two of five offspring of X J in group J had impairments, but both of these had at least one offspring with impairments. Two of three offspring of both 518 and YL in group M had impairments. Most striking was the lack of any impairments found in group P. Glucose Intolerance in Macaca mulatta I 157 DISCUSSION There are multiple etiologies for NIDDM in human beings. Commonly, NIDDM occurs in older people; about 80% are overweight. Many are considered insulinresistant. A current scenario for NIDDM supports a tissue refractiveness to insulin, involving either the receptor binding or metabolic defects distal to the receptors [Roth, 1980; Olefsky, 1981; Kolterman et al, 1981; Skyler, 19821. However, fasting or response hyperinsulinemia is not a consistent finding associated with IGT or diabetes [Defronzo et al, 19831. Fasting insulin levels may remain within normal limits. Impairment in the acute secretory phase of insulin release is often noticeable in the early stages preceding diabetes; more severe carbohydrate impairment is associated with a significant decrease in the amount of insulin secreted Fajans, 1981; Luft et al, 19811. Insight into the various causes of NIDDM requires identification of specific subpopulations, as well as recognition of the many metabolic alterations occurring during the progression toward overt diabetes. Availability of suitable experimental animals can facilitate studies on NIDDM. Unfortunately, there are few animals with known heritable conditions analogous to human NIDDM [Howard, 19841. Among diabetic rodents, the db and ob mouse strains are perhaps the closest to human NIDDM in the ways that specific metabolic abnormalities develop [Coleman, 19821. These mice become obese and hyperinsulinemic, and show moderate to severe carbohydrate intolerance depending on genetic backgrounds [Coleman & Hummel, 19731. Diet can be used in some circumstances to exacerbate carbohydrate intolerance [Leiter et al, 19831. The most intriguing data on the natural development of diabetes in monkeys can be gleaned from a series of papers by Hamilton and co-workers [Maller & Hamilton, 1968; Hamilton, 1972; Hamilton et al, 1972; Hamilton & Ciaccia, 19781. Caged, sedentary, well-fed Macaca mulatta were followed as they aged and became obese, hyperinsulinemic, and eventually diabetic. The potential for producing diabetic primates has generally not been realized because there have not been suffkient numbers or they have not been monitored sufficiently long. Also, a satisfactory breeding situation has never been established for ongoing, in-depth studies and for provision of monkeys to other investigators studying diabetes. The Cay0 Santiago rhesus macaques have been under continuous observation since 1956. Because new monkeys have been introduced only through births, the population is relatively homogeneous and matrilineal relationships have been established. Obesity has been documented, and there have been anecdotal comments of apparent overt diabetes. The recent reduction of the Cay0 Santiago population through removal of monkeys to the CPRC Sabana Seca Field Station has made many of them available for biomedical research. One of our primary objectives was to establish whether these monkeys did have carbohydrate impairment and fasting or insulin secretory abnormalities, as the early observations suggested, and whether such abnormalities would be associated with their natal group, matrilines, age, or body morphometry, especially obesity. We have identified substantial numbers of macaques with hypo- or hyperinsulinemia and IGT. Just as there are many causaI factors for diabetes in humans, impairments in these monkeys likely relate to a variety of circumstances. Genetics does appear to play some role in the appearance of impairments. Abnormalities appeared commonly in groups M and J. Impairments were more frequent among several sibling units than among others in these groups. The absence of impairments among group P monkeys further supports the selective effects of inheritance on carbohydrate intolerance among these monkeys. 158 / Howard, Kessler, and Schwartz Morphometric measurements relative to body mass (MQI and weight) did reveal greater size of impaired monkeys. The Modified Quetelet Index, Obesity Index Rh, and abdominal skinfold measurements have been found to correlate strongly with body fat values derived from tritiated water determinations in monkeys [Walker et al, 1984; Jen et al, 19851. Skinfold measurements were also greater in impaired than in nonimpaired monkeys, but differences lacked statistical significance; problems associated with experimental variations in establishing skinfold thicknesses have been noted [Bray, 19761. Age could not be established as a major contributor to impairments, although the selection of monkeys in this first phase of the study may have masked age effects. Impairments were equal between females and males. Some of the iv-GITs were conducted during January or February, when many females were pregnant, and in May, when mothers were lactating. Differences in glucose clearance, fasting insulin levels, and/or insulin secretory responses associated with pregnancy in monkeys have previously been noted, although results were not consistent among investigators [Pitkin et al, 1970; Mintz et al, 1972; Meis et al, 19821. Rather than differentiate among results obtained during early or late pregnancy, we chose to examine data from all pregnant females as one category. We had previously reported gestational diabetes in a n aged pregnant female, who also had IGT postpartum [Kessler et al, 19851. Some of the females in this current group with apparent abnormalities during pregnancy may eventually be found to have been gestationally diabetic, as were the three reported here. However, gestational diabetes does not likely account for a significant number of the abnormalities among the females. Certainly the percentage of abnormalities among nonpregnant females was greater than among pregnant females, although results were not significantly different. The presence of gestational diabetes in this initial examination merely adds further credence to our postulate that IGTs have various causes within this group of rhesus macaques. An additional concern was whether the observed abnormalities were due to inherent metabolic aberrations or whether they were stress-induced. Although monkeys were initially in a seminatural, free-ranging environment, conditions on Cay0 Santiago cannot be considered stress-free, eg, social and sexual competition, aggression, and food availability can be major contributors to stress. The stresses of the new situation at Sabana Seca would include caging conditions affecting freedom of movement, handling, sedation, etc. Acute stress can impair carbohydrate clearance [Honjo et al, 1976; Ausman & Gallina, 1978; Streett & Jonas, 19821, presumably through augmentation of catecholamine secretion, which inhibits the acute insulin response [Miller & Soeldner, 19691. Certain sedatives and anesthetics can cause significant insulin secretory changes, although ketamine .HC1 seems to cause only minimal changes in glucose clearance or in the insulin response [Kemnitz & Kraemer, 1982; Brady & Koritnik, 19851. Monkeys examined with iv-GIT immediately after removal from Cay0 Santiago and again later showed minimal changes. The current results do not support imposed stress as a major contributor to the observed carbohydrate impairments. The prevalence of ICA has direct bearing on delineation of subpopulations of impaired monkeys. The cause of diabetes in Celebes black macaques [Sulawesi crested macaques] (Macaca nigra) is a n islet lesion in which secretory cell deterioration occurs concurrently with deposition of the protein amyloid [Howard, 1972; Howard, 19781. As the secretory cells deteriorate, released antigens induce ICA formation; the ICA can be used as a marker for the islet lesion and beta cell loss in Macaca nigra [Howard & Fang, 19841. Among these Macaca mulatta, the presence of ICA did correlate with increased glucagon concentrations. Such a n increase would be expected in light of recent results [Howard, 19861 that showed that quantities of alpha cells increased during progression of the islet lesion in Macaca nigra. In the current work, we wanted to examine Cay0 Santiago monkeys for NIDDM in which Glucose Intolerance in Mucucu rnuluttu I 159 a n islet lesion did not play a role, ie, in which causes for diabetes are associated with genetics, diet, obesity, receptor defects, and old age. More than 70% of the Cayo Santiago monkeys are ICA negative and thus are less likely to have a n islet lesion as the cause for impairments. Our results can be placed in the context of the asynchronous metabolic changes that occur during progression toward diabetes. Sequential changes for monkeys with NIDDM, excluding those with abnormalities due to a n islet lesion or pregnancy, have been noted by other investigators [Hamilton & Ciaccia, 1978; Hansen & Bodkin, 19851 and have been further corroborated in our studies. Hyperinsulinemia or other insulin abnormalities, whether fasting or secretory, can be apparent before there is overt carbohydrate intolerance; many of the hyperinsulinemic monkeys were normoglycemic and had normal carbohydrate clearance. Presumably, in those monkeys as in human beings, the chronic milieu of insulin secretion is sufficient to prepare the cells for metabolic clearance before arrival of the glucose bolus, ie, cells are adequately primed so that glucose clearance is seemingly unrelated to insulin secretion. As the effects of genetics, diet, and obesity contribute further to the continuation of hyperinsulinemia, impaired carbohydrate clearance or moderate fasting hyperglycemia may appear. With time, the beta cells lose their ability to respond with adequate insulin secretion and there is severe fasting hyperglycemia and marked carbohydrate intolerance. Our survey allowed identification of many monkeys with tendencies toward further deterioration. Some of these monkeys will likely progress to a n overtly diabetic state. Through recognition of subpopulations of monkeys with different causes for carbohydate impairment and in different stages of development toward diabetes, it will be possible to utilize these macaques to gain a better understanding of their progression toward diabetes and to establish analogies with similar aspects of the human disease. CONCLUSIONS 1. Over 30% of the Macaca mulatta removed from Cay0 Santiago as well as others now housed at Sabana Seca (aged 6 to > 2 0 years) had evidence of carbohydrate intolerance. 2. Carbohydrate impairments, mainly decreased glucose clearance in iv-GlTs, but also some fasting hyperglycemia, were found in 15% of the monkeys. 3. Insulin aberrations of fasting hypo- or hyperinsulinemia and secretory response abnormalities of either minimal or excessive insulin during iv-GTTs were present i n 27%of the monkeys. 4. Male and nonpregnant female monkeys with impairments had significantly greater Modified Quetelet Indexes and body weight than did their nonimpaired counterparts. 5. The prevalence of impairments does relate to matrilines; impairments appeared often in groups J and M, whereas group P monkeys had no significant impairments. 6. Neither sex nor islet cell antibodies were associated with the prevalence of impairments. 7. Age as a contributing factor could not be delineated in this study. 8. Acute stress was not likely a contributor to the carbohydrate impairments. 9. Monkeys exhibited several metabolic abnormalities analogous to those observed during development of non-insulin-dependent diabetes mellitus in human beings. ACKNOWLEDGMENTS The work described in this article, Publication No. 1447 from the Oregon Regional Primate Research Center (ORPRC), was supported by Animal Resources Branch Grant RR-00163 and General Research Support Grant RR-05694, both from 160 / Howard, Kessler, and Schwartz the Division of Research Resources, National Institutes of Health. The ORPRC is fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC). Work in the ORPRC laboratories was supported by U.S. Public Health Service Grants AM-21982, AG-2281, and RR-05694. At the CPRC, work was supported by U.S. Public Health Services grant RR-01293 to the University of Puerto Rico. Joann Wolff, Tonya Van Bueren, and Fred Feil a t the ORPRC and Sammy Martinez, Janis Gonzalez, and the staff of the CPRC provided valuable technical assistance. REFERENCES Ausman, L.M.; Gallina, D.L. Response to tes mellitus. 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