Basement membrane thickness in muscle capillaries of normal and spontaneously diabetic Macaca nigra.

The thickness of the capillary basement membrane of femoral muscle was examined in normal and spontaneously diabetic Macaca nigra. Thickness correlated significangly with the degree and severity of diabetes; greater thickness was associated with decreased glucose tolerance, decreased serum insulin, and increased glucose and triglyceride. In normal monkeys, the average basement membrane thickness was 696 A and in diabetic monkeys, 837 A, The minimum basement membrane thickness was 524 A in normal monkeys and 634 A in diabetics. Normals differed significantly from diabetic monkeys.

Insular amyloidosis and diabetes mellitus in Macaca nigra.

Amyloid in the islets of Langerhans increases with increasing severity of diabetes mellitus in Macaca nigra. The amount of insular amyloid was quantified, and diabetic monkeys averaged eight times more islet amyloid than did normal monkeys. The quantity of insular amyloid correlated significantly with glucose clearance in intravenous glucose tolerance tests and with serum glucose, triglycerides, immunoreactive insulin, and prebetallipoprotein measured after an overnight fast. As with human beings, insular amyloid appeared to be more prevalent in aging monkeys. The results support the hypothesis that the interrelated islet pathologic and metabolic events, which result in the appearance of insular amyloid concomitant with islet cell necrosis, may contribute more to maturity-onset diabetes in aging individuals than has been heretofore realized.

Nonhuman primates as models for the study of human diabetes mellitus.

Nonhuman primates have been used for a variety of studies on diabetes mellitus. Spontaneous, natural forms of diabetes have been well documented in several species; there are limited data on numerous other species that indicate diabetes or a diabetes-like syndrome. The causes and manifestations of spontaneous diabetes, their prevalence, and their severity vary among species. Diabetes has also been induced in nonhuman primates with streptozotocin, alloxan, hypothalamic lesions, or pancreatectomy. The extent and severity of metabolic and hormonal abnormalities vary according to the method of induction, the individual monkey, and the species. Metabolic, hormonal, and pathologic abnormalities present in human diabetics also occur in monkeys with either spontaneous or induced diabetes. Hyperglycemia and impaired glucose clearance are common, lipid concentrations are elevated, and hemoglobin A1c concentrations are increased in hyperglycemic monkeys. Monkeys may have fasting hypo- or hyperinsulinemia; insulin responses are often impaired in glucose tolerance tests. Glucagon concentrations may be increased. Aortic atherosclerosis, muscle capillary microangiopathies, cataracts, and glomerulosclerosis have been documented. Primate size and longevity allow longitudinal studies with procedures that may not be feasible in smaller animals or in human beings. Nonhuman primates may be the models of choice for studies on selected aspects of diabetes and its secondary complications.

Correlations of hemoglobin A1c and metabolic status in nondiabetic, borderline diabetic, and diabetic Macaca nigra.

Glycosylated hemoglobin A1c (HbA1c) increases from 2.6% in nondiabetic Macaca nigra to 7.9% in diabetic monkeys. Monkeys without overt hyperglycemia but with impaired glucose clearance, impaired insulin secretion, and increased postprandial glucose have a significant increase in glycosylation to 3.5%. HbA1c. Two different forms of hemoglobin are electrophoretically distinguishable in this species due to an amino acid change in the globin beta-chain. Colorimetric analysis established that glycosylation of the different hemoglobin forms was related only to metabolic status. Percentages of glycosylated hemoglobins indicate the inability of monkeys with varying severity of metabolic abnormalities to adequately maintain glucose homeostasis.

Changes in islet cell composition during development of diabetes in Macaca nigra.

The islets of Langerhans in sections from the pancreas tail of Macaca nigra were stained by antiserum to insulin, glucagon, or somatostatin. The area of stained cells per total area of the islets was determined by a computerized photometric method. Insulin of the beta cells occupied 77% of the islet area in nondiabetic (ND) monkeys and decreased to 62% in monkeys in the earliest stages of metabolic deterioration, i.e., hormonally impaired (HI) monkeys. At the later stage of borderline diabetes (BD), monkeys had only 39% of the islet area occupied by insulin and the area was diminished to less than 1% in diabetic (D) monkeys. Islets in HI monkeys had an unusual pattern in which only the beta cells in the periphery of islets were stained. Glucagon in the alpha cells stained 7% of the islet area in ND monkeys, but the area was almost doubled to 13% in HI monkeys; the percentage decreased to about 5% in BD and 3% in D monkeys. Somatostatin accounted for 5% of the islet area in ND monkeys, was slightly greater at 7% in HI monkeys, and decreased to 3% in BD and 2% in D monkeys. Alterations in percentages of secretory cells correlated with several of the metabolic and clinical changes.

Islet cell cytoplasmic antibodies in Macaca nigra.

Islet cell antibodies (ICA) have been measured in mature Macaca nigra. Of 30 nondiabetic monkeys, 26 (87%) were ICA-negative; of 43 monkeys with evidence of mild to severe hormonal or glycemic abnormalities, 39 (91%) were ICA-positive. Pancreatic islets were examined from biopsy and autopsy sections to assess cell deterioration and amyloid infiltration. No ICA were found in 13 of 18 (72%) monkeys with no evidence of amyloid, whereas 30 of 35 (86%) monkeys with islet amyloid and concurrent cell deterioration were ICA-positive. Association of ICA with metabolic and islet abnormalities was significant at P less than or equal to 0.001. ICA were specific for the islet cells in pancreatic sections; plasma preincubated with insulin, glucagon, or acetone extracts of tissues retained their ICA-positive reaction. The relationships of ICA in older monkeys to the islet lesion and to metabolic abnormalities could be relevant to similar situations in aging diabetic persons.

Cardiovascular complications.

This report summarizes the current state of knowledge concerning the cardiovascular system in various animal models of diabetes and presents their major strengths and weaknesses for studying the important research questions in the field. Nonhuman primates have many desirable features for studies on the macrovascular and cardiac complications of the disease as well as risk factor alterations, but their availability, cost, and maintenance present practical disadvantages. The spontaneous rodent models of diabetes currently are not considered very useful for cardiovascular research, but they have not been well characterized with respect to most aspects of their cardiovascular system. Alloxan-diabetic rabbits offer some promise for examining the effects of diabetes on atherogenesis, lipoprotein metabolism, and cardiomyopathy, but additional research is required to validate their usefulness. Insufficient data are available on canine and swine models of diabetes to judge their merits for cardiovascular research. The Task Force recommends: (1) additional longterm investigations to determine the extent and severity of cardiovascular complications in the well-characterized rodent models and in diabetic rodents with multiple risk factor abnormalities; (2) further studies on the macrovascular disease and lipoprotein abnormalities of the alloxan-diabetic rabbit and the development of rabbit colonies with spontaneous diabetes; (3) increased emphasis on such potentially important but neglected areas of research in diabetic animals as the intramyocardial circulation, adventitial blood vessels, blood pressure, platelet function, blood coagulation, blood rheology, and autonomic nervous function; (4) long-term studies on the influence of control of hyperglycemia and of insulin therapy on cardiovascular complications in diabetic animals; and (5) encouragement of use of diabetic nonhuman primates for cardiovascular research and institution of measures to increase their supply and availability by expanding current colonies, screening newly imported animals for diabetes, and establishing a visiting scientist's program allowing investigators to study diabetic primates at resource centers.

Metabolic and underlying causes of diabetes mellitus.

It is emphasized that animal models should be used to study specific genotypic or phenotypic expressions associated with diabetes rather than assuming a single animal model can reflect diverse forms of the human disease. Diabetic and normal animals are reviewed on the basis of their usefulness as models of genetic, viral, and chemically induced diabetes, including the often associated immune phenomena. Characteristics of spontaneously diabetic animals with and without obesity are also described with an emphasis on both genetics and metabolic derangements. Recommendations for future animal experimentation include: more longitudinal studies evaluating the role of sex, prenatal environment, diet, and viral or chemical attack on B-cell function; characterization of the immune phenomena associated with B-cell lesions (and insulitis) in diabetic and immunologically incompetent lines; clarification of relationships between obesity and islet-cell function with emphasis on the role of fuel metabolism, vitamins, and minerals; and, finally, the development of new models with specific genetic aberrations placed in normal or diabetic lines.

Aortic atherosclerosis in normal and spontaneously diabetic Macaca nigra.

Aortic atherosclerosis is minimal in normal Macaca nigra; development of atherosclerosis correlates with increasing severity of diabetes mellitus. The extent of aortic involvement (plaque plus sudanophilia) was quantified and compared with metabolic and clinical parameters. Increasing atherosclerosis correlated with decreasing ability to clear glucose in a tolerance test (P less than 0.01), decreasing insulin (P = 0.02), and increasing glucose (P less than 0.01) and triglycerides (P less than 0.01). A diabetic index, established as a summation of several metabolic measurements, correlated with atherosclerosis at P less than 0.001. On the average, involvement of the thoracic aorta was about 3-fold greater than in the abdominal portion; involvement reached over 40% in severely diabetic monkeys. Atherosclerosis development is unique in these monkeys since they consume a natural ration low in fat and cholesterol. Serum cholesterol did not correlate with diabetes or artherosclerosis. Increasing age alone was associated with slight sudanophila, some intima-media thickening, and occasional small lesions. However, only with increasing severity of diabetes was there significant atherosclerosis.

Effects of ADP and epinephrine on macaque and human platelets. Implications for studies on human atherosclerosis.

The ranges for near-threshold ADP concentrations for the aggregation of macaque and human citrated platelets overlapped. The minimum concentrations of epinephrine, 0.05 microM to 1.0 microM, that at least doubled the aggregation response at threshold ADP concentrations were comparable for macaque and human citrated platelets. Epinephrine (1.0 microM to 10 mM) alone never aggregated macaque citrated platelets. Biphasic aggregation occurred with both macaque and human citrated platelets. The addition of heparin to a final concentration of 2.2 units/ml had no effect on the threshold ADP concentrations or the sensitivity of macaque or human citrated platelets to epinephrine. One microM phentolamine eliminated the potentiating effect of 1 microM epinephrine on ADP-induced aggregation of macaque and human citrated platelets. The threshold concentrations of ADP for macaque platelets were sharply reduced when heparin was used as an anticoagulant rather than citrate. However, epinephrine induced a similar increase in aggregability with both citrated and heparinized platelets, 0.55 +/- 0.09 SEM% and 0.44 +/- 0.09 SEM%, respectively. These data indicate that macaque and human platelets behave in a similar manner in response to ADP and that epinephrine potentiates the ADP-induced aggregation of macaque and human platelets equally well.

Lipoprotein patterns in nondiabetic, borderline diabetic, and diabetic Macaca nigra.

Lipoproteins were isolated by sequential ultracentrifugation, and the concentrations and compositions were determined in nondiabetic (ND), borderline diabetic (BD), and diabetic (D) Macaca nigra males consuming a chow ration. The total concentrations and components of the VLDL and IDL increased significantly with metabolic deterioration (P less than 0.01). Concentrations and components of LDL increased in the BD and D monkeys, but changes were not statistically significant. The HDL2 and HDL3 particles were virtually unchanged among the three different metabolic groups. The VLDL was the major carrier of the triglycerides, especially in D monkeys. Cholesterol was present predominantly in the LDL. The LDL-cholesterol to HDL-cholesterol ratio increased in the BD and D monkeys, owing mainly to increases in the LDL-cholesterol content. Apoprotein antisera showed apoprotein B in the VLDL, IDL, and LDL, apoprotein E in the VLDL and IDL, and apoprotein A-I in the HDL2 and HDL3 fractions. Because Macaca nigra consume a nonatherogenic, low-cholesterol, low-fat ration, the changes in lipoproteins, particularly in VLDL and IDL, are attributable to metabolic alterations associated with diabetes.

Correlations of aortic histology with gross aortic atherosclerosis and metabolic measurements in diabetic and nondiabetic Macaca nigra.

We studied the aortic histology of 28 Macaca nigra males and females, from 6 to more than 20 years old, normal and manifesting various degrees of spontaneous diabetes. Correlations of several metabolic and hormonal indicators of diabetes severity with gross and microscopic findings in the aortas demonstrated direct associations with the severity of atherosclerosis. Mild to relatively severe aortic lesions were present. These monkeys showed many changes similar to those observed in medium and large arteries of diabetic humans. Intimal proliferation, prominent extracellular fibers as part of the intimal thickening, and lipid deposition--mostly in extracellular locations--were particularly evident. Significant relationships were observed when glucose clearance, insulin secretion, and fasting glucose levels were correlated with all aortic microscopic findings. Cholesterol concentrations had no correlation with the histological state of the aortas, and triglyceride levels correlated only with aortic lipid content and intimal thickness. Aortic pathologic changes increased with age; diabetics had significantly greater changes than nondiabetics. Macaca nigra can be useful in the study of how diabetes affects the development of atherosclerosis without the influence of an atherogenic diet.

Metabolism of arachidonic acid by macaque platelets. Implications for studies on atherosclerosis.

The metabolism of [1-14C]arachidonic acid [( 1-14C]AA) by washed platelets from macaques and human subjects was investigated. The results were as follows: At substrate levels of 1 microM, similar amounts of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), prostaglandin D2 (PGD2), and thromboxane A2 (TXA2), measured as thromboxane B2 (TXB2), were produced from [1-14C]AA by platelets from rhesus, Celebes black, and cynomolgus macaques and humans. An increase in the AA concentration from 1 microM to 20 microM decreased the TXB2: PGD2 ratio (aggregator: antiaggregator) from greater than 5 to less than 2 in all series. In the human series, the ratio decrease was due to an increase in PGD2 production; in the macaque series, PGD2 production increased and TXB2 production decreased. Under basal conditions and at 1 microM AA concentrations, the amounts of prostaglandins and thromboxanes produced by platelets from male and female rhesus macaques were the same. An increase in substrate concentration from 1 microM to 20 microM AA decreased TXB2 production and increased PGD2 production to the same extent in platelets from male and female rhesus macaques. Imidazole increased prostaglandin production and decreased TXB2 production by platelets from both male and female rhesus macaques. The TXB2: PGD2 ratios were reduced below 1.5; there was no difference between the ratios in the two series. In the presence of 1 mM imidazole, greater amounts of prostaglandins and thromboxanes were produced in the male than in the female series. These data indicate that macaque's platelets are a suitable model for the study of AA metabolism in human platelets.

Atherosclerosis and insulin in primates with diabetes mellitus.

The most commonly available primate models of diabetes mellitus are of the insulin-dependent type and are attained through beta cell ablation techniques. Noninsulin-dependent primate models are less common since the animals must have a genetic predisposition to diabetes. Few studies have been conducted on lipid or vascular abnormalities associated with diabetes in primates. Diabetes develops spontaneously in Macaca nigra as the result of a lesion in the islets of Langerhans. As secretory cells are gradually lost, mild to moderate hyperglycemia, impaired glucose clearance, acute insulin release, hyperglucagonemia, and chronic hypoinsulinemia develop. Overtly diabetic monkeys require insulin therapy and thus alternate between hypoinsulinemia and hyperinsulinemia. The development of aortic atherosclerosis correlates positively with the severity of metabolic impairment. Lipid deposition is primarily extracellular and there is a paucity of foam cells. The very low density and intermediate-density lipoprotein fractions increase significantly, the low-density lipoprotein fraction increases slightly, and the high-density lipoprotein fractions remain essentially unchanged. Because these monkeys are maintained on a nonatherogenic chow ration, the effects of diabetes, per se, on vascular sclerosis can be evaluated.

Immunohistochemical study of islet amyloid in diabetes mellitus.

Amyloid was isolated from islets of amyloidotic pancreata of monkey and human beings by solubilization of non-amyloid materials from the pancreas and digestion of contaminating collagen and elastin. The resulting pellet was estimated to be greater than 90% pure islet amyloid. Antibodies specific for monkey islet amyloid and for monkey and human liver amyloid A (AA) were raised in rabbits. Immunohistochemical reaction using the peroxidase antiperoxidase method demonstrated that amyloidotic pancreas reacted with both anti-AA and anti-islet amyloid antibodies. Although the antibodies are specific toward antigens, they cross-react with tissues from human and monkeys. The immunochemical results suggest the possibility that more than one kind of amyloid is associated with islet amyloidosis, but that a significant portion of the islet amyloid is related to AA. Preliminary chemical analysis indicated that islet amyloid is enriched with hexosamines while AA contains both hexosamines and hexoses. Establishment of the islet amyloid composition(s) can give insight into its source and its role in diabetes in Macaca nigra and human beings.

Reaction patterns of islet-cell autoantibodies in Macaca nigra.

Circulating islet-cell autoantibodies (ICAAs) that reacted specifically with cytoplasmic components have been found in the blood of prediabetic Macaca nigra. The three distinct reaction patterns observed involved the majority of islet cells throughout the islet; a moderate number of cells, mainly at the islet periphery and around the vasculature; and a few cells scattered throughout the islet. Pancreas sections incubated with sera containing ICAAs followed with peroxidase-conjugated antibody were then reacted with anti-insulin, antiglucagon, or antisomatostatin antisera. The pattern associated with most of the islet cells was shown to be reactive to beta cells and was termed B-ICAA; the pattern with cells at the periphery was identified as alpha cells (A-ICAA); and the scattered cells contained somatostatin (D-ICAA). None of the three islet hormones were able to block ICAA reaction after overnight incubation, so the ICAAs are not anti-islet hormone antibodies. The varied reactions with antigens of different secretory cells indicate release of a variety of immunogens from islet cells as they necrose and cause the formation of different ICAAs.

Potentiation with epinephrine of macaque platelet aggregation by other agonists: implications for studies on human atherosclerosis.

The effects of low concentrations of epinephrine on the aggregation of macaque and human platelets by arachidonic acid (AA), collagen, and thrombin were studied. When epinephrine (0.05 to 1 microM) was added to macaque or human citrated or macaque heparinized platelets, either before or after the addition of near-threshold concentrations of AA, significant increases in aggregability were always seen. Epinephrine alone did not aggregate macaque platelets from citrated blood. When near-threshold concentrations of collagen or thrombin were present in the medium, low concentrations of epinephrine (0.05 to 0.50 microM) potentiated the aggregation of macaque and human citrated platelets and macaque heparinized platelets. The P values for the addition of epinephrine were less than 0.01 in all series. The ability of low epinephrine concentrations to potentiate aggregation of macaque platelets by other agonists is of particular significance because in humans the most important effect of epinephrine on platelets in vivo is probably the potentiation, by low concentrations, of aggregation induced by other aggregatory agents normally present in the blood in low concentrations.

An improved apparatus for mounting retinal vasculature.

An apparatus for mounting retinal vasculature substantially reduced the time required to mount the capillary bed, minimized trauma to the vessels, and consistently resulted in preparations that allowed maximal viewing of retinal capillaries.

Obesity is an obligatory component for development of type II diabetes in predisposed monkeys: a testable hypothesis.

Monkeys have been extensively studied with respect to the development of obesity and diabetes. Segregation of the two disorders on a genetic basis has not been possible.

Abnormalities in insulin secretion and glucose clearance among Macaca mulatta examined on Cayo Santiago.

Macaca mulatta on Cayo Santiago (CS) were examined with intravenous glucose tolerance tests (IV-GTT) for evidence of abnormalities in glucose clearance and insulin secretion. About 10% of the 98 monkeys had impaired glucose clearance associated with impaired insulin secretion. Another 6% had either fasting or secretory hyperinsulinemia with slightly increased rates of glucose clearance, and 20% had low insulin secretion, but no significant changes in glucose clearance. Results were compared to those obtained with CS-derived monkeys tested at Sabana Seca (SS). Glucose clearance per amount of insulin secreted was 40% more effective among CS macaques than among those at SS. There were no differences in weight between impaired and control macaques on CS. Effects of genetics, physical activity, and food consumption can be studied among these macaques and results related to similar metabolic abnormalities in prediabetic and diabetic human beings.