Glucose Tolerance in Obesity
Animal models of obesity are highly associated with observations of reduced glucose tolerance (61), and in the non-human primate this glucose intolerance precedes overt type 2 diabetes, as recently reviewed (62). Glucose tolerance to an intravenous glucose infusion ranged in monkeys from a glucose disappearance rate of 5 %/min to 2%/min without any change in fasting plasma glucose. When tolerance dropped below 2%, fasting plasma glucose began to be elevated (61). Thus, there is a powerful compensatory response which, despite declining glucose uptake rates, maintains plasma glucose at normal levels in the obese non-human primate.
Longitudinal in vivo studies in rhesus monkeys have shown that as obese monkeys begin to make the final transition from impaired insulin sensitivity and impaired glucose tolerance at the whole body to overt type 2 diabetes, increasing fasting plasma glucose very closely parallels increasing basal hepatic glucose production (63). In a related event, as hyperinsulinemia progresses to high levels, hepatic extraction of insulin (the proportion of the insulin presented to the liver which is removed by the liver) declines (64).
quently generally declines as fasting plasma glucose begins to rise in the early stages of overt diabetes (notably shown in Figure 14.4) (67). Nevertheless, at the time of diagnosis of diabetes, insulin levels are usually still elevated above normal, and ^ cell responsiveness to glucose is completely absent, as recently reviewed (62).
Under basal fasting conditions in both monkeys and humans, insulin is secreted with a 10 to 14 minute oscillating periodicity (68, 69), and the amplitude of this periodic secretory output increases with obesity and hyperinsulinemia. This amplification of the secretory output in obesity may play a protective role, or alternatively, may signal disturbed ^ cell functioning and regulation in the prodrome to diabetes. Further, this periodic secretory pattern is completely disrupted by 48 hours of fasting or by the development of diabetes, even when basal insulin levels are above or within the normal range (70). Oscillations are also lost by an exogenous infusion of glucose raising glucose levels to about 6 mmol (71). The presence of oscillations in plasma levels of insulin may thus be viewed as a reflection of a 'contented' beta cell (71).
Beta cell insulin secretion is minimally affected during the early stages of obesity, as shown by the comparison of changes in body fat and in fasting plasma insulin for monkey A-7 illustrated in Figure 14.2. Insulin sensitivity, measured at the whole body by the euglycemic hyperinsulinemic clamp, remained normal during that period as well, while fat mass more than doubled. The increase in insulin secretion and the decline in insulin sensitivity that took place in this monkey after age 12 were closely related, and have been observed in a larger group of monkeys (65). Thus, changes in adiposity per se were not directly associated with changes in insulin secretion or insulin sensitivity. In most monkeys and in most humans, increased adiposity appears play a role as an apparent facilitator or permissive factor for the declines in whole body insulin action and the increase in insulin output. Both basal fasting levels of insulin and ^ cell responsiveness to a glucose stimulus increase in the early stages of the transition from 'simple' obesity to insulin-resistant prediabetic obesity (66). Insulin secretion subse
Tissue Specificity in the Sequence of Appearance of Defective Insulin Action
There is a wide range of insulin sensitivity in monkeys with normal glucose tolerance (24,72), including obese monkeys with or without insulin resistance.
We tend to think of insulin sensitivity, or its converse, insulin resistance, as a single entity, regardless of how or where it is measured. This is unfortunate and has resulted from the paucity of studies in which whole body insulin-resistance and resistance at each of the major insulin sensitive tissues have been measured simultaneously. Most commonly, so-called 'normals' (usually age and/or weight matched) are compared to so-called diabetics (individuals with significant hyperglycemia). Under these groupings, normals are normal in both whole body and tissue determinations and diabetics are 'resistant' at both the whole body and at various tissues. More detailed analysis has shown in rhesus monkeys that whole body insulin resistance, probably principally reflective of skeletal muscle insulin resistance, develops in obesity in parallel with hy-perinsulinemia (72), and well before the appearance of insulin resistance at adipose tissue and at liver (63). Resistance in the latter two tissues seems to be directly associated with the progression of individuals from obese with hyperinsulinemia to overtly diabetic.
Insulin Action on Glycogen Metabolism in
Skeletal Muscle, Adipose Tissue, and Liver
Obese hyperinsulinemic monkeys had a significant decline in insulin-mediated change in glycogen syn-thase activity in skeletal muscle (73). This defect appeared early at about the same time as the increasing insulin secretion noted above, that is, at the same time as ^ cell hyper-responsiveness developed. Obese and insulin resistant monkeys also had significantly higher insulin stimulated glucose 6-phos-phate concentrations compared to normal monkeys, suggesting that a step distal to glucose 6-phosphate is a major contributor to reduced insulin-mediated glucose disposal and reduced insulin action on glycogen synthase activity (74).
In adipose tissue, by contrast, both basal and insulin-stimulated total activities of both glycogen synthase and glycogen phosphorylase were increased above normal in obese hyperinsulinemic monkeys (75). Insulin action to increase glycogen synthase independent activity was reduced in obese hyperinsulinemic monkeys compared to the normal monkeys. Specifically, in normal monkeys insulin stimulation induced a 100% increase in glycogen synthase independent activity over basal levels compared to a 50% increase in obese hyperin-sulinemic monkeys (and no increase in diabetic monkeys) (75).
At the liver of monkeys, a different picture of defects in insulin action with obesity has emerged. Glycogen synthase activation and glycogen phos-phorylase inactivation by insulin (in a reciprocal fashion) were significant in the liver of both normal lean monkeys (76) and obese hyperinsulinemic monkeys under the condition of a euglycemic hy-perinsulinemic clamp (76). In obese insulin-resistant monkeys, under the same conditions, glycogen syn-thase (GS) total activity was lower under basal conditions compared to the lean young animals. Nevertheless, total GS activity was significantly increased by insulin stimulation in the liver of insulin-resistant monkeys. Both the basal GS independent ac tivity and the insulin-stimulated independent activity of the insulin-resistant monkeys were higher in the latter group compared to the lean animals (77). Thus, insulin action at the liver was found to be strong in monkeys that were otherwise determined to be insulin resistant at muscle and adipose tissue, as well as resistant at the whole body level. This would accord with the normal hepatic glucose production of insulin-resistant obese monkeys (increased only in diabetics where insulin's suppression of hepatic glucose production is significantly impaired (63)).
PREVENTION OF OBESITY: LESSONS FROM ANIMAL MODELS
Although the causes of obesity in the vast majority of humans (99%) and in all obese non-human primates are unknown, studies in rodents and in nonhuman primates have unequivocally demonstrated that calorie restraint, sufficient to prevent significant increase in total body fat, prevents obesity (by definition), but more importantly, prevents most obesity-associated diseases. Excess body fat or altered energy balance clearly plays a facilitative role in many of the comorbidities of obesity. While we continue the search for the underlying causes of obesity, animal models show that even without knowing the causes, interventions to reduce the degree of obesity can have very strong positive consequences for many obesity-associated diseases, as well as for overall reduction in mortality and extension of life span.
Trowell and Burkitt's studies of epidemiological changes in modernizing societies showed that obesity is the first of the 'diseases of civilization' to emerge in the longitudinal picture (78). As such obesity is clearly the earliest target for intervention to halt a wide range of non-communicable diseases of modern and modernizing societies. Gracey has termed this defined cluster of diseases the New World syndrome (79), and has included within its sphere obesity, type 2 diabetes, hypertension, dys-lipidemia, and cardiovascular disease (also termed the metabolic syndrome X (80)) (with the addition of cigarette smoking and alcohol abuse).
The World Health Organization has commented extensively on the societal factors which have accompanied or induced the changes leading to the New World syndrome, and which have led to identification of obesity as a global epidemic (25). The WHO Report cited such components of modernization as the development of market economies, reliance on imported non-traditional foods, increasing urbanization, changing occupational structures, increasing socioeconomic status, increases in animal fat and animal protein intake, decreases in vegetable fat and vegetable protein intake, reduction in total and specifically complex carbohydrates, and increases in sugar intake. The net effect of these factors might be viewed as providing an unrestrained environment in which genetic potential becomes fully expressed. Alternatively, the environment may be interacting in such a way as to be detrimental to 'normal' gene expression.
As noted below, evidence from animal models strongly indicates that these diseases are not independent. As in diseases of human civilization, the sequence of the appearance in animals starts with obesity. The prevention of obesity can prevent or greatly reduce all of the others, and as a further consequence, greatly reduce morbidity and mortality. Because the benefits of obesity prevention have to date been difficult to attain in humans, examination of the data from animal models of obesity prevention can be informative for the human condition and the potential importance of obesity mitigation.
Primary prevention includes all measures aimed at reducing the incidence or preventing the occurrence of a disease and its complications or of reducing the risk of disease. Secondary prevention includes the measures introduced to mitigate the consequences of a disease, slow its progression, and reduce its associated morbidities following the early diagnosis of the disease. For example, primary prevention of obesity in animals is achieved when the development of increased body fat is completely prevented, usually by calorie restriction, while secondary prevention is introduced when weight reduction or body fat ablation of an already obese animal has been instituted.
Successful prevention interventions rely upon an understanding of the natural history of a disease, together with the identification of sufficiently powerful and successful methods for preventing the disease. In the case of obesity prevention in humans a significant number of risk factors for obesity have been identified, and a modest amount is known about the natural history of obesity (as established in this volume). In humans, however, only a few methods of limited applicability (primarily surgical approaches) have been identified which truly modify the course of the disease, generally mitigating its consequences after obesity has reached severe stages (81). In rodents and in non-human primates, by contrast, deeper understanding of the aging-related changes in body composition and of the factors increasing risk for obesity have been developed. In addition, the usual laboratory environment, with its readily available constraints and manipulability, has enabled the introduction of powerful non-surgical approaches to obesity prevention, thus allowing assessment of its consequences and its likely implications for humans. Current efforts in humans are principally limited to identification of high-risk individuals for 'lifestyle' changes, and environmental manipulations. The power of these interventions is extremely limited at this time. Nevertheless, studies in non-human primates demonstrate that the successful prevention of obesity has far-reaching consequences and extraordinarily high potential for positive impact on human health.
Efficacy of Primary Prevention of Obesity in Rodents and Non-human Primates
The longest ongoing study of calorie restriction in non-human primates, initiated when the monkeys were fully adult at about age 10, continues at this time and has already shown the powerful effects of obesity prevention in preventing or greatly postponing morbidity and increasing average lifespan. Further, simply instituting primary prevention of obesity (by calorie titration on an individual animal basis) has provided the most powerful means known to prevent the development of overt diabetes (82). In the restricted animals, there is no diabetes, while in the ad libitum fed animals diabetes rate exceeds 30%, and the obesity rate exceeds 50% (82). Calorie restriction also results in the prevention or significant postponement of many features that normally contribute to cardiovascular risk, including many diabetes-associated metabolic dysfunctions, such as reduced dyslipidemia, and improved blood pressure profile (83). As a result of this disease prevention, there appears to be excellent potential to extend maximal lifespan as well. This, however, must await further extension of the study as the monkeys are now in their late 20s and the maximal reported lifespan in captivity for the rhesus monkey is 40 years (83).
The extension of average life expectancy and the potential for extension of maximal lifespan raise the possibility that, as in rodents, calorie restriction in non-human primates exerts an anti-aging effect which is separate from and in parallel with the anti-disease effects so clearly already demonstrated (83). The mechanisms by which such anti-aging effects of obesity prevention are achieved are, as yet, unknown. However, several such potential mechanisms are under study in rodents as well as in nonhuman primates. In rodents, a reduction in mitochondrial oxidative damage during aging has been found with calorie restriction (84). Examination of differential gene expression in rodents with and without calorie restriction has indicated a marked reduction in the stress response and a lower expression of a number of metabolic genes in the calorie-restricted group (85).
Improvement in glucose tolerance, dyslipidemia and blood pressure, and prevention of diabetes do not appear to require maximal leanness. In one reported study, body fatness in calorie-restricted monkeys has been maintained at levels ranging from 10 to 22%, a normal range for non obese adult monkeys (83). Whether levels below this (excessive leanness) will further improve health or will in fact be detrimental to health will await the reports of other studies of calorie restriction in non-human primates (86).
Fat distribution may also be playing a role, as calorie restriction in non-human primates results in improved body fat distribution (reduced fat in the abdominal region) which is directly associated with the reduced overall body fat content (87).
Prevention of Hyperleptinemia
The hyperleptinemia associated generally with obesity in humans and monkeys as noted above (38)
is prevented by calorie restriction with the prevention of obesity, and the normalization of leptin levels could potentially be advantageous, although this is speculative at this point.
Prevention of Obesity: Effects on
Glucoregulation, Insulin Secretion, and Insulin Action
In rodents as well as in rhesus monkeys, calorie restriction results in maintenance of normal fasting plasma glucose levels, and normal glucose tolerance (82). Both fasting plasma glucose and fasting plasma insulin are lower relative to control ad libitum fed monkeys, but are not reduced relative to normal lean young adult monkeys (88,89).
In monkeys, dietary restriction produces significantly higher in vivo insulin action compared to ad libitum fed monkeys of the same age (90). Aging and obesity-associated insulin resistance appear to be mitigated by long-term restraint on calories in monkeys, and the same is likely to be true in humans. The development of type 2 diabetes that is prevented by calorie restriction and obesity prevention may be accomplished by this sustaining of insulin action (82), presumably particularly in skeletal muscle.
In non-human primates, glycogen synthase activity in skeletal muscle is increased by calorie restriction (91). The mechanism by which this effect is achieved is unknown. In some of those monkeys, however, there was an unexpected decrease in glycogen syn-thase fractional activity with insulin stimulation, a greater increase in skeletal muscle glucose 6-phos-phate, and the greatest increase in glycogen phos-phorylase activity with insulin. These unusual responses were associated with a relatively lower whole body glucose uptake rate compared to the other calorie-restricted monkeys. There was an unexpected increase in the glucose 6-phosphate Ka of skeletal muscle glycogen synthase, indicating phosphorylation (rather than dephosphorylation) of glycogen synthase in response to insulin (91). These changes may be involved in the anti-diabetogenic properties of caloric restriction.
Obesity, Dyslipidemia, and its Prevention by Calorie Restriction
Dyslipidemia: Hypertriglyceridemia and Low HDL Cholesterol
Monkeys, like humans, frequently develop dys-lipidemia in middle age, including hypertrig-lyceridemia and reduced HDL cholesterol levels (92), and this dyslipidemia is highly associated with the presence of obesity, with or without diabetes. Pharmaceutical agents which alter dyslipidemia in humans do so in monkeys as well (93,94).
Prevention of Dyslipidemia by Calorie Restriction to Prevent Obesity
Abnormalities in plasma lipid levels are virtually entirely prevented by calorie restriction in non-human primates (95,96). Both the reduction in plasma triglyceride levels relative to ad libitum fed controls, and the increase in the HDL2b subfraction, which is associated with reduction in atherosclerotic risk, were principally accounted for by the reduction in body mass and the associated improvement in glucoregulation noted above (96). Calorie restriction in rhesus monkeys, while producing no change in plasma LDL cholesterol concentrations, reduced the molecular weight of the LDL particles, and reduced their triglyceride and phospholipid content, together with reduced proteoglycan binding (97).
Restraining Calories to Prevent Obesity: Effects on Energy Expenditure
line shown is for the ad libitum monkeys only. This reduced energy expenditure was present even when the energy expenditure rate was adjusted for differences in body weight, body surface area, or lean body mass (98).
In the same study of long-term calorie restriction in adult rhesus monkeys, thyroxine (T4) was reduced and the free thyroxine index tended to be lower, without change in triiodothyronine (T3).
Calorie restraint in adult monkeys has been reported to result in lower body temperature (99). However, this was not observed in a group of long-term older calorie-restricted monkeys (98).
When rhesus monkeys were studied after 10 years of adult onset calorie restriction with long-term stabilized body weight, total daily energy expenditure was reduced compared to ad libitum fed monkeys, as shown in Figure 14.8. Note that the regression
Physical activity of the calorie-restricted monkeys was greater than of the similar aged ad libitum monkeys (who were considerably fatter), but activity did not differ between calorie-restricted older monkeys and similar body weight younger adult animals (98).
The reduced energy expenditure during calorie restriction is therefore not due to a reduced activity level as might have been suspected.
Secondary prevention, or mitigation of already existing obesity for the purpose of reducing the negative health consequences of obesity, is also important. Behavioral and environmental manipulations are widely applied but only modestly successful. Surgical treatments are of limited application. Pharmacological approaches are expanding and currently animal models are contributing to the examination of new anti-obesity agents.
New Pharmacological Studies in Animal Models of Obesity
Beta 3 Adrenergic Receptor Agonists to Increase Energy Output from Adipose Tissue and Reduce Fat Mass
Although ft adrenergic agonists have been under study in rodents for more than 20 years, only in the past several years has the uniqueness of the primate (human and non-human) receptor been identified. Currently, agents under study are specific for the human receptor and are being extensively tested in non-human primates whose receptor is very similar the human sequence(100). The receptor sequence in the rhesus monkey is shown in Figure 14.9. Studies of agonists in monkeys have been reviewed recently (101). Such agonists have been shown to be active at the non-human primate receptor (102), acutely producing lipolysis and metabolic rate elevation and increased UCP1 expression in brown adipose tissue. To date, however, none has been reported to produce a reduction in body weight. This may be due to an insufficient number of receptors on the adipose tissue of humans. Recent studies have shown an increase in the expression of the mitochondrial uncoupling proteins (UCP2 and 3), and possible increase in the number of brown adipocytes (103). These agonists also seem to have lipid-lowering and insulin-sensitizing effects. In young rats a agent has led to reduced body mass and adiposity which was blunted in older rats (104).
GLP-1 and exendin-4. GLP-1 (glucagon-like polypeptide-1) has been studied in non-human primates (101), but its short half-life has precluded it from being considered for clinical use. Another amino acid peptide with 53% sequence similarity to GLP-1, exendin-4, has been shown to have prolonged glucose lowering action in vivo in obese nonhuman primates and in rodent models of obesity and diabetes (105).
Thiazolidinediones. Thiazolidinediones, a class of insulin sensitizers, have been examined in animal models of obesity and of diabetes, including application to non-human primates (101). In general they improve insulin sensitivity and lower plasma glucose levels in some, but not all prediabetic and diabetic subjects (106). They also reduce hypertri-glyceridemia. Recently a new mechanism of action of this class has been reported, the enhancement of glycogen synthase activity in skeletal muscle, possibly accounting for some of the apparent insulin-sensitizing effects observed in the whole body (107). Figure 14.10 shows the results of a study in which the thiazolidinedione R-102380 was administered for 6 weeks with measurements made before and at the end of the dosing period. Glycogen synthase activity was measured in skeletal muscle biopsies obtained under basal and insulin-stimulated conditions (before and during a euglycemic clamp). All four monkeys studied showed an increase in insulin action to increase glycogen synthase independent activity as well as fractional activity (independent divided by total activity). Insulin-sensitizers are likely to continue to be a focus of expanded efforts to mitigate the health consequences of obesity and to slow or prevent the development of diabetes.
Leptin administration. The administration of leptin to either rodents or to humans with leptin deficiency has been shown to reverse this form of obesity (108). The leptin-deficient obese subjects lost significant adipose tissue mass, and reproductive function was restored (108). When administered
to non-human primates without any abnormality in the leptin axis, peripheral leptin had no effect on food intake or body weight (109). However, when leptin was administered to the normal monkeys intracerebroventricularly there appeared to be a delayed reduction in food intake the next day. Leptin administered to the obese Psammomys obesus sand rat failed to affect body weight, body fat, or adipose gene expression (110), although there were some gene expression changes induced by leptin in lean control animals.
Further studies of possible central mechanisms of leptin action are needed, but the hope for a quick 'obesity fix' is unlikely to be realized by the administration of leptin to most obese persons.
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