Insulin secretion in obese and non-obese NIDDM

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Diabetes Research and Clinical Practice 28 Suppl. (1995) S27-S37

Insulin secretion in obese and non-obese NIDDM Erol Cerasi", Rafael Nesher, Michal Gadot, David Gross, Nurit Kaiser Department of Endocrinology and Metabolism, Hadassah University Hospital, Po. Box 12000, 91120 Jerusalem, Israel

Abstract Both the insulin response to glucose and the sensitivity to insulin show large variation in the normal population. Many subjects have either a markedly low insulin response or low sensitivity to insulin, with nevertheless normal glucose tolerance. For such subjects to become diabetic, insulin secretion or insulin action must further deteriorate with time, or other factors are added which tip the balance towards diabetes. Most evidence to date indicates that reduced f3-cell responsiveness and reduced insulin sensitivity co-exist in subjects prior to developing NIDDM. Both insulin secretion and insulin action are genetically controlled and influenced by intrauterine and neonatal factors. Insulin secretion and insulin action vary inversely in a closely linked manner; inability to fully compensate for changes in one variable mar generate a functional deficit in glucose homeostasis. Subjects combining low functions would run a proportionately larger risk of decompensating the glucose tolerance and be more vulnerable, in terms of diabetes susceptibility, to factors that further reduce insulin output or insulin action. Careful analysis of existing data prompts us to ascribe a dominating role to the impairment of insulin secretion in the pathogenesis of IGT and NIDDM. Patients with NIDDM also exhibit increased proportions of pro insulin and pro insulin conversion intermediates. We used hyperinsulinaemic diabetic and non-diabetic Psammomys obesus to study the possible relationship between steady-state pancreatic insulin stores and the proportion of proinsulin-related pep tides in the plasma and the pancreas. A marked increase in these peptides was associated with 90% reduction in insulin stores of the pancreas. After food deprivation, the depletion of pancreatic insulin in the diabetic animals was partially corrected, and the proinsulin z'insulin ratio normalized. In contrast, non-diabetic psammomys showed only 50% reduction in pancreatic insulin stores under non-fasting conditions, with no change in proinsulinyinsulin ratio. These findings suggest that in the diabetic Psammomys obesus, pancreatic capacity for storage/production of insulin is limited; the metabolic consequences of this limitation are amplified by increased secretory demand secondary to insulin resistance, thus facilitating the establishment of hyperglycaemia, which may in itself further exacerbate the pancreatic dysfunction. Keywords:

Non-insulin-dependent diabetes mellitus; Insulin secretion; Psammomys obesus; Hyperglycaemia

* Corresponding author, Tel.: + 972 2 776788; Fax: + 972 2 420740. 0168-8227/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved. SSDI0168-8227(95)01083·P

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1. Introduction

NIDDM patients present substantial levels of immunoreactive insulin (IRI) in the circulation. The significance of this finding has been an object for controversy among diabetologists: are NIDDM patients normoinsulinaemic, hypoinsulinaemic, or rather hyperinsulinaemic? The latter opinion has become the dominating one over recent years, and except for severe NIDDM, where consensus seems to indicate the presence of insulin deficiency, it has been regarded as a proof for insulin resistance being the main pathogenic factor in NIDDM. There are several reasons for these conflicting interpretations; the most important one relates to the difficulty in assessing quantitatively the in vivo insulin secretion. 2. Assessment of insulin secretion in vivo methodological problems It is difficult to evaluate insulin secretion in man. First, insulin release in vivo is estimated from peripheral insulin levels, with the assumption that the clearance of the hormone is constant under all conditions, which is not the case [1]. Methods exist for calculating the insulin secretion with the help of C-peptide administration [2] or mathematical modelling [3,4]; however, they are impractical and therefore they have not been used extensively. Second, the relationship between stimulus intensity and insulin response is difficult to quantify, and therefore rarely taken into consideration. Plasma insulin is often measured after oral glucose or a meal, when vagal stimulation and gut incretins like GLP-I synergistically amplify the insulin response to glucose [5-7]. This synergism can be of a formidable magnitude; thus, in a dose-response study with oral glucose, it was observed that incremental differences in blood glucose of as little as 1 mmoljl were sufficient to augment the insulin response by 50%-70% in healthy subjects [8] (see also Fig. 2). Interpreted against this background, 'normal' insulin levels in a hyperglycaemic patient should indicate severe impairment of the insulin secretion. Glucose vs. insulin dose-response curves have been constructed from acute experiments in

28 Suppl. (1995) 527-537

healthy subjects [9]; however, it is difficult to relate the insulin levels in diabetics to such curves since the effect of long-term induced hyperglycaemia in normal subjects has not been investigated. Finally, prolonged stimulation with glucose induces a state of potentiation in the islet, which in time amplifies the insulin response (see below): according to the intensity and duration of the stimulation, the degree of amplification may reach 200-400% [10-12] (see also Fig. 3). Disregard for this mechanism may lead to gross misinterpretation of the plasma insulin data. 3. Basal insulin release in NIDDM Beta-cells do not passively leak out hormone; therefore the basal (fasting) insulin level is the result of active, regulated release which, like the stimulated secretion, is dependent on the blood glucose concentration. In NIDDM, basal insulin levels are variable but usually within the range of the control population [13]. However, since these patients are hyperglycaemic, their 'basal' state in reality corresponds to a situation of {3-cell stimulation. Accordingly, insulin-induced normoglycaemia resulted in markedly diminished 'basal' plasma insulin values in diabetic patients [14]. Furthermore, from comparison with the steadystate plasma insulin level obtained in control subjects rendered hyperglycaemic by glucose infusion, it could be concluded that the basal insulin secretion in diabetics was diminished to the same extent as the stimulated release [15]. It has therefore been proposed that the basal insulin concentration in NIDDM is the result of {3-cell adaptation to hyperglycaemia, whose level reflects the steady-state of the feedback regulatory system [16]. 4. Acute insulin response to glucose The kinetic aspects of the insulin response to glucose can best be evaluated with i.v. administration of the sugar (IVGTT or hyperglycaemic clamps) which, although unphysiologic, avoids interference with incretin factors (see above). The most pronounced defect of the insulin response in NIDDM is observed during the first minutes of stimulation, the first-phase release being markedly

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reduced or entirely absent (Fig. 1). Even in mild diabetics, the decreased and delayed appearance of the insulin response is striking. This pattern is observed both in obese and lean NIDDM, as well as in persons with glucose intolerance (IGT) only; however, the higher the fasting blood glucose of the patient, the flatter the insulin response to glucose (for a full review, see Ref. 17). Secondphase insulin response can be quite pronounced in IGT and mild NIDDM (see Fig. 3); this is the consequence of the priming effect of glucose, which amplifies the secretion rate after prolonged stimulation (see below). In advanced NIDDM also, the second-phase of the response is markedly reduced. Glucose dose-response studies in NIDDM show that the maximal capacity of first-phase insulin response is decreased [18]. Second-phase response was found to increase at very high blood glucose levels, the dose-response curve thus showing a right-shift; these kinetic aspects are illustrated in Fig. 2. Since the insulin response to oral glucose corresponds mainly to second-phase release, a similar K rn change was noted [8] (Fig. 2B). Very similar findings were obtained in vitro

in islets isolated from Acomys cahirinus, a rodent model of NIDDM: compared to rat islets, the Vrnax of the first-phase response was reduced, while the second-phase response showed increased K rn [19]. The kinetics of insulin release from perifused islets of Acomys is presented in Fig. 3. It is of interest to note that obesity, which is accompanied by increased insulin secretion in the control subjects, has a similar effect in NIDDM; this is shown in Fig. 2A: when the glucose-insulin dose-response relationships in obese and lean diabetics with a similar hyperglycaemia were compared, the obese patients showed a 2-3-fold increase in the Vrnax of the insulin response, which was identical to the Vrnax differences between matched obese and lean controls [18]. Also in the acomys model, diet-induced obesity resulted in augmentation of the insulin response to glucose, the response however remaining subnormal [20]. In addition to these changes in the gross aspects of insulin release, more subtle malfunctions of the ,B-cell can be demonstrated in NIDDM: the normal oscillatory nature of insulin secretion is modified in diabetics, the 13-14-min cycle be-

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Fig. I. Plasma insulin response to glucose infusion. Blood glucose was maintained at around 20 mmoljl by i.v. bolus and continuous infusion of glucose between 0 and 60 min. Representative examples of lean and obese healthy volunteers (CONTROL) and mild Type II diabetic patients (NIDDM). Note the scale difference in the Y-axis of right-hand panel. In many diabetics, glucose administration induces an initial reduction in the plasma insulin levels (see 5-min value in obese patient).

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Fig. 2. Dose-effect relationship of glucose-induced insulin response in control subjects and NIDDM patients. (A) Increasing amounts of glucose were injected i.v. within 2 min at time 0, the plasma glucose and insulin levels followed during 60 min, and the integrated incremental values plotted. The insulin response reflects mainly the first-phase of hormone secretion. Note the Y-axis scale difference in the right-hand panel. (B) Left-hand panel (i.v.) presents results obtained after 60 min of i.v. glucose infusion at increasing doses in lean controls and NIDDM patients. Blood glucose levels, and the incremental plasma insulin values achieved are shown (second-phase response). Right-hand panel (oral) depicts results obtained at 30 min by oral administration of increasing glucose doses in lean healthy and diabetic subjects. Here, blood glucose levels are plotted on a logarithmic scale.

ing lost in favour of irregular bursts of secretion [21], 5. Priming effect of glucose in NIDDM In addition to its acute releasing effect, glucose generates a state of potentiation in the {3-cell,

which expresses itself by the amplification of the insulin response to subsequent stimulations [12]. In a series of experiments, where a 1-h glucose infusion was repeated following a 40-60-min rest period, it could be demonstrated that the insulin response to the second stimulus was greatly amplified in subjects with NIDDM or IGT (Fig. 4),

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Time (min) Fig. 3. Glucose-induced insulin release in Acomys cahirinus. Islets were isolated from glucose-intolerant acomys, placed in perifusion chambers, and equilibrated with 3.3 mmoljl glucose from - 30 to 0 min. The islets were then stimulated with 16.7 mmoljl glucose during two 30-min periods, between 0-30 and 40-70 min, separated by lO-min perifusion with 3.3 mmoljl glucose. Note the absence of first-phase response during the first stimulation, and its restoration during the second glucose stimulus (priming effect of glucose).

the sensitivity for the potentianng effect of glucose being similar to that in control subjects [22]. As illustrated in Fig. 3, the conservation of glucose-induced potentiation in a functionally defi-

cient islet was strikingly demonstrated in the acomys model: in vitro priming of acomys islets by glucose resulted in the marked amplification of the response, a substantial first-phase release now becoming evident [23]. Thus, while the acute insulin-releasing effect of glucose is greatly reduced in NIDDM, the ability of the sugar to generate potentiation is retained and may even partly correct the secretory defect. Consequently, plasma insulin levels measured early or late during glucose stimulation may convey quite different information on ,a-cell function if this mechanism is ignored. As an example, when the 120-min plasma insulin values during OGrr in NIDDM and IGT are measured, results are higher than normal, suggesting hyperfunction of the islet [24]. However, this is due to the generation of a strong signal for potentiation by the higher glucose levels throughout the test at a time when blood glucose is still high enough to stimulate insulin secretion. If early (30 min) time-points are chosen, the insulin response to OGrr shows a linear fall from normal over IGT to NIDDM. Thus, provided the plasma insulin data is interpreted with full reference to the physiology of insulin secretion, it becomes clear that ,a-cell responsive-

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Fig. 4. Priming effect of glucose on insulin secretion (time-dependent potentiation). A bolus and continuous glucose infusion was repeated at intervals of 60 min (during 0-60 and 120-180 min, blood glucose plateaus around 15 mrnolyl) in a healthy subject (control) and in a patient with impaired glucose tolerance OGT). Note that the markedly reduced first-phase insulin response of the subject with IGT was almost entirely corrected after the glucose priming.

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ness to glucose is lower than normal in IGT, and more so in NIDDM [8,17,25].

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6. Proinsulin-related peptides in NIDDM

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In patients with NIDDM, the plasma ratio of proinsulin-related peptides to insulin is elevated [26-28]. This contributes to the hyperglycaemia, since proinsulin has low biological activity compared to insulin; thus, true hypoinsulinaemia is more prevalent in NIDDM patients than previously realised [29]. We investigated [30] the mechanisms leading to hyperproinsulinaemia in Psammomys obesus, a desert gerbil which develops obesity and insulin resistance when maintained on laboratory chow [31]. Plasma samples from hyperinsulinaemic psammomys were analyzed by HPLC, the results being summarized in Fig. 5. The IRI in the plasma of non-diabetic psammomys consisted predominantly of intact insulin. In contrast, diabetic psammomys exhibited considerable increase in the relative amounts of proinsulin and split products, intact insulin being undetectable in some animals. On average, the relative amount of proinsulin and conversion intermediates was much higher in the diabetic as compared to the non-diabetic group (Fig. 5). Fig. 6 shows that the pancreatic content of total IRI in non-fasted diabetic psammomys was less than 10% of that in fasted animals, with an elevated

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Fig. 5. Plasma levels of insulin and proinsulin-rclated peptides in non-fasted diabetic and non-diabetic Psammomys obesus. Insulin and proinsulin-related peptides were resolved by HPLC. The graph presents results as a percent of total immunoreactive insulin determinations. Black area: insulin; hatched area: proinsulin; white area: proinsulin conversion intermediates.

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Fig. 6. Pancreatic content of insulin and proinsulin-related peptides in Psammomys obesus. Pancreatic extracts were resolved by HPLC. Proinsulin and conversion intermediates are presented together. Open bars: fed animals; hatched bars: overnight fasted animals.

proinsulinjinsulin ratio. These findings suggest that the rise in proinsulin to insulin ratio in the plasma of diabetic psammomys results from the high demand for insulin driven by hyperglycaemia, leading to depletion of pancreatic insulin stores and probably to enhanced release of immature granules rich in proinsulin-related peptides. Acute hyperstimulation of f3-cells in man, produced by a 3-h infusion of glucose, together with tolbutamide and glucagon, was not sufficient to augment the pro insulin to insulin ratio [32]. As

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for chronic hyperstimulation, it is reasonable to assume that hyperglycaemia per se also plays a role, since in contrast to NIDDM, in insulin resistant obese subjects despite the chronically increased load on the insulin producing machinery, the proportion of proinsulin remains normal [33]. It has to be stressed that elevated secretion of proinsulin-related peptides in NIDDM has a major impact on the fasting IRI level (due to the lower clearance rate of proinsulin); this necessitates caution in the interpretation that a high fasting IRI concentration is the marker of insulin resistance in NIDDM. As to the insulin response to glucose, since the decrease of total plasma IRI in IGT and NIDDM, corrected for blood glucose level and time of stimulation, is so pronounced, an increase in the proportion of proinsulin is of little relevance to the present discussion.

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7. Is the deficient insulin response specific for glucose?

Fig. 7. Synergistic interaction between blood glucose level and i.v. glucagon on plasma insulin response. Glucagon (0.5 rng) was injected rapidly at time 0, and the first-phase plasma insulin response measured. The test was performed in NIDDM patients with a fasting plasma glucose level around 8 mmoljl (diabetic), and in control subjects at their normal fasting glucose (around 4 mmoljl; control) and during a glucose clamp (around 8 mmol z'l: control hyperglycaemic).

Most NIDDM patients who fail to secrete insulin on glucose administration do respond to non-glucose secretagogues such as arginine, glucagon, isoproterenol, etc. This observation has led to the acceptance that the {3-cell defect in NIDDM is restricted to non-recognition of glucose [34]. However, the situation is somewhat more complex: the seemingly normal insulin response of the diabetic to agents that act synergistically with glucose is due to the hyperglycaemia of the patient. This is illustrated in Fig. 7, which shows that when control subjects were hyperglycaemia-matched to patients by a glucose infusion, their insulin response to glucagon was several-fold greater than that obtained in the NIDDM patients. Similar results were obtained with arginine [15]. Such studies could suggest that the diabetic {3-cell defect includes the glucagon and arginine stimuli, and therefore is not specific for glucose. However, these agents are inactive in the absence of glucose, they are amplifiers of glucose-induced insulin release. Indeed, arginine augmented the insulin level by a factor of 2-4 at all glucose concentrations, equally in controls, and NIDDM and IGT patients [35].

Sulfonylureas also require glucose to amplify the insulin output, most of the effect being observed at medium-high levels of glucose in vitro as well as in vivo (for review, see Ref. 36). In moderately hyperglycaemic NIDDM patients, near-normal acute insulin responses to intravenously administered sulfonylurea were observed; however, when combined with a glucose dose-response study, the ability of sulfonylureas to increase the sensitivity of the {3-cell for glucose was found limited [37]. Under chronic therapeutic conditions, sulfonylureas seem to retain their stimulatory action: the insulinogenic effect of glucose was increased by 50%-100% in NIDDM patients treated with sulfonylureas for 3-6 months [38,39]. To summarize, much of the data available does suggest that the deficient insulin response in NIDDM is specific for glucose. Most other insulin secretagogues depend on glucose in order to activate secretion, the final insulin response being the result of synergism with the glucose effect. Glucose-induced insulin release being diminished in NIDDM, even when normally amplified by non-

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glucose secretagogues, the final result is a low insulin response. 8. Insulin response during the development of NIDDM

There is a consensus regarding the role of reduced insulin responsiveness as a significant risk factor for the transition from IGT to overt NIDDM; this has been shown both in large population studies [40] and in the follow-up of gestational diabetics [41,42]. In contrast, the situation is controversial regarding the earlier stages of the development of glucose intolerance (so called prediabetic stage). Most authors favour the view that prediabetic individuals are hyperinsulinaemic: epidemiological studies show that insulin resistance and hyperinsulinaemia predict NIDDM (summarized in [43]), and the follow-up of a high risk group, the offspring of two diabetic parents, indicated that those with initial insulin resistance and hyperinsulinaemia show a higher incidence of IGT and diabetes several decades later [44,45]. It is questionable, however, whether such subjects do have over-functioning f3-cells: first-phase insulin response to glucose was either within normal limits or slightly above normal despite insulin resistance [45]. This contrasts with the mean 3-fold increase of the insulin response found in equally insulin resistant non-diabetic obese subjects, or women in third trimester pregnancy [18,46]. Even under short-term conditions (e.g, nicotinic acid administration for 2 weeks [47]), normal f3-cells are fully capable of adjusting to the increased secretory demands. Unfortunately, the magnitude of the glycaemic 'overdrive' in insulin resistance is difficult to quantify. Attempts have been made to correlate quantitatively the insulin response to the subject's insulin sensitivity [4,48,49], however these methods have not gained wide use. In contrast to the above 'hyperinsulinism hypothesis', several observations suggest that a decreased f3-cell function exists prior to the impairment of the glucose tolerance. Thus, in healthy identical twins of diabetic patients, the insulin response was as decreased as in the diabetic twin [50,51]. In women with a past history of gestational diabetes, insulin response to glucose was sub-

normal [41,42]. We found reduction in first-phase insulin response also in a population of lean normal subjects [3]. Low insulin responses exist also in children [52], which concords with the significant heritability and stability of the insulin response [53,54]. The incidence of IGT and NIDDM was 4.5-fold greater over a 5-15 year follow-up in such subjects with initially low insulin responses [55,56]. It is our belief, therefore, that IGT, and later NIDDM, develop mainly on the basis of impaired insulin responsiveness. 9. Possible mechanisms for the impaired insulin secretion Many steps in the stimulus-secretion coupling of glucose-induced insulin release have been examined, and often found defective, in islets from diabetic animals or in some types of NIDDM. These include the glucose transporter Glut 2, the glucokinase, the adenylate cyclase system, futile cycling at the level of glucose - glucose-6-phosphate, various mitochondrial deficiencies, and the f3-cell amylin. It is beyond the scope of this review to summarize the vast literature in this area. Interested readers are referred to a recent short but comprehensive review by Malaisse [57]. 10. Conclusions Both the insulin response to glucose and the sensitivity to insulin show a large variation in the normal population [3,48,49,58]. Thus, there exist substantial numbers of subjects with either a markedly low insulin response or low sensitivity to insulin, with nevertheless normal glucose tolerance. For such subjects to become diabetic, either insulin secretion or insulin action deteriorates further with time, or additional factors tip the balance towards diabetes. Most evidence to date indicates that reduced f3-cell responsiveness and reduced insulin sensitivity co-exist in subjects prone to develop NIDDM. Both insulin secretion and insulin action are genetically controlled [53,59] and influenced by intrauterine and neonatal factors [60,61]. Insulin secretion and insulin action vary inversely in a closely linked manner; inability to fully compen-

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sate for changes in one variable may generate a functional deficit in glucose homeostasis. Therefore, ,a-cell function and insulin action should be considered on a gradual scale of risks, where subjects combining low functions would run a proportionately larger risk of decompensating the glucose tolerance. In addition, it seems reasonable to assume that subjects whose functional capacity is in the lower end of the normal spectrum, both regarding insulin production and insulin action, would be more vulnerable in terms of diabetes susceptibility to factors that further reduce the function (age, environmental toxins, amylin deposition [62], 'glucose toxicity', etc. for the ,a-cell; age, obesity, decreased physical activity, etc. for insulin action). Although these conclusions may equally apply to insulin secretion and to insulin action, careful analysis of existing data prompts us to ascribe a dominating role to the impairment of insulin secretion in the pathogenesis of IGT and NIDDM. Acknowledgements

The studies summarized in this paper were supported by grants from the Wolfson Foundation, the Juvenile Diabetes Foundation International, and the Israel Science Foundation.

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