Diabetes mellitus has been reviewed as a group of conditions with impaired function of one or more portions of a feed-back system involving the release and utilization of insulin. It is hoped that this may form a useful scheme by which we can study and understand a number of complex metabolic states which we must still collectively refer to as diabetes mellitus

Prout, Thaddeus E.

VCU Scholars Compass

14 The State of Insulin m the Blood* THADDEUS E. PROUT Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland In discussing the state of insulin in the blood, I should like to review briefly the regulation of insulin secretion and what is known about its manner of synthesis and release. Since some of the insulin in the blood may be in an ineffective or inactive state, I shall refer to active insulin as efjective insulin. METABOLITE STIMULATORS The stimulus acting on the pan-creas that controls the level of effective insulin in the blood is probably related to more than one metabolite. For many years glucose was considered the only substance capable of insulin stimulation, but recently it has been shown that keto-acids (Mebane and Madison, 1962) and certain amino acids such as leucine and arginine (Floyd et al., 1965; Merimee et al., 1965) can also stimulate insulin release. There is need to learn whether this has physiological significance be-fore assigning it a specific role in the checks and balances of human metabolism. Intravenous studies using arginine will be followed by oral feedings of appropriate animal proteins to determine whether or not there will be an increase in the level of serum insulin without a rise in blood sugar. * The schema presented here is the result of much discussion involving all members of the endocrine division at the Johns Hopkins Hospital. Dr. Kenneth Zierler, Dr. David Rabino-witz, .and Dr. Thomas Merimee are especially cited in the formulation of · these concepts. SENSING-RELEASING MECHANISMS The manner in which glucose or other metabolites bring about the secretion of insulin is unknown. It is reasonable to postulate that a sensing device must be present that recognizes the amount of the spe-cific stimulator present and in-forms the pancreas that insulin is needed. This immediately raises the possibility that a pathologic state could exist wherein a faulty sensing device would fail to inform the pancreas of the need, and insulin would not be secreted despite the presence of normal quantities of insulin in the pancreas. Studies with the sulfonylurea drugs may per-haps be interpreted as bearing on this question. It is clear that insulin can exist within the islet cells quite unaware of the hyperglycemic blood flowing past and that sulfonylurea drugs can bring about the release of this insulin. These drugs may activate a sensing device. It is equally possible that they may ac-tivate a releasing mechanism for we cannot separate these two postu-lated functions clinically. In indi-viduals with adequate pancreatic stores of insulin that are not re-sponsive to elevated levels of glu-cose, the sulfonylurea drugs bring about a more normal response to the stimuli. There is increasing evi-dence that there is carbohydrate intolerance that advances with age (Andres, 1966, in press) and again it seems likely that this relative in-sensitivity comes about through a failure either of the sensing device MCV QUARTERLY 2(1): 14·18, 1966 or releasing mechanism. Perhaps other conditions which alter insulin release, such as the glucose intoler-ance following fasting, may relate to these mechanisms. SYNTHESIS AND STORAGE Although we can say little about the mechanisms of release of in-sulin, we are indebted to the beau-tiful studies by Lacy ( 1961) with the electron microscope for an ex-position of the migration of the insulin granule across the beta-cell to the cell surface. On reaching the cell surface, the envelope of the beta granule appears to coalesce with the surface membrane where upon the beta granule is discharged into the extracellular space. At this point, unfortunately, the resolution of the electron microscope does not allow further physical view of the discharged granule. After dis-charge of the previously stored insulin, empty sacks are again seen in the beta cell; these are fre-quently surrounded in part by a protein believed to be ribonuclear protein. The ribonuclear protein granules clustered about the sack slowly disappear as the amorphous material fills the empty sack, and it is easy to presume that we have witnessed the synthesis of insulin on a template. The amorphous ma-terial in the sack is next seen to condense into a storage form that is typical of the beta granules pre-viously discharged. The discharge of these granules in response to known stimuli such as glucose and sulfonylurea drugs adds further to the circumstantial evidence that we are in fact witnessing the synthesis and release of insulin. Randle and his associates (see Coore and Randle, 1964) have been able to show that insulin can be released from pancreatic slices in vitro by the addition of glucose to the medium. In this system, epinephrine inhibits the release of insulin by both glucose and the sul-fonylureas. Perhaps the most im-portant observation has been that T. E. PROUT he has been able to prevent the re-lease of insulin from the pancreas by the addition of manoheptulose when glucose is added to the me-dium, but manoheptulose does not prevent the release of insulin under the stimulus of the sulfonylureas. This is the first time that anyone has been able to separate the re-lease mechanisms associated with glucose from those related to the sulfonylureas and parenthetically this suggests that the release mech-anism rather than the sensing device is the site of action of the sulfony-lureas in human subjects. CIRCULATING INSULIN In considering the state of in-sulin in the blood, we must deal with certain direct questions: What is its form? How is it transported? And what is the material being measured which we call "insulin?" These are fundamental questions, but our answers must be incom-plete. From the work of Sanger (1960) we have the precise structure of the insulin molecule, and the molecular weight is known to be approxi-mately 6,000. It has not been pos-sible to be certain of the form taken by circulating insulin. It probably circulates as a double molecule or dimer with a molecu-lar weight of 12,000, but it may circulate in aggregates of three or four molecules (Prout, 1963). There is some evidence that the insulin may be transported in the blood attached to a carrier protein, but this cannot be definitely proven at this time. Insulin is measured in biologic fluids on the basis of its biologic effect or its immunologic properties. In general, there are two types of biological assay, one measuring the effect of glucose uptake or glycogen deposition in a rat hemi-diaphragm and the other measuring the incor-poration of C" from labelled glu-cose into glycogen or CO. by the rat epididymal fat pad. Both of these tests measure a net biologi-cal effect which may be altered as a result of several modifying fac-tors either potentiators or inhibi-tors. For this reason, the activity thus measured is referred to as the "insulin-like activity"' or ILA. The immunoassay of Berson and Yalow ( 1960) identifies immunologically active insulin quite specifically and with considerable sensitivity; it is, of course, able to tell us nothing about the biological activity of the thing that is measured. About these two types of assay have grown up two schools of thought which di-vide the investigators interested in this field . Faced with this dilemma, it is of some comfort to consider the num-ber of ways in which the ILA as measured by the diaphragm and the immunoassay give parallel results (table 1). By both tests the blood insulin is low in the fasting sub-jects but rises after glucose or after administration of the sulfonylureas. By both tests the blood insulin is high in patients with insulin secret-ing islet cell tumors. In obese pa-tients both assay techniques reveal increased insulin levels. The levels may be high by both assay meth-ods in certain adult diabetes, and most importantly immunologically active insulin and ILA as tested by the rat hemi-diaphragms are both TABLE l Similarities in the Assay of Insulin in Whole Serum by the Rat Diaphragm and by the Immunoassay . l) Law insulin levels in blood of fasting pa-tients 2) Rise of insulin after glucose administra-tion 3) Rise of insulin after sulfonylurea admin-istration 4) High levels of insulin In hyperinsulinlsm 5) Elevated levels of Insulin in some obese subjects 6) Suppre~lon of Insulin in vitro and in vivo by specific Insulin antibodies 7) Disappearance of Insulin ofter pancrea-tec.tomy 8) Absence of Insulin in serum of patients in diabetic keto-acidosis 15 suppressed by anti-insulin anti-bodies either in vitro or in vivo. Finally, the insulin measured by either method disappears when the pancreas has been removed. In these respects both assays would appear to be measuring the same material. The observation that insulin-like activity measured by the fat pad was several fold that measured by the diaphragm or the immuno-assay turned attention to the ques-tion of whether or not a circulating form of insulin existed which had lost, or perhaps had never had, immunologic identity. By means of a cation exchange resin column, Antoniades and his associates ( 1964) separated a form of insulin-like activity that could be measured initially by the fat pad but not by the diaphragm or immunoassay. This was called "bound" insulin as opposed to the "free" form. Others have found insulin-like activity that is "atypical" (Samaan et al., 1963) or "non-suppressible" (Froesch et al., 1963) because the activity un-like that of crystalline insulin is not inhibited by anti-insulin anti-sera added to the test medium. 'Ve can conveniently consider together these various types of "bound," "complexed," "non-suppressible," or "typical" insulins and contrast them to insulin that is measurable by the other methods (table 2). One difficult problem confront-ing the proponents of atypical in-sulin in that some "atypical" insulin seems to persist in the blood for considerable periods of time after pancreatectomy (Goldberg and Egdahl, 1961). Before attempting to give clinical relevance to ILA of the "atypical" type, a second observation requires physiologic explanation. We are asked, for example, to believe that ILA meas-ured only by the fat pad is circulat-ing in excess quantities in patients with decompensated diabetes in whom the principle evidence of decompensation is failure of insulin to act normally upon fat synthesis (Steinke et al., 1961). These criti-16 STATE OF INSULIN IN THE BLOOD TABLE 2 Differences Between "Typical" and "Atypical" insulin "Typical" "Atypical" Assay Measured by diaphragm Measured by fat-pad fat-pad and immunoassay Effect of Inhibited Not inhibited antiserum Effect of pan- Disappears Remains createctomy Origin Pancreas Unknown K eto-aci dos is Absent Present in large quantity Other unresolved Biologic activity of in- Cannot be converted questions sulin measured immuno- reproducibly to logically is not known typical form TABLE 3 The Feed-back Mechanism of Insulin Synthesis and Release Metabolite stimulator l Sensing-releasing device Pancreatic synthesis and storage Cellular response Metabolite utilization t Insulin-cell receptor linkage t Insulin in biologic fluids cisms have lead many to deny that "atypical" insulin has any real rela-tionship to true pancreatic insulin. Despite these difficulties, it is theo-retically possible that some form of insulin may exist which is differ-ent from the insulin measured im-munologically and by its biologic effects on muscle and further study of the problem will be necessary. A FEED-BACK MECHANISM We can now picture the glucose insulin regulating mechanism as a feed-back system (table 3). A met-abolic stimulator, usually glucose, is measured by a sensing device which tells the pancreatic cell to release and re-synthesize insulin in relation to metabolic need. After circulating insulin has arrived in the blood, it is picked up by a specific cell receptor and initiates a cellular response. The cellular response not only serves a metabolic need but also removes from the blood some of the metabolic stimulator that initiated the cycle. As a result of the fall in the metabolite, no fur-ther stimulation for insulin release is given, and the feed-back mecha-nism is satisfied. Various other fac-tors can presumably inhibit or po-tentiate the different steps of this process. In this framework the diabetes with which we have been most familiar in the past is associated with failure of insulin release from the pancreas. This is most typically exemplified as the pancreatic ex-haustion of juvenile diabetes. It is also true of the pancreas that has been destroyed by pancreatitis, hemochromatosis, or removed by the surgeon's knife. It may be seen where pancreatic exhaustion results from metabolic stresses such as hyperthyroidism, multiple pregnan-cies, or long-standing obesity. In all cases the pancreas is no longer able to respond to demand, and clinical diabetes is seen. In true pancreatic exhaustion the sulfonylureas are, of course, ineffective. We should also note that a state T. E. PROUT of functional low insulin output exists where there is either dulling of the sensing device or interfer-ence with insulin release; either of these mechanisms impairs the ability of the pancreas to deliver insulin on demand. In these in-stances, the sulfonylureas release insulin. Epinephrines and the thi-azides inhibit release of insulin. The new concept that must be brought into this scheme is the clinical situation in which metabolic abnormalities of the type seen in diabetes are present in spite of high circulating levels of insulin. A state of impaired carbohydrate intolerance in association with a high level of plasma insulin can be understood if the insulin present is ineffective. It is also possible to recognize the clinical syndrome in which normal high levels of insulin compete with antagonists such as growth hormone, acromegaly, or adrenal steroids. A similar though less well defined impairment of in-sulin is seen in association with obesity. Here again we may see impaired carbohydrate tolerance in the presence of high circulating levels of insulin (Karam et al., 1963). In this state it appears likely that the impairment to in-sulin function is located primarily at the level of cellular response. The same may be true in carbo-hydrate induced hypertriglyceri-demia, a condition closely allied to diabetes but not necessarily true diabetes. There is some evidence that the lipoatrophic form of dia-betes may be characterized by high levels of circulating insulin that are rendered ineffective by an insulin antagonist (Louis et al., 1965). "Prediabetes" may take one of two forms. It may be a state in which high levels of insulin are put out to overcome peripheral impair-ment or circulating antagonists but in which carbohydrate intolerance cannot be demonstrated by ordi-nary techniques. This state is readily understood within the present framework. "Prediabetes" may also be present in a patient who shows 17 evidence of the "complications" of diabetes before carbohydrate toler-ance is lost. There is some evidence that different tissues react to differ-ent levels of insulin and by pre-sumption that levels of insulin ade-quate for the metabolism of some tissue would be ineffective for others. This may well be the rea-son why it is possible to see de-generative complications in the retina that is typical of diabetic retinopathy in some patients who have no carbohydrate intolerance. The same may be true of patients who develop vascular disease in the absence of alterations in carbo-hydrate utilization. Perhaps the future study will allow us to dem-onstrate that tissue sensitivity in such patients is the cause of the degenerative complications. There is abundant evidence that compli-cations may be seen in the absence of carbohydrate impairment, and we can no longer accept the fre-quently stated belief that control of carbohydrate metabolism in dia-betes will prevent all other forms of the "complications." Much of the theme presented here can be verified, but much of it still remains speculative. It is probably possible to place all vari-ants of the diabetic state some-where within this cycle so that dia-betes will eventually become better understood as a complex metabolic disorder involving all tissues and all metabolites rather than the simple derangement of carbohy-drate utilization. SUMMARY Diabetes mellitus has been re-viewed as a group of conditions with impaired function of one or more portions of a feed-back sys-tem involving the release and utili-zation of insulin. It is hoped that this may form a useful scheme by which we can study and understand a number of complex metabolic states which we must still collec-tively refer to as diabetes mellitus. 18 STATE OF INSULIN IN THE BLOOD REFERENCES ANDRES, R. Aging, glucose-tolerance, and diabetes-a proposal. In The Endocrine and Aging. L. Gitman (ed.) Springfield, Ill.: Charles C. Thomas, 1966 (in press). ANTONIADES, H. N., J. A. BOUGAS, R. CAMERINI-DAVALOS, AND H . M. PYLE. Insulin regulatory mecha-nisms and diabetes mellitus. Dia-betes 13: 230-240, 1964. COORE, H. G., AND P. J. RANDLE. Regulation of insulin secretion studied with pieces of rabbit pan-creas incubated in vitro. Biochem. J. 93: 66-78, 1964. FLOYD, J . C., JR., S. s. FAJANS, R . F. KNOPF, J. RULL, AND J. W. CONN. Stimulation of insulin secretion by amino acids. Clin. R es. 13 : 322, 1965 ( abstr). FROESCH, E. R., H . Bi.iRGI, E. B. RANSEJER, P . BALLY, AND A. LAB-HART. Antibody-suppressible and nonsuppressible insulin-like activi-ties in human serum and their phys-iologic significance. An insulin assay with adipose tissue of in-creased precision and specificity. J. Clin. Invest. 42 : 1816-1834, 1963. GOLDBERG, H. L. AND R. H. EGDAHL. Studies suggesting the extra-pan-creatic production of substances with insulin-like activity. Fed. Proc. 20: 190, 1961 (abstr). KARAM, J. H ., G. M. GRODSKY, AND P. H. FORSHAM. Excessive insulin response to glucose in obese sub-jects as measured by immunochemi-cal assay. Diabetes 12: 197, 1963. LACY, P. E. Electron microscopy of the beta cells of the pancreas. Am. J. Med. 31 : 851-859, 1961. LOUIS, L. H., J. w. CONN, AND M. c. MINNICK. Isolation of a peptide from bovine adeno-hypophysis which induces hyperglycemia and insulin resistance in men and dogs. Diabetes 14: 445, 1965 (abstr.). MEBANE, D. AND L. L. MADISON. The hypoglycemic effect of ketone bod-ies. J. C/in. Invest. 41: 1383-1384, 1962. MERIMEE, T. J., D . A. LILLICRAP, AND D. RABINOWITZ. Effect of arginine on serum-levels of human growth-hormone. Lancet 2: 688-670, 1965. PROUT, T. E. The chemical structure of insulin in relation to biological activity and to antigenicity. Metab-olism 12: 673-686, 1963. SAMAAN, N., R . FRASER, AND W. J. DEMPSTER. The "typical" and "atyp-ical" forms of serum insulin. Dia-betes 12: 339-348, 1963. SANGER, F. Chemistry of insulin. Brit. Med. Bull. 16: 183-188, 1960. STEINKE, J., K. W. TAYLOR, AND A. E. RENOLD. Insulin and insulin antag-onists in the serum of untreated juvenile diabetes. Studies with iso-lated rat diaphragm and rat adipose tissue. Lancet 1: 30-31, 1961. YALOW, R. S. ANDS. A. BERSON. Im-munoassay of endogenous plasma insulin in man. J. Clin. Invest. 39: 1157-1175, 1960. 

The State of Insulin in the Blood

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The State of Insulin in the Blood

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