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The role of amylin in the insulin resistance of non-insulin-dependent diabetes mellitus
TIBS 15-AUGUST1990
TALKINGPOINT DIABETES M~I.I.ITUS is a common metabolic disorder which is defined by the presence of chronically elevated levels of blood glucose (hyperglycaemia). Dietary carbohydrate is absorbed into the bloodsteam mainly in the form of glucose. The pancreatic hormone insulin stimulates the rapid clearance of glucose from the blood by stimulating glucose oxidation, the conversion of glucose to glycogen in skeletal muscle, to triacylglycerol in liver and adipose tissue, and also by suppression of hepatic glucose production. Insulin, therefore, plays a fundamental role in maintaining blood glucose levels within the physiological range. Insulin-dependent (type I) diabetes results from an autoimmune-mediated destruction of pancreatic ~-cells with consequent loss of insulin production, and resulting in hyperglycaemia. People with this disease usually have an absolute requirement for insulin replacement therapy in order to ensure survival. In marked contrast, non-insulin-dependent (type II) diabetes mellitus (NIDDM) is often characterized by hyperglycaemia in the presence of higher-than-normal levels of plasma insulin (hyperinsulinaemia) L2. Thus, in N1DDM,tissue processes which control carbohydrate metabolism have decreased sensitivity to insulin. Likewise, impaired sensitivity of carbohydrate metabolism to insulin is usually present in obesity and essential hypertension, which are metabolic states frequently associated with NIDDM. Progression of the NIDDM state is associated with increasing concentrations of blood glucose and a relative decrease in the rate of glucose-induced insulin secretion~; however, fasting levels of insulin remain elevated compared with levels in non-obese normal people 1.2. This brief description of NIDDM may be augmented with details from recent review articles~.2.
B, Leighton is at the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX$ 3QU, UK. J. S. Cooper is at the Amylin Corporation, 9373 Town Center Drive, La Jolla, San Diego, CA 92121, USA.
Decreased responsiveness of glucose metabolism to insulin in skeletal muscle and the liver (insulin resistance or insensitivity) is characteristic of many conditions, including non-insulin-dependent (type II) diabetes mellitus. Most current work in this area centres on the hypothesis that the primary defect is an impairment of insulin binding and/or transduction of the insulin signal in affected tissues. However, studies imply that defects in the post-insulin receptor signaling pathways are of primary importance in the causation of insulin resistance. Amylin, a novel pancreatic hormone, secreted along with insulin from the pancreatic ~cells, can modulate insulin effects, to produce insulin resistance in skeletal muscle and liver.
Pathogenic biochemical lesions in nonInsulin-dependent diabetes mellitus The primary lesion responsible for the pathogenesis of NIDDM remains unknown, although it is one of the most common metabolic disorders affecting mankind. About 70% of people with NIDDM are obese, and a similar percentage have essential hypertension. Age related factors are also thought to be important. Thus, impairment of glucose tolerance normally begins in the third decade of life3, and there is a sharp rise in the incidence of NIDDM in people over the age of 45. Studies carried out over a number of years with Pima Indians 4 (an isolated ethnic population with a high prevalence of NIDDM) and with a colony of primates s (Macaca mulatta) which develop a spontaneous NIDDM-Iike syndrome, strongly suggest that insulin resistance is a major determinant of the decline of glucose tolerance. Use of the euglycaemic/hyperinsulinaemic clamp 1.2 (in which blood glucose is kept at a physiological level by glucose infusion in the face of concomitant infusion of low or high doses of insulin) in non-diabetic subjects has shown that skeletal muscle is the major site of insulin-stimulated glucose disposal (50-70%). A recent non-invasive study
~) 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
has also demonstrated that more than 70% of a [1-13Clglucose load is incorporated into glycogen in skeletal muscleE In N1DDM patients, there is a marked decline in insulin-mediated glucose disposal into skeletal muscle and muscle glycogen synthase activity7. If defective insulin binding, or signaling mediated via the insulin receptor was the cause of insulin resistance, there would be an equivalent impairment of all forms of intracellular insulin-stimulated glucose metabolism. Oxidative and non-oxidative glucose disposal (mainly storage of glucose as glycogen) should, therefore, be affected equally. However, the euglycaemic clamp studies of NIDDM subjects show that decreases in oxidative and non-oxidative glucose disposal contribute about 30% and 70%, respectively, to the impairment of insulin-mediated glucose disposal in skeletal muscle2,8. This suggests that defects in the insulin signalling pathway distal to the insulin receptor operate to cause the insulin resistant state in muscle in NIDDM. More evidence in support of this conclusion is presented below.
Insulin sensitivity in skeletal muscle Much current research on the biochemical basis of NIDDM reflects the view that defects in insulin-mediated 295
TIBS 15-AUGUST1990
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5~ E
4~ 3~
0
2p ~ 1 /
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ib
ido
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Log concn, insulin (pU ml-~)
Rgure 1 The relationship between glycogen synthesis and insulin concentration in incubated soleus muscle [control (0)], and the effects of the presence of amylin (lnM) (e) 3° and of the conditions of aging (..)3 and obesity (A) 11.
control of carbohydrate metabolism in target tissues are the predominate cause of insulin resistance°. Insulin binds to the extracellular ~-subunit of its plasma membrane bound receptor (a heterotetramer of two ~- and two transmembrane ~-subunits) to trigger the signal transduction pathway~°. The ~-subunit contains an intracellular domain with tyrosine kinase activity. Insulin binding may induce signaling through initiation of a phosphorylation cascade via endogenous tyrosine kinase-mediated phosphorylation of cytosolic substrates. Alternatively, insulin binding may generate a second messenger (perhaps derived from inositol phosphate glycans) through alteration of the conformation of the ~-subunit which activates a GTP binding protein (G protein) and/or specific phos-
pholipases. However, despite vigorous research into abnormalities in these elements of the signaling pathway in the insulin resistance in obesity and NIDDM, it remains unclear that it is a specific intrinsic defect in the insulin receptor and/or signal transduction mechanism that exists, or is the pathogenic lesion in these states. Recently, various animal models have been examined3,u-~s in an effort to uncover the mechanisms that cause insulin resistance in skeletal muscle. These animal models exhibit hyperinsulinaemia and a decline in glucose tolerance and are of relevance to the study of insulin resistance in diabetes, as they closely resemble the pre-diabetic state. The dose-related effects of insulin on various aspects of glucose metabolism have been measured either in vivo TM in perfused hind-limb preparations or in isolated incubated muscle preparations n-~s (see Fig. 1). The sensitivity of many metabolic processes to insulin (the concentration of hormone required to stimulate any process halfmaximally) can be measured in such preparations. Importantly, there is close agreement between in vitro ~7 and in vivo ~6 measurements of insulin sensitivity. In muscle, insulin stimulates flux through at least three key non-equilibrium processes: facilitated glucose transport; glycogen synthase (which regulates glycogen synthesis); pyruvate dehydrogenase (which contributes to the regulation of glucose oxidation in the Krebs cycle). All these processes can be precisely measured in isolated incubated muscle preparations, in which the rate of glycolysis largely reflects glucose transport. In most insulin resistant states, with the exception of obesityn, the sensitivity of the glucose transporter to insulin in skeletal muscle is either mod-
Table I. Degrees of insulin resistance In skeletal muscle In different conditions Condition
Glycogen synthesisa
Glycolysis
Ref.
4, 4, 4, 4, 4,
no change
3
Obesity (Zucker rats)
4, 4, 4, 4,
4, 4, 4, 4, 4,
11
Glucocorticoid excess
4, 4, 4, 4,
4,
12
Hyperthyroidism (5 days)
4, 4, 4, 4, 4,
no change
13
Uraemia
no change
4,
14
4, 4,
no change
15
Aging
Endotoxaemia
aEach arrow denotes a decrease of insulin sensitivity (of approximately 100 IlU m1-1) relative to control muscle, Glycolysis reflects glucose transport 3. 296
estly decreased or unaltered (see Table I). In all conditions (except uraemia ~4) there is a dramatic decrease in the sensitivity of glycogen synthesis to insulin. The finding that different insulin-mediated processes respond to insulin in differing degrees in these conditions suggests that the mechanisms causing insulin resistance in skeletal muscle are not the result of a simple defect of insulin action.
Amylinand diabetes Amylin is the major peptide component of islet amyloid found in the pancreases of people with NIDDM~8J9. Deposits of islet amyloid have been reported in more than 90% of subjects with NIDDM2°, and also in more than 75% of diabetic Pima lndians2L Originally called diabetes-associated peptide ~9, amylin22 has now been isolated and completely characterized as a 37 amino acid peptide 19. A similar peptide isolated and incompletely sequenced from insulinoma-associated amyloid has been called insulinoma/islet amyloid polypeptide (IAPP)2°. Amylin is synthesized in pancreatic [~-cells23. It is located in secretory granules in ~-cells24. Amylin is co-secreted with insulin from isolated islets of Langerhans 25, and the isolated perfused rat pancreas 26, in response to nutrient secretagogues such as arginine and glucose. A high content of amylin is present in human pancreatic tissue (ninefold higher than glucagon 27) and the rate of secretion of amylin in the rat islets of Langerhans is between 10 and 37% of that of insulin 26. Molecular cloning studies indicate that rat and human amylin are synthesized from a precursor, preproamylin, which contains a signal peptide and a small prohormone-like sequence containing mature amylin23,28. Furthermore, the amylin amino acid sequence is well conserved among various species 29and is about 50% identical to the sequence of the 37 amino acid neuropeptide, calcitonin gene related peptide (CGRP). There is no sequence conservation over residues 20-29 in amylin and CGRP and it is this region in human amylin that is considered to be responsible for formation of amyloid fibrils TM (twisted, paired helical filaments formed by peptides in an antiparallel, ~-pleated sheet configuration). Presently, there is little published data on the levels of circulating amylin in insulin-resistant people or animals, but this information will become available
TIBS 15-AUGUST1990 in the near future with the development of amylin-specific radioimmunoassays 26. However, indirect evidence, particularly the association with chronically increased insulin secretion present in NIDDM, strongly suggests that hypersecretion of amylin is the defect which leads to the formation of islet amyloid masses, which could in turn produce the secondary islet cell failure characteristic of advanced NIDDM. Amylin is a potent inhibitor of insulin-stimulated rates of glycogen synthesis in isolated incubated rat skeletal muscle preparations 3° (see Fig. 1), but it does not affect insulinstimulated glucose metabolism in adipocytes2E Amylin has also been shown to cause both peripheral and hepatic insulin resistance when administered to rats during hyperinsulinaemic euglycaemic glucose clamp conditionsaL Similar effects have been observed both in vitro30,32and in v i [ o 3] with CGRE In a metabolic state such as NIDDM, which is characterized by relative hyperinsulinaemia and probable hyperamylinaemia, these properties of amylin could stimulate increased rates of lipogenesis from carbohydrate and hence increased triacylglycerol deposition in adipose tissue, leading in turn to obesity. Therefore, we believe that the obesity which frequently accompanies NIDDM is a result of the disordered metabolic state, rather than a predisposing factor for diabetes as has previously been postulated. These findings further suggest that varying degrees of hyperamylinaemia and hyperinsulinaemia could combine to contribute to the metabolic processes underlying obesity in other disease states. The effects of pancreatic human amylin, and of aging and obesity in animal models, on insulin-stimulated rates of glycogen synthesis in skeletal muscle preparations are compared in Fig. 1. Amylin inhibits both non-insulin-mediated and insulin-mediated glucose disposal 3°. Thus skeletal muscles incubated in the presence of amylin require a 100-fold greater concentration of insulin to obtain the same response as do control muscles incubated with a physiological concentration of insulin (100 IJU ml-~ or 0.7 nM). Amylin at 1 nM was found to cause the same degree of insulin resistance in skeletal muscle as found in incubated muscle preparations isolated from obese ~ and aging 3 animals. Both of the latter are established models of insulin-resistant conditions
BLOOD LIVER
Amylir Insulin: PANCREAS
SKELETAL MUSCLE
Lactate i
I:lgure 2 A model for the putative role for amylin, which is co-secreted with insulin26, in the regulation of glucose metabolism in the liver and skeletal muscle. The broken line indicates the sites of disruption of the post-insulin receptor signal in liver and muscle. which resemble states associated with the development of NIDDM. The similarity between the nature and extent of the insulin resistance produced in isolated skeletal muscle, compared with that in muscles isolated from the two animal models, suggests to us that increased circulating amylin may underlie the levels of peripheral insulin resistance found in these conditions. Four characteristic abnormalities exist in patients with NIDDML2,~8.These are (1) impaired insulin secretion, (2) peripheral insulin resistance, (3) elevated rates of hepatic glucose production, and (4) islet amyloid formation. Available evidence on amylin activity supports the hypothesis that amylin is involved in the pathogenesis of NIDDM~a,3°. Thus, the sequence of amylin isolated from islet amyloid was identical to that encoded by the normal gene ~9,28. Amylin causes insulin resistance in skeletal muscle both in vitro 3° and in vivo al. Moreover, as amylin does not influence the binding of insulin to its receptor (Cooper et al., unpublished) the inhibition of insulin-stimulated glycogen synthesis in skeletal muscle in vitro by amylin must occur via modulation of post-insulin-receptor
responses s°, as occurs in NIDDM. The modulation of insulin sensitivity by amylin produces changes similar to those observed in various insulin-resistant animal models, in which post-insulin receptor defects account for the decreased sensitivity to insulin in skeletal muscle. Amylin has also been reported to cause increased rates of hepatic glucose output in vivo 3~ and to decrease rates of insulin secretion from isolated islets of Langerhans (see Ref. 18).
Physiological and pathological roles for amylin As mentioned above, amylin has about 50% sequence identity to the neuropeptide CGRP]9. Exogenous CGRP can affect a variety of processes including inhibition of insulin, gastric acid and amylase secretion. CGRP is a potent vasodilator and has positive chronotropic and ionotropic effects in atrial tissue 33. CGRP shares with amylin the ability to inhibit insulin-stimulated rates of glycogen synthesis in skeletal muscle32. More interestingly, CGRP shows specific binding in rat liver plasma membranes 34 and stimulates adenylate cyclase activity35. However, the content of CGRP in the liver is below 297
TIBS15-AUGUST1990 detectable limits s6 (Leighton and Foot, unpublished). This suggests that CGRP itself cannot regulate hepatic metabolism. Amylin, however, is secreted into the portal venous blood supplying the liver, and is well placed to regulate metabolism in the liver by binding to the putative hepatic CGRP binding sites. This is likely to be the system through which amylin exerts its action to increase the rate of hepatic glucose production. Amylin could increase the activity of the indirect pathway necessary for hepatic glycogen synthesis (i.e. the conversion of glucose to C3-intermediates [e.g. lactate] with subsequent reconversion of C3 intermediates to glycogen in the liver via the gluconeogenic pathwayST). After feeding, the elevated level of blood glucose stimulates both the rate of insulin secretion and hepatic glycogen synthase activity (thereby promoting hepatic glycogen deposition). In theory, flux through the gluconeogenic pathway after a meal should be inhibited by the high levels of insulin reaching the liver, which in turn should decrease the rate of hepatic glycogen synthesis. However, amylin, co-secreted with insulin 2~, may function in normal physiology to temporarily decrease the sensitivity of some hepatic processes to insulin (e.g. the gluconeogenic pathway) and thereby favour formation of glycogen via the indirect pathway. Such an action may favour hepatic glucose output. Indeed it will be interesting to establish the role of amylin in the daily changes in peripheral and hepatic insulin sensitivity that occur in both rat and man ~7. Although CGRP is located in both sensory nerves (unmyelinated C fibres) and motor end plate in skeletal muscles2 as well as CGRP-containing nerve fibres there is no innervation of the bulk of skeletal muscle fibres. Thus, the bloodborne hormone, amylin, will be the agent actually interacting at the membrane to decrease the effects of insulin. Consequently we see CGRP and amylin as a pair of regulators, a neurotransmitter and a hormone respectively, somewhat similar to noradrenaline, the sympathetic neurotransmitter, and adrenaline, the adrenal medullary hormone. The different physiological roles of CGRP and amylin will reflect their availability to responsive end-organs as well as differential sensitivity to possible receptor sub-types to the two related, but distinct (and distinctly evolutionary conserved38), ligands. As with other hormones, we will
298
expect to find disease states associated 8 Golay, A., DeFronzo, R. A., Ferrannini, E., Simonson, D. C., Thorin, D., Acheson, K., with deficient or excess secretion of Thi(~baud, D., Curchod, B., J~quir, E. and Felber, amylin. For example, increased rates of J. P. (1988) Diabetologia 31, 585-591 glucose turnover (i.e. conversion of glu- 9 Draznin, B., Melmed, S. and LeRoith, D. (1989) Insulin Action, Alan R. Liss cose to lactate in peripheral tissues 10 Rosen, O. M. (1987) Science 237, 1452-1458 with subsequent glucose synthesis from R. A. J., Budohoski, L., McManus, B. lactate in the liver, or Cori cycle activ- 11 Challiss, and Newsholme, E. A. (1984) Biochem. J. 221, ity, see Fig. 2), increases in the secretion 915-917 rates of insulin, and probably amylin, 12 Leighton, B., Challiss, R. A. J., Lozeman, F. J. and Newsholme, E. A. (1987) Biochem. J. 246, and decreased sensitivity of muscle 551-554 glycogen synthesis to insulin are all fea13 Dimitriadis, G. D., Leighton, B., Vlachonikolis, I., tures of endotoxaemia ~s and hyperthyParry-Billings, M., Challiss, R. A. J., West, D. roidism 13. Amylin, in combination with and Newsholme, E. A. (1988) Biochem. J. 253, 87-92 other catabolic hormones, may cause P. P., Hunt, W. A., Johnson, C. M., enhanced Cori cycle activity by pro- 14 Stein, DeFronzo, R. A. and Smith, J. (1989) moting increased lactate formation from Metabolism 38, 562-567 glucose in muscle and by promoting 15 Leighton, B., Dimitriadis, G. D., Parry-Billings, M., Bond, J., DeVasconcelos, P. R. L. and increased rates of glucose formation Newsholme, E. A. (1989) Clinical Sci. 77, from lactate in the liver (see Fig. 2). 61-67
Summary Non-insulin-dependent diabetes mellitus, as well as other conditions associated with insulin resistance, will result from hypersecretion of a hormone with the demonstrated effects of amylin. Thus, the newly discovered hormone amylin, which works in collaboration with insulin to direct the disposal of fuel molecules in liver, skeletal muscle and adipose tissue, can underlie some of the major, unexplained disorders of fuel metabolism, including non-insulindependent diabetes mellitus and obesity.
Acknowledgements We acknowledge contributions of many colleagues including Mr A. C. Willis, Dr A. J. Day, Ms E. A. Foot, Dr E. A. Newsholme, Dr M. Parry-Billings, Dr G. D. Dimitriadis, Dr R. A. J. Challiss and Dr E J. Lozeman and to thank Drs Anne Roberts, T. J. Rink and S. Brenner for thoughtful comments on the manuscript.
References Space limitations have prevented citation of all the available literature. More references are cited in the following literature. 1 Waldh~usl, W. K. and Bratusch-Marrain, P. (1987) Diabetes/Metabolism Rev. 3, 79-109 2 DeFronzo, R. A. (1988) Diabetes 37, 667-687 3 Leighton, B., Dimitriadis, G. D., Parry-Billings, M., Lozeman, F. J. and Newsholme, E. A. (1989) Biochem. J. 261, 383-387 4 Lillioja, S., Mort, D. M., Howard, B. V., Bennett, P. H., Yki-J~rvinen, H., Freymond, D., Nyomba, B., Zurlo, F., Swinburn, B. and Bogardus, C. (1988) New Engl. J. Med. 318, 1217-1225 5 Bodkin, N. L,, Metzger, B. L. and Hansen, B. C. (1989) Am. J. Physiol. 256, E676-E681 6 Jue, T., Rothman, D. L., Shulman, G. I., Tavitian, B. A., DeFronzo, R. A. and Shulman, R. G. (1989) Proc. Natl Acad. Sci. USA 86, 4489-4491 7 Young, A. A., Bogardus, C., Wolfe-Lopez, D. and Mott, D. M. (1988) Diabetes 37,303-308
16 James, D. E., Jenkins, A. B. and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574 17 Leighton, B., Kowalchuk, J. M., Challiss, R. A. J. and Newsholme, E. A. (1988) Am. J. Physiol.
255, E41-E45 18 Cooper, G. J. S., Day, A. J., Willis, A. C.,
Roberts, A. N., Reid, K. B. M. and Leighton, B. (1989) Biochim. Biophys. Acta 1014, 247-258 19 Cooper, G. J. S., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B. and Reid, K. B. M. (1987) Proc. Natl Acad. Sci. USA 84, 8628-8632 20 Westermark, P., Wernstedt, C., Wilander, E., Hayden, D. W., O'Brien, T. D. and Johnson, K. H. (1987) Proc. Natl Acad. Sci. USA 84, 3881-3885 21 Clark, A,, Saad, M. F., Nezzer, T., Uren, C., Knowler, W. C., Bennett, P. H. and Turner, R. C. (1989) Diabetic Med. 6, (Suppl. 1) A9 22 Cooper, G. J. S., Leighton, B., Dimitriadis, G. D., Parry-Billings, M., Kowalchuk, J. M., Howland, K., Rothbard, J. B., Willis, A. C. and Reid, K. B. M. (1988) Proc. Natl Acad. Sci. USA 85, 7763-7766 23 Leffert, J. D., Newgard, C. B., Okamato, H., Milburn, J. L. and Luskey, K. L. (1989) Proc. Natl Acad. Sci. USA 68, 3127-3130 24 Lukinius, A., Wilander, E., Westermark, G. T., Engstrom, U. and Westermark, P. (1989) Diabetologia 32, 240-244 25 Kanatsuka, A., Makino, H., Ohsawa, Tokuyama, Y., Yamaguchi, T., Yoshida, S. and Adachi, M. (1989) FEBS Lett. 259, 199-201 26 Ogawa, A., Harris, V., McCorkle, S. K., Unger, R. H. and Luskey, K. L. (1990) ./. Clin. invest. 85, 973-976 27 Nakazato, M., Asai, J., Kangawa, K., Matsukura, S. and Matsuo, H. (1989) Biochem. Biophys. Res. Commun. 164, 394-399 28 Roberts, A. N., Leighton, B., Todd, J. A., Cockburn, D., Schofield, P. N., Sutton, R., Holt, S., Boyd, Y., Day, A. J., Foot, E. A., Willis, A. C., Reid, K. B. M. and Cooper, G. J. S. (1989) Proc. Natl Acad. Sci. USA 86, 9662-9666 29 Nishi, M., Chart, S. J., Nagamatsu, S., Bell, G. I. and Steiner, D. F. (1989) Proc. Natl Acad. Sci. USA 86, 5738--5742 30 Leighton, B. and Cooper, G. J. S. (1988) Nature 335, 632-635 31 Molina, J. M., Cooper, G. J. S., Leighton, B. and Olefsky, J. M. (1990) Diabetes 39, 260-265 32 Leighton, B., Foot, E. A., Cooper, G. J. S. and King, J. M. (1989) FEBS Lett. 249, 357-361 33 Yamaguchi, A., Chiba, T., Okimura, Y., Yamatani, T., Morishita, T., Nakamura, A., Inui, T., Noda, T. and Fujita, T. (1988) Biochem. Biophys. Res. Commun. 152, 383-391
TIBS 1 5 - A U G U S T 1 9 9 0 34 Yamaguchi,A., Chiba, T., Yamatani,T., Inui, T., Morishita, T., Nakamura, A., Kadowaki, S., Fukase, M. and Fujita, T. (1988) Endocrinology 123, 2591-2596 35 Saito, A., Kimura, S. and Goto, K. (•986)
Am. J. Physiol. 250, H693-H696 36 Okimura, Y., Chihara, K., Abe, H., Kita, T., Kashio, Y., Sato, M. and Fujita, T. (1987) Regul. Pept. 17, 327-337 37 Newgard, C. B., Moore, S. V., Foster, D. W. and
McGarry, J. D. (1984) J. Biol. Chem. 259, 6948-6963 38 Cooper, G. J. S., Leighton, B., Willis, A. C. and Day, A. J. (1989) Prog. Growth Factor Res. 1, 96-102
LETTERS PQQ in plants It was an article in TIBS 1 that first drew my attention, and, no doubt, that of others, to the existence and structure of pyrroloquinoline quinone, PQQ. Since then the journal has kept us well informed of developments concerning this newest, unexpected addition to the family of oxidoreductive coenzymes. The latest contributions from experts in the field z-4, emphasize the difficulties in detecting PQQ unambiguously, and of estimating it reliably in different tissues, especially mammalian tissues: they characterize it as an 'elusive coenzyme'. However these articles made scant reference to PQQ in plant tissues, and I would like to comment briefly, as an 'outsider', on this aspect. This aspect may be of interest or even of importance if PQQ ever achieves the status of a vitamin. A plant enzyme that, since the discovery of PQQ, has been suspected of containing this cofactor is the diamine oxidase of pea and other legume seeds. The pea enzyme was worked on extensively in the 1960s by (among others) the late P. J. Mann and his colleague J. M. Hill at Rothamsted s,6. It was shown to have a bright pink colour and, like the pink amine oxidase of bovine serum which was demonstrated to contain PQQ in 19847, shown to contain copper and to be inhibited by carbonyl compounds such as hydrazine derivatives. Like the serum enzyme, it was at one time thought that its carboxyl cofactor was a pyridoxal derivative, although diligent attempts to detect pyridoxal compounds in hydrolysates by Hill and Mann were unsuccessful. It was very pleasing, therefore, when Glatz and his co-workers 8, in an optical and electrochemical study of the enzyme and of hydrolysates made from it, produced evidence for the presence of PQQ or something very like it. Citro et al. 9 have more recently showed that the diamine oxidase of lentil seedlings reacts specifically and quantifiably with an antibody raised against gelatin-bound PQQ. The evidence presented by these two groups of workers for the presence of PQQ would seem to satisfy the critical scrutiny of Dr Duine 3. In addition,
there is evidence, suggestive rather than conclusive, that an N-methyl putrescine oxidase from tobacco roots may also be a quinoprotein l°. It is still a little surprising that organisms have managed to adapt such a reactive species as an o-quinone group into a cofactor with a regulated oxidoreductive role. Plant biochemists are probably more conscious of the potential reactivity of o-quinones than are most of their 'bacterial' and 'animal' colleagues. In many leaf and vegetable extracts, o-quinones are formed adventitiously by the enzymic and nonenzymic oxidation of dihydroxyphenols present in such tissues u. It is their polymerization that is the basis of the 'browning' of these plant extracts, but their reaction with proteins, for example, often makes the extraction of unmodified enzymes, organelles and viruses from these tissues difficult and even impossible ]2. Indeed, part of the way in which the potential reactivity conferred by the o-quinone group is attenuated in PQQ, is that the positions in the quinone ring that are adjacent to the carbonyl groups, and which are highly susceptible to nucleophilic substitution ]1, are already blocked by fused pyrrole and pyridine rings.
Propagation of an error: sheet structures So Edison has finally let the cat out of the bag. In his recent article on textbook errors 1, he points out the widespread use of the diagram of [3-sheet structures taken from the 1951 paper of Pauling and Corey2, which happens to be drawn with D-amino acids. I remember in the 1960s Howard Schachman challenging his graduate classes to spot the 'deliberate mistake' in the Pauling-Corey paper. (At the time he gave the opinion it was probably a reversing error in the printing process.) I have to confess a personal failure but since then have repeated the
References 1 Duine, J. A. and Frank, J. (1981) Trends Biochem. Sci. 6, 278-280 2 Gallop, P. M., Paz, M. A., Fluckiger, R. and Kagan, H. M. (1989) Trends Biochem. Sci. 14, 343--346 3 Duine, J. A. (1990) Trends Biochem. Sci. 15, 96 4 Gallop, P. M. (1990) Trends Biochem. Sci. 15, 96-97 5 Mann, P. J. G. (1961) Biochem. J. 79, 623-631 6 Hill, J. M. and Mann, E J. G. (1964) Biochem. J. 91, 171-182 7 Lobenstein-Verbeck,C. L., Jongejan,J. A., Frank, J. and Duine, J. A. (1984) FEBS Lett. 170, 305-309 8 Giatz, Z., Kovar,J., Macholan, L. and Pec, P. (1987) Biochem. J. 242, 603-606 9 Citro, G., Verdina, A., Galati, R., Floris, G., Sabatini, S. and Finazzi-Agro,A. (1989) FEBS Lett. 247, 201-204 10 Davies, H. M., Hawkins, D. J. and Smith, L. A. (1989) Phytochemistry 28, 1573-1578 11 Pierpoint, W. S. (1971)in Rothamsted Experimental Station Report 1970, Part 2, pp. 199-218 12 Jervis, L. and Pierpoint, W. S. (1989) J. Biotechnol. 11,161-198
W. S. PIERPOINT 4 Wheatlock Head, Redbourne, Herts. AL3 7HS, UK.
same challenge to my own students every year in an attempt to make them at least look at some source references. For 20 years I have not had to dip into my pocket for a prize. It will be interesting to continue the effort for a few more years if only to see if the students take another bit of advice, to keep up to date with TIBS!
References 1 Edison,A. S. (1990) Trends Biochem. Sci. 15, 216-217 2 Pauling, L. and Corey,R. B. (1951) Proc. Natl Acad. Sci. USA 37, 729-740
JOHN M. WRIGGLESWORTH King's College London, Biomolecular Sciences Division, Campden Hill Road, London W8 7AH, UK.
299
TALKINGPOINT DIABETES M~I.I.ITUS is a common metabolic disorder which is defined by the presence of chronically elevated levels of blood glucose (hyperglycaemia). Dietary carbohydrate is absorbed into the bloodsteam mainly in the form of glucose. The pancreatic hormone insulin stimulates the rapid clearance of glucose from the blood by stimulating glucose oxidation, the conversion of glucose to glycogen in skeletal muscle, to triacylglycerol in liver and adipose tissue, and also by suppression of hepatic glucose production. Insulin, therefore, plays a fundamental role in maintaining blood glucose levels within the physiological range. Insulin-dependent (type I) diabetes results from an autoimmune-mediated destruction of pancreatic ~-cells with consequent loss of insulin production, and resulting in hyperglycaemia. People with this disease usually have an absolute requirement for insulin replacement therapy in order to ensure survival. In marked contrast, non-insulin-dependent (type II) diabetes mellitus (NIDDM) is often characterized by hyperglycaemia in the presence of higher-than-normal levels of plasma insulin (hyperinsulinaemia) L2. Thus, in N1DDM,tissue processes which control carbohydrate metabolism have decreased sensitivity to insulin. Likewise, impaired sensitivity of carbohydrate metabolism to insulin is usually present in obesity and essential hypertension, which are metabolic states frequently associated with NIDDM. Progression of the NIDDM state is associated with increasing concentrations of blood glucose and a relative decrease in the rate of glucose-induced insulin secretion~; however, fasting levels of insulin remain elevated compared with levels in non-obese normal people 1.2. This brief description of NIDDM may be augmented with details from recent review articles~.2.
B, Leighton is at the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX$ 3QU, UK. J. S. Cooper is at the Amylin Corporation, 9373 Town Center Drive, La Jolla, San Diego, CA 92121, USA.
Decreased responsiveness of glucose metabolism to insulin in skeletal muscle and the liver (insulin resistance or insensitivity) is characteristic of many conditions, including non-insulin-dependent (type II) diabetes mellitus. Most current work in this area centres on the hypothesis that the primary defect is an impairment of insulin binding and/or transduction of the insulin signal in affected tissues. However, studies imply that defects in the post-insulin receptor signaling pathways are of primary importance in the causation of insulin resistance. Amylin, a novel pancreatic hormone, secreted along with insulin from the pancreatic ~cells, can modulate insulin effects, to produce insulin resistance in skeletal muscle and liver.
Pathogenic biochemical lesions in nonInsulin-dependent diabetes mellitus The primary lesion responsible for the pathogenesis of NIDDM remains unknown, although it is one of the most common metabolic disorders affecting mankind. About 70% of people with NIDDM are obese, and a similar percentage have essential hypertension. Age related factors are also thought to be important. Thus, impairment of glucose tolerance normally begins in the third decade of life3, and there is a sharp rise in the incidence of NIDDM in people over the age of 45. Studies carried out over a number of years with Pima Indians 4 (an isolated ethnic population with a high prevalence of NIDDM) and with a colony of primates s (Macaca mulatta) which develop a spontaneous NIDDM-Iike syndrome, strongly suggest that insulin resistance is a major determinant of the decline of glucose tolerance. Use of the euglycaemic/hyperinsulinaemic clamp 1.2 (in which blood glucose is kept at a physiological level by glucose infusion in the face of concomitant infusion of low or high doses of insulin) in non-diabetic subjects has shown that skeletal muscle is the major site of insulin-stimulated glucose disposal (50-70%). A recent non-invasive study
~) 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00
has also demonstrated that more than 70% of a [1-13Clglucose load is incorporated into glycogen in skeletal muscleE In N1DDM patients, there is a marked decline in insulin-mediated glucose disposal into skeletal muscle and muscle glycogen synthase activity7. If defective insulin binding, or signaling mediated via the insulin receptor was the cause of insulin resistance, there would be an equivalent impairment of all forms of intracellular insulin-stimulated glucose metabolism. Oxidative and non-oxidative glucose disposal (mainly storage of glucose as glycogen) should, therefore, be affected equally. However, the euglycaemic clamp studies of NIDDM subjects show that decreases in oxidative and non-oxidative glucose disposal contribute about 30% and 70%, respectively, to the impairment of insulin-mediated glucose disposal in skeletal muscle2,8. This suggests that defects in the insulin signalling pathway distal to the insulin receptor operate to cause the insulin resistant state in muscle in NIDDM. More evidence in support of this conclusion is presented below.
Insulin sensitivity in skeletal muscle Much current research on the biochemical basis of NIDDM reflects the view that defects in insulin-mediated 295
TIBS 15-AUGUST1990
I
~7r
5~ E
4~ 3~
0
2p ~ 1 /
0L
ib
ido
1obo ioooo
Log concn, insulin (pU ml-~)
Rgure 1 The relationship between glycogen synthesis and insulin concentration in incubated soleus muscle [control (0)], and the effects of the presence of amylin (lnM) (e) 3° and of the conditions of aging (..)3 and obesity (A) 11.
control of carbohydrate metabolism in target tissues are the predominate cause of insulin resistance°. Insulin binds to the extracellular ~-subunit of its plasma membrane bound receptor (a heterotetramer of two ~- and two transmembrane ~-subunits) to trigger the signal transduction pathway~°. The ~-subunit contains an intracellular domain with tyrosine kinase activity. Insulin binding may induce signaling through initiation of a phosphorylation cascade via endogenous tyrosine kinase-mediated phosphorylation of cytosolic substrates. Alternatively, insulin binding may generate a second messenger (perhaps derived from inositol phosphate glycans) through alteration of the conformation of the ~-subunit which activates a GTP binding protein (G protein) and/or specific phos-
pholipases. However, despite vigorous research into abnormalities in these elements of the signaling pathway in the insulin resistance in obesity and NIDDM, it remains unclear that it is a specific intrinsic defect in the insulin receptor and/or signal transduction mechanism that exists, or is the pathogenic lesion in these states. Recently, various animal models have been examined3,u-~s in an effort to uncover the mechanisms that cause insulin resistance in skeletal muscle. These animal models exhibit hyperinsulinaemia and a decline in glucose tolerance and are of relevance to the study of insulin resistance in diabetes, as they closely resemble the pre-diabetic state. The dose-related effects of insulin on various aspects of glucose metabolism have been measured either in vivo TM in perfused hind-limb preparations or in isolated incubated muscle preparations n-~s (see Fig. 1). The sensitivity of many metabolic processes to insulin (the concentration of hormone required to stimulate any process halfmaximally) can be measured in such preparations. Importantly, there is close agreement between in vitro ~7 and in vivo ~6 measurements of insulin sensitivity. In muscle, insulin stimulates flux through at least three key non-equilibrium processes: facilitated glucose transport; glycogen synthase (which regulates glycogen synthesis); pyruvate dehydrogenase (which contributes to the regulation of glucose oxidation in the Krebs cycle). All these processes can be precisely measured in isolated incubated muscle preparations, in which the rate of glycolysis largely reflects glucose transport. In most insulin resistant states, with the exception of obesityn, the sensitivity of the glucose transporter to insulin in skeletal muscle is either mod-
Table I. Degrees of insulin resistance In skeletal muscle In different conditions Condition
Glycogen synthesisa
Glycolysis
Ref.
4, 4, 4, 4, 4,
no change
3
Obesity (Zucker rats)
4, 4, 4, 4,
4, 4, 4, 4, 4,
11
Glucocorticoid excess
4, 4, 4, 4,
4,
12
Hyperthyroidism (5 days)
4, 4, 4, 4, 4,
no change
13
Uraemia
no change
4,
14
4, 4,
no change
15
Aging
Endotoxaemia
aEach arrow denotes a decrease of insulin sensitivity (of approximately 100 IlU m1-1) relative to control muscle, Glycolysis reflects glucose transport 3. 296
estly decreased or unaltered (see Table I). In all conditions (except uraemia ~4) there is a dramatic decrease in the sensitivity of glycogen synthesis to insulin. The finding that different insulin-mediated processes respond to insulin in differing degrees in these conditions suggests that the mechanisms causing insulin resistance in skeletal muscle are not the result of a simple defect of insulin action.
Amylinand diabetes Amylin is the major peptide component of islet amyloid found in the pancreases of people with NIDDM~8J9. Deposits of islet amyloid have been reported in more than 90% of subjects with NIDDM2°, and also in more than 75% of diabetic Pima lndians2L Originally called diabetes-associated peptide ~9, amylin22 has now been isolated and completely characterized as a 37 amino acid peptide 19. A similar peptide isolated and incompletely sequenced from insulinoma-associated amyloid has been called insulinoma/islet amyloid polypeptide (IAPP)2°. Amylin is synthesized in pancreatic [~-cells23. It is located in secretory granules in ~-cells24. Amylin is co-secreted with insulin from isolated islets of Langerhans 25, and the isolated perfused rat pancreas 26, in response to nutrient secretagogues such as arginine and glucose. A high content of amylin is present in human pancreatic tissue (ninefold higher than glucagon 27) and the rate of secretion of amylin in the rat islets of Langerhans is between 10 and 37% of that of insulin 26. Molecular cloning studies indicate that rat and human amylin are synthesized from a precursor, preproamylin, which contains a signal peptide and a small prohormone-like sequence containing mature amylin23,28. Furthermore, the amylin amino acid sequence is well conserved among various species 29and is about 50% identical to the sequence of the 37 amino acid neuropeptide, calcitonin gene related peptide (CGRP). There is no sequence conservation over residues 20-29 in amylin and CGRP and it is this region in human amylin that is considered to be responsible for formation of amyloid fibrils TM (twisted, paired helical filaments formed by peptides in an antiparallel, ~-pleated sheet configuration). Presently, there is little published data on the levels of circulating amylin in insulin-resistant people or animals, but this information will become available
TIBS 15-AUGUST1990 in the near future with the development of amylin-specific radioimmunoassays 26. However, indirect evidence, particularly the association with chronically increased insulin secretion present in NIDDM, strongly suggests that hypersecretion of amylin is the defect which leads to the formation of islet amyloid masses, which could in turn produce the secondary islet cell failure characteristic of advanced NIDDM. Amylin is a potent inhibitor of insulin-stimulated rates of glycogen synthesis in isolated incubated rat skeletal muscle preparations 3° (see Fig. 1), but it does not affect insulinstimulated glucose metabolism in adipocytes2E Amylin has also been shown to cause both peripheral and hepatic insulin resistance when administered to rats during hyperinsulinaemic euglycaemic glucose clamp conditionsaL Similar effects have been observed both in vitro30,32and in v i [ o 3] with CGRE In a metabolic state such as NIDDM, which is characterized by relative hyperinsulinaemia and probable hyperamylinaemia, these properties of amylin could stimulate increased rates of lipogenesis from carbohydrate and hence increased triacylglycerol deposition in adipose tissue, leading in turn to obesity. Therefore, we believe that the obesity which frequently accompanies NIDDM is a result of the disordered metabolic state, rather than a predisposing factor for diabetes as has previously been postulated. These findings further suggest that varying degrees of hyperamylinaemia and hyperinsulinaemia could combine to contribute to the metabolic processes underlying obesity in other disease states. The effects of pancreatic human amylin, and of aging and obesity in animal models, on insulin-stimulated rates of glycogen synthesis in skeletal muscle preparations are compared in Fig. 1. Amylin inhibits both non-insulin-mediated and insulin-mediated glucose disposal 3°. Thus skeletal muscles incubated in the presence of amylin require a 100-fold greater concentration of insulin to obtain the same response as do control muscles incubated with a physiological concentration of insulin (100 IJU ml-~ or 0.7 nM). Amylin at 1 nM was found to cause the same degree of insulin resistance in skeletal muscle as found in incubated muscle preparations isolated from obese ~ and aging 3 animals. Both of the latter are established models of insulin-resistant conditions
BLOOD LIVER
Amylir Insulin: PANCREAS
SKELETAL MUSCLE
Lactate i
I:lgure 2 A model for the putative role for amylin, which is co-secreted with insulin26, in the regulation of glucose metabolism in the liver and skeletal muscle. The broken line indicates the sites of disruption of the post-insulin receptor signal in liver and muscle. which resemble states associated with the development of NIDDM. The similarity between the nature and extent of the insulin resistance produced in isolated skeletal muscle, compared with that in muscles isolated from the two animal models, suggests to us that increased circulating amylin may underlie the levels of peripheral insulin resistance found in these conditions. Four characteristic abnormalities exist in patients with NIDDML2,~8.These are (1) impaired insulin secretion, (2) peripheral insulin resistance, (3) elevated rates of hepatic glucose production, and (4) islet amyloid formation. Available evidence on amylin activity supports the hypothesis that amylin is involved in the pathogenesis of NIDDM~a,3°. Thus, the sequence of amylin isolated from islet amyloid was identical to that encoded by the normal gene ~9,28. Amylin causes insulin resistance in skeletal muscle both in vitro 3° and in vivo al. Moreover, as amylin does not influence the binding of insulin to its receptor (Cooper et al., unpublished) the inhibition of insulin-stimulated glycogen synthesis in skeletal muscle in vitro by amylin must occur via modulation of post-insulin-receptor
responses s°, as occurs in NIDDM. The modulation of insulin sensitivity by amylin produces changes similar to those observed in various insulin-resistant animal models, in which post-insulin receptor defects account for the decreased sensitivity to insulin in skeletal muscle. Amylin has also been reported to cause increased rates of hepatic glucose output in vivo 3~ and to decrease rates of insulin secretion from isolated islets of Langerhans (see Ref. 18).
Physiological and pathological roles for amylin As mentioned above, amylin has about 50% sequence identity to the neuropeptide CGRP]9. Exogenous CGRP can affect a variety of processes including inhibition of insulin, gastric acid and amylase secretion. CGRP is a potent vasodilator and has positive chronotropic and ionotropic effects in atrial tissue 33. CGRP shares with amylin the ability to inhibit insulin-stimulated rates of glycogen synthesis in skeletal muscle32. More interestingly, CGRP shows specific binding in rat liver plasma membranes 34 and stimulates adenylate cyclase activity35. However, the content of CGRP in the liver is below 297
TIBS15-AUGUST1990 detectable limits s6 (Leighton and Foot, unpublished). This suggests that CGRP itself cannot regulate hepatic metabolism. Amylin, however, is secreted into the portal venous blood supplying the liver, and is well placed to regulate metabolism in the liver by binding to the putative hepatic CGRP binding sites. This is likely to be the system through which amylin exerts its action to increase the rate of hepatic glucose production. Amylin could increase the activity of the indirect pathway necessary for hepatic glycogen synthesis (i.e. the conversion of glucose to C3-intermediates [e.g. lactate] with subsequent reconversion of C3 intermediates to glycogen in the liver via the gluconeogenic pathwayST). After feeding, the elevated level of blood glucose stimulates both the rate of insulin secretion and hepatic glycogen synthase activity (thereby promoting hepatic glycogen deposition). In theory, flux through the gluconeogenic pathway after a meal should be inhibited by the high levels of insulin reaching the liver, which in turn should decrease the rate of hepatic glycogen synthesis. However, amylin, co-secreted with insulin 2~, may function in normal physiology to temporarily decrease the sensitivity of some hepatic processes to insulin (e.g. the gluconeogenic pathway) and thereby favour formation of glycogen via the indirect pathway. Such an action may favour hepatic glucose output. Indeed it will be interesting to establish the role of amylin in the daily changes in peripheral and hepatic insulin sensitivity that occur in both rat and man ~7. Although CGRP is located in both sensory nerves (unmyelinated C fibres) and motor end plate in skeletal muscles2 as well as CGRP-containing nerve fibres there is no innervation of the bulk of skeletal muscle fibres. Thus, the bloodborne hormone, amylin, will be the agent actually interacting at the membrane to decrease the effects of insulin. Consequently we see CGRP and amylin as a pair of regulators, a neurotransmitter and a hormone respectively, somewhat similar to noradrenaline, the sympathetic neurotransmitter, and adrenaline, the adrenal medullary hormone. The different physiological roles of CGRP and amylin will reflect their availability to responsive end-organs as well as differential sensitivity to possible receptor sub-types to the two related, but distinct (and distinctly evolutionary conserved38), ligands. As with other hormones, we will
298
expect to find disease states associated 8 Golay, A., DeFronzo, R. A., Ferrannini, E., Simonson, D. C., Thorin, D., Acheson, K., with deficient or excess secretion of Thi(~baud, D., Curchod, B., J~quir, E. and Felber, amylin. For example, increased rates of J. P. (1988) Diabetologia 31, 585-591 glucose turnover (i.e. conversion of glu- 9 Draznin, B., Melmed, S. and LeRoith, D. (1989) Insulin Action, Alan R. Liss cose to lactate in peripheral tissues 10 Rosen, O. M. (1987) Science 237, 1452-1458 with subsequent glucose synthesis from R. A. J., Budohoski, L., McManus, B. lactate in the liver, or Cori cycle activ- 11 Challiss, and Newsholme, E. A. (1984) Biochem. J. 221, ity, see Fig. 2), increases in the secretion 915-917 rates of insulin, and probably amylin, 12 Leighton, B., Challiss, R. A. J., Lozeman, F. J. and Newsholme, E. A. (1987) Biochem. J. 246, and decreased sensitivity of muscle 551-554 glycogen synthesis to insulin are all fea13 Dimitriadis, G. D., Leighton, B., Vlachonikolis, I., tures of endotoxaemia ~s and hyperthyParry-Billings, M., Challiss, R. A. J., West, D. roidism 13. Amylin, in combination with and Newsholme, E. A. (1988) Biochem. J. 253, 87-92 other catabolic hormones, may cause P. P., Hunt, W. A., Johnson, C. M., enhanced Cori cycle activity by pro- 14 Stein, DeFronzo, R. A. and Smith, J. (1989) moting increased lactate formation from Metabolism 38, 562-567 glucose in muscle and by promoting 15 Leighton, B., Dimitriadis, G. D., Parry-Billings, M., Bond, J., DeVasconcelos, P. R. L. and increased rates of glucose formation Newsholme, E. A. (1989) Clinical Sci. 77, from lactate in the liver (see Fig. 2). 61-67
Summary Non-insulin-dependent diabetes mellitus, as well as other conditions associated with insulin resistance, will result from hypersecretion of a hormone with the demonstrated effects of amylin. Thus, the newly discovered hormone amylin, which works in collaboration with insulin to direct the disposal of fuel molecules in liver, skeletal muscle and adipose tissue, can underlie some of the major, unexplained disorders of fuel metabolism, including non-insulindependent diabetes mellitus and obesity.
Acknowledgements We acknowledge contributions of many colleagues including Mr A. C. Willis, Dr A. J. Day, Ms E. A. Foot, Dr E. A. Newsholme, Dr M. Parry-Billings, Dr G. D. Dimitriadis, Dr R. A. J. Challiss and Dr E J. Lozeman and to thank Drs Anne Roberts, T. J. Rink and S. Brenner for thoughtful comments on the manuscript.
References Space limitations have prevented citation of all the available literature. More references are cited in the following literature. 1 Waldh~usl, W. K. and Bratusch-Marrain, P. (1987) Diabetes/Metabolism Rev. 3, 79-109 2 DeFronzo, R. A. (1988) Diabetes 37, 667-687 3 Leighton, B., Dimitriadis, G. D., Parry-Billings, M., Lozeman, F. J. and Newsholme, E. A. (1989) Biochem. J. 261, 383-387 4 Lillioja, S., Mort, D. M., Howard, B. V., Bennett, P. H., Yki-J~rvinen, H., Freymond, D., Nyomba, B., Zurlo, F., Swinburn, B. and Bogardus, C. (1988) New Engl. J. Med. 318, 1217-1225 5 Bodkin, N. L,, Metzger, B. L. and Hansen, B. C. (1989) Am. J. Physiol. 256, E676-E681 6 Jue, T., Rothman, D. L., Shulman, G. I., Tavitian, B. A., DeFronzo, R. A. and Shulman, R. G. (1989) Proc. Natl Acad. Sci. USA 86, 4489-4491 7 Young, A. A., Bogardus, C., Wolfe-Lopez, D. and Mott, D. M. (1988) Diabetes 37,303-308
16 James, D. E., Jenkins, A. B. and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574 17 Leighton, B., Kowalchuk, J. M., Challiss, R. A. J. and Newsholme, E. A. (1988) Am. J. Physiol.
255, E41-E45 18 Cooper, G. J. S., Day, A. J., Willis, A. C.,
Roberts, A. N., Reid, K. B. M. and Leighton, B. (1989) Biochim. Biophys. Acta 1014, 247-258 19 Cooper, G. J. S., Willis, A. C., Clark, A., Turner, R. C., Sim, R. B. and Reid, K. B. M. (1987) Proc. Natl Acad. Sci. USA 84, 8628-8632 20 Westermark, P., Wernstedt, C., Wilander, E., Hayden, D. W., O'Brien, T. D. and Johnson, K. H. (1987) Proc. Natl Acad. Sci. USA 84, 3881-3885 21 Clark, A,, Saad, M. F., Nezzer, T., Uren, C., Knowler, W. C., Bennett, P. H. and Turner, R. C. (1989) Diabetic Med. 6, (Suppl. 1) A9 22 Cooper, G. J. S., Leighton, B., Dimitriadis, G. D., Parry-Billings, M., Kowalchuk, J. M., Howland, K., Rothbard, J. B., Willis, A. C. and Reid, K. B. M. (1988) Proc. Natl Acad. Sci. USA 85, 7763-7766 23 Leffert, J. D., Newgard, C. B., Okamato, H., Milburn, J. L. and Luskey, K. L. (1989) Proc. Natl Acad. Sci. USA 68, 3127-3130 24 Lukinius, A., Wilander, E., Westermark, G. T., Engstrom, U. and Westermark, P. (1989) Diabetologia 32, 240-244 25 Kanatsuka, A., Makino, H., Ohsawa, Tokuyama, Y., Yamaguchi, T., Yoshida, S. and Adachi, M. (1989) FEBS Lett. 259, 199-201 26 Ogawa, A., Harris, V., McCorkle, S. K., Unger, R. H. and Luskey, K. L. (1990) ./. Clin. invest. 85, 973-976 27 Nakazato, M., Asai, J., Kangawa, K., Matsukura, S. and Matsuo, H. (1989) Biochem. Biophys. Res. Commun. 164, 394-399 28 Roberts, A. N., Leighton, B., Todd, J. A., Cockburn, D., Schofield, P. N., Sutton, R., Holt, S., Boyd, Y., Day, A. J., Foot, E. A., Willis, A. C., Reid, K. B. M. and Cooper, G. J. S. (1989) Proc. Natl Acad. Sci. USA 86, 9662-9666 29 Nishi, M., Chart, S. J., Nagamatsu, S., Bell, G. I. and Steiner, D. F. (1989) Proc. Natl Acad. Sci. USA 86, 5738--5742 30 Leighton, B. and Cooper, G. J. S. (1988) Nature 335, 632-635 31 Molina, J. M., Cooper, G. J. S., Leighton, B. and Olefsky, J. M. (1990) Diabetes 39, 260-265 32 Leighton, B., Foot, E. A., Cooper, G. J. S. and King, J. M. (1989) FEBS Lett. 249, 357-361 33 Yamaguchi, A., Chiba, T., Okimura, Y., Yamatani, T., Morishita, T., Nakamura, A., Inui, T., Noda, T. and Fujita, T. (1988) Biochem. Biophys. Res. Commun. 152, 383-391
TIBS 1 5 - A U G U S T 1 9 9 0 34 Yamaguchi,A., Chiba, T., Yamatani,T., Inui, T., Morishita, T., Nakamura, A., Kadowaki, S., Fukase, M. and Fujita, T. (1988) Endocrinology 123, 2591-2596 35 Saito, A., Kimura, S. and Goto, K. (•986)
Am. J. Physiol. 250, H693-H696 36 Okimura, Y., Chihara, K., Abe, H., Kita, T., Kashio, Y., Sato, M. and Fujita, T. (1987) Regul. Pept. 17, 327-337 37 Newgard, C. B., Moore, S. V., Foster, D. W. and
McGarry, J. D. (1984) J. Biol. Chem. 259, 6948-6963 38 Cooper, G. J. S., Leighton, B., Willis, A. C. and Day, A. J. (1989) Prog. Growth Factor Res. 1, 96-102
LETTERS PQQ in plants It was an article in TIBS 1 that first drew my attention, and, no doubt, that of others, to the existence and structure of pyrroloquinoline quinone, PQQ. Since then the journal has kept us well informed of developments concerning this newest, unexpected addition to the family of oxidoreductive coenzymes. The latest contributions from experts in the field z-4, emphasize the difficulties in detecting PQQ unambiguously, and of estimating it reliably in different tissues, especially mammalian tissues: they characterize it as an 'elusive coenzyme'. However these articles made scant reference to PQQ in plant tissues, and I would like to comment briefly, as an 'outsider', on this aspect. This aspect may be of interest or even of importance if PQQ ever achieves the status of a vitamin. A plant enzyme that, since the discovery of PQQ, has been suspected of containing this cofactor is the diamine oxidase of pea and other legume seeds. The pea enzyme was worked on extensively in the 1960s by (among others) the late P. J. Mann and his colleague J. M. Hill at Rothamsted s,6. It was shown to have a bright pink colour and, like the pink amine oxidase of bovine serum which was demonstrated to contain PQQ in 19847, shown to contain copper and to be inhibited by carbonyl compounds such as hydrazine derivatives. Like the serum enzyme, it was at one time thought that its carboxyl cofactor was a pyridoxal derivative, although diligent attempts to detect pyridoxal compounds in hydrolysates by Hill and Mann were unsuccessful. It was very pleasing, therefore, when Glatz and his co-workers 8, in an optical and electrochemical study of the enzyme and of hydrolysates made from it, produced evidence for the presence of PQQ or something very like it. Citro et al. 9 have more recently showed that the diamine oxidase of lentil seedlings reacts specifically and quantifiably with an antibody raised against gelatin-bound PQQ. The evidence presented by these two groups of workers for the presence of PQQ would seem to satisfy the critical scrutiny of Dr Duine 3. In addition,
there is evidence, suggestive rather than conclusive, that an N-methyl putrescine oxidase from tobacco roots may also be a quinoprotein l°. It is still a little surprising that organisms have managed to adapt such a reactive species as an o-quinone group into a cofactor with a regulated oxidoreductive role. Plant biochemists are probably more conscious of the potential reactivity of o-quinones than are most of their 'bacterial' and 'animal' colleagues. In many leaf and vegetable extracts, o-quinones are formed adventitiously by the enzymic and nonenzymic oxidation of dihydroxyphenols present in such tissues u. It is their polymerization that is the basis of the 'browning' of these plant extracts, but their reaction with proteins, for example, often makes the extraction of unmodified enzymes, organelles and viruses from these tissues difficult and even impossible ]2. Indeed, part of the way in which the potential reactivity conferred by the o-quinone group is attenuated in PQQ, is that the positions in the quinone ring that are adjacent to the carbonyl groups, and which are highly susceptible to nucleophilic substitution ]1, are already blocked by fused pyrrole and pyridine rings.
Propagation of an error: sheet structures So Edison has finally let the cat out of the bag. In his recent article on textbook errors 1, he points out the widespread use of the diagram of [3-sheet structures taken from the 1951 paper of Pauling and Corey2, which happens to be drawn with D-amino acids. I remember in the 1960s Howard Schachman challenging his graduate classes to spot the 'deliberate mistake' in the Pauling-Corey paper. (At the time he gave the opinion it was probably a reversing error in the printing process.) I have to confess a personal failure but since then have repeated the
References 1 Duine, J. A. and Frank, J. (1981) Trends Biochem. Sci. 6, 278-280 2 Gallop, P. M., Paz, M. A., Fluckiger, R. and Kagan, H. M. (1989) Trends Biochem. Sci. 14, 343--346 3 Duine, J. A. (1990) Trends Biochem. Sci. 15, 96 4 Gallop, P. M. (1990) Trends Biochem. Sci. 15, 96-97 5 Mann, P. J. G. (1961) Biochem. J. 79, 623-631 6 Hill, J. M. and Mann, E J. G. (1964) Biochem. J. 91, 171-182 7 Lobenstein-Verbeck,C. L., Jongejan,J. A., Frank, J. and Duine, J. A. (1984) FEBS Lett. 170, 305-309 8 Giatz, Z., Kovar,J., Macholan, L. and Pec, P. (1987) Biochem. J. 242, 603-606 9 Citro, G., Verdina, A., Galati, R., Floris, G., Sabatini, S. and Finazzi-Agro,A. (1989) FEBS Lett. 247, 201-204 10 Davies, H. M., Hawkins, D. J. and Smith, L. A. (1989) Phytochemistry 28, 1573-1578 11 Pierpoint, W. S. (1971)in Rothamsted Experimental Station Report 1970, Part 2, pp. 199-218 12 Jervis, L. and Pierpoint, W. S. (1989) J. Biotechnol. 11,161-198
W. S. PIERPOINT 4 Wheatlock Head, Redbourne, Herts. AL3 7HS, UK.
same challenge to my own students every year in an attempt to make them at least look at some source references. For 20 years I have not had to dip into my pocket for a prize. It will be interesting to continue the effort for a few more years if only to see if the students take another bit of advice, to keep up to date with TIBS!
References 1 Edison,A. S. (1990) Trends Biochem. Sci. 15, 216-217 2 Pauling, L. and Corey,R. B. (1951) Proc. Natl Acad. Sci. USA 37, 729-740
JOHN M. WRIGGLESWORTH King's College London, Biomolecular Sciences Division, Campden Hill Road, London W8 7AH, UK.
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