The Incretin Approach for Diabetes Treatment
`Modulation of Islet Hormone Release by GLP-1 Agonism
`
`Jens Juul Holst and Cathrine Ørskov
`
`Glucagon-like peptide (GLP)-1 is a gut hormone that
`stimulates insulin secretion, gene expression, and ␤-cell
`growth. Together with the related hormone glucose-
`dependent insulinotropic polypeptide (GIP), it is re-
`sponsible for the incretin effect, the augmentation of
`insulin secretion after oral as opposed to intravenous
`administration of glucose. Type 2 diabetic patients typ-
`ically have little or no incretin-mediated augmentation
`of insulin secretion. This is due to decreased secretion
`of GLP-1 and loss of the insulinotropic effects of GIP.
`GLP-1, however, retains insulinotropic effects, and the
`hormone effectively improves metabolism in patients
`with type 2 diabetes. Continuous subcutaneous admin-
`istration greatly improved glucose profiles and lowered
`body weight and HbA1c levels. Further, free fatty acid
`levels were lowered, insulin resistance was improved,
`and ␤-cell performance was greatly improved. The nat-
`ural peptide is rapidly degraded by the enzyme dipepti-
`dyl peptidase IV (DPP IV), but resistant analogs as well
`as inhibitors of DPP IV are now under development, and
`both approaches have shown remarkable efficacy in
`experimental and clinical studies. Diabetes 53 (Suppl.
`3):S197–S204, 2004
`
`THE INCRETIN EFFECT IN TYPE 2 DIABETES
`It is now recognized that inadequate secretion of insulin
`may be a very early element in the development of type 2
`diabetes and that its progression is due to declining ␤-cell
`function (1–3). The ␤-cell defect is partly due to loss of
`␤-cells, but the loss, which may amount to 50% in ad-
`vanced type 2 diabetes (4), does not seem to parallel the
`dysfunction. This raises the possibility that the dysfunc-
`tion could at least be partly due to dysregulation. Thus,
`dysfunction of the autonomic innervation of the islets
`could be responsible, perhaps particularly with respect to
`the early insulin response to meal (5,6). Endocrine regu-
`lation of islet function could also be involved. Up to
`
`From the Departments of Medical Physiology and Medical Anatomy, The
`Panum Institute, University of Copenhagen, Copenhagen, Denmark.
`Address correspondence and reprint requests to Jens Juul Holst, MD,
`Departments of Medical Physiology and Medical Anatomy, The Panum
`Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. E-
`mail: holst@mfi.ku.dk.
`Received for publication 12 March 2004 and accepted in revised form 24
`May 2004.
`This article is based on a presentation at a symposium. The symposium and
`the publication of this article were made possible by an unrestricted educa-
`tional grant from Servier.
`J.J.H. has received honoraria from Novartis and is a paid consultant for
`Novo Nordisk and Amylin.
`DPP IV, dipeptidyl peptidase IV; GIP, glucose-dependent insulinotropic
`polypeptide; GLP, glucagon-like peptide.
`© 2004 by the American Diabetes Association.
`
`two-thirds of the insulin normally secreted in relation to
`meal intake is thought to be due to the insulinotropic
`actions of the so-called incretin hormones. The incretin
`effect is defined as the increased stimulation of insulin
`secretion elicited by oral as compared with intravenous
`administration of glucose under similar plasma glucose
`levels. Indeed, patients with type 2 diabetes have been
`demonstrated to exhibit an almost total loss of incretin
`effect (7). Therefore, it could be hypothesized that defi-
`cient incretin function plays an essential contributory role
`in the pathogenesis of type 2 diabetes. Deficient incretin
`effect could be due to impaired secretion of the incretin
`hormones as well as to impaired effects on islet function.
`Furthermore, if a defect is identified, a therapy based on
`substitution of the defective element might be devised.
`
`THE INCRETIN HORMONES
`Today, there is general agreement that the two most
`important incretin hormones are glucose-dependent insu-
`linotropic polypeptide (GIP), formerly known as gastric
`inhibitory polypeptide, and glucagon-like peptide (GLP)-1
`(8,9). Both are potent insulinotropic hormones released by
`oral glucose as well as ingestion of mixed meals.
`GIP. GIP is a peptide of 42 amino acids belonging to the
`glucagon-secretin family of peptides, the members of
`which have pronounced sequence homology, particularly
`in the NH2-terminus. It is processed from a 153–amino acid
`precursor (10), but specific functions for other fragments
`of the precursor have not been identified. The GIP recep-
`tor has been cloned and is related to the receptors for the
`other members of the glucagon-secretin family. It is ex-
`pressed in the islets and also in the gut, adipose tissue,
`heart, pituitary, adrenal cortex, and several regions of the
`brain (11). GIP is secreted from specific endocrine cells,
`so-called K cells, with highest density in the duodenum but
`found in the entire small intestinal mucosa (12). Secretion
`is stimulated by absorbable carbohydrates and by lipids.
`GIP secretion is therefore greatly increased in response to
`meals, resulting in 10- to 20-fold elevations of the plasma
`concentration (13).
`Interaction of GIP with its receptor on the ␤-cells causes
`elevation of cAMP levels, which in turn increases the
`intracellular calcium concentration and enhances exocy-
`tosis of insulin-containing granules by a mechanism distal
`to the elevation of calcium (14).
`GLP-1. GLP-1 is a product of the glucagon gene (15). It is
`expressed not only in pancreatic ␣-cells but also in the
`L-cells of the intestinal mucosa, one of the most abundant
`endocrine cells of the gut (16). Here the primary transla-
`tion product proglucagon is not cleaved to produce gluca-
`
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`INCRETINS IN DIABETES TREATMENT
`
`gon like in the islets but to release from its COOH-terminal
`part the two glucagon-like peptides GLP-1 and GLP-2 (17),
`both showing ⬃50% sequence homology with glucagon.
`GLP-1 secretion is stimulated by the presence of nutrients
`in the lumen of the gut (9), and its secretion throughout
`the day is highly correlated to the release of insulin (18).
`GLP-1 is one of the most potent insulin-releasing sub-
`stances known, exceeding that of GIP (19). Like GIP it
`interacts with a G protein– coupled receptor on the ␤-cells,
`which causes accumulation of cAMP, and most if not all of
`the subsequent effects seem to be secondary to this (14).
`Which is the most important incretin hormone? The
`incretin function of GIP, first suggested by Dupre et al.
`(20), was confirmed in detailed clamp studies by Andersen
`et al. (21) and has been probed in immunoneutralization
`studies (22) and, more recently, in studies employing a
`fragment, GIP (7-30)amide, which turns out to be a GIP
`receptor antagonist (23). Both treatments reduced insulin
`responses to oral glucose and impaired glucose tolerance.
`Mice with a targeted deletion of the GIP receptor gene
`become glucose intolerant (24,25). Using the mimicry
`approach, in which endogenous concentrations are mim-
`icked by intravenous infusion, it could be shown that the
`elevated GIP concentrations elicited by oral glucose can
`completely account for the accompanying augmented in-
`sulin release (26). The glucose intolerance of the GIP
`receptor knockout mice is not severe, but these mice may
`compensate by hypersecretion of other insulinotropic
`factors. Nevertheless, it is evident that GIP is not the only
`incretin hormone.
`Immunoneutralization experiments
`clearly showed that intestinal extracts contain potent
`insulinotropic agents in addition to GIP (27). Also, inves-
`tigations by Lauritsen and colleagues (28,29) in patients
`with resections of different parts of the small intestine, or
`in celiac disease, showed that the incretin effect does not
`correlate to GIP secretion, and that the distal small
`intestine releases an additional incretin. In all probability,
`this additional incretin is GLP-1. Like GIP, it is strongly
`insulinotropic in mimicry experiments (30), and antago-
`nists of the GLP-1 receptor show that GLP-1 is responsible
`for a substantial part of the insulin response to oral
`glucose (31,32). In agreement with these observations,
`mice with a targeted deletion of the GLP-1 receptor
`become glucose intolerant and may develop fasting hyper-
`glycemia (33). GIP secretion and pancreatic sensitivity to
`GIP are augmented in these knockout mice (34), suggest-
`ing that acute ablation of the GLP-1 activity could have
`even more extensive effects.
`Thus, the evidence for an important incretin function for
`both hormones is quite strong. However, GIP has been
`reported to be insulinotropic only at elevated glucose
`levels and to be less potent than GLP-1. GLP-1 on the other
`hand circulates in much lower (up to 10-fold) concentra-
`tions than GIP. Vilsboll et al. (35), therefore, recently
`reinvestigated the insulinotropic effects of the two hor-
`mones infused at rates that would result in physiological
`elevations of their concentration; glucose concentrations
`were clamped at fasting or slightly elevated levels in order
`to mimic the prandial situation. At their physiological
`postprandial concentrations, the two hormones had simi-
`lar and highly significant insulinotropic effects at fasting
`glucose as well as at 6 mmol/l, whereas at 7 mmol/l GLP-1
`
`was somewhat more effective. Thus, both hormones nor-
`mally contribute to the incretin effect in humans from the
`beginning of the meal (because increases in their concen-
`trations are seen already after 5–10 min). Together, the
`two hormones appear to act in an additive manner. Thus,
`when GIP and GLP-1 infusions (which separately provided
`similar insulin responses) were combined, the resulting
`response amounted to approximately the sum of the
`individual responses (36). Recently, mice with a double
`knockout of the GIP and GLP-1 receptors have been
`generated (37,38). The results obtained in these animals
`are consistent with an additive effect of the two hormones
`on glucose tolerance. In none of the studies did the double
`knockout animals develop fasting hyperglycemia, and
`their insulin sensitivity was normal. Interestingly, the
`potent GLP-1 receptor agonist exendin-4 (AGONISTS OF THE
`GLP-1 RECEPTOR: EXENDIN 4), which in control animals pro-
`foundly lowered blood glucose, had no effect in the double
`knockouts, suggesting that it acts exclusively via incretin
`receptors. Furthermore, valine pyrrolidide and SYR
`106124, inhibitors of dipeptidyl peptidase IV (DPP IV) (see
`below), the enzyme responsible for the initial metabolism
`of GLP-1 and GIP, which lower blood glucose and improve
`glucose tolerance in both GLP-1 and GIP single receptor
`knockout mice, had no effects in the double knockouts,
`suggesting that the effects of these inhibitors are exerted
`mainly via enhancing GIP and GLP-1 survival, without
`important involvement of other substrates.
`
`INCRETINS IN TYPE 2 DIABETES
`Given that GIP and GLP-1 together are responsible for the
`incretin effect in healthy subjects, it is possible to analyze
`the incretin defect in patients with type 2 diabetes. Theo-
`retically, the defect could be due to impaired secretion or
`accelerated metabolism of the incretin hormones; alterna-
`tively, the effect of the hormones could be compromised.
`There are many publications on the secretion of GIP in
`type 2 diabetes, and both increased, normal, and de-
`creased secretion have been reported (39). In a recent
`study in patients with type 2 diabetes covering a wide
`clinical spectrum of the disease, Toft-Nielsen et al. (40),
`using a highly specific COOH-terminal GIP assay, found
`near-normal fasting levels and meal responses with no
`correlations between metabolic parameters and GIP re-
`sponses. In the same study, a very significant impairment
`of GLP-1 secretion was observed. By multiple regression
`analysis, the impairment was found related to impaired
`␤-cell function. In a previous study in a small group of
`identical twins discordant for type 2 diabetes, the GLP-1
`response was lower in the diabetic twin (41), whereas in
`first-degree relatives of diabetic individuals,
`the 24-h
`GLP-1 profiles were normal (42), probably indicating that
`impaired secretion is a consequence rather than a cause of
`diabetes. GLP-1 is metabolized extremely rapidly by the
`ubiquitous enzyme DPP IV, which cleaves off two amino
`acid residues from the NH2-terminus and renders the
`metabolite [designated GLP-1 (9-36)amide] inactive (43).
`This occurs with such rapidity that a steady state is never
`obtained. In fact, only ⬃10 –15% of the hormone reaches
`the systemic circulation and thereby the pancreas in intact
`biologically active form. In agreement with this, the circu-
`lating concentrations of the intact hormone are much
`lower than those of the metabolite, but in patients with
`
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`type 2 diabetes during meal intake, intact hormone con-
`centrations are lower than in control subjects (44). Differ-
`ences in elimination cannot explain this (45), which must
`therefore be due to decreased secretion. Physiologically,
`the extensive and even local degradation of GLP-1 sug-
`gests that its main activities are exerted locally before the
`peptide is degraded. There is indeed substantial evidence
`that the peptide normally acts via activation of sensory
`neurons, which then via central reflexes influence central
`and gastrointestinal functions, including insulin secretion
`(46 – 48).
`The complex mechanism of action of the hormone
`makes it difficult to estimate the impact of decreased L-cell
`secretion for ␤-cell function, because with intravenous
`infusion one can only mimic the part of its actions that is
`exerted via the endocrine route. Activation of the sensory
`neural pathway probably requires higher concentrations,
`as probably found locally around nerve endings in the
`intestine. Impaired secretion of GLP-1, therefore, is likely
`to contribute significantly to the impaired incretin effect in
`type 2 diabetes.
`Regarding the effect of the hormones, a dramatic differ-
`ence emerges. Nauck et al. (19) studied the effects of
`intravenous infusions of GIP and GLP-1 in moderate type
`2 diabetic patients and matched control subjects and
`found that the insulinotropic effect of GIP was almost lost
`in patients, whereas the insulin response to GLP-1 was
`similar to control. Similar results were obtained by Elahi
`et al. (49). In a recent study of advanced type 2 diabetic
`patients (50), GIP infusion was incapable of enhancing
`second-phase insulin secretion (and glucose turnover)
`during hyperglycemic clamp, whereas with GLP-1 the
`insulin response to hyperglycemia was completely re-
`stored. Although supraphysiological GLP-1 concentrations
`in these studies restored the glucose responsiveness of
`␤-cells, this does not imply that the ␤-cell sensitivity to
`GLP-1 is normal. On the contrary, as shown by Kjems et al.
`(51), ␤-cell sensitivity may be considerably impaired, and
`this may further aggravate the impact of impaired GLP-1
`secretion in type 2 diabetic patients.
`
`THE INCRETIN APPROACH IN THE TREATMENT OF
`TYPE 2 DIABETES
`Based on the two defects identified, the decreased secre-
`tion of GLP-1 and the loss of second phase stimulation of
`insulin secretion by GIP, it could be hypothesized that
`GLP-1 (but not GIP) could be used for diabetes treatment
`as a substitution therapy. Indeed, GLP-1 administration
`has been shown to be highly effective in the treatment of
`type 2 diabetes, causing marked improvements in glyce-
`mic profile, insulin sensitivity, and ␤-cell performance, in
`addition to reducing weight (52). As discussed above, the
`hormone is metabolized rapidly by DPP IV, and the native
`peptide therefore cannot be used clinically. Instead, resis-
`tant analogs of the hormone (or agonists of the GLP-1
`receptor) are currently developed, along with DPP IV
`inhibitors demonstrated to protect the endogenous hor-
`mone and enhance its activity. Agonists currently in clin-
`ical development include both albumin-bound analogs of
`GLP-1 and exendin-4, a lizard peptide. Clinical studies with
`exendin have been carried out for ⬎6 months and indi-
`cated efficacy in patients inadequately treated with oral
`
`J.J. HOLST AND C. ØRSKOV
`
`antidiabetic agents. Orally active DPP IV inhibitors, suit-
`able for once-daily administration, have demonstrated
`similar efficacy. Diabetes therapy based on GLP-1 receptor
`activation, therefore, seems very promising. Obviously, it
`will not be possible to evaluate the drawbacks or benefits
`of the individual agents until the results of extensive
`clinical trials are available. However, on the basis of the
`already available data, it is possible to discuss apparent
`strengths and weaknesses of the various approaches.
`Diabetes treatment with native GLP-1. How effective
`are agonists of the GLP-1 receptor for diabetes treatment?
`It has been demonstrated repeatedly that intravenous
`infusion of GLP-1 at doses of 1–1.2 pmol 䡠 kg⫺1 䡠 min⫺1 can
`normalize fasting plasma glucose in type 2 diabetic indi-
`viduals, even in long-term insulin-treated patients with
`poor residual ␤-cell capacity (53). In a detailed study of a
`large group of patients, Toft-Nielsen et al. (54) found that
`GLP-1 infusion had the largest glucose-lowering effect in
`the patients with the highest fasting plasma glucose val-
`ues; on the other hand, normalization required many hours
`and euglycemia was not reached even after 7 h (but
`glucose levels were still declining). It can be hypothesized
`that in patients with very little residual ␤-cell capacity,
`GLP-1 infusion will not be able to induce euglycemia in the
`fed state. However, virtually complete normalization of
`plasma glucose during infusion of GLP-1 overnight and
`during subsequent meals was reported by Rachman et al.
`(55); possibly these patients had a milder disease than
`those of Toft-Nielsen’s study. The most important set of
`data in this respect was presented by Larsen et al. (56 –58).
`They infused GLP-1 continuously to a group of type 2
`diabetic patients for 7 days at four dose rates: 4, 8, 16, and
`24 ng 䡠 kg⫺1 䡠 min⫺1 (4 ng 䡠 kg⫺1 䡠 min⫺1corresponds to app.
`1.2 pmol 䡠 kg⫺1 䡠 min⫺1). The two highest doses were
`poorly tolerated and were discontinued. This actually
`defines the therapeutic window for native GLP-1. There
`was a pronounced clinical effect of both the 4-ng and 8-ng
`infusion rates and no side effects. The full effect on
`glycemia was obtained after a few hours, and there was no
`further improvement during the subsequent 7 days. How-
`ever, blood glucose levels were not completely normal-
`ized, with fasting and intermeal levels of ⬃7 mmol/l. In this
`group of patients, therefore, it appears that GLP-1 at the
`highest tolerated dose did not acutely reestablish euglyce-
`mia. Zander et al. (52) used portable insulin pumps to
`deliver 4.8 pmol 䡠 kg⫺1 䡠 min⫺1 GLP-1 continuously for 6
`weeks in a group of uncontrolled, obese type 2 diabetic
`individuals. No changes were observed in the saline-
`treated group, whereas in the GLP-1 group fasting and
`average plasma glucose concentrations were lowered by
`⬃5 mmol/l, HbA1c decreased by 1.2%, free fatty acids were
`significantly lowered, and the patients had a significant
`and gradual weight loss of ⬃2 kg. In addition, insulin
`sensitivity almost doubled, and insulin secretion capacity
`(measured using a 30 mmol/l glucose clamp ⫹ arginine)
`greatly improved. There were no significant side effects. In
`spite of the pronounced hypoglycemic effect (5 mmol/l),
`however, normal glucose values were not obtained (fast-
`ing levels ⬃10 mmol/l, somewhat
`lower toward the
`evening). This could be due the fact that the dose selected
`was not necessarily maximal (note that the dose was not
`associated with side effects). Further studies using the
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`INCRETINS IN DIABETES TREATMENT
`
`same technique indicated that a higher infusion rate might
`be more effective (59). Also, subcutaneous infusion may
`not be optimal; very high levels of the metabolite GLP-1
`(9-36)amide are generated during the infusion, and al-
`though this metabolite has been reported to have insulin-
`independent glucose-lowering properties (60), it also acts
`as a GLP-1 receptor antagonist (61). From these studies, it
`can be concluded that subacute GLP-1 treatment has not
`yet been documented to induce euglycemia in typical type
`2 diabetic subjects. It would be unreasonable to expect
`anything else from the GLP-1 receptor agonist described
`below as long as additional effects of these have not been
`identified.
`Agonists of the GLP-1 receptor: exendin 4. The best-
`studied GLP-1 receptor activator is exendin 4. Its intrinsic
`potency toward the GLP-1 receptor seems to be similar to
`that of GLP-1. It may have effects in addition to those
`elicited by activation of the GLP-1 receptor (62), but
`studies in mice with GLP-1 receptor deletion do not
`support this (38). In vivo, however, it is much more potent
`than GLP-1 due to slower degradation. Amino acid no. 2 is
`substituted with Gly (while GLP-1 has Ala), and because of
`this, exendin 4 is resistant to DPP IV (63). However, this
`does not in itself prolong the survival of the molecule in
`the body very much (from a half-life of 2 min to ⬃4 min)
`(63), but exendin is also eliminated much more slowly
`than the GLP-1 metabolite (or position 2–substituted ana-
`logs) mainly because of a slower renal elimination with a
`clearance of 1.8 ml 䡠 kg⫺1 䡠 min⫺1 similar to the normal
`glomerular filtration rate (64). Otherwise, exendin 4 ap-
`pears to act in humans in a manner identical to GLP-1 (64).
`The clinical usefulness of exendin 4 was evaluated in a
`proof-of-concept phase 2 study (65). Exendin 4 (now
`named AC2993 or exenatide) was injected subcutaneously
`two to three times daily for 4 weeks in patients already
`treated with metformin, sulfonylurea, or both.
`In all
`groups, there was a reduction in HbA1c of 0.7–1.1%. The
`most common adverse effect was transient mild to mod-
`erate nausea. Mild hypoglycemia was reported in about a
`third of the patients also treated with sulfonylurea. This
`finding was not substantiated by measurements but could
`reflect a partial uncoupling of the glucose dependency of
`the insulinotropic actions of GLP-1 by sulfonylurea (66).
`On the other hand, the incidences also illustrate the
`potency of this combination (66). Recently, in phase 3
`studies, exenatide was given twice daily initially in doses
`of 5 ␮g for 1 month and subsequently at 5 or 10 ␮g per
`injection for 5 months (see Amylin Corporation’s Web site
`[67]). This approach apparently reduced the tendency to
`cause initial nausea. Mild hypoglycemia was noted in 35%
`of the patients also treated with sulfonylurea. The average
`drop in HbA1c was 1% from ⬎8%, and values ⬍7% were
`observed in 40 – 46% (at 10 ␮g). A most interesting finding
`was significant weight loss, also observed in an open-label
`study (68), where a gradual weight loss was seen over a
`period of 26 weeks. It can be concluded that exenatide
`provides considerable additional glycemic control, even in
`patients inadequately treated with oral antidiabetic agents,
`and also causes weight loss, which can be predicted to
`provide further improvements of metabolism (69).
`It
`should be noted that twice-daily injection of exenatide
`does not provide full 24-h exposure of the GLP-1 recep-
`
`tors, considered important for full antidiabetic effect of at
`least intravenously administered GLP-1 (58), and this may
`explain the apparently less conspicuous effect on fasting
`plasma glucose (a drop of only 1 mmol/l over 26 weeks)
`with exenatide compared with native GLP-1 given contin-
`uously. A more pronounced and gradual fall in HbA1c was
`observed, but the decline flattened out during the last 3
`months. This may indicate that mean 24-h plasma glucose
`was lowered relatively more than fasting plasma glucose.
`HbA1c levels after 1 year of treatment have been reported
`(67) and appear not to have declined further over the last
`half year. Again, this may reflect the lack of 24-h exposure
`with the current treatment regimen. In agreement with
`this, Amylin has announced that a slow-release formula-
`tion is in development. It should also be noted that
`exenatide has not been clinically evaluated in mono-
`therapy, but only as an adjunct to existing therapies,
`where native GLP-1 has been shown to exert additive or
`even synergistic effects.
`Albumin-bound GLP-1 derivatives. Another approach
`has been to bind a GLP-1 analog to albumin in order to
`exploit the slow elimination kinetics of this molecule.
`NovoNordisk developed an acylated derivative of GLP-1,
`which binds noncovalently to albumin; Conjuchem cre-
`ated an analog of GLP-1, which after injection establishes
`a covalent bond with albumin; and Human Genome Sci-
`ences generated a fusion protein consisting of a DPP
`IV–resistant GLP-1 analog covalently bound to human
`albumin.
`The NovoNordisk compound NN2211 consists of native
`GLP-1,
`in which a C16 acyl chain is attached via a
`glutamoyl spacer to lysine residue no. 26 (while Lys34 was
`substituted by Arg). The compound shows slow release
`from the subcutaneous injection site and binds to albumin,
`which renders the molecule resistant to DPP IV and allows
`at least the bound fraction to escape renal elimination.
`This has resulted in a half-life in healthy and type 2
`diabetic subjects of 10 –12 h following a single subcutane-
`ous injection (70) and thereby adequate 24-h exposure
`after single injection. In addition, in chronic treatment the
`large postinjection concentration excursions caused by
`less long-lived analogs, which may be associated with side
`effects such as nausea, are likely to be avoided (71). The
`analog itself is equipotent with GLP-1 at the cloned human
`GLP-1 receptor. Because of its extensive half-life, the
`analog has proven suitable for studies in rodents, in which
`native GLP-1 barely has effects because of extensive
`metabolism. Thus, in rats with ␤-cell deficiencies, NN2211
`(now also called Liraglutide) had marked antihyperglyce-
`mic effects and greatly delayed development of diabetes.
`There were also significant effects on ␤-cell mass and food
`intake (72). Similar effects were observed in diabetic ob/ob
`and db/db mice (73). NN2211 also induced lasting and
`reversible weight loss in both normal and obese rats (74).
`This analog, like GLP-1 or exendin, inhibited fatty acid–
`and cytokine-induced apoptosis in primary ␤-cells (75,76).
`In clinical studies, NN2211 effectively reduced fasting as
`well as meal-related (12 h postinjection) glycemia by
`modifying insulin secretion, delaying gastric emptying, and
`suppressing prandial glucagon secretion (70). In a blinded
`phase 2 study involving 3 months of daily injections (77),
`ascending doses of NN2211 in monotherapy were com-
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`pared with glimepiride. It was concluded that the com-
`pound improved glycemic
`control
`comparably
`to
`glimepiride. Weight was maintained with a tendency to
`decrease, and the risk of hypoglycemia was very low.
`Antibodies against NN2211 could not be detected. This
`analog clearly possesses favorable pharmacokinetic prop-
`erties. The clinical results obtained so far are derived from
`studies in patients with very mild diabetes. The glucose-
`lowering effect was maximal after 1 week, and there was
`no further lowering over the next ⬃3 months.
`Other agonists. The Conjuchem compound CJC-1131 is
`composed of a D-Ala8 –substituted GLP-1 molecule with a
`linker and a reactive moiety attached to the COOH-
`terminus. After injection in vivo, this molecule conjugates
`covalently to Lys34 of albumin and thereby acquires the
`half-life of albumin. The CJC-1131–albumin conjugate
`binds to the GLP-1 receptor and activates cAMP with a
`potency similar to that of GLP-1 (78). It lowered blood
`glucose in wild-type mice but not in mice with a GLP-1
`receptor knockout. Blood glucose levels were lower in
`CJC-1131–treated db/db mice even 1 week after discontin-
`uation of the compound. It also increased pancreatic
`insulin mRNA levels and markers of ␤-cell proliferation
`(78). In recent studies in human volunteers, elimination
`half-lives of 9 –14 days were found (79), and in studies
`presented at the International Diabetes Federation Con-
`gress in 2003, the compound was reported to have dose-
`dependent effects on glycemia lasting at least 48 h and up
`to 8 days in some patients. Phase 2 clinical studies are
`currently ongoing. The compound is theoretically of con-
`siderable interest, because it seems to demonstrate 1) that
`molecules as large as albumin can get access to the
`relevant GLP-1 receptors and activate them and 2) that
`they can do so presumably without intracellular internal-
`ization. The very long half-life of the molecule suggests
`that it might be useful for administration once weekly. The
`company claims that the conjugate is not antigenic. The
`apparent retention of high biological activity in spite of
`the presence of the albumin moiety contrasts strikingly to
`the NN 2211 compound, the intrinsic activity of which is
`clearly lowered by albumin binding (by ⬃2–3 orders of
`magnitude), suggesting that the site of attachment to
`albumin is of importance and that the linker position and
`length in 2211 could be suboptimal.
`Agonists and ␤-cell survival. The long-term results
`obtained with GLP-1 analogs raise the question whether
`long-term GLP-1 treatment in humans is truly associated
`with a ␤-cell protective effect. Such an effect should have
`translated into a gradual lowering of fasting plasma glu-
`cose concentrations, which has not been observed. On the
`other hand, therapy for 3 months (NN 2211) may not be
`sufficient, and regarding exenatide, its pharmacokinetics
`are probably suboptimal. It must also be taken into
`account that for exenatide, control group results have not
`been reported (possibility of impairment?).
`
`INHIBITION OF GLP-1 DEGRADATION: DPP IV
`INHIBITORS
`The use of inhibitors of DPP IV was suggested on the
`background of the extreme DPP IV–mediated degradation
`of GLP-1 in patients with diabetes (80,81). It was demon-
`strated that with available inhibitors, it was possible to
`
`J.J. HOLST AND C. ØRSKOV
`
`completely protect exogenous and endogenous GLP-1
`from degradation and thereby greatly enhance its insuli-
`notropic activity (82). Numerous subsequent studies have
`indicated that administration of orally active DPP IV
`inhibitors markedly improve metabolism in animal models
`of glucose intolerance. For example, in mice rendered
`glucose intolerant by high-fat diets, the inhibitor valine
`pyrrolidide almost doubled the plasma levels of intact
`bioactive GLP-1, augmented insulin secretion, and virtu-
`ally normalized the considerably impaired glucose toler-
`ance (83). Using the Probiodrug inhibitor, P3298,
`Pospisilik et al. (84) reported sustained improvements
`over 12 weeks in fasting glucose, glucose tolerance, insulin
`sensitivity, and ␤-cell responsiveness to glucose in dia-
`betic Zucker fatty rats. Using the Ferring inhibitor FE
`999011, Sudre et al. (85) demonstrated that chronic inhi-
`bition of DPP IV markedly delayed development of diabe-
`tes in the Zucker diabetic fatty rat. It is of considerable
`interest that rats with a mutation in the DPP IV gene,
`resulting in inactivation of the catalytic subunit of the
`enzyme, do not develop glucose intolerance with aging
`(86), while a DPP IV inhibitor (DPP728) improved glucose
`tolerance in aging control rats. Similarly, mice lacking DPP
`IV, unlike wild-type animals, are refractory to the devel-
`opment of obesity and hyperinsulinemia upon high-fat
`feeding and protected against streptozotocin-induced loss
`of ␤-cell mass and hyperglycemia (87). These effects seem
`to be due to elevated levels of GLP-1, and importantly
`indicate that absence of enzyme activity does not result in
`an untoward phenotype.
`Proof of concept of the use of DPP IV inhibitors for
`diabetes treatment was obtained by Ahren et al. (88) using
`the Novartis DP728 inhibitor. Administered two to three
`times daily for 4 weeks, the inhibitor caused significant
`lowering of fasting as well as postprandial plasma glucose
`and significantly lowered HbA1c. There were very few and
`mild side effects. Novartis has subsequently introduced
`another inhibitor, LAF 237, which has a longer duration of
`action and which is, therefore, suitable for once-daily
`administration. This compound was demonstrated to have,
`given as a single 100-mg dose, very similar effects to DP
`728 administered two to three times daily (89). Notably,
`there were no significant side effects as noted with DP 728,
`which had been suspected to be possible consequences of
`DPP IV inhibition. There are many other substrates for
`DPP IV than GLP-1 (90), but it seems that the extreme
`degradation of GLP-1 makes it a preferential