Biochemical and Biophysical Research Communications 308 (2003) 207–213
`
`Breakthroughs and Views
`
`BBRC
`
`www.elsevier.com/locate/ybbrc
`
`Glucose-dependent insulinotropic polypeptide analogues and
`their therapeutic potential for the treatment of obesity-diabetes
`
`Victor A. Gault,* Peter R. Flatt, and Finbarr P.M. O’Harte
`
`School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine BT52 1SA, Northern Ireland, UK
`
`Received 17 June 2003
`
`Abstract
`
`Glucose-dependent insulinotropic polypeptide (GIP) is a key incretin hormone, released postprandially into the circulation in
`response to feeding, producing a glucose-dependent stimulation of insulin secretion. It is this glucose-dependency that has attracted
`attention towards GIP as a potential therapeutic agent for the treatment of type 2 diabetes. A major drawback to achieving this goal
`has been the rapid degradation of circulating GIP by the ubiquitous enzyme, dipeptidylpeptidase IV (DPP IV). However, recent
`studies have described a number of novel structurally modified analogues of GIP with enhanced plasma stability, insulinotropic and
`antihyperglycaemic activity. The purpose of this article was to provide an overview of the biological effects of several GIP modi-
`fications and to highlight the potential of such analogues in the treatment of type 2 diabetes and obesity.
`Ó 2003 Elsevier Inc. All rights reserved.
`
`Keywords: Dipeptidylpeptidase IV; Glucagon-like peptide-1; Glucose-dependent insulinotropic polypeptide; GIP analogues; Insulin secretion;
`Obesity; Type 2 diabetes
`
`Glucose-dependent insulinotropic polypeptide (GIP)
`is a 42 amino acid gastrointestinal hormone secreted
`from enteroendocrine K-cells in response to food and
`nutrient absorption [1]. Although initially characterized
`for its ability to inhibit histamine-induced gastric acid
`secretion, the primary role of GIP as an incretin hor-
`mone moderating glucose-induced insulin secretion is
`widely recognized [2]. In addition to its actions on the
`pancreatic beta cell, GIP is also known to exert various
`extrapancreatic effects, which further enhance its glucose
`lowering ability. In particular, GIP has been shown to
`augment insulin-dependent inhibition of glycogenolysis
`in the liver [3] and to exert stimulatory effects on glucose
`uptake and metabolism in muscle [4]. Furthermore,
`functional GIP receptors have been identified on
`adipocytes [5] and have been shown to stimulate glucose
`transport
`[6],
`increase fatty acid synthesis [7], and
`stimulate lipoprotein lipase activity [8]. More recent
`studies have shown GIP to stimulate beta cell mito-
`genesis and inhibit apoptosis [9,10]. Consequently, this
`
`* Corresponding author. Fax: +44-28-7032-4965.
`E-mail address: va.gault@ulster.ac.uk (V.A. Gault).
`
`0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved.
`doi:10.1016/S0006-291X(03)01361-5
`
`wide spectrum of biological activities has sparked the
`recent interest in GIP as a novel therapeutic candidate
`for the treatment of type 2 diabetes [11].
`
`Solutions to difficulties posed by GIP as a therapeutic
`agent
`
`Currently there are two main concerns in attempting
`to utilize GIP as a potential therapeutic agent. First, the
`native peptide has a short biological half-life in the cir-
`culation (approximately 3–5 min), due primarily to
`degradation by the ubiquitous enzyme, dipeptidylpep-
`tidase IV (DPP IV; EC 3.4.14.5). After release into the
`circulation, the native peptide (GIP 1-42) is rapidly hy-
`drolysed at the amino terminus removing Tyr1–Ala2 to
`produce the truncated metabolite GIP(3-42) [12]. This
`major degradation product was initially thought to be
`inactive [13], however, recent observations have shown it
`to function as a GIP receptor antagonist in vivo [14]. To
`circumvent degradation, DPP IV inhibitors are cur-
`rently being developed for in vivo administration. Sev-
`eral positive effects on glycaemic control have been
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`
`reported [15–17], but the effects of long-term interfer-
`ence on the metabolism of a plethora of other peptide
`substrates for DPP IV are as yet unknown [18]. Con-
`ceivably, a more attractive approach than widespread
`non-specific DPP IV inhibition involves the synthesis of
`specific GIP analogues modified at the enzyme cleavage
`site [11].
`Another concern regarding the use of GIP in diabetes
`therapy stems from a study in which GIP was shown to
`exhibit marked reduction of insulinotropic activity in
`type 2 diabetic subjects [19]. Indeed, several studies have
`shown blunted insulin responses to GIP infusion in type
`2 diabetes, albeit with differing degrees of beta cell re-
`sistance [19–22]. However, type 2 diabetes is associated
`with a global defect in insulin secretion not merely re-
`stricted to GIP, and encompassing other factors, in-
`cluding glucose and GLP-1 [23,24]. It has also been
`shown that beta cell sensitivity to GIP in type 2 diabetic
`patients improves with glyburide treatment [25]. Muta-
`tions of GIP receptors are rare in diabetes [21,26] and it
`is evident that any speculated abnormalities in GIP re-
`ceptor binding can be overcome by enzyme-resistant
`analogues of GIP [11,27–33]. Furthermore, a recent
`study has shown that an N-terminally modified ana-
`logue of GIP, Tyr1-glucitol-GIP, evokes a substantially
`larger and more protracted insulin response to oral
`glucose than the native GIP in type 2 diabetic patients
`[34].
`
`The attractiveness and potential of stable GIP analogues
`
`A number of significant features indicate that en-
`zyme-resistant analogues of GIP have unique potential
`for diabetes therapy. First, GIP is the major physio-
`logical
`incretin as indicated by greater hormone re-
`sponses to feeding [35] and comparative studies using
`either receptor antagonists [36,37] or receptor knockout
`mice [38,39]. Second, structural modification of the
`sister incretin, GLP-1, invariably compromises biologi-
`cal activity at the GLP-1 receptor [40–44], whereas the
`opposite is true for many analogues of GIP [11,27–33].
`Third, any effect of GIP on increasing glucagon con-
`centrations is only observed at normal glucose concen-
`trations [45,46] and thus is irrelevant following feeding
`or in treating type 2 diabetes [47]. Fourth, in contrast to
`GLP-1, GIP lacks significant effects on gastric emptying
`and is therefore well tolerated by human subjects. This
`has become an increasing and possibly damning diffi-
`culty with the use of GLP-1 and its analogues in pa-
`tients, as evidenced by increased gastrointestinal side
`effects and nausea [48]. This, therefore, focuses increas-
`ing attention on the antidiabetic potential of GIP. To
`date, several studies have been published examining the
`in vitro activities of a range of GIP fragments and an-
`alogues [27,49–54]. Moreover, as reviewed in the fol-
`
`lowing sections, the in vivo antidiabetic potential of a
`family of selective designer human GIP 1-42 analogues
`(Fig. 1) modified at positions Tyr1 [11,28–30], Ala2 [31–
`33], and Glu3 [37,55] have been tested in animal models
`of type 2 diabetes and obesity.
`
`Effects of modifications at position Tyr1
`
`Several novel Tyr1-modified analogues of GIP have
`been developed, modelled on previous studies with the
`glucagon-secretin family of gastrointestinal peptides and
`knowledge of the substrate-binding specificity of DPP IV
`(Fig. 1). These analogues include N-acetyl-, N-Fmoc-, N-
`glucitol-, N-palmitate-, and N-pyroglutamyl-GIP [11,27–
`30]. All analogues modified at Tyr1 exhibited complete
`resistance to DPP IV with in vitro half-lives greater than
`24 h compared with 2.3 h for native GIP (Table 1), as il-
`lustrated for N-acetyl-GIP in Figs. 2A–B. This is in
`agreement with DPP IV substrate-binding specificity,
`which predicts the requirement for a bulky N-terminal
`amino acid (such as tyrosine in the case of GIP) possessing
`a free protonated a-amino group. By attaching amino
`acids or functional groups to the N-terminus of GIP, Tyr1
`becomes unprotonated, therefore, removing the explicit
`prerequisite required for DPP IV to act [18].
`In assessing the biological activity of each analogue in
`vitro, cAMP formation in human GIP-receptor trans-
`fected cells [56] and insulinotropic activity in clonal
`BRIN-BD11 cells [57] was measured. All of the Tyr1-
`modified analogues
`studied exhibited an increased
`potency (2- to 10-fold increase in EC50 values) in stim-
`ulating cAMP production compared to native GIP
`(Table 1; Fig. 2C). Furthermore, the Tyr1-modified an-
`alogues exhibited significantly increased insulin secre-
`tory responses (1.1- to 1.4-fold) compared to the native
`peptide in BRIN-BD11 cells,
`indicative of improved
`biological activity (Table 1; Fig. 2D). However, subtle
`differences could be observed within this group, as both
`N-Fmoc- and N-palmitate-GIP appeared to be moder-
`ately (14–20%) less potent in vitro than N-acetyl-, N-
`glucitol-, and N-pyroglutamyl-GIP.
`In determining the insulin releasing and antihyper-
`glycaemic potential of the GIP analogues in vivo, we
`used ob/ob mice, an extensively studied animal model of
`spontaneous obesity and diabetes. Characteristically
`these mice exhibit hyperphagia, marked obesity, mod-
`erate hyperglycaemia, and severe hyperinsulinaemia
`[28]. All of the Tyr1-modified analogues were noticeably
`superior at stimulating insulin release (2.0- to 2.5-fold)
`and lowering blood glucose (1.4- to 1.9-fold) compared
`with native GIP (Table 1). Furthermore, it could be
`observed that N-acetyl-, N-glucitol-, and N-pyroglut-
`amyl-GIP were slightly more potent than both N-Fmoc-
`and N-palmitate-GIP. Of the Tyr1-modified analogues
`tested, N-acetyl-GIP [29] appeared to be the most
`
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`V.A. Gault et al. / Biochemical and Biophysical Research Communications 308 (2003) 207–213
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`209
`
`Fig. 1. A diagrammatic representation of the 42 amino acid sequence of human glucose-dependent insulinotropic polypeptide (GIP) with the solid
`arrow indicating the site of enzymatic degradation by dipeptidyl peptidase IV (DPP IV). The coloured amino acid residues (1–3) indicate the
`analogues synthesized and tested in animal models of obesity-diabetes [11,27–33], based on the entire human GIP 1-42 sequence.
`
`impressive (Table 1). As shown in Fig. 3, this stable
`analogue significantly augmented the plasma insulin
`response and curtailed the glycaemic excursion follow-
`ing conjoint administration with glucose to obese dia-
`betic ob/ob mice. The ability of N-acetyl-GIP to
`overcome the severe insulin resistance and beta cell
`defect (including poor response to native GIP) in this
`animal model is notable and affords proof of concept
`
`that such analogues offer potential as future therapeutic
`agents for type 2 diabetes.
`
`Effects of modifications at position Ala2
`
`A series of Ala2-substituted analogues of GIP have
`also been synthesized and tested for their DPP IV
`
`Table 1
`Summary of the biological properties of designer human GIP analogues modified at the N-terminal Tyr1, Ala2 or Glu3 amino acid residues
`
`DPP IV
`half-life
`(h)
`
`cAMP
`production EC50
`(nmol/liter)
`
`Plasma glucose
`AUC (% GIP max
`response)
`
`Plasma insulin
`AUC (% GIP max
`response)
`
`Modification
`
`Peptide
`
`Native hormone
`
`GIP
`
`Tyr1-modification
`
`Ala2-substitution
`
`N-Acetyl-GIP
`N-Glucitol-GIP
`N-Pyroglutamyl-GIP
`N-Palmitate-GIP
`N-Fmoc-GIP
`
`(Gly2)GIP
`(Ser2)GIP
`(Abu2)GIP
`(Sar2)GIP
`
`Maximal insulin
`response
`(% GIP max)
`100  3.1
`127  4.0
`141  8.1
`118  1.6
`122  3.3
`112  2.5
`107  6.4
`140  4.7
`62  0.6
`42  3.8
`52  2.2
`(Pro3)GIP
`Glu3-substitution
`>24
`Nil
`160
`43
`DPP IV half-lives were calculated by plotting the percentage of intact peptide remaining after incubation with DPP IV (n ¼ 3) versus incubation
`time. EC50 values were calculated from cAMP dose–response curves (n ¼ 3) in human GIP-receptor transfected CHL cells using Graph pad Prism.
`Maximal insulin response values in BRIN-BD11 cells (n ¼ 8) were calculated relative to the maximal percentage GIP response. Plasma glucose and
`insulin AUC values from ob/ob mice (n ¼ 7–8) were calculated and recorded as a percentage of the maximal GIP response. Values represent
`means SEM. Data taken from [11,27–33].
`
`2.3
`
`>24
`>24
`>24
`>24
`>24
`
`>8
`4.8
`1.9
`1.6
`
`18.2
`
`1.86
`2.03
`2.67
`10.0
`9.4
`
`15.0
`14.9
`38.5
`54.6
`
`100
`
`62
`57
`61
`69
`69
`
`81
`75
`94
`106
`
`100
`
`251
`236
`224
`206
`208
`
`144
`126
`79
`96
`
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`
`Fig. 2. (A,B) Representative HPLC profiles obtained after incubation of native GIP (A) and N-acetyl-GIP (B) with DPP IV for 2 h. Reaction
`products were separated on a Vydac C-18 column and peaks corresponding to intact GIP, N-acetyl-GIP, and GIP(3-42) are indicated. (C) Dose-
`dependent production of cAMP by native GIP and N-acetyl-GIP upon binding to CHL cells stably transfected with the human GIP receptor. (D)
`Dose-dependent effects of GIP and N-acetyl-GIP on insulin secretion from BRIN-BD11 cells. Values are means  SEM (n ¼ 3–8). **P < 0:01,
`***P < 0:001 compared to control. DP < 0:05 and DDDP < 0:01 compared to native GIP. Data taken from [29].
`
`Fig. 3. Plasma glucose and insulin responses of 18 h fasted obese diabetic (ob/ob) mice after intraperitoneal administration of glucose alone (2 g/kg
`body weight; open circles), or in combination with either 25 nmol/kg GIP (solid squares) or N-acetyl-GIP (open triangles). Values are means SEM
`(n ¼ 7–8). *P < 0:05, **P < 0:01, and ***P < 0:001 compared to glucose alone. DP < 0:05, DDP < 0:01, and DDDP < 0:001 compared to GIP. Data
`taken from [29].
`
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`211
`
`stability and biological activity (Fig. 1). These include
`(Abu2)GIP,
`(Gly2)GIP,
`(Sar2)GIP,
`(Ser2)GIP, and
`(DD-Ala2)GIP [31–33]. Substitution of Ala2 with either
`2-aminobutyric acid (Abu) or sarcosine (Sar) did not
`appear to confer any increased resistance against DPP
`IV, as both analogues followed similar or slightly ac-
`celerated patterns of degradation to the native peptide
`(Table 1) [32]. Indeed, both of these substitutions im-
`paired biological activity compared with native GIP
`when tested using the in vitro insulin secretion and
`cAMP models. Surprisingly though, both analogues
`displayed antihyperglycaemic and insulinotropic activity
`comparable to native GIP when administered to obese
`diabetic ob/ob mice (Table 1) [32].
`In sharp contrast, substitution with either a glycine
`(Gly) or serine (Ser) residue for Ala2 [31] produced more
`stable analogues with significantly prolonged DPP IV
`half-lives compared with native GIP (Table 1). Unlike
`the Abu and Sar substitutions, both (Gly2)GIP and
`(Ser2)GIP displayed enhanced abilities to elevate cAMP
`(1.2-fold increase in EC50 values) and stimulate insulin
`secretion (1.2- to 1.4-fold) in vitro (Table 1). When
`tested in obese diabetic ob/ob mice, both analogues ex-
`hibited significantly improved insulinotropic activity
`(1.3- to 1.5-fold) and antihyperglycaemic activity (1.2- to
`1.3-fold) compared with native GIP (Table 1), further
`supporting the idea that DPP IV resistant analogues of
`GIP may prove useful in the treatment of type 2 diabetes
`[31].
`Using a similar approach, the research group of
`Pederson and McIntosh [33] recently investigated sub-
`stitution of the LL-alanine in position 2 of GIP with
`DD-alanine. This enzyme resistant analogue exhibited
`moderately reduced biological activity in vitro, although
`it significantly decreased the glycaemic excursion (1.6-
`fold) in fa/fa VDF Zucker rats with an efficacy similar to
`that seen with either (Gly2)GIP or (Ser2)GIP [31].
`However, while several of these Ala2-substituted ana-
`logues demonstrated significantly improved biological
`activity compared with native GIP, their efficacy was
`not as impressive as that of the Tyr1-modified analogues
`(Table 1).
`
`Effects of modification at position Glu3
`
`Substitution of Glu3 in GIP with a proline (Pro)
`residue [55] produced a novel GIP analogue, (Pro3)GIP
`(Fig. 1), completely resistant to DPP IV (Table 1).
`Surprisingly though, (Pro3)GIP inhibited GIP-stimu-
`lated cAMP production and insulin secretion with high
`sensitivity and specificity in vitro (Table 1). Further-
`more, studies using ob/ob mice showed that (Pro3)GIP
`effectively and specifically countered the insulin releasing
`and antihyperglycaemic actions of the native peptide
`in vivo [37,55], reminiscent of the effects of the major
`
`DPP IV degradation product, GIP(3-42) [14]. (Pro3)GIP
`has also recently been utilized to demonstrate that GIP
`is the major physiological
`incretin, accounting for
`approximately 80% of nutrient-induced enteroinsular
`pancreatic beta cell stimulation [37].
`Interestingly, the therapeutic potential of such a se-
`lective GIP receptor antagonist has recently been dem-
`onstrated in a study by Miyawaki and colleagues [58],
`where they showed that GIP plays a central role in lipid
`metabolism and in the development of both genetically
`inherited and diet-induced obesity. Thus, growing evi-
`dence supports the long-held view that GIP is an im-
`portant
`factor directly linking over-nutrition to fat
`deposition, obesity, and glucose intolerance [59,60].
`
`Conclusion
`
`Structural modification of GIP at Tyr1, Ala2 or Glu3
`resulted in analogues with greatly increased, moderately
`increased or antagonistic biological properties, respec-
`tively, both in vitro and in vivo. The Tyr1-modified
`analogues, especially N-acetyl-GIP, exhibit a substan-
`tially enhanced potency and duration of action com-
`pared to native GIP. Accordingly, these novel agents
`provide the basis for exploration to realize the potential
`of GIP in diabetes therapy. However, despite the tre-
`mendous potential for GIP analogues in the treatment
`of diabetes-obesity, their peptidic nature effectively rules
`out the option of straightforward oral administration.
`Therefore,
`in order to further develop rational drug
`design for GIP,
`information on appropriate delivery
`systems,
`three-dimensional
`structure and molecular
`interactions of the peptide with its receptor are essential.
`
`Acknowledgments
`
`We dedicate this paper to Professor Vincent Marks who has been a
`great inspiration and long-term enthusiast of GIP. The authors’ work
`was supported financially by University of Ulster Research Strategy
`Funding and the Research and Development Office of Health and
`Personal Social Services for Northern Ireland.
`
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