A long-acting GDF15 analog causes robust, sustained weight loss and reduction of food intake in an obese nonhuman primate model.

Growth Differentiation Factor-15 (GDF15) is a circulating polypeptide linked to cellular stress and metabolic adaptation. GDF15's half-life is ~3 h and activates the glial cell line-derived neurotrophic factor family receptor alpha-like (GFRAL) receptor expressed in the area postrema. To characterize sustained GFRAL agonism on food intake (FI) and body weight (BW), we tested a half-life extended analog of GDF15 (Compound H [CpdH]) suitable for reduced dosing frequency in obese cynomolgus monkeys. Animals were chronically treated once weekly (q.w.) with CpdH or long-acting GLP-1 analog dulaglutide. Mechanism-based longitudinal exposure-response modeling characterized effects of CpdH and dulaglutide on FI and BW. The novel model accounts for both acute, exposure-dependent effects reducing FI and compensatory changes in energy expenditure (EE) and FI occurring over time with weight loss. CpdH had linear, dose-proportional pharmacokinetics (terminal half-life ~8 days) and treatment caused exposure-dependent reductions in FI and BW. The 1.6 mg/kg CpdH reduced mean FI by 57.5% at 1 week and sustained FI reductions of 31.5% from weeks 9-12, resulting in peak reduction in BW of 16 ± 5%. Dulaglutide had more modest effects on FI and peak BW loss was 3.8 ± 4.0%. Longitudinal modeling of both the FI and BW profiles suggested reductions in BW observed with both CpdH and dulaglutide were fully explained by exposure-dependent reductions in FI without increase in EE. Upon verification of the pharmacokinetic/pharmacodynamic relationship established in monkeys and humans for dulaglutide, we predicted that CpdH could reach double digit BW loss in humans. In summary, a long-acting GDF15 analog led to sustained reductions in FI in overweight monkeys and holds potential for effective clinical obesity pharmacotherapy.

Design, synthesis and preclinical evaluation of bio-conjugated amylinomimetic peptides as long-acting amylin receptor agonists.

Pramlintide is an equipotent amylin analogue that reduces food intake and body weight in obese subjects and has been clinically approved as an adjunctive therapy for the treatment of adult diabetic patients. However, due to its extremely short half-life in vivo, a regimen of multiple daily administrations is required for achieving clinical effectiveness. Herein is described the development of prototypical long-acting pramlintide bioconjugates, in which pramlintide's disulfide-linked macrocycle was replaced by a cyclic thioether motif. This modification enabled stable chemical conjugation to a half-life extending antibody. In contrast to pramlintide (t1/2 < 0.75 h), bioconjugates 35 and 38 have terminal half-lives of ∼2 days in mice and attain significant exposure levels that are maintained up to 7 days. Single dose subcutaneous administration of 35 in lean mice, given 18-20 h prior to oral acetaminophen (AAP) administration, significantly reduced gastric emptying (as determined by plasma AAP levels). In a separate study, similar administration of 35 in fasted lean mice effected a reduction in food intake for up to 48 h. These data are consistent with durable amylinomimetic responses and provide the basis for further development of such long-acting amylinomimetic conjugates for the potential treatment of obesity and associated pathologies.

Potent Sodium/Glucose Cotransporter SGLT1/2 Dual Inhibition Improves Glycemic Control Without Marked Gastrointestinal Adaptation or Colonic Microbiota Changes in Rodents.

The sodium/glucose cotransporters (SGLT1 and SGLT2) transport glucose across the intestinal brush border and kidney tubule. Dual SGLT1/2 inhibition could reduce hyperglycemia more than SGLT2-selective inhibition in patients with type 2 diabetes. However, questions remain about altered gastrointestinal (GI) luminal glucose and tolerability, and this was evaluated in slc5a1-/- mice or with a potent dual inhibitor (compound 8; SGLT1 Ki = 1.5 ± 0.5 nM 100-fold greater potency than phlorizin; SGLT2 Ki = 0.4 ± 0.2 nM). 13C6-glucose uptake was quantified in slc5a1-/- mice and in isolated rat jejunum. Urinary glucose excretion (UGE), blood glucose (Sprague-Dawley rats), glucagon-like peptide 1 (GLP-1), and hemoglobin A1c (HbA1c) levels (Zucker diabetic fatty rats) were measured. Intestinal adaptation and rRNA gene sequencing was analyzed in C57Bl/6 mice. The blood 13C6-glucose area under the curve (AUC) was reduced in the absence of SGLT1 by 75% (245 ± 6 vs. 64 ± 6 mg/dl⋅h in wild-type vs. slc5a1-/- mice) and compound 8 inhibited its transport up to 50% in isolated rat jejunum. Compound 8 reduced glucose excursion more than SGLT2-selective inhibition (e.g., AUC = 129 ± 3 vs. 249 ± 5 mg/dl⋅h for 1 mg/kg compound 8 vs. dapagliflozin) with similar UGE but a lower renal glucose excretion threshold. In Zucker diabetic fatty rats, compound 8 decreased HbA1c and increased total GLP-1 without changes in jejunum SGLT1 expression, mucosal weight, or villus length. Overall, compound 8 (1 mg/kg for 6 days) did not increase cecal glucose concentrations or bacterial diversity in C57BL/6 mice. In conclusion, potent dual SGLT1/2 inhibition lowers blood glucose by reducing intestinal glucose absorption and the renal glucose threshold but minimally impacts the intestinal mucosa or luminal microbiota in chow-fed rodents.

Unique pharmacology of a novel allosteric agonist/sensitizer insulin receptor monoclonal antibody.

OBJECTIVE: Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. METHODS: A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. RESULTS: In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. CONCLUSION: Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology.

Novel Monoclonal Antibody Is an Allosteric Insulin Receptor Antagonist That Induces Insulin Resistance.

A hallmark of type 2 diabetes is impaired insulin receptor (IR) signaling that results in dysregulation of glucose homeostasis. Understanding the molecular origins and progression of diabetes and developing therapeutics depend on experimental models of hyperglycemia, hyperinsulinemia, and insulin resistance. We present a novel monoclonal antibody, IRAB-B, that is a specific, potent IR antagonist that creates rapid and long-lasting insulin resistance. IRAB-B binds to the IR with nanomolar affinity and in the presence of insulin efficiently blocks receptor phosphorylation within minutes and is sustained for at least 3 days in vitro. We further confirm that IRAB-B antagonizes downstream signaling and metabolic function. In mice, a single dose of IRAB-B induces rapid onset of hyperglycemia within 6 h, and severe hyperglycemia persists for 2 weeks. IRAB-B hyperglycemia is normalized in mice treated with exendin-4, suggesting that this model can be effectively treated with a GLP-1 receptor agonist. Finally, a comparison of IRAB-B with the IR antagonist S961 shows distinct antagonism in vitro and in vivo. IRAB-B appears to be a powerful tool to generate both acute and chronic insulin resistance in mammalian models to elucidate diabetic pathogenesis and evaluate therapeutics.

State-Dependent Allosteric Inhibition of the Human SLC13A5 Citrate Transporter by Hydroxysuccinic Acids, PF-06649298 and PF-06761281.

In the liver, citrate is a key metabolic intermediate involved in the regulation of glycolysis and lipid synthesis and reduced expression of the hepatic citrate SLC13A5 transporter has been shown to improve metabolic outcomes in various animal models. Although inhibition of hepatic extracellular citrate uptake through SLC13A5 has been suggested as a potential therapeutic approach for Type-2 diabetes and/or fatty liver disease, so far, only a few SLC13A5 inhibitors have been identified. Moreover, their mechanism of action still remains unclear, potentially limiting their utility for in vivo proof-of-concept studies. In this study, we characterized the pharmacology of the recently identified hydroxysuccinic acid SLC13A5 inhibitors, PF-06649298 and PF-06761281, using a combination of 14C-citrate uptake, a membrane potential assay and electrophysiology. In contrast to their previously proposed mechanism of action, our data suggest that both PF-06649298 and PF-06761281 are allosteric, state-dependent SLC13A5 inhibitors, with low-affinity substrate activity in the absence of citrate. As allosteric state-dependent modulators, the inhibitory potency of both compounds is highly dependent on the ambient citrate concentration and our detailed mechanism of action studies therefore, may be of value in interpreting the in vivo effects of these compounds.

AKAP150 participates in calcineurin/NFAT activation during the down-regulation of voltage-gated K(+) currents in ventricular myocytes following myocardial infarction.

The Ca(2+)-responsive phosphatase calcineurin/protein phosphatase 2B dephosphorylates the transcription factor NFATc3. In the myocardium activation of NFATc3 down-regulates the expression of voltage-gated K(+) (Kv) channels after myocardial infarction (MI). This prolongs action potential duration and increases the probability of arrhythmias. Although recent studies infer that calcineurin is activated by local and transient Ca(2+) signals the molecular mechanism that underlies the process is unclear in ventricular myocytes. Here we test the hypothesis that sequestering of calcineurin to the sarcolemma of ventricular myocytes by the anchoring protein AKAP150 is required for acute activation of NFATc3 and the concomitant down-regulation of Kv channels following MI. Biochemical and cell based measurements resolve that approximately 0.2% of the total calcineurin activity in cardiomyocytes is associated with AKAP150. Electrophysiological analyses establish that formation of this AKAP150-calcineurin signaling dyad is essential for the activation of the phosphatase and the subsequent down-regulation of Kv channel currents following MI. Thus AKAP150-mediated targeting of calcineurin to sarcolemmal micro-domains in ventricular myocytes contributes to the local and acute gene remodeling events that lead to the down-regulation of Kv currents.

AKAP150 contributes to enhanced vascular tone by facilitating large-conductance Ca2+-activated K+ channel remodeling in hyperglycemia and diabetes mellitus.

RATIONALE: Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes mellitus may arise from impaired large-conductance Ca(2+)-activated K(+) (BKCa) channel function. The scaffolding protein A-kinase anchoring protein 150 (AKAP150) is a key regulator of calcineurin (CaN), a phosphatase known to modulate the expression of the regulatory BKCa ß1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes mellitus is unknown. OBJECTIVE: To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa ß1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes mellitus. METHODS AND RESULTS: We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/nuclear factor of activated T-cells c3 (NFATc3) activation, and resistance artery constriction in hyperglycemic animals on high-fat diet. Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. d-glucose-dependent suppression of BKCa channel ß1 subunits required Ca(2+) influx via voltage-gated L-type Ca(2+) channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, high-fat diet mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa ß1 subunits and attenuated high-fat diet-induced elevation in arterial blood pressure. CONCLUSIONS: Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes mellitus.

Anchored phosphatases modulate glucose homeostasis.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic ß-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-ß-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in ß-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

Epac2: a molecular target for sulfonylurea-induced insulin release.

Sulfonylurea drugs are used in type 2 diabetes mellitus therapy to induce release of endogenous insulin from pancreatic beta cells. They act on sulfonylurea receptors, which are the regulatory subunits of adenosine triphosphate (ATP)-sensitive potassium ion (K(ATP)) channels and cause channel closure to trigger exocytosis. Epac2 was identified as an intracellular target for sulfonylurea drugs, providing a potential nonelectrogenic signaling component to the mechanism of action for these agents. Commonly used sulfonylureas such as tolbutamide and glibenclamide induced Epac2 activation with distinct kinetic profiles. Epac2(-/-) mice failed to respond to sulfonylureas, suggesting that both sulfonylurea receptors and Epac2 are necessary for the action of these drugs. These data require that the cellular and physiological effects of drugs that alter the open state of the K(ATP) channel be reassessed.

Diamyd, an alum-formulated recombinant human GAD65 for the prevention of autoimmune diabetes.

Diamyd Medical AB is developing Diamyd (GAD-65), an alum formulation of a full-length recombinant human glutamic acid decarboxylase 65 for subcutaneous injection, for the potential prevention and treatment of type 1 diabetes (T1DM) or latent autoimmune diabetes (LADA) in adults. Phase II clinical trials indicated that Diamyd was safe and well tolerated in patients with T1DM or LADA. Diamyd is currently in phase II/III and III clinical trials for T1DM.

MyRIP anchors protein kinase A to the exocyst complex.

The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery.

Increased oxygen radical formation and mitochondrial dysfunction mediate beta cell apoptosis under conditions of AMP-activated protein kinase stimulation.

AMP-activated protein kinase influences cellular metabolism, glucose-regulated gene expression, and insulin secretion of pancreatic beta cells. Its sustained activation by culture at low glucose concentrations or in the presence of 5-aminoimidazole-4-carboxamide riboside (AICAR) was shown to trigger apoptosis in beta cells. This study shows that both low glucose- and AICAR-induced apoptosis are associated with increased formation of mitochondrial superoxide-derived radicals and decreased mitochondrial activity. Mitochondrial dysfunction was reflected by an increased oxidized state of the mitochondrial flavins (FMN/FAD) but not of NAD(P)H. It was accompanied by suppression of glucose oxidation and glucose-induced insulin secretion, while palmitate oxidation appeared unaffected. When the cellular accumulation of superoxide-derived radicals was quenched by the ROS scavengers vitamin E, N-acetylcysteine, or the SOD-mimetic compound MnTBAP, apoptosis was significantly inhibited. Both low glucose and AICAR also elevated the expression of BH3-domain-only Bcl-2 antagonists, and induced caspase-3 activation, causing caspase-dependent truncation of Bcl-2. Overexpression of recombinant human Bcl-2 prevented caspase-3 activation, endogenous Bcl-2 processing, and apoptosis, but did not attenuate oxygen radical formation, AMPK activation, or JNK phosphorylation. We conclude that apoptosis by prolonged AMPK activation in beta cells results from enhanced production of mitochondria-derived oxygen radicals and onset of the intrinsic mitochondrial apoptosis pathway, followed by caspase activation and Bcl-2 cleavage which may amplify the death signal.

In depth analysis of the N-terminal bioactive domain of gastric inhibitory polypeptide.

Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal regulator of insulin release and glucose homeostasis following a meal. Strategies have been undertaken to delineate the bioactive domains of GIP with the intention of developing small molecular weight GIP mimetics. The molecular cloning of receptors for GIP and the related hormone GLP-1 (glucagon-like peptide-1) has allowed examination of the characteristics of incretin analogs in transfected cell models. The current report examines the N-terminal bioactive domain of GIP residing in residues 1-14 by alanine scanning mutagenesis and N-terminal substitution/modification. Further studies examined peptide chimeras of GIP and GLP-1 designed to localize bioactive determinants of the two hormones. The alanine scan of the GIP(1-14) sequence established that the peptide was extremely sensitive to structural perturbations. Only replacement of amino acids 2 and 13 with those found in glucagon failed to dramatically reduce receptor binding and activation. Of four GIP(1-14) peptides modified by the introduction of DP IV-resistant groups, a peptide with a reduced bond between Ala2 and Glu3 demonstrated improved receptor potency compared to native GIP(1-14). The peptide chimera studies supported recent results on the importance of a mid-region helix for bioactivity of GIP, and confirmed existence of two separable regions with independent intrinsic receptor binding and activation properties. Furthermore, peptide chimeras showed that binding of GLP-1 also involves both N- and C-terminal domains, but that it apparently contains only a single bioactive domain in its N-terminus. Together, these results should facilitate development of incretin based therapies using rational drug design for potential treatment of diabetes.

Prior in vitro exposure to GLP-1 with or without GIP can influence the subsequent beta cell responsiveness.

Glucagon-like peptide-1 (7-36) amide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) potentiate glucose-induced insulin release when present at the time of nutrient stimulation. This study examines whether they can also influence rat beta cell responsiveness to subsequent stimulations. When rat beta cells were cultured for 24 h with 1 nM GLP-1, they progressively desensitized to subsequent GLP-1 stimuli, as evidenced by cellular cAMP production. This GLP-1-induced desensitization did not occur when the incretin was only present during three periods of 1 h at 10 mM glucose that alternated with 6-9 h culture at 3 mM glucose. After these 24h, the beta cells exhibited the same secretory response to glucose (10 mM) and GLP-1 (10 nM at 10 mM glucose), whether GLP-1 was present during the pulses or not. Similarly the presence of 1 nM GIP during these one hour pulses did not influence subsequent secretory responses to glucose and GLP-1. However, when both GLP-1 and GIP, each at 0.5 nM, were added to the one hour pulses, they not only amplified insulin release during the pulses, as was the case with their single addition, but also increased the secretory response to a subsequent stimulation by glucose and GLP-1. These data distinguish between a desensitization effect of a prolonged exposure to GLP-1 and a positive priming effect of a discontinuous exposure to a combination of GLP-1 plus GIP. They may have to be taken into account in drug treatment strategies aiming the mimicking of physiologic patterns in the regulation of insulin release.

Plasticity of the beta cell insulin secretory competence: preparing the pancreatic beta cell for the next meal.

It is well established that the acute rise in plasma glucose and in the incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (7-36) amide (GLP-1), as occurs during a meal, is of pivotal importance in regulating the minute-to-minute output of insulin from pancreatic beta cells. In addition to this well studied acute effect, both glucose and incretin hormones have been recently observed to determine the future secretory responsiveness of the cells. Such plasticity of the insulin secretory competence would imply that glucose and incretins not only act during the present meal, but also help to prepare the beta cells to function during the subsequent meal. Evidence supporting this hypothesis is growing as a result of physiological studies of cultured beta cells (either primary cells or beta cell lines), as well as from an increasing number of large-scale gene expression studies, exploring transcriptional and post-transcriptional events in genes regulated by glucose and incretins. On the basis of this hypothesis, one can speculate that genetic or environmental disturbances of plasticity of the insulin secretory competence is one aspect of beta cell dysfunction that can contribute to the aetiology of type 2 diabetes.

Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are gut-derived incretins that potentiate glucose clearance following nutrient ingestion. Elimination of incretin receptor action in GIPR(-/-) or GLP-1R(-/-) mice produces only modest impairment in glucose homeostasis, perhaps due to compensatory upregulation of the remaining incretin. We have now studied glucose homeostasis in double incretin receptor knockout (DIRKO) mice. DIRKO mice exhibit normal body weight and fail to exhibit an improved glycemic response after exogenous administration of GIP or the GLP-1R agonist exendin-4. Plasma glucagon and the hypoglycemic response to exogenous insulin were normal in DIRKO mice. Glycemic excursion was abnormally increased and levels of glucose-stimulated insulin secretion were decreased following oral but not intraperitoneal glucose challenge in DIRKO compared with GIPR(-/-) or GLP-1R(-/-) mice. Similarly, glucose-stimulated insulin secretion and the response to forskolin were well preserved in perifused DIRKO islets. Although the dipeptidyl peptidase-IV (DPP-IV) inhibitors valine pyrrolidide (Val-Pyr) and SYR106124 lowered glucose and increased plasma insulin in wild-type and single incretin receptor knockout mice, the glucose-lowering actions of DPP-IV inhibitors were eliminated in DIRKO mice. These findings demonstrate that glucose-stimulated insulin secretion is maintained despite complete absence of both incretin receptors, and they delineate a critical role for incretin receptors as essential downstream targets for the acute glucoregulatory actions of DPP-IV inhibitors.

[Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.