Glucagon-like peptide 1 (GLP-1)

Background The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent β-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. Scope of review In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. Major conclusions Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders

In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases.

Maintenance of adequate glucose metabolism is a prerequisite for human health, and pathological failure to buffer against prolonged episodes of hypo- and/or hyperglycemia can result in severe microvascular disease, metabolic damage, coma, and death. Unsurprisingly, before the discovery and commercialization of insulin in the 1920's, juvenile-onset diabetes, with its paucity of endogenous insulin, was a disease with only a few years between a patient's diagnosis and premature demise. The discovery of insulin and its ability to lower blood glucose transformed juvenile-onset (type-1) diabetes from a fatal to a manageable disease. However, early on, it was noted that insulin derived from pancreatic extracts [ 1 ] or as crude insulin preparations [ 2 ] sometimes first elevated blood glucose and then later decreased blood glucose levels. The increase in blood glucose, which peaked around 20 min after the administration, was believed to be caused by a toxic fraction resulting from suboptimal insulin purification [ 2 ]. The same toxic fraction was thought to be responsible for local skin irritations and abscesses that were sometimes observed in patients treated with these formulations [ 2 ]. These observations spurred efforts to optimize the isolation and purification of insulin from tissue homogenates. Aiming to develop a fast and inexpensive method for commercial insulin purification, in 1923, Charles Kimball and John Murlin precipitated a pancreatic fraction that, after evaporation and reconstitution in water, had a robust hyperglycemic effect when injected into rabbits and dogs [ 3 ]. Because the fraction was incapable of decreasing blood glucose, Kimball and Murlin hypothesized that the hyperglycemic effect resulted from a secreted factor, one that antagonizes insulin's hypoglycemic effect. The factor was named ‘ the gluc ose agon ist’ , or “ glucagon ” [ 3 ]. Over the subsequent decades, substantial research efforts were directed toward unravelling the molecular underpinnings of glucose regulation by the two opposing pancreatic hormones (as reviewed in [ 4 , 5 ]). Among the numerous major discoveries made in this regard was the finding that the hyperglycemic effect of glucagon resides in its ability to act in the liver to stimulate glycogenolysis and gluconeogenesis [ [6] , [7] , [8] , [9] , [10] , [11] , [12] ] despite its seemingly paradoxical ability to stimulate insulin secretion in humans reported by Ellis Samols in 1965 [ 13 ]. In the early 1950's, Earl Sutherland and Christian de Duve demonstrated that pancreatic glucagon production was abolished when the function of islet α-cells was compromised by treatment with either cobalt or synthalin A but was preserved upon alloxan-induced impairment of the exocrine pancreatic acinar and islet β-cells [ 14 , 15 ]. Subsequent histological studies by Claes Hellerstrom and Bo Hellman divided α-cells into α 1 -and α 2 -subtypes, but it was not until the early 1960s that convincing evidence was provided that α 2 -cells were the source of glucagon [ 16 ]. This conclusion was based on suppression of α 2 -cell numbers in rats and guinea pigs by administration of exogenous glucagon [ 17 , 18 ] plus the strong staining in α 2 -cells for tryptophan, which is an amino acid present in glucagon. Later, α 1 -cells were shown by Bo Hellman and Ake Lernmark to be the source of an inhibitor of insulin secretion [ 19 ]. This was eventually established as somatostatin. In 1959, Roger Unger generated and characterized the first glucagon-detecting antibody and thus paved the path to the development of the first radioimmunoassays (RIA) to detect glucagon in blood and tissue samples [ 20 ]. It was subsequently found by Ellis Samols and Vincent Marks in 1966 [ 21 ] and confirmed by others that glucagon-like immunoreactivity was also present in extra-pancreatic tissues, in particular in the intestine [ [22] , [23] , [24] , [25] , [26] , [27] ]. Notably, the circulating glucagon-like material was also detected in 1967 by Samols and Marks in pancreatectomized humans [ 21 ] and in the following year by Roger Unger and Isabel Valverde in dogs [ 27 ]. This eliminated the pancreas as the origin of this glucagon-like immunoreactivity. Unger then demonstrated in 1968 that intraduodenal, but not intravenous, glucose administration increases circulating glucagon-like immunoreactivity [ 27 ], implying that the intestine secretes this glucagon-like material. The intestinal glucagon-like material was heterogeneous, comprising several fractions of different molecular size and with apparently distinct biological actions relative to pancreatic-derived glucagon [ 27 , 28 ]. The intestinal glucagon-immunoreactive material did not induce hyperglycemia when injected into dogs and was devoid of glycogenolytic effects in the isolated perfused rat liver [ 27 ]. However, analogous to the effects of glucagon, the intestinal material stimulated the release of insulin, suggesting that the intestinal glucagon-like

Preproglucagon (Gcg) is expressed in pancreatic α-cells, in enteroendocrine L-cells throughout the gut, predominantly in the distal ileum and colon, and in a population of neurons in the nucleus tractus solitarii (NTS) of the brainstem [ 52 , [84] , [85] , [86] , [87] , [88] , [89] ]. Diphtheria toxin-induced ablation of preproglucagon-positive neurons in the NTS demonstrated that this small population of neurons is the primary source of endogenous GLP-1 in the brain [ 90 ]. Cleavage sites within the proglucagon molecule together with expression of specific prohormone convertase enzymes determines which smaller peptide molecules/hormones are formed, including glicentin (aa 1–69), glicentin-related pancreatic polypeptide (GRPP; aa 1–30), glucagon (aa 33–61), oxyntomodulin (OXM; aa 33–69), the major proglucagon fragment (MPGF; aa 72–158), and the glucagon-like peptides 1 (GLP-1; aa 72–107/108) and 2 (GLP-2; aa 126–158) ( Figure 1 ) [ [51] , [52] , [53] , 91 ]. Several of the PGDPs have important and well-defined (pharmacological) effects on systemic metabolism, including the modulation of food intake and satiety (GLP-1, glucagon, oxyntomodulin), regulation of fluid homeostasis (water intake and urine excretion) (GLP-1) [ 92 ], thermogenesis (glucagon), lipid metabolism (GLP-1, glucagon, GLP-2), gastrointestinal motility (glucagon, GLP-1, GLP-2), and gastric emptying (glucagon, GLP-1, GLP-2). Glucagon released from pancreatic α-cells and GLP-1 released from intestinal L-cells have opposing effects on blood glucose. Therefore, the expression and cleavage of proglucagon and secretion of the various PGDPs must be precisely controlled in a cell-specific process. The expression of Gcg in the pancreas, intestine, and brain is under the control of a single promoter and is initiated from an identical transcription start codon ( Figure 2 ). The rodent Gcg promoter, along with its adjacent DNA control/enhancer elements, is located within the 2.5 kb 5′-flanking region of the Gcg transcription start [ 89 , 93 , 94 ]. In rodents, the ∼1.3 kb 5′-flanking sequence is sufficient to direct transgene expression to Gcg + cells in the brain and the pancreas [ 95 ] but extension of this region to include ∼2.5 kb is required to target Gcg + cells in the intestine [ 96 ], including evolutionarily preserved sequences in the first intron [ 97 ]. Figure 2 Schematic on the tissue-selective processing of proglucagon in the pancreatic islets. Schematic on the transcriptional regulation of preproglucagon (PPG) in the pancreatic islets. Pax6: paired box 6; CDX2/3: caudal type homeobox 2/3; MafB: MAF bZIP transcription factor B; cMaf: c-Maf inducing protein; NKX2.1: NK2 homeobox 1; PDX1: pancreatic and duodenal homeobox 1; Pax4: paired box 4; CRE: cAMP response element; CREB: cAMP response element binding protein; PPG: preproglucagon; HNF3: hepatocyte nuclear factor 3; Isl1: ISL LIM homeobox 1; Preb: prolactin element binding. For further explanations, please see text. Figure 2 The cell-specific expression of Gcg is orchestrated by a series of homeodomain proteins that bind to specific cis-acting elements in the Gcg promoter and/or enhancer region to either stimulate or inhibit Gcg promoter activity [ 94 , [98] , [99] , [100] ]. The rat Gcg promoter comprises at least 5 cis-acting elements (G 1 – G 5 ) plus a cAMP response element (CRE), all of which are located within the 2.5 kb region upstream of the Gcg transcription start [ 89 , 94 , [101] , [102] , [103] ]. In α-cells, the TATA box, as well as the adjacent G1 and G4 elements, represent the minimal promoter which is essential for Gcg expression while the elements G5, G2, G3, and CRE represent a more distal located enhancer region [ 94 , [101] , [102] , [103] ] ( Figure 2 ). Signaling events leading to the stimulation of Gcg expression in α-cells include heterodimerization of the transcription factor paired box protein 6 (Pax6) with cellular muscular aponeurotic fibrosarcoma (c-Maf), MAF bZIP transcription factor B (MafB) or caudal type homeobox 2/3 (Cdx2/3), and consequent binding of these heterodimers to the G1 element ( Figure 2 ) [ 98 , [104] , [105] , [106] ]. Pax6 can also bind to the G3 element [ 100 ], and it plays a key role in regulating Gcg expression and α-cell development, because mice lacking Pax6 fail to produce glucagon-producing α-cells [ 107 ]. Pax6 also stimulates Gcg expression in the enteroendocrine cells of the gastrointestinal epithelium [ 108 ]. Mice homozygous for a dominant negative Pax6 mutation (SEYNeu) have repressed Gcg expression in enteroendocrine cells in the small and large bowel and absence of immunoreactive GLP-1 and GLP-2 [ 109 ]. Further supporting the role of Pax6 in regulating intestinal Gcg expression, adenoviral overexpression of Pax6 enhances Gcg promoter activity and Gcg expression in intestinal enteroendocrine cells such as the secretin tumor cell line-1 (STC-1) and cells derived from colonic tumors of transgenic mice expressing large T antigen

Native GLP-1 has a very short half-life, which, depending on the species, is around 1–2 min [ [182] , [183] , [184] ] and results from two causes: (a) the action of the enzyme dipeptidylpeptidase-4 (DPP-4) and (b) renal elimination. DPP-4 cleaves GLP-1 (7-36amide) and GLP-1 (7–37) at the N-terminal dipeptide to generate GLP-1 (9-36amide) or GLP-1 (9–37), low affinity ligands for the GLP-1 receptor [ [185] , [186] , [187] , [188] ]. Both these intact forms as well as inactivated GLP-1 metabolites are also rapidly cleared from the circulation via the kidneys. In mice, the enzyme neprilysin additionally rapidly degrades the metabolites, making GLP-1 difficult to measure in this species [ 189 ]. While GLP-1 degradation is unaffected by kidney function, the clearance of both GLP-1, and to a greater extent its inactive metabolites, is delayed in patients with renal insufficiency [ 184 ]. DPP-4 exists in two forms; i.e., it is a membrane-spanning cell surface protein and a circulating protein, and both forms have actions that extend beyond its proteolytic activity [ 190 ]. In the intestine, DPP-4 is highly expressed in the enterocyte brush border and in endothelial cells [ 191 ]. Consequently, as discussed in a comprehensive review [ 152 ], a large portion of intestinal GLP-1 is already degraded in the capillaries of the distal gut with an estimated ∼25% of active GLP-1 reaching the liver and only ∼10–15% reaching the general circulation [ 152 , 176 , 183 , 191 ]. Pharmacological inhibition or genetic reduction of DPP-4 activity preserves much higher circulating levels of intact GLP-1 [ 191 , 192 ], and this was demonstrated to potentiate the insulinotropic effect of GLP-1 in anesthetized pigs (eventually leading to the development of DPP-4 inhibitors for clinical use) [ 193 ]. When administered i.v., i.p., or s.c. in rats, GLP-1 (7–36 amide) has a half-life of 0.8–4.7 min, 0.6–13.5 min and 4.6–7.1 min, respectively [ 194 ]. Substantial evidence indicates that the DPP-4-generated GLP-1 metabolites (GLP-1 (9-36amide) and GLP-1 (9–37)) have no major role in regulating glucose metabolism [ [195] , [196] , [197] ]. However, one report of an experiment in obese humans suggested that GLP-1 (9-36amide) is a weak insulin secretagogue [ 198 ], and administration of GLP-1 (9-36amide) improves glucose handling without affecting insulin secretion in anesthetized pigs and in humans [ 199 , 200 ]. GLP-1 (9-36amide) also improves cardiac output in the post-ischemic mouse heart when administered during reperfusion, and it affects vasodilation in mesenteric arteries in mice [ 201 ]. This scenario contrasts with the apparent lack of effect of high doses of GLP-1 (9-36amide) on glucoregulation in ob/ob mice or on cognitive function in high-fat fed mice [ 202 , 203 ]. Indeed, there is evidence that truncated GLP-1 (9-36amide) has no effect on glucose clearance or insulin secretion in healthy humans [ 196 ]. In fact, the peptide acts as a weak GLP-1R antagonist, clearly counteracting the biological effects of GLP-1 (7-36amide) in vitro [ 204 ].

The cellular mechanisms underlying glucose-stimulation of GLP-1 secretion from L-cells are, at least in part, similar to the stimulation of insulin secretion in the islets. In enteroendocrine GLUTag cells, glucose, and fructose dose-dependently increase GLP-1 secretion through closure of ATP-sensitive K ATP channels and subsequent membrane depolarization ( Figure 3 ) [ 247 , 249 , 265 ]. Glucose-induced membrane depolarization entails opening of voltage-dependent Ca 2+ (VDC) channels, and the resulting Ca 2+ influx then triggers vesicular exocytosis and secretion of GLP-1 into the circulation ( Figure 3 ) [ 266 ]. Underlining the role of the K ATP channels in mediating this process, glucose-stimulated Ca 2+ entry and GLP-1 secretion are mimicked upon treatment of GLUTag cells with the K ATP channel inhibitor tolbutamide [ 265 ]. While the importance of K ATP channel activity in mediating GLP-1 release has been confirmed in vitro , its relevance for GLP-1 secretion in vivo is less clear. While sulphonylureas potently promote insulin secretion in type-2 diabetic patients via inhibition of K ATP channel activity [ [267] , [268] , [269] , [270] ], there is no clear evidence that sulphonylureas affect GLP-1 secretion in humans (as reviewed in [ 266 ]). However, the Kir6.2/SUR1 channel complex of the K ATP channel is present in human L-cells [ 271 ] and K ATP channel subunits and glucokinase are highly expressed in murine L-cell populations [ 266 ]. In summary, while L-cell depolarization is crucial for GLP-1 secretion, the role of the Kir6.2/SUR1 channel complex of the K ATP channels for mediating this process in vivo warrants clarification. Monosaccharides demonstrated to stimulate GLP-1 secretion include glucose, galactose, and fructose [ 239 , 247 ]. Low concentrations of glucose or methyl-α-glucopyranoside stimulate L-cell electrical activity and promote GLP-1 secretion via sodium-glucose cotransporter (SGLT1)-dependent induction of small inward currents ( Figure 3 ) [ 247 ]. Preventing luminal glucose absorption by blockade of SGLT1 reduces GLP-1 secretion in the isolated perfused canine ileum [ 248 ] and in the rat ileum [ 178 ] and impairs glucose-stimulation of GLP-1 release in GLUTag cells [ 247 ]. Notably, the glucose transporter-2 (GLUT2) has been implicated in glucose-stimulated GLP-1 secretion, as demonstrated by an impaired GLP-1 response to oral glucose in mice deficient for GLUT2, and this is accompanied by reduced glucose-stimulated insulin secretion and impaired glucose tolerance [ 272 ]. However, pharmacological inhibition of active, sodium-coupled glucose transport impaired glucose-stimulated GLP-1 secretion in vitro , whereas inhibition of facilitative GLUT-mediated glucose transport was without effect [ 273 ]. In summary, glucose uptake into the L-cells seems to be mediated via both, GLUT2 and SGLT1 ( Figure 3 ) [ [274] , [275] , [276] ], with the electrogenic SGLT1 mediated uptake being of particular importance for stimulus secretion coupling. Downstream of glucose-mediated membrane depolarization, vesicular exocytosis of GLP-1 is orchestrated in a Ca 2+ dependent manner involving a cellular machinery like that in β-cells [ 277 , 278 ]. Fructose stimulation of GLP-1 secretion has been demonstrated in rats, mice, humans, and GLUTag cells [ 249 , 279 ]. When given orally, fructose is a far less potent GLP-1 secretagogue relative to an isocaloric load of glucose [ 249 ]. Similar findings are reported in humans upon intragastric infusion of glucose and fructose at doses that are matched for sweetness [ 279 ]. A possible role of intestinal sweet-taste receptors in glucose-stimulated GLP-1 secretion remains uncertain. In isolated murine and human L-cell cultures, glucose, and the artificial sweetener sucralose, each stimulates GLP-1 secretion with diminished glucose stimulation of GLP-1 secretion in mice lacking α-gustducin, an integral sweet-taste receptor element [ 280 ]. In contrast, there is no effect of sucralose on GLP-1 secretion in primary L-cell cultures in other reports [ 281 , 282 ], and infusion of artificial sweeteners does not affect glucose-stimulated GLP-1 secretion in healthy human volunteers [ 283 , 284 ].

Protein and amino acid stimulation of GLP-1 secretion has been demonstrated in murine primary colonic L-cell cultures [ 260 , 302 ], in GLUTag cells [ 256 , 259 ], in human NCI-H716 cells [ 251 , 303 ] and in the isolated perfused rat ileum or colon [ 250 , 304 ] as well as in vivo in mice [ 305 ], rats [ 256 , 306 ], and humans [ 307 , 308 ]. In healthy human volunteers, a diet with 30% kcal from protein (40% carbohydrates, 30% fat) causes greater GLP-1 secretion than a diet with 10% kcal protein (60% carbohydrates, 30% fat) [ 308 ]. Individual amino acids stimulating GLP-1 secretion include glutamine, asparagine, phenylalanine, and glycine, with glutamine and glycine being the most potent [ [258] , [259] , [260] ]. When orally administered, glutamine also increases circulating GLP-1 and insulin in lean, obese and type-2 diabetic individuals [ 307 ]. In human NCI-H716 cells, stimulation of GLP-1 secretion was also demonstrated for leucine, isoleucine, valine, skimmed milk, casein, and whey [ 303 ]. l -Arginine, a potent insulin secretagogue [ 309 ], also stimulates GLP-1 release from isolated rat intestine, and, when given orally, augments GLP-1 and insulin levels and improves glucose tolerance in mice, effects that are absent in GLP-1R KO mice [ 305 ]. Meat hydrolysate stimulates GLP-1 secretion from NCI-H716 cells, an effect that is not related to changes in proglucagon expression [ 253 ]. This stimulatory effect can be blocked by pretreatment with the p38 inhibitor SB203580, the PI3 kinase inhibitor wortmannin or the MEK1/2 inhibitor U0126 [ 310 ]. The corn protein zein stimulates GLP-1 secretion in GLUTag cells and in the small intestine of anesthetized rats [ 256 ], and it stimulates GLP-1 secretion when administered either orally [ 228 ] or directly into the ileum [ 257 ]. Intraluminal administration of peptones stimulate GLP-1 secretion in the isolated perfused rat ileum [ 250 ] but not upon ileal perfusion in healthy human volunteers [ 210 ]. Pectin stimulates GLP-1 secretion in the isolated perfused rat colon [ 304 ] but not in the isolated rat ileum [ 250 ]. The low molecular fraction of wheat protein hydrolysate (LWP) increases GLP-1 secretion in both GLUTag cells and when directly administered in rats [ 306 ]. In rats, the LWP-induced GLP-1 secretion further improves glucose tolerance and enhances insulin secretion, an effect that is blocked by pre-administration of the GLP-1R antagonist exendin (9–39) [ 306 ]. Protein-stimulation of GLP-1 secretion has also been demonstrated in humans, with similar GLP-1 responses upon uptake of whey, casein, gluten or cod protein [ [311] , [312] , [313] ]. Protein-induction of GLP-1 secretion seems to be dose-dependent, as demonstrated by uptake of isocaloric diets comprising 14%, 25%, or 50% of energy coming from proteins [ 314 ]. The molecular mechanisms underlying protein stimulation of GLP-1 secretion include activation of Ca 2+/ calmodulin-dependent kinase II [ 306 ]. Substantial evidence supports that peptide-mediated GLP-1 secretion is a Ca 2+ sensitive process and involves L-cell signaling via the Ca 2+ sensing receptor (CaSR) and the peptide transporter 1 (PEPT1) [ 302 ]. Consistent with this, glycine-sarcosine (Gly-Sar) stimulation of GLP-1 secretion from purified murine L-cell cultures is blocked in the absence of extracellular Ca 2+ and is inhibited upon treatment with the L-type Ca 2+ -channel blocker nifedipine [ 302 ]. Oligopeptide stimulation of GLP-1 release is impaired upon treatment of L-cell cultures with a CaSR antagonist and is ameliorated in mice deficient for the peptide transporter 1 (PEPT1) [ 302 ]. Aromatic amino acids such as phenylalanine, however, also interact with GPR142 [ 315 ].

As discussed above, in addition to enteroendocrine L-cells, GLP-1 is also produced in a discrete set of non-TH-positive neurons in the caudal portions of the NTS [ 86 , 148 , [346] , [347] , [348] ], and these hindbrain GCG + positive neurons are the primary source of endogenous brain GLP-1 [ 90 ]. Either peripheral administration of leptin [ 349 ] or gastric balloon distention [ 350 ] acutely activates GLP-1-producing neurons in the NTS, as assessed by cFos immunoreactivity. Direct electrical stimulation of the NTS evokes glutamatergic excitatory post-synaptic currents (EPSCs) in GCG + positive neurons [ 351 ]. Generation of mice that express eYFP under control of the Gcg promoter has enabled the isolation and characterization of NTS GCG + neurons in ex vivo tissue slices [ 351 ]. Electrophysiological whole-cell voltage- and current-clamp recordings in horizontal or coronal brainstem slices has revealed a rapid leptin-induced depolarization of these NTS GCG + neurons, thus confirming the ability of leptin to directly stimulate central GLP-1 secretion [ 351 ]. Of note, the hindbrain GCG + neurons lack the GLP-1 receptor such that they cannot be directly activated by peripherally-derived GLP-1 [ 351 ]. In addition, neither electrophysiological administration of PYY, melanotan II, nor ghrelin stimulates these neurons in isolated NTS brain slices [ 351 ]. In contrast, leptin [ 351 ], CCK, and epinephrine [ 352 ] stimulate Gcg neurons. In the NTS, neurons expressing GLP-1 also express the leptin receptor [ 351 , 353 ]. Electrical stimulation of the solitary tract indicates that PPG neurons in the NTS are second-order neurons that receive direct input from vagal afferents. Thus, peripheral endocrine signals, such leptin or GLP-1, can via activation of vagal afferents trigger central activation of PPG neurons in the NTS [ 351 ]. CCK-induced firing of the NTS GCG + neurons can be blocked by treatment with the glutamate receptor antagonist DNQX or by inhibition of α1-adrenergic signaling [ 352 ]. Consistent with these findings, peripherally administered CCK induces cFos immunoreactivity in GLP-1-producing neurons of the hindbrain vagal complex of the NTS [ 354 ], and surgical vagal deafferentation reduces CCK-induced NTS neuronal cFos activation by approximately 50% [ 355 ]. Thus, these neurons are able to sense and respond to a variety of peripheral signals that help to regulate both short and long-term energy balance. Indeed, similar to chronic blockade of CNS GLP-1R, viral knockdown of GLP-- expressing neurons in rats increases body mass, and specifically body adiposity [ 356 ]. In a recent report, chemogenetic stimulation of Gcg neurons reduced food intake without conditioning avoidance, and this occurred when the animals were fed or fasted or were fed chow or HFD [ 357 ]. On the other hand, acute chemogenetic inhibition of these neurons did not increase ad lib feeding but did increase refeeding after a fast and blocked stress-induced hypophagia [ 90 ]. In summary, the glutamatergic GLP-1-producing neurons in the NTS are activated by multiple peripheral signals and regulate many aspects of feeding behavior. This CNS GLP-1 system does not seem to be activated by peripherally-secreted (endogenous) GLP-1 and therefore may be distinct from the peripheral GLP-1 system.

The rat and human GLP-1R protein comprises 463 amino acids with 90% sequence homology between these species [ 359 , 360 , 364 ]. GLP-1 binding to and activation of GLP-1R is a complex process that is comprehensively summarized in previous review articles [ 359 , 401 ]. As a class B GPCR, GLP-1R comprises seven transmembrane helices (TMH) interconnected by intracellular loops, with a C-terminal intracellular domain and a large (∼120 amino acid) N-terminal extracellular domain (ECD) [ 401 ]. Upon biosynthesis of the receptor in the endoplasmic reticulum (ER), the N-terminal ECD of GLP-1R (and of all other class-B GPCRs) contains a short leader sequence encoding for a signal peptide. This signal peptide is of crucial importance for translocation of the receptor across the ER as well as for trafficking of the receptor to the cell surface [ 359 , [401] , [402] , [403] , [404] ]. Underlining the importance of the signal peptide in receptor trafficking, blocking the signal peptide through site-directed mutagenesis causes retention of the receptor within the ER [ 404 ]. Following translocation of the receptor across the ER, the signal peptide is enzymatically cleaved by a peptidase, leaving behind the full length GLP-1R with the N-terminal helix at the beginning of the ECD and four β-strands forming two antiparallel sheets that are connected through three disulfide bonds between six cysteine (Cys) residues [ 401 ]. The disulfide bond between Cys 1 and Cys 3 connects the N-terminal α-helix of the receptor to the first β-sheet, while the disulfide bond between Cys 2 and Cys 5 connects two β-sheets and the disulfide bond between Cys 4 and Cys 6 holds the central β-sheets in proximity to the C-terminal domain of the receptor [ 401 , [405] , [406] , [407] , [408] , [409] ]. Several studies have aimed to identify the functional GLP-1 motifs orchestrating its interaction with GLP-1R [ [410] , [411] , [412] ]. Analysis of the crystal structure of GLP-1R linked to GLP-1 suggests that certain residues in the α-helical region of GLP-1 interact with residues in the N-terminal ECD of GLP-1R [ 407 , 409 ]. These data agree with reports demonstrating a crucial role of the GLP-1R ECD for recognition by GLP-1 [ 405 , [413] , [414] , [415] , [416] ]. Although these studies convincingly demonstrate the importance of the GLP-1R ECD for ligand binding, this interaction seems less important for ligand-induced activation of the receptor [ 401 ]. Accordingly, exendin (9–39) and exendin-4 have similar ligand binding affinities to the human GLP-1R, yet exendin (9–39) is not able to activate the receptor [ 363 ]. Furthermore, despite a low affinity to bind GLP-1R, [Ser(2)]exendin (1–9) is nonetheless able to activate the receptor with low potency [ 417 ]. Taken together, the ligand motifs required for receptor binding seem to differ from those motifs required for receptor activation. Indeed, for most (if not all) members of the glucagon receptor family, the activation of the receptor requires interaction of the N-terminal residues of the ligand with the transmembrane helices and extracellular loops of the receptor [ 401 , [418] , [419] , [420] ]. A two-domain model has emerged and suggests that GLP-1 binds via its α-helical and C-terminal motifs to the N-terminal ECD of GLP-1R followed by the activation of GLP-1R through binding of the N-terminal residues of GLP-1 with the transmembrane helices and extracellular loops of the receptor [ 401 , 421 , 422 ]. A model for ligand-induced receptor conformation has also been proposed based on comparative molecular simulations using GLP-1R bound to positive and negative allosteric modulators [ 423 ]. Based on this model, a negative allosteric modulator binds and deactivates GLP-1R through binding between the cleft of the GLP-1R helices VI and VII, thereby pushing helix VI to an inactive state that prevents association of the receptor with the G protein [ 423 ]. A positive allosteric modulator binds and activates GLP-1R through binding between helix V and VI, thereby creating an intercellular binding site for the G protein [ 423 ]. Similar conclusions have been reached by analysis of the GLP-1 bound receptor conformation using cryo-electron microscopy [ 424 ] and by analyzing the crystal structure of GLP-1R bound to truncated peptide agonist [ 425 ].

Upon ligand binding, GPCRs located on the cell surface transduce the extracellular signal to the interior of the cell. At some point, the ligand-induced receptor activation has to be terminated, and the sensitivity of the receptor to be activated by its ligand has to be restored. Data related to the molecular mechanisms leading to termination of GLP-1R activity (desensitization) and subsequent GLP-1R resensitization are incompletely understood and partially conflicting. Desensitization of GPCRs is generally achieved by two families of serine/threonine kinases, the second-messenger-dependent protein kinases and the receptor-specific G protein-coupled receptor kinases (GRKs) [ 436 , 437 ]. After ligand-induced receptor activation, these kinases phosphorylate the receptor. This induces intracellular recruitment of arrestin, which then binds to the GPCR and uncouples the receptor from its heterotrimeric G proteins [ 436 , 437 ]. Another potential mode of receptor deactivation is the translocation of the ligand–receptor complex to intracellular compartments (endosomes), such that the now intracellular receptor is physically dissociated from the membrane [ 436 , 437 ]. While such ligand-induced receptor internalization has been demonstrated for many GPCRs (including GLP-1R), its importance for receptor deactivation is controversial and is potentially receptor specific. While some studies support a role of receptor internalization in ligand-induced receptor desensitization [ 438 , 439 ], no such role has been demonstrated for the β2 adrenergic receptor (β 2 -ADR) [ [440] , [441] , [442] , [443] ] or the receptors for angiotensin 1A [ 444 ], D1 dopamine [ 445 ], m2mACh [ 446 ], neurokinin 1 [ 447 ], histamine H2 [ 448 ], secretin [ 449 ], or GLP-1 [ [450] , [451] , [452] ]. However, although it may not be relevant for receptor inactivation, receptor internalization is nevertheless important for receptor resensitization. A commonly proposed model is that the phosphorylated and arrestin-bound receptor, along with a previously membrane-bound G protein-coupled receptor phosphatase (GRP), is engulfed into the maturing endosome. The acidic pH of the endosome (pH∼5–6) then causes a change in the receptor conformation that enables the GRP to bind and dephosphorylate the receptor. Following subsequent dissociation of the ligand and the arrestin, the re-sensitized receptor either travels back to the plasma membrane or is degraded in the lysosomes [ 437 , 453 ]. Ligand-induced internalization and recycling of GLP-1R has been demonstrated in Chinese hamster lung fibroblasts [ 453 ], in rat insulinoma cells [ 429 , 453 , 454 ], and in rat pancreatic BRIN BD11 cells [ 452 ]. In vitro studies using 125I-labeled GLP-1 (7-36amide) in rat insulinoma cells indicate that internalization of the ligand-GLP-1R complex is saturable and time- and temperature-dependent [ 454 ]. At the plasma membrane, GLP-1R recruitment of arrestin is observed as early as 1 min after stimulation with exendin-4 [ 452 ]. Surface re-expression of GLP-1R after ligand-induced GLP-1R endocytosis appears with a half-time of 15 min with no GLP-1R endocytosis upon treatment of cells with exendin (9–39) [ 453 ]. Notably, in the endosomes, the receptor–ligand complex co-localizes with adenylate cyclase. Pharmacological inhibition of GLP-1R endocytosis by dynasor attenuates cAMP formation in BRIN BD11 pancreatic β-cells and lowers PKA substrate phosphorylation, resulting in a lesser magnitude of glucose-stimulated insulin secretion [ 450 ]. These data are consistent with reports that the internalized GLP-1R is to a certain extent still capable of stimulating insulin secretion from pancreatic β-cells [ 451 ]. Continued cAMP generation after GLP-1R sequestration (internalization) is accompanied by joint translocation of the activated Gαs subunit along with GLP-1R into the endosome [ 452 ]. Interestingly, generation of cAMP by the internalized GLP-1R persists after recruitment of arrestin, which indicates that arrestin recruitment is not essential for GLP-1R desensitization [ 452 ]. In contrast to these findings, siRNA mediated knockdown of β-arrestin in INS-1 cells attenuates GLP-1-induced phosphorylation of CREB, ERK1/2, and IRS-2 and diminishes the insulinotropic effect of GLP-1 in conditions of low (2.5 mM) and high glucose (25 mM) [ 429 ]. These studies suggest that recruitment of β-arrestin following ligand-induced activation of GLP-1R is important for the insulinotropic effect of GLP-1. However, knockdown of β-arrestin has minimal effect on GLP-1R internalization or desensitization [ 429 ]. In one recent report, exendin-4 analogs were modified on their N-terminal end to modulate GLP-1R trafficking and/or signaling [ 455 ]. The insulinotropic efficacy of these biased-signaling molecules was inversely correlated to the receptor internalization; i.e. insulin release was greatest using molecules that retain GLP-1R at the cell surface [ 455 ]. In summary, accumulating e

Induction of glucose-dependent insulin secretion by GLP-1 has been demonstrated in vivo in numerous species including rodents [ [468] , [469] , [470] , [471] ] and humans [ 83 ] as well as in the isolated perfused pancreas of rats [ 81 , [472] , [473] , [474] ], dogs [ 473 ], pigs [ 80 , 475 ], and humans [ 77 ], and in isolated islets [ 476 ] and cultured pancreatic cell lines [ 82 , [370] , [371] , [372] , [373] , 472 , [477] , [478] , [479] ]. GLP-1 (7–37) is roughly 100-fold more potent than glucagon to stimulate the secretion of insulin [ 480 ]. Nevertheless, intra-islet glucagon levels are much higher than circulating levels, and studies in mice highlight the importance of glucagon, acting through the GLP-1 receptor in the control of glucose-stimulated insulin secretion [ 173 , 365 ]. The key importance of GLP-1 and GIP in stimulating insulin secretion under conditions of hyperglycemia is the finding that oral glucose-induced insulin secretion is more impaired in mice that lack both incretin hormone receptors [ [481] , [482] , [483] ]. 8.1 Acute insulinotropic effects of GLP-1 In β-cells, binding of GLP-1 to its receptor leads to activation of adenylate cyclase (AC) and subsequently to an increase in cAMP ( Figure 5 ) [ 82 ]. Overexpression of GLP-1R in rat insulinoma RIN1046-38 cells results in elevated levels of basal cAMP [ 484 ]. While both GLP-1 (1–37) and GLP-1 (7–37) stimulate insulin secretion, GLP-1 (7–37) is efficacious at a lower dose. At a dose of 5 μM, both GLP-1 (1–37) and GLP-1 (7–37) increase cAMP levels in RIN1046-38 cells but at a dose of 5 nM, only GLP-1 (7–37) enhances cAMP levels [ 82 ]. Consistent with the increase in cAMP, GLP-1 stimulation of insulin mRNA is substantially greater upon treatment of RIN1046-38 cells with GLP-1 (7–37) relative to treatment with GLP-1 (1–37) [ 82 ]. Further supporting the greater insulinotropic potency of GLP-1 (7–37) relative to GLP-1 (1–37), GLP-1 (7–37) stimulates insulin release in the perfused rat pancreas at a concentration of 50 pM whereas treatment with GLP-1 (1–37), even at a dose of 0.5 μM, has no effect on insulin secretion [ 81 ]. Underlining the role of cAMP in the insulinotropic effect of GLP-1, enhanced cAMP hydrolysis through overexpression of the cyclic nucleotide phosphodiesterase 3B (PDE3B) diminishes GLP-1-induced insulin secretion [ 485 ]. Insulin and IFG-1 increase levels of PDE3B, and treatment of hamster clonal β-cells (HIT-T15) with IGF-1 deteriorates the insulinotropic effect of GLP-1 [ 486 ]. Transgenic mice overexpressing PDE3B under control of the rat insulin 2 promoter are glucose intolerant and have impaired insulin secretion following an intravenous glucose challenge [ 487 ]. In summary, there is substantial evidence indicating that cAMP is an important second messenger driving the acute insulinotropic effect of GLP-1. Figure 5 Schematic on GLP-1 mediated insulin secretion in the β-cell. GLUT1/2: glucose transporter 1/2; AC: adenylate cyclase; PKA: protein kinase A; Epac2: exchange protein activated by cAMP; Pdx-1: pancreatic and duodenal homeobox 1; CICR: calcium-induced calcium release. For further explanations, please see text. Figure 5 The GLP-1-induced rise in cAMP leads to activation of PKA and enhanced signaling via exchange proteins directly activated by cAMP (Epac) ( Figure 5 ) [ [488] , [489] , [490] ]. cAMP activates PKA, which phosphorylates the β 2 subunit of the L-type VDC channels and potentially the Kir6.2 and SUR1 subunits of the K ATP channels [ 491 ]. This consequently increases the sensitization of the K ATP channels to ATP, leading to closure of the K ATP channels, depolarization of the cell membrane, and opening of VDC channels ( Figure 5 ). The subsequent Ca 2+ influx promotes exocytosis of the insulin granules and acute secretion of insulin into the circulation. At the same time, the GLP-1-activated PKA inhibits voltage-gated K + (K v )-channels, preventing membrane repolarization and boosting Ca 2+ influx via prolonged opening of the VDC channels ( Figure 5 ) [ 492 ]. Inhibition of PKA in isolated islets attenuates GLP-1-induced insulin secretion [ 490 ]. The acute insulinotropic effect of GLP-1 does not fully depend on PKA signaling [ 493 ]. Up to 50% of the GLP-1-induced insulin release can be mediated through signaling via Epac [ 489 , 494 , 495 ]. Similar to PKA, members of the Epac family contain an evolutionarily conserved cAMP binding domain which enables the members of this family (Epac1 and Epac2) to regulate diverse biological functions in a cAMP-dependent manner [ 496 ]. Both Epac1 and Epac2 are expressed in the pancreas [ 495 , 497 ]. In β-cells, the Epac proteins stimulate Ca 2+ release from the endoplasmic reticulum (ER), increasing insulin secretion by increasing the intracellular pool of Ca 2+ [ 488 ]. cAMP activates Epac2 through direct binding. In high glucose conditions where there is an enhanced Ca 2+ influx into the β-cells via the VDC channels, Epac2 opens RYR Ca 2+ chan

The prevalence of type 2 diabetes correlates with excess body weight and also increases with age [ 525 ]. The progression to type-2 diabetes is invariably associated with a decline in functional β-cell mass [ [526] , [527] , [528] , [529] , [530] ]. Decreased β-cell proliferative capacity is also age-dependent in rodents and humans [ 526 , [531] , [532] , [533] , [534] , [535] , [536] , [537] ]. The replication rate of human β-cells is greatest in young childhood and puberty but declines with increasing age [ 526 , 531 , 533 , 536 ]. Collectively, these observations suggest that age-related changes in β-cell neogenesis and replication might be causally linked to the development of type 2 diabetes [ 525 , 532 , 538 ]. A role for β-cell dedifferentiation has also been proposed [ 539 ]. Agonists of the GLP-1 receptor improve glycemic control via both their acute insulinotropic action and, under certain circumstances, also by chronic action to preserve β-cell mass through stimulation of β-cell proliferation and inhibition of apoptosis ( Figure 7 ) [ [540] , [541] , [542] , [543] , [544] , [545] ]. While inconsistent results have been reported, the treatment duration, age of the animal, and species under investigation as well as diet-composition might affect the ability of GLP-1R agonism to improve β-cell replication [ 540 ]. GLP-1R regulation of β-cell proliferation and apoptosis seems to engage mechanisms that include signaling via Pdx1. Exendin-4 stimulates β-cell proliferation and inhibits β-cell apoptosis in wildtype mice but not in mice with β-cell specific inactivation of Pdx1 [ 542 ]. GLP-1R agonism also promotes β-cell growth and survival by stimulating the expression of the insulin receptor substrate 2 (Irs2) via mechanisms that include activation of CREB [ 546 ]. Irs2 is a substrate of the IGF1 and insulin receptor tyrosine kinases that promote growth, function and survival of the β-cells [ 547 ]. Consistent with this role of Irs2, increased expression of Irs2 in β-cells improves insulin secretion in obese mice and protects against STZ-induced β-cell destruction (as reviewed in [ 547 ]). Mice deficient for Irs2 [ 544 ], or transgenic mice deficient in CREB activity [ 546 ], are hyperglycemic due to severe β-cell destruction and enhanced β-cell apoptosis. Exendin-4 improves Irs2 function by enhancing CREB phosphorylation in vitro and in vivo [ 546 ]. Chronic administration of exendin-4 is unable to prevent β-cell loss in mice that are lacking Irs2 [ 544 ], demonstrating the requirement of Irs2 to GLP-1-mediated β-cell plasticity. Stimulation of β-cell proliferation secondary to stimulating GLP-1R is usually only observed in young animals (during periods characterized by preserved proliferative capacity), and not in older rodents [ 548 ]. Solid clinical proof of slowed diabetes progression or enhanced β-cell mass has not been provided in type-2 diabetic patients treated with any GLP-1 receptor agonist [ 549 ]. Figure 7 Schematic on the metabolic effects of GLP-1. The shown effects include direct and indirect GLP-1 effects on metabolism. For further explanations, please see text. Figure 7 The anti-apoptotic effect of GLP-1R agonism has been demonstrated in vivo in mice [ 543 , 550 , 551 ] and rats [ 552 , 553 ] as well as in several rodent [ 550 , 551 ] and human [ 554 , 555 ] cell lines, purified rat β-cells [ 543 ], and humans [ 556 ]. STZ-induced β-cell apoptosis is reduced upon administration of exendin-4 [ 543 ] or GLP-1 (7–36 amide) [ 551 ] and treatment with exendin-4 diminishes hyperglycemia induced by STZ [ 543 ]. STZ-induced β-cell apoptosis is accelerated in mice deficient for the GLP-1 receptor [ 543 ]. In isolated rat β-cells, treatment with exendin-4 reduces the apoptotic effect induced by treatment with pro-apoptotic cytokines, such as IL1b, TNFa, and interferon gamma [ 543 ]. In mouse pancreatic βTC-6 cells, liraglutide enhances β-cell survival via stimulation of anti-apoptotic signaling mechanisms that include stimulation of PI3 kinase-dependent phosphorylation of AKT, leading to inactivation of the pro-apoptotic protein BAD and silencing of FoxO1 [ 550 ]. An innovative approach to assess human β-cell replication in vivo was recently established by the group of Alvin Powers [ 557 ]. In this model, human β-cells were grafted under the renal capsule of lean diabetic immunodeficient NOD scid gamma mice. Using this model, exendin-4 was found to enhance β-cell proliferation in juvenile but not adult human islets [ 557 ]. Notably, the age-dependent decline in the exendin-4 induced β-cell proliferation was not related to changes of GLP-1R expression, as also indicated by preservation of exendin-4-induced insulin secretion in the adult islets [ 557 ]. However, in juvenile but not adult human islets, exendin-4 stimulated calcineurin/NFAT signaling and enhanced expression of proliferation-promoting factors such as NFATC1, FOXM1, and CCNA1 [ 557 ]. [ 557 ]. Collectively, these data suggest that the

GLP-1 also decreases blood glucose by suppressing glucagon secretion ( Figure 7 ) [ 475 , 573 , 574 ]. GLP-1 inhibition of glucagon secretion has been demonstrated in vivo in numerous species including mice [ 575 ], dogs [ 576 ], and humans [ 516 , 517 , 573 , [577] , [578] , [579] , [580] , [581] ], as well as in the isolated perfused pancreas of rats [ 473 , 474 , 582 ], dogs [ 473 ], and pigs [ 475 ] and in isolated intact murine islets [ 583 ]. Clamp studies in patients with type 2 diabetes indicate that GLP-1 inhibition of glucagon secretion is equally important for lowering blood glucose as GLP-1 stimulation of insulin release [ 578 ]. The mechanisms underlying GLP-1 suppression of glucagon secretion are complex. In the isolated perfused pig pancreas, GLP-1 dose-dependently stimulates the secretion of somatostatin [ 475 ] which is a potent suppressor of glucagon secretion [ 584 ]. Somatostatin inhibits glucagon secretion via paracrine mechanisms and, when blocked, stimulates glucagon release in isolated rat islets [ 585 , 586 ]. In the isolated perfused rat pancreas, co-infusion of GLP-1 with a specific somatostatin receptor 2 (SSTR2) antagonist (PRL-2903) abolishes the GLP-1-induced suppression of glucagon secretion [ 582 ]. While these data point to a prominent role of somatostatin in mediating GLP-1 suppression of glucagon secretion, treatment of isolated murine islets with the SSTR2 antagonist CYN154806 does not fully inhibit GLP-1's ability to suppress glucagon secretion [ 583 ]. Consistent with the conclusion that GLP-1 suppression of glucagon secretion does not fully depend on somatostatin, GLP-1 effects on glucagon secretion can be mimicked by forskolin-induced changes in cAMP. Accordingly, treatment of isolated murine islets with low concentrations of forskolin (1–10 nM) suppresses glucagon secretion by up to 60%, while high concentrations of forskolin (0.1–10 μM) stimulate glucagon release [ 583 ]. Notably, the PKA inhibitor 8-Br-Rp-cAMPS attenuates the inhibitory effect of GLP-1 on glucagon secretion, suggesting that GLP-1 suppression of glucagon secretion is PKA-dependent [ 583 ]. In intact murine islets, blockade of N-type Ca 2+ channels with ω-conotoxin, but not L-type Ca 2+ channels using nifedipine, abolishes low glucose (1 mM) stimulation of glucagon secretion and blunts the GLP-1-mediated inhibitor effects. In summary, these data indicate that GLP-1 may inhibit α-cell glucagon secretion via PKA-dependent modulation of N-type Ca 2+ channel activity in addition to its paracrine action via somatostatin [ 583 ]. GLP-1 may also inhibit glucagon secretion indirectly via its insulinotropic effect on the β-cells. Accordingly, as described in recent review articles [ 4 , 574 ], GLP-1 stimulates both the δ-cells to secrete somatostatin and the β-cells to secrete insulin, amylin, zinc, and GABA, all of which suppress the release of glucagon. In α-cell-derived IN-R1-G9 cells, insulin inhibits glucagon release via activation of phosphatidylinositol 3-kinase (PI3K). Inhibition of PI3K by wortmannin offsets the ability of insulin to suppress glucagon secretion [ 587 ]. In α-cells, insulin further enhances translocation of GABA-A receptors [ 588 ], and GABA released from β-cells enhances glucose inhibition of glucagon secretion [ 589 ]. Insulin is co-crystalized with Zn 2+ in the secretory granules of the β-cells [ 590 , 591 ] and Zn 2+ is co-secreted with insulin under conditions of hyperglycemia [ 592 , 593 ]. Zn 2+ acts in a paracrine manner on the α-cells to inhibit the secretion of glucagon [ [593] , [594] , [595] ]. Interestingly, disruption of the intrapancreatic infusion of Zn 2+ but not of Zn 2+ free insulin accelerates glucagon secretion in rats made diabetic by STZ, indicating that Zn 2+ but not insulin is the main stimulus underlying the Zn 2+ -insulin inhibition of glucagon secretion [ 595 ]. Consistent with this, glucagon secretion is inhibited upon treatment of α-TC cells with Zn 2+ [ 596 ]. As demonstrated in isolated rat α-cells, intact islets and the perfused rat pancreas, Zn 2+ inhibits pyruvate-induced glucagon secretion via opening of K ATP channels and inhibition of α-cell electrical activity [ 593 ]. In summary, there is credible evidence indicating that Zn 2+ that is co-secreted with insulin from the β-cells plays a prominent role in inhibiting release of glucagon [ [593] , [594] , [595] ]. Amylin, which is also co-secreted with insulin, also affects glucagon release from the α-cells. Amylin dose-dependently suppresses arginine-mediated glucagon secretion in rats [ 597 ] whereas pharmacological inhibition of amylin signaling enhances glucagon secretion [ 598 ]. Pramlintide, a synthetic amylin receptor agonist, improves glycemic control in diabetic patients via inhibition of postprandial glucagon secretion and inhibition of gastric emptying [ 599 , 600 ]. Interestingly, amylin does not affect glucagon secretion in isolated islets [ 601 ] nor in the perfused rat pancreas [ 602

Accumulating evidence indicates that some cardiac effects of GLP-1 (7-36amide) are actually mediated via the DPP-4-generated GLP-1 (9-36amide) and its smaller degradation products and, thus, are independent of GLP-1R signaling. At a dose of 0.3 nmol/L, pretreatment with GLP-1 (7-36amide) improves recovery of LVDP after ischemia/reperfusion (I/R) injury in both WT and GLP-1R KO mice [ 201 ]. Interestingly, a >10 fold greater dose of exendin-4 is needed relative to GLP-1 (7-36amide) to achieve a similar effect in WT mice but still with lower efficacy relative to GLP-1 (7-36amide) in GLP-1R KO mice [ 201 ]. The superior cardioprotective effect of GLP-1 (7-36amide) relative to exendin-4 and the preservation of efficacy of GLP-1 (7-36amide) but not exendin-4 in GLP-1R KO mice suggest that some of the cardioprotective effects of GLP-1 (7-36amide) are independent of GLP-1R signaling and are potentially mediated via the DPP-4-cleaved GLP-1 metabolite GLP-1 (9-36amide) [ 201 ]. Interestingly, when mice are pretreated with either GLP-1 (7-36amide) or GLP-1 (9-36amide), only GLP-1 (7-36amide) improves LVDP. However, when administered during reperfusion, both GLP-1 (7-36amide) and GLP-1 (9-36amide) improve functional recovery from I/R injury in both WT and GLP-1R KO mice [ 201 ]. Consistent with these data, while exendin-4 but not GLP-1 (9–36) has infarct limiting potential in the isolated rat heart, exendin-4 and GLP-1 (9–36) both improve left ventricular performance during reperfusion [ 626 ]. The infarct-limiting potential of exendin-4 is abolished by treatment with exendin (9–39), thus indicating that this cardioprotective effect is GLP-1R-dependent [ 626 ]. Treatment of isolated mouse cardiomyocytes with either exendin4 or GLP-1 (9-36amide) increases phosphorylation of AKT and ERK [ 631 ], both of which are known to positively affect cardiomyocyte growth and survival [ 632 , 633 ]. Collectively, these data indicate that pharmacologic administration of GLP-1 (9-36amide) may have a functional role to improve recovery from I/R injury via GLP-1R-independent mechanisms, whereas GLP-1 (7-36amide) but not GLP-1 (9-36amide) affects cardiac contractility via GLP-1R signaling [ 201 ]. Further supporting a cardiometabolic role of GLP-1 (9-36amide), both GLP-1 (7-36amide) and GLP-1 (9-36amide), but not exendin-4, promote vasodilation in isolated and ex vivo cultured mesenteric blood vessels [ 201 ], i.e., the vasodilatory effect of GLP-1 (7-36amide) was not different relative to that of GLP-1 (9-36amide) and was likewise preserved in GLP-1R KO mice [ 201 ]. Whether the low endogenous concentration and the restricted pharmacokinetic action profile of endogenous GLP-1 metabolites are sufficient to elicit such effects under physiological circumstances remains questionable.

One of the most frequently described extrapancreatic effects of pharmacokinetically-optimized GLP-1R analogs is their ability to lower body weight via centrally regulated inhibition of food intake ( Figure 7 ). Suppression of food intake by GLP-1R agonism has been demonstrated in numerous species including mice [ [465] , [466] , [467] , 665 , 666 ], rats [ 667 , 668 ], birds [ 669 , 670 ], pigs [ 671 , 672 ], non-human primates [ 673 , 674 ], and humans [ [675] , [676] , [677] , [678] ]. The anorexigenic effect of GLP-1R agonism has been demonstrated in numerous clinical studies and fMRI analysis support that GLP-1 inhibition of food intake is centrally mediated in healthy individuals and patients with type-2 diabetes [ [679] , [680] , [681] , [682] , [683] , [684] ]. 13.1 Effect of peripheral administration of GLP-1 analogs on food intake Peripheral administration of GLP-1 analogs decreases body weight via suppression of food intake [ [465] , [466] , [467] , 665 , 685 ]. Depending on the molecule (native GLP-1 or a longer-acting GLP-1 analog) and the route of administration, GLP-1 and its analogs seem to engage different signaling pathways to either directly or indirectly transmit the signal to reduce food intake to the CNS. Central GLP-1R appear to be necessary for the anorectic effect of peripherally administered GLP-1R agonists in rodents [ [686] , [687] , [688] ]. When administered s.c., liraglutide fails to inhibit eating in mice possessing a CNS-specific deletion of GLP-1R [ 688 ]. Another report indicates that i.v. infusion of liraglutide inhibits eating in rats by directly activating hypothalamic Pomc/Cart neurons in the ARC, without activating GLP-1-producing neurons in the hindbrain, and independent of GLP-1R signaling in vagal afferents, the area postrema (AP) and the PVN [ 686 ]. In rats, central (i.c.v.) administration of exendin (9–39) was also sufficient to attenuate the anorexigenic effect of i.p. injected liraglutide and exendin-4 [ 687 ], and bilateral neurochemical lesions of the lateral parabrachial nucleus (IPBN) with ibotenic acid blunted the anorexigenic effect of peripherally administered exendin-4 [ 689 ]. When applied either s.c. or i.p., exendin-4 and liraglutide induced acute neuronal activation (measured by activation of cFos) in PVN, AP, and NTS [ 686 , 690 ]. Interestingly, the short-term anorexigenic effects of i.p. injected low-dose liraglutide and exendin-4 were also attenuated by subdiaphragmatic vagal deafferentation [ 687 , 691 ], but longer-term effects of these compounds on food intake did not depend on intact vagal afferents [ 691 ]. All of these findings are consistent with the concept that GLP-1 receptor agonists inhibit eating and reduce body weight primarily by acting directly in the brain, and that various brain areas are involved in mediating the eating-inhibitory effects of GLP-1 receptor agonists. This presumably reflects the ability of long-acting GLP-1 analogs to enter the brain at areas with an incomplete BBB, such as the median eminence. While these data collectively emphasize the importance of neuronal GLP-1R for the food intake inhibition by long-acting GLP-1 analogs, it is unclear whether and to what extend this accounts for physiological doses of intestinal-derived native GLP-1. When infused into the hepatic portal vein, GLP-1 (7-36amide) increased cFos expression in the NTS, the AP, and the central nucleus of the amygdala, but not in the hypothalamic ARC or PVN [ 692 ], i.e., native GLP-1 appears to induce an activation pattern in the brain that is different from the pattern induced by long-acting GLP-1 receptor agonists. While peripherally administered GLP-1 seemingly crosses the blood brain barrier (BBB) via simple diffusion, the half-life of GLP-1 is less than 2 min, leading to disagreement regarding whether peripherally derived GLP-1 is what activates CNS GLP-1R under physiological conditions. In rats, i.v. administration of GLP-1 at high physiological doses stimulates vagal afferents, and this is blocked by pretreatment with exendin (9–39) [ 693 ]. While this activation appears to be involved in the insulinotropic and gastric emptying inhibiting effects of exogenous GLP-1 [ 693 ], it does not appear to be crucial for the effect of i.v. administered native GLP-1 on eating [ 694 ], because GLP-1 infusions into the hepatic portal vein did not lose their potency to reduce food intake after subdiaphragmatic vagal deafferentation in rats [ 692 , 694 ]. Interestingly, however, the food intake inhibition following i.p. administration of a supposedly “physiological” dose of GLP-1 (7-36amide) depends on the integrity of abdominal vagal afferents [ 694 ], indicating that peripheral GLP-1 can inhibit eating via at least two partly separate pathways. Consistent with a role of vagal afferents and vagal afferent GLP-1 receptors in the eating-inhibitory effect of peripheral endogenous GLP-1, virus-mediated knockdown of GLP-1R in vagal afferent neurons increased

The molecular underpinnings regulating GLP-1 inhibition of food intake and body weight loss are complex. Undoubtedly, brain GLP-1R signaling is crucial for the anorexigenic effect of exogenously administered long-acting GLP-1R agonists, as demonstrated by failure of liraglutide to suppress food intake in CNS-specific GLP-1R KO mice [ 688 ]. Consistent with this, i.c.v. administration of exendin (9–39) is sufficient to attenuate the anorexigenic effect of peripherally administered liraglutide and exendin-4 [ 687 ]. However, CNS-specific GLP-1R KO mice have normal body mass and composition whether on a chow or HFD [ 688 ], suggesting that these receptors may be necessary for the pharmacological response to GLP-1 but not the physiological control of body weight. However, data obtained in KO animals generally have to be regarded with caution since compensatory mechanisms, which limit interpretations and affect study outcomes, might develop. Depending on the route of administration (and the GLP-1R agonist used), peripherally administered GLP-1 analogs seem either to act directly in the hypothalamus and hindbrain, both areas with incomplete BBB, or else on vagal afferents to transmit the signal to the hindbrain which then projects to other key feeding areas in the brain. Hindbrain GLP-1R activation enhances phosphorylation of PKA and MAPK while decreasing the activity of AMPK in the NTS, and inhibition of PKA/MAPK activity by administration of Rp-cAMP or UO126 attenuates food intake inhibition of exendin-4 administered into the 4th ventricle [ 706 ]. These data suggest that hindbrain GLP-1R activation suppresses food intake via PKA/MAPK-induced inhibition of AMPK. The anorexigenic effect of GLP-1R agonism involves the glucose-dependent silencing of central AMPK action. Activity of AMPK is increased under conditions of fasting [ 707 ] and is decreased under conditions of enhanced glucose utilization [ [708] , [709] , [710] ]. Consistent with AMPK's role as a stress- and nutrient-responsive protein kinase, pharmacological, physiological (e.g. fasting-induced), or genetic activation of AMPK stimulates food intake whereas AMPK inhibition decreases food consumption [ 707 , 711 ]. Central administration of GLP-1 reduces the activity (phosphorylation) of AMPK [ 712 , 713 ] and treatment of mouse hypothalamic GT1-7 cells with exendin-4 stimulates glycolysis and decreases phosphorylation of AMPK [ 714 ]. Pharmacological activation of AMPK by administration of 5-aminoimidazole-4-carboxamide-1-β- d –ribofuranoside (AICAR) in either the 3rd [ 714 ] or the 4th ventricle [ 706 ] attenuates the anorectic effect of exendin-4 injected into the same ventricles. Consistent with these data, i.c.v. administration of the glycolysis inhibitor 2-deoxyglucose (2-DG) likewise increases activity of AMPK and blunts food intake suppression by i.c.v.-injected exendin-4 [ 714 ]. In summary, compelling evidence indicates that the anorexigenic effect of central GLP-1 receptor activation is partially mediated by central stimulation of glycolysis and concomitant silencing of AMPK activity. Accordingly, pharmacological activation of AMPK in the VMH blunts food intake suppression by i.c.v. exendin-4 [ 714 , 715 ] and attenuates central GLP-1 effects on brown and inguinal white adipose tissue [ 666 ]. The anorexigenic effect of GLP-1 seems to be transduced via GLP-1R signaling in several areas of the brain, including both hypothalamic nuclei and the hindbrain. Microinjection of exendin-4 into the NTS reduces intake of palatable high-fat diet and inhibits food reward behavior [ 716 ], and hindbrain inhibition of GLP-1R via administration of exendin (9–39) into the 4th ventricle increases food intake [ 717 ]. The anorexigenic and weight-lowering effects of liraglutide are blunted upon genetic ablation of GLP-1R in glutamatergic neurons, yet fully preserved when GLP-1R is deleted in GABAergic neurons [ 718 ]. Peripheral administration of GLP-1 (7-36amide) enhances neuronal activity in the VMH [ 719 , 720 ], and direct administration of GLP-1 into the LH, VMH, or DMH of rats acutely decreases food intake [ 721 ]. Similar findings were reported in rats that received direct infusion of GLP-1 (7-36amide) into the PVN [ 722 , 723 ]. In rats, liraglutide decreases food intake when directly injected into the ARC, LHA, or PVN while direct administration into the VMH decreases body weight without affecting food intake, but increases the expression of Ucp1 in the brown and inguinal white adipose tissue [ 666 ]. The acute effects of exendin-4 to reduce food intake and to improve glucose handling are preserved upon knockdown of GLP-1R in the VMH [ 715 ], indicating that the effects of central GLP-1R activation on eating and BAT thermogenesis are mediated by at least partially separate brain areas and circuitries.

Central GLP-1 regulation of food intake is not restricted to GLP-1R signaling in the hypothalamus and hindbrain and is not limited to modulation of homeostatic feeding, i.e. food intake to maintain energy homeostasis. GLP-1 also affects non-hunger related (hedonic) feeding by targeting brain areas involved in reward, motivation and addiction, such as the VTA, NAcc, lateral septum [ 701 , 741 , 744 , 745 ], and PVT [ 704 ]. These mesolimbic regions express GLP-1R [ 347 ] and receive projections from PPG neurons in the NTS [ 354 , 741 , 744 ]. Peripheral administration of exendin-4 [ 691 ] or direct administration of GLP-1 into the core of the NAcc [ 744 ] increases cFos expression in this region, and direct activation of NAcc GLP-1R reduces food intake while its direct inhibition by exendin (9–39) induces hyperphagia [ 744 ]. Food intake inhibition by NAcc GLP-1R is unrelated to taste avoidance [ 744 ] and does not induce pica [ 701 , 741 ]. Administration of exendin-4 into the shell of the NAcc decreases sucrose reward behavior in the progressive ratio (PR) operant conditioning test [ 701 ]. Consistent with this, GLP-1R agonism decreases a variety of reward behaviors in rodents, including the motivation to lever press for food reward in an operant conditioning test [ 746 ], alcohol intake [ 747 , 748 ], alcohol seeking behavior [ 747 ], amphetamine-induced conditioned place preference (Amp-CPP) [ 748 ], and hedonic feeding [ 748 ]. The inhibitory effect of GLP-1R analogs on reward behaviors is blunted in mice with CNS-specific deletion of GLP-1R [ 748 ]. Several lines of evidence indicate that NAcc GLP-1R activation decreases food intake by reducing food palatability. In satiated rats given a choice between chow and high-fat diet (HFD), exendin-4 preferentially reduces consumption of the more palatable HFD [ 741 ]. In rats, intra-NAcc injection of exendin (9–39) increases the size of a sucrose meal and accelerates the licking frequency during the first meal, an indicator of increased palatability [ 749 ]. In summary, GLP-1R agonism decreases homeostatic and hedonic feeding, and modulates food intake not only via the hypothalamus and the hindbrain but also via signaling in the mesolimbic system, in which GLP-1R activation affects reward behavior and palatability.

GLP-1 inhibition of water intake was first reported by Mads Tang-Christensen in 1996 [ 92 ] and was later confirmed by others [ [755] , [756] , [757] , [758] , [759] , [760] ]. GLP-1 inhibition of water consumption is independent of food intake [ 758 ] and is blocked by pretreatment with exendin (9–39) [ 92 ]. In rats, inhibition of drinking by GLP-1 (7–36 amide) is dose-dependent and occurs within 30 min following its central (i.c.v.) or peripheral (i.p.) administration [ 92 ]. In one study in healthy humans, i.v. infusion of GLP-1 decreased water consumption by 36% in response to i.v. infusion of hypertonic saline (5% NaCl) [ 755 ]. Peripheral but not central administration of GLP-1 fails to inhibit angiotensin (ANG) II-induced drinking in rats that have been treated with monosodium glutamate (MSG) to induce damage of the ARC and the sensory circumventricular organs, suggesting that peripherally administered GLP-1 effects drinking behavior via CNS-dependent mechanisms [ 760 ]. GLP-1 affects renal function by decreasing water intake as well as by stimulating the excretion of urine and sodium ( Figure 7 ). The natriuretic and diuretic effect of GLP-1R agonism was first shown in 1996 [ 92 ] and was subsequently confirmed in rodents [ 642 , 660 , 761 ] and in healthy, obese, and type-2 diabetic humans [ 661 , 663 ]. While the acute natriuretic effect of GLP-1 has been solidly confirmed in humans [ [762] , [763] , [764] ], 12 wk treatment of T2DM patients with liraglutide or the DPP-4 inhibitor sitagliptin had no effect on markers of renal function, including glomerular filtration rate, renal plasma flow, albumin-to-creatine ratio, and excretion of sodium, potassium, and urea [ 765 ]. These data suggest that that GLP-1's effects on renal function might be transient and vanish upon more chronic treatment. In one study in T2DM patients, the natriuretic effect of liraglutide persisted after 3 wk treatment [ 662 ]. In rats, GLP-1 induced natriuresis and diuresis is accompanied by increased renal plasma flow and glomerular filtration rate [ 660 , 761 ]. Denervation of the kidney results in failure of GLP-1 to increase glomerular filtration rate and decreases the natriuretic and diuretic effect of GLP-1 [ 761 ]. These data suggest that the natriuretic and diuretic effect of GLP-1 might be due to inhibition of sodium reabsorption in the proximal tubule and emphasize that GLP-1 requires intact renal innervation to increase glomerular filtration. Studies in rodents and humans indicate that GLP-1R is expressed in the renal vasculature [ 392 , [766] , [767] , [768] ]. However, treatment of isolated porcine proximal tubular cells with GLP-1 reduced sodium re-absorption [ 768 ]. Consistent with this, GLP-1, exendin-4, and the DPP-4 inhibitor sitagliptin reduced proximal tubular sodium reabsorption in rats [ 660 , 761 , 769 , 770 ] and humans [ 663 ]. In the renal proximal tubule, GLP-1 reduced Na + /H + exchanger isoform 3 (NHE3)-mediated bicarbonate reabsorption [ 660 ]. Consistent with this, exendin-4 decreased NHE3-mediated sodium-dependent pH recovery in porcine kidney LLC-PK 1 cells [ 771 ]. Inhibition of NHE3 activity by GLP-1R agonism seems to be mediated via cAMP activation of PKA, which phosphorylates NHE3 at its Ser552 and Ser605 residues [ 660 , 771 , 772 ]. In rodents [ 660 , 761 ] and humans [ 663 , 763 ], GLP-1R agonism increases renal lithium clearance, a marker for proximal tubular sodium reabsorption. These data suggest that the diuretic and natriuretic effects of GLP-1 include changes in renal hemodynamics and reduced sodium reabsorption due to PKA-dependent downregulation of NHE3 activity in the renal proximal tubule. GLP-1 might also affect kidney function via its cardiovascular effects. In rats, infusion of GLP-1 increases renal blood flow and the glomerular filtration rate [ 660 , 761 ], and this effect is potentially mediated via GLP-1's ability to increase vasodilation of the afferent renal arteriole. In wildtype but not GLP-1R KO mice, liraglutide stimulates the secretion of natriuretic peptide (ANP) from the atrial cardiomyocytes and liraglutide fails to affect natriuresis and vasorelaxation in mice deficient for ANP [ 640 ].

In addition to its direct and vagally mediated insulinotropic action on β-cells, GLP-1 also improves postprandial glucose handling by inhibition of gastric emptying, decelerating the rate at which glucose is absorbed into the circulation ( Figure 7 ) [ 573 , [789] , [790] , [791] ]. Inhibition of gastric motility and gastric acid secretion following treatment with GLP-1R agonists has been demonstrated in mice [ 792 ], rats [ [793] , [794] , [795] ], dogs [ [796] , [797] , [798] ], pigs [ 799 , 800 ], and humans [ 573 , [789] , [790] , [791] , 799 , [801] , [802] , [803] , [804] , [805] ]. In rats, inhibition of gastric emptying occurs after peripheral or central administration of GLP-1 [ 795 ], and this effect is mediated via hindbrain GLP-1R signaling [ 806 ]. Vagal afferent GLP-1 receptor knockdown accelerates gastric emptying [ 518 ] and vagal afferent denervation [ 795 ] or peripheral administration of exendin (9–39) [ 794 , 795 ] blocks the effect of centrally or peripherally administered GLP-1 on gastric emptying. These data are consistent with the failure of GLP-1 to inhibit gastric acid secretion in vagotomized human subjects [ 807 ] as well as the failure of GLP-1 to inhibit vagally-induced motility in the isolated perfused pig antrum [ 800 ]. These data suggest that GLP-1-mediated inhibition of gastric motility includes vagal afferents, as well as GLP-1R dependency in the periphery and the brain [ 795 ]. Inhibition of adrenergic signaling through combined administration of phentolamine and propanolol abolishes the inhibitory effect of GLP-1 on gastric motility, suggesting that GLP-1 inhibits gastric emptying via adrenergic signaling [ 794 ]. In fasted rats, co-administration of GLP-1 and GLP-2 leads to a greater inhibition of small bowel motility relative to either monotherapy [ 793 ]. Exendin-4 fails to affect gastric emptying in GLP-1R KO mice [ 792 ]. In healthy human volunteers, intravenous GLP-1 decelerates gastric emptying after the first meal to a greater extent than after a second meal, indicating that GLP-1 inhibition of gastric emptying undergoes rapid desensitization [ 803 ]. In overweight humans, liraglutide 3 mg solidly inhibits gastric emptying after 5wk of treatment, but the efficacy markedly decreases after 16wk of treatment, yet with still significant effect relative to placebo treated controls [ 808 ]. In rats, liraglutide- but not exendin-4-induced inhibition of gastric emptying is markedly diminished after 14 wk of treatment [ 742 ]. Because gastric emptying is a prerequisite for glycemic rises after meals, which in turn trigger insulin secretion, deceleration of gastric emptying in response to exogenous GLP-1 leads to lesser post-meal glucose excursions and reduced insulin secretory responses [ 809 ]. This has led to doubts about the importance of an incretin role for pharmacological GLP-1 administration, because incretin hormones should lead to a net increase in post-nutrient insulin secretory responses [ 810 ].

GLP-1R expression has also been demonstrated in the rat [ 347 , 821 ] and mouse [ 822 ] hippocampus, a brain region implicated in spatial learning and memory [ 823 , 824 ]. In rats, central GLP-1R agonism improves several aspects of learning and memory, as demonstrated by improved performance in the Morris water maze test and by enhanced latency in the passive avoidance test [ 417 ]. GLP-1's improvement of learning and memory can be blocked by pretreatment with exendin (9–39), and is absent in mice deficient for the GLP-1 receptor [ 417 ]. Targeted hippocampal restoration of GLP-1R expression in otherwise GLP-1R-deficient mice using AAV-mediated gene transfer reverses GLP-1 improvement of learning and memory in the passive avoidance test [ 417 ]. Central GLP-1R signaling has also been demonstrated to have neuroprotective effects. GLP-1 and exendin-4 enhance differentiation and neurite outgrowth in rat pheochromocytoma (PC12) cells and in human neuroblastoma SK-N-SH cells [ 825 ]. The neuroprotective effect of GLP-1R agonism is similar to that conferred by nerve growth factor (NGF) and is blocked when PC12 cells are co-incubated with exendin (9–39) [ 825 ]. Both GLP-1 and exendin-4 protect cultured hippocampal neurons against glutamate-induced apoptosis [ 826 ]. The time and severity of seizures induced by systemic administration of the neurotoxin kainic acid is enhanced in GLP-1R KO mice relative to WT controls whereas the destructive effect of kainic acid is ameliorated in the GLP-1R KO mice upon targeted AAV-mediated restoration of hippocampal GLP-1R expression [ 417 ]. The molecular mechanisms underlying the neuroprotective effects of central GLP-1R agonism are, at least in part, mediated through the ability to increase cAMP formation and subsequently to enhance activation of PI3-kinase and ERK. GLP-1R agonism also increases cAMP levels in cultured hippocampal neurons [ 826 ] and in PC12 cells [ 825 ]. Pharmacological inhibition of either PI3-Kinase or ERK blocks the stimulatory effect GLP-1 and exendin-4 on neurite outgrowth in PC12 cells [ 825 ]. In PC12 cells, GLP-1 stimulation of neurite outgrowth is only partially silenced by treatment with the PKA inhibitor H89, indicating that cAMP-mediated activation of PI3K and ERK following GLP-1R agonism does not fully depend on PKA signaling, at least in these cells [ 825 , 827 ]. Huntington's disease (HD) is characterized by severe neurodegeneration that is caused by a mutation of the huntingtin protein (HTT) [ 828 ]. The neurotoxicity and neurodegeneration induced by the mutant HTT protein has been attributed to enhanced oxidative stress and accumulation of HTT due to inappropriate autophagic HTT clearance [ 829 ]. The prevalence of T2DM is elevated in patients with HD [ 830 ], suggesting that impaired insulin sensitivity might be a causal factor contributing to neurodegeneration in HD patients [ 830 , 831 ]. Overexpression of mutant HTT impairs insulin signaling and stimulates neuronal apoptosis in human SK-N-MC neuronal cells [ 831 ]. Treatment of SK-N-MC cells with liraglutide improves insulin sensitivity and enhances cell viability, potentially by mechanisms that include amelioration of neuronal glucotoxicity, improvement of oxidative stress and less aggregation of the mutant HTT through stimulation of AMPK-mediated autophagy [ 831 ]. Alzheimer's disease (AD) is characterized by neurodegeneration of cholinergic neurons in the hippocampus. In a rat model of neurodegeneration, GLP-1 and exendin-4 ameliorated neurodegeneration by reducing ibotenic acid-induced depletion of cholinergic cell bodies, as demonstrated by preservation of choline acetyltransferase immunoreactivity in basal forebrain cholinergic neurons [ 826 ]. In rats, administration of GLP-1R agonists into the hippocampus further prevented impairment of spatial learning and memory induced by central administration of amyloid β (Aβ), a protein considered to play a causative role in impairment of learning and memory in Alzheimer disease [ 832 , 833 ]. Consistent with this, chronic GLP-1R agonism has been demonstrated in several mouse models of AD to prevent the development of memory deficits [ [834] , [835] , [836] ]. Clinical studies evaluating the effect of GLP-1R agonism on neurodegeneration associated with AD are scarce. AD is frequently reported to be accompanied by decreased glucose transport across the BBB and 6 month treatment of AD patients with liraglutide improved BBB glucose transfer relative to placebo controls [ 837 ]. In a double blind placebo controlled study, 12 week treatment with liraglutide in patients at risk for AD did not show cognitive differences between the study cohorts [ 838 ]. Other clinical trials evaluating the neuroprotective effects of GLP-1R agonism on AD are currently ongoing [ 839 ] GLP-1 analogs have also proven successful for the treatment of Parkinson's Disease (PD). PD is typically characterized by degeneration of dopaminergic neurons, a condition that can

The exogenous supplementation of native hormones, commonly purified from tissue homogenates, has led to numerous, seminal discoveries, most prominently the identification of insulin in 1921 [ 875 ] followed by the discovery of glucagon in 1923 [ 3 ]. Despite numerous such groundbreaking discoveries, including identifying the intestine as a source of incretins [ 56 ], this practice did not necessarily identify hormones capable of sufficiently lowering body weight. Its anorexigenic and insulinotropic action make GLP-1 an appealing candidate for the treatment of obesity and diabetes. The pharmacological value of native GLP-1, however, is limited by a short half-life [ 182 , 185 , 876 ] and dose-limited adverse gastrointestinal effects [ 734 , 877 ]. A common approach to enhance the therapeutic benefits of GLP-1-based pharmacology is therefore to improve its pharmacokinetics. Biochemical modification of the native peptide has enhanced exposure and time of action to improve the metabolic efficacy at tolerable doses. Furthermore, these optimized molecules allow for less frequent administration, which places less burden on the patient and improved compliance. 20.1 Optimized GLP-1 monoagonists Several structurally refined GLP-1 derivatives with improved bioavailability and sustained action have been developed for the treatment of T2DM [ 878 ]. These drugs include exenatide (Byetta®, short-acting; and Bydureon®, long-acting, AstraZeneca), lixisenatide (Lyxumia®, Sanofi), liraglutide (Victoza®; Novo Nordisk), dulaglutide (Trulicity®; Eli Lilly & Co), albiglutide (Tanzeum®, GlaskoSmithKline), and semaglutide (Ozempic®, Novo Nordisk) [ 878 ]. Exenatide (Byetta® and Bydureon®, AstraZeneca), is a synthetic 39-amino acid peptide (first discovered as exendin-4 in the saliva of the gila monster, Heloderma suspectum) ( Figure 8 ) [ 879 ]. The first 30 amino residues of exendin-4 have ∼53% sequence homology to mammalian GLP-1, whereas the C-terminal nonapeptide extension has no similarities. Unlike GLP-1, exendin-4 has a glycine at the second amino acid position at the N-terminus, which protects the peptide from DPP-4-mediated degradation and inactivation. In addition, exendin-4 differs from mammalian GLP-1 by a series of amino acids in the central and C-terminal domains that include Leu10, Lys12, Gln13, Met14, Glu16, Glu17, Tyr19, Arg20, Leu21, Glu24, Lys27, Asn28, and Gly30. The C-terminal end of exendin-4 is extended relative to GLP-1 by 9 amino acids. The C-terminal extension supports a secondary structure by formation of a tryptophan cage and improves potency at the GLP-1 receptor [ 880 ]. When administered either i.v., i.p., or s.c. in rats, the half-life of exendin-4 is 18–41 min, 125–174 min, and 90–216 min, respectively [ 194 ]. Relative to GLP-1 (7–36 amide), the bioavailability of exendin-4 is nearly doubled from 36 to 67% for GLP-1 to 74–76% for exendin-4, with decelerated plasma clearance from 35 to 38 ml/min to 4 – 8 ml/min [ 194 ]. These pharmacokinetic parameters support twice-daily administration in humans. In patients with T2DM, exenatide decreases fasting and post-prandial levels of blood glucose [ [881] , [882] , [883] , [884] ], slows gastric emptying [ 885 ], reduces cardiovascular risk factors [ 886 ] and decreases body weight accompanied by suppression of food intake [ [885] , [886] , [887] ]. The placebo-subtracted weight loss from baseline induced by exenatide is typically in the range of low single-digit percentages. After 82 wk of treatment of T2DM patients with exenatide, the placebo-subtracted weight loss from baseline is approximately 5% [ 886 ]. A once-weekly formulation of exenatide has been developed by Amylin Pharmaceuticals and has been marketed by AstraZeneca since 2012 under the trade name Bydureon®. The primary ingredient in the formulation that allows for extended-release is polyD, l -lactide-co-glycolic acid. Together with the other excipients of the formulation, the pharmacokinetics of exenatide are improved by a mechanism involving the formation of a depot of microspheres at the injection site, which slows dissipation from the injection site [ 888 ]. Compared to classical exenatide given twice daily (BID, the once-weekly (QW) formulation at a 2 mg dose level provides a relatively stable plasma concentration at steady-state (∼300 pg/ml) because of the more continuous delivery of the drug, with equal (or slightly improved) efficacy on glycemic control and body weight after 24 and 30 wk of treatment [ 888 ]. Figure 8 Timeline of GLP-1R agonists approved by the FDA for the treatment of diabetes. Numbers in parenthesis reflect the half-life of the molecules. For further explanations, please see text. Figure 8 Lixisenatide (Adlyxin®, Sanofi) ( Figure 8 ) is an analogue of exenatide in which the proline at position 38 is omitted and six sequential lysine residues are added to the C-terminus [ 889 ]. Lixisenatide has comparable pharmacokinetic properties relative to exenatide and is, wit

Another more recently developed concept of polypharmacology is to combine some of the sequences of several structurally related hormones into a single entity of enhanced potency and sustained action. The concept underlying these unimolecular multi-agonists is that a single molecule with complementary signaling through more than one receptor, each of which offers beneficial effects on systems metabolism, would maximize metabolic outcomes at tolerable doses. This strategy has refined the toolbox of weight loss pharmacology, and several molecules with agonism at key metabolic receptors are, as reviewed below, currently in clinical evaluation. One might ask why a unimolecular multi-agonist is believed to be superior over the adjunct administration of the independent hormones? Apart from the possibility that acquiring regulatory approval is seemingly easier for a single molecule relative to a co-mixture of hormones, it must be considered that each molecule in a co-mixture has its own unique pharmacokinetic profile. Simultaneously injected hormones might differ in their half-life and time of action due to differences in rates of absorption, distribution, metabolism, and clearance, as well as the potential for drug–drug interaction. While these differences might compromise the optimal metabolic outcome of a co-mixture of separate molecule entities, a single molecule might bypass some of these limitations. With a single molecule approach, the ratio of activities of the constituents, however, will be fixed, whereas individual combination therapies present the potential benefit of being titratable to allow more optimal ratio between doses of the agonists. This is particularly important when one of the components has a narrow therapeutic window. 20.3.1 GLP-1 dual- and triple-agonists The possibility of using glucagon as a constituent of a multi-functional single molecule for the treatment of chronic metabolic diseases was initially counterintuitive due to its established role in raising blood glucose as part of the counter-regulatory physiological response to defend against insulin-induced hypoglycemia. However, the catabolic qualities of glucagon, notably its effect to decrease hepatic fat and circulating lipids, could be leveraged for therapeutic benefit in metabolic diseases if its diabetogenic action were mitigated. The prospect emerged of using GLP-1 to buffer the hyperglycemic effect of glucagon while providing complementary mechanisms to lower body weight, most notably the effects of GLP-1 to suppress appetite to coincide with the energy expenditure effect of glucagon. Co-infusion of GLP-1 and glucagon to human subjects resulted in decreased food intake, likely secondary to GLP-1 action, and increased energy expenditure, suggested to be driven by glucagon action [ 783 , 785 ]. Chronic co-administration of selective mono-agonists to obese non-human primates resulted in significantly more body weight loss when compared to the individual treatments, thus demonstrating the translation of these acute effects observed in humans and the chronic effects observed in primates [ 673 ]. The structural similarity of GLP-1 and glucagon as well as of their respective receptors, along with the inherent low-potency promiscuity of these hormones at the other respective receptors (apart from the pancreas, where glucagon has recently been demonstrated to also act on GLP-1R [ 173 ]), suggested the possibility of integrating both activities into a sequence-intermixed single molecule hybrid. Preclinical proof of concept of enhanced body weight lowering relative to GLP-1 mono-therapy without impaired glycemic control was first demonstrated by two independent research efforts. Both of these GLP-1/glucagon co-agonists, one based on a glucagon-derived backbone [ 922 ] and the other based on an oxyntomodulin-derived sequence [ 923 ], had superior weight loss in obese rodents relative to matched analogs that were selective in activity towards GLP-1R. Since these initial reports, other medicinal chemistry strategies have been employed to design unique GLP-1/glucagon co-agonists, including the use of exendin-4 derived sequences [ 924 ], resulting in a rich pipeline of candidates that are advancing through clinical trials [ 925 ]. Two unique compounds have been evaluated in Phase 2 trials (SAR425899 and MEDI0382), and although dual-agonism has produced promising results on body weight and glycemic control in diabetic patients [ 926 , 927 ], it is not apparent that these co-agonists are superior to selective GLP-1R agonists. The original rationale for combining GLP-1 and GIP, the other predominant incretin hormone, was to leverage the individual islet effects of each hormone to impart superior glycemic control. Co-infusion of GLP-1 and GIP to healthy human subjects has resulted in increased insulinotropic effects [ 928 ]. However, controversy still exists as to whether it is best to therapeutically promote or inhibit GIP action for beneficial

Depending on the molecule (native or recombinant long-acting) and the route of administration, GLP-1 analogs have broad pleiotropic action on metabolism. Among the numerous beneficial effects mediated by GLP-1R agonists are regulation of blood glucose, lowering of body weight via inhibition of food intake and the decrement of gastric motility, stimulation of cell proliferation, reduction of inflammation and apoptosis, improvement of cardiovascular function, and neuroprotection. Recombinant GLP-1R analogs with improved pharmacokinetics and sustained action are successful in the treatment of type-2 diabetes, and there is considerable and ongoing effort to further optimize their action profile to improve therapeutic outcome and patient compliance. Novel peptides that combine the pharmacology of GLP-1 with that of other gut peptides are in clinical evaluation for the treatment of diabetes and obesity. While long-term clinical studies are ongoing, we can be carefully optimistic that improved GLP-1-based pharmacology can one day be safely applied to further lower body weight relative to currently available GLP-1R agonists.