Incretin secretion: direct mechanisms
The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are secreted from gastro-intestinal K- and L-cells, respectively, and play an important role in post-prandial blood glucose regulation. They do this by direct stimulation of the pancreatic β-cell, accounting for some 25-70% of postprandial insulin secretion in healthy subjects. In patients with type 2 diabetes (T2D, however, this effect is greatly reduced or lost due to a combination of severely impaired or eliminated insulinotrophic effect of GIP and reduced meal stimulated GLP-1 secretion. This suggests that the therapeutic potential of GIP for the treatment for T2D is limited, whereas GLP-1 based treatments have been on the market since 2005. Research is now pursuing novel approaches to utilize the effects of GLP-1 for T2D treatment. A combinatorial approach by which the activity of the major enzyme responsible for incretin degradation (dipeptidyl peptidase-4) is inhibited (drugs are already on the market) while the secretion of endogenous GLP-1 secretion is stimulated at the same time may prove particularly rewarding. In this section we review current knowledge on the mechanisms for direct activation of GIP and GLP-1 secretion.
Direct mechanisms of incretin secretion
The endocrine K- and L-cells are located in the intestinal epithelium and are morphologically described as cone-shaped cells with apical cytoplasmic processes equipped with microvilli extending into the gut lumen. While this suggested that the incretin producing cells might be able to directly sense nutrients, it was only after the development of the GLP-1 producing cell lines GLUTag, STC-1 and NCI-H716 that evidence supporting this assumption was provided and the underlying pathways began to be unraveled. Later, the development of transgenic mice that express fluorescent markers under control of the pre-pro-GIP and proglucagon promoter allowed studies on primary murine K- and L-cells, respectively . Through studies on these models and by later verification in in vivo models, it is now recognized that K- and L-sense and secrete their hormonal content in response to carbohydrates (glucose and fructose), lipids, and amino acids and perhaps proteins and bile acids. The underlying molecular mechanisms involved are nutrient specific as described below.
Glucose: Glucose is a potent stimulator of both GIP and GLP-1 secretion in humans. After gastric bypass the GLP-1 response to glucose intake is greatly exaggerated and it appears that all of the dramatic effects of gastric bypass on the secretion of the gut hormones can be explained by the effects of glucose alone. Glucose stimulates GLP-1 secretion by sodium-coupled uptake through the sodium-glucose transporter 1 (SGLT1), which transports one glucose molecule together with two sodium ions. This causes cell membrane depolarization, opening of voltage-gated calcium channels (V-type) with influx of extracellular calcium and activation of the exocytotic machinery. In addition, intracellular metabolism of glucose has been shown to stimulate GLP-1 secretion. In this case, the mechanisms involved appear to be uptake thorough SGLT1 and/or GLUT2 (the latter of which is electro-neutral), metabolism to ATP, closure of ATP-sensitive potassium channels (KATP), which prevent the normal leak of potassium ions out of the cell and causes cell depolarization. The relative contributions these two pathways is a matter of debate, but SGLT1 appears to be the driving force for release as drug-inhibition or genetic knock out of this transporter eliminates glucose-stimulated GLP-1 secretion . For GIP, glucose seems to stimulate secretion by similar pathways, although the mechanism has been less well investigated. However, similar to GLP-1, SGLT1 seems to be the driving force for secretion. Fructose has been shown to stimulate GLP-1 secretion but it appears to be a less potent GLP-1 stimulator than glucose, perhaps because fructose is taken up in an electro-neutral manner (through GLUT5) and therefore is thought to stimulate secretion exclusively by KATP-channel closure. Interestingly, fructose does not stimulate secretion of GIP , although K-cells would be expected to respond to fructose as they both express GLUT5 and the KATP channel subunits Kir6.2 and SUR1. This enigma remains unsolved.
In contrast to carbohydrates, lipids seem to stimulate incretin secretion by depolarization-independent pathways namely through activation of distinct G-protein coupled receptors (GPR). Short chain fatty acids (SCFA) are formed by the microbiological degradation of complex carbohydrates in the distal small intestine and large intestine and stimulates GIP and GLP-1 secretion in rodent models by activation of GPR41 (FFA3) or GPR43 (FFAR2) which link to either Gq or Gi/0 pathways (GPR41) or exclusively to Gi/0 (GPR43). By doing so, SCFA stimulates secretion by activation of phospholipase C which converts PIP2 into IP3 and DAG, causing mobilization of calcium from intra-cellular stores (IP3) and activation of conventional (c) and novel (n) PKC isoforms (DAG), which are thought to directly activate the secretory machinery (see fig. 1). The stimulatory effects of SCFA in humans are, however, less clear. Long-chain fatty acids (LCFA) stimulate GLP-1 and GIP secretion secretion by activation of the Gq-coupled receptors GPR40 and GPR120, thus stimulating secretion as outlined above. Also the intermediate products of lipid metabolism oleylethanolamide (OEA) and 2-oleoylglycerol (2-OG) have been shown to stimulate GLP-1 secretion. In this case, binding to the Gαs-linked GPR119 receptor underlies secretion, meaning that the exposure to secretion coupling is by activation of adenylate cyclase and production of cAMP. cAMP elevation then stimulates secretion by activation of PKA-dependent pathways and perhaps also through activation of the nucleotide exchange proteins 2 directly activated by cAMP (Epac2), supposedly stimulating secretion by increasing the rapid releasable vesicle pool through vesicle recruitment. Perhaps this effect is best demonstrated by that the addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) powerfully stimulates GIP and GLP-1 in rodent models.
Figure 1Figure 1. Mechanisms of nutrient-stimulated GLP-1 secretion. (I) Glucose stimulates GLP-1 secretion by cell depolarization through sodium coupled uptake via SGLT1 and by intracellular metabolism to ATP, causing closure of KATP channels (subsequent to SGLT1 and perhaps GLUT2 mediated uptake). The depolarization activates voltage-gated calcium channels with influx of extra-cellular calcium, causing activation of the exocytotic machinery. Fructose also stimulates GLP-1 secretion, but in this case only the ATP-dependent pathway is involved, as the fructose is taken up electro-neutrally by GLUT5. (II). Short-chain fatty acids (SCFA) and long-chain fatty acids (LCFA) stimulates GLP-1 secretion by activation of distinct G-protein-coupled receptors coupled to the Gq-subunit (SCFA: GPR41 and 43, LCFA: GPR40 and 120). In this case secretion is stimulated by activation of PLC-dependent pathways which convert PIP2 into IP3 and DAG and thereby causes mobilization of calcium from intra-cellular stores (IP3) and activation of PKC (DAG). The lipid metabolites OEA and 2OG stimulate GLP-1 secretion by activation of the Gαs-linked GPR119 receptor, hence coupling exposure to secretion through activation of adenylate cyclase, elevation of cAMP and activation of PKA-dependent pathways and perhaps through activation of EPAC2. Bile acids may stimulate GLP-1 secretion through activation of the Gαs-coupled TGR5 receptor, stimulating secretion as described above. Abbreviations; LCFA: Long-chain fatty acids, SCFA: Short chain fatty acids, PLC: Phospholipase-C, PKA: Phosphokinase-A, SGLT1: sodium-glucose transporter 1, OEA: oleylethanolamide, 2-OG: 2-oleoylglycerol.
Protein and amino acids
Compared to carbohydrates and lipids, the effects of protein and amino acids on incretin hormone secretion are less well characterized. Glutamine has, however, been shown to stimulate both GLP-1 and GIP secretion from healthy, obese and diabetic subjects , although it was a relatively weak stimulus for GIP secretion. In the GLUTag cell line, a number of amino acids stimulate GLP-1 secretion, including glutamine. The mechanisms of secretion is thought to be via sodium-coupled uptake and through elevation of intracellular cAMP, perhaps by activation of an unidentified Gαs-coupled receptor. Different food proteins (e.g. whey and corn and meat hydrolysates) also stimulate GLP-1 and GIP secretion in L-cell models and from humans, but their capacity to stimulate secretion differs. The mechanisms responsible are still unclear, but proton coupled uptake through Pep1, sodium-coupled uptake and activation of GPR93 has been suggested to be involved.
Other directly activated mechanisms of incretin secretion
Bile acids are released from the gallbladder upon meal intake to facilitate the formation of micelles and absorption of fat. From the small intestine bile acids are returned to the liver through the portal circulation (enterohepatic circulation). A few studies have recently shown that bile acids stimulate release of GLP-1 in humans and L-cell models, but the role of bile acids for GLP-1 secretion remains unsettled as other human studies shows no effect. The mechanisms involved appear to be activation of the Gαs-coupled GPBAR (TGR5) receptor, thus stimulating secretion by PKA-dependent pathways as outlined above. Furthermore, it may be speculated that bile acids could stimulate secretion by sodium-coupled uptake through the NTCP and ASBT transporters, which are largely responsible for hepatic and intestinal bile acid uptake. Effects of bile acids on GIP secretion have to our knowledge not been described. Other stimuli shown to directly stimulate GLP-1 or GIP secretion includes the myokine IL-6 and several different artificial sweeteners, but for the later the current balance of evidence does not favor any effect on secretion in intact physiological systems.
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