Incretins
Insulin

Stefano Rousset 28/03/14

Can T2DM be partially considered an intestinal disease? An insight into incretins functions.

Physiological functions of incretins

It is known that at least 50% of insulin secretion following a meal is due to the so-called incretin effect, mediated by the intestinal secretion of two hormones responsible for a potent insulinotropic action.
The two hormones responsible for this effect are GIP (gastric inhibitory peptide) and GLP-1 (glucagon-like peptide 1).

Synthesis and degradation.
GIP is synthesized and released in the bloodstream from the K-cells particularly abundant in the duodenum and jejunum. His precursor is a protein composed of 153 amino acids whose gene is localized on the long arm of chromosome 17; the action of proconvertase 1/3 determines the synthesis of the biologically active hormone composed of 42 amino acids. GIP synthesis reaches his peak some minutes after the ingestion of food and it is regulated by the arrival of nutrients in the gut; in particular, fatty acids, sucrose, galactose and fructose are important stimuli for synthesis and secretion of GIP, while amino acids represent a weaker stimulus. Once in the bloodstream, GIP is rapidly degraded in 5 to 7 minutes by the action of dipeptidyl-peptidase-4.

GLP-1 is synthesized from the L-cells localized in the small intestine and ascending colon. The gene is localized on the long arm of chromosome 2 and encodes for a 180-amino acids protein, proglucagon, which is converted in the active form GLP-1 by the actions of proconvertase 1/3 (an isoform active in the intestinal L-cells); this process is different from the one that takes place in the pancreatic alpha-cells, where the action of a different set of proconvertase ( PC2 and 7B2, a chaperon protein) leads to the synthesis of the hormone glucagon.

Active GLP-1 presents two different isoforms, and the most important (80% of total) is the C-terminal amidated form, GLP-1 (7-36) amide, while the other one is GLP-1 (7-37).
L-cells are endocrine intestinal cells in direct contact with nutrients present in the gut through the microvilli on the luminal surface, but also with vagus nerve terminations and with the microvasculature through the basolateral membrane. As a consequence, nutrients as well as endocrine and neural stimuli are capable of activating secretions in this cells: GIP determines activations of vagus nerve which in turn determines the secretion of GLP-1, but an important role is carried out by some other neurotransmitters and peptides such as acetylcholine, CGRP, GABA, CCK, leptin and insulin, while somatostatin has an inhibitory role.
The main stimulus for the secretion of GLP-1 is a meal rich in carbohydrates and fats, but also simple sugars, amino acids, fatty acids and fibres could stimulate synthesis and release of this hormone. The mechanism involved in the lipid-dependent secretion of GLP-1 requires the binding to specific GPCRs (G-protein coupled receptors), while long-chain fatty acids stimulate the release of CCK from the neuro-endocrine I cells, and CCK in turn stimulates the secretion of GLP-1. Another mechanism requires the activations of biliary-receptor TGR5. Talking about glucose, it determines secretion of GLP-1 with a mechanism similar to that used by the gustative cells in the tongue: indeed, L-cells present sweet-sensitive receptors, like gustducin, capable of inducing cell depolarization and release of GLP-1.
GLP-1 secretion is biphasic, similar to that of insulin, presenting a peak after 5-15 minutes from the ingestion of food followed by a second longer secretive phase in 30 to 60 minutes. As well as GIP, GLP-1 is rapidly inactivated in 1 to 2 minutes by the action of dipeptidyl-peptidase-4.

Receptors.
GIP receptor is a glycoprotein encoded by a gene localized on the long arm of chromosome 19 and expressed in the alpha and beta pancreatic cells, but also in some other organs and tissues. It is a G-protein-coupled-receptor, responsible for the activation of transduction signals that in turn lead to the activation of a lot of different kinase protein (PKA, PI-3K, PKB, MAPK, PLA2) which determine the insulinotropic effect of GIP.

GLP-1 receptor is encoded by the glp-1r gene situated on the short arm of chromosome 6, it is composed of 463 amino acids and it is expressed in the alpha and beta pancreatic cells, in the heart, kidney, gastrointestinal tract, skin, pituitary, hypothalamus, hippocampus and cerebral cortex. It is a GPCR made up of an N-terminal extracellular domain which presents the binding site for GLP-1, seven alpha-helical transmembrane domain and a C-terminal intracellular domain responsible for signal transduction thanks to the association with at least three different isoforms of the Gsα protein, which lead to the activation of different kinase-protein, such as PKA, PKC, PI-3K, Epac2 and MAPK.

Actions.
GIP, through the activation of his receptor on the beta-cell in the pancreas, determines the activation of PKA, which in turn catalyzes the phosphorylation of regulatory proteins like GLUT2, SUR1, alfa-SNAP and others ion channels; this events determine an increase and strengthening of insulin release induced by cell depolarization. The same effect is achieved also by the activation of PLA2 and the consequent release of arachidonic acid.
Furthermore, GIP enhances expression of insulin and other enzymes-codifying genes, such as GLUT-1 and hexokinase, which are involved in the first phase of glucose catabolism, a process that eventually leads to insulin secretion. Other evidences suggest a role of GIP as an anti-apoptotic and growth factor: this result is obtained thanks to the activation of transduction pathways which include PKA/CREB, MAPK, PI-3K/PKB.

GLP-1 is one of the most powerful molecules capable of stimulating insulin secretion, due to the activation of PKA and Epac2, responsible for a huge number of final effect:

• Phosphorylation of SUR1 subunit of the ATP-sensitive K+ channels, and consequent depolarization and release of insulin-containing granules.
• Inhibition of voltage-dependent K+ channels with consequent inhibition of repolarization.
• Phosphorylation of L-type Ca2+ channels and increase of intracellular calcium concentration.
• Phosphorylation of vescicle-associated proteins like synapsin-1 and RIM proteins, facilitating exocytosis of insulin granules.
• Increased expression of GLUT2 and glucokinase.

GLP-1 also determines an increase in insulin synthesis, through several mechanism:

• Stabilization of insulin mRNA.
• Increased transcription and biosynthesis of insulin gene, through a boost of PDX-1 intracellular concentration (PDX-1 is the most important insulin transcription factor) and activation of others TF capable of binding to and activating the insulin gene promoter. GLP-1 induces nuclear translocation of PDX-1 phosphorylated form, which associates with histone acetyltransferase p300 determining hyperacetilation of histone H4 and induction of insulin gene transcription.

GLP-1 stimulates beta-cell proliferation and acts as an anti-apoptotic factor:

• Activation of IRS2/PI-3K/PKB pathway leads to activation of cyclin and cdk (cyclin dependent kinase) and consequent increase of proliferation and cell survival.
• Activation of CREB determines expression of IRS2 and cyclin D1 codifying genes (this are proteins that promote cellular proliferation).
• Activation of PKB and MAPK leads to a decrease of caspase-3 activity and inhibition of apoptosis.
• Activation of CREB in a cAMP/PKA-dependent manner causes an up-regulation of Bcl-2 and Bcl-Xl (anti-apoptotic factors).
• Pleiotropic effects are also achieved through activation of Wnt signalling in a cAMP/PKA-dependent manner, with disassembling of the beta-catenin phosphorylation complex, so that beta-catenin manages to escape proteasome-dependent degradation and forms the complex beta-catenin/TCF2 which works as a transcription factor promoting cell proliferation.
• Activation of PI-3K/PKB transduction pathway with consequent phosphorylation and nuclear exclusion of FoxO1, a transcription factor that acts as PDX-1 inhibitor.

GLP-1 also binds to his receptor on the pancreatic alpha-cells, inhibiting synthesis and secretion of glucagon.

The Role of Incretins in Glucose Homeostasis and Diabetes Treatment, 2008

New insight into the mechanisms underlying the function of the incretin hormone glucagon-like peptide-1 in pancreatic β-cells: the involvement of the Wnt signaling pathway effector β-catenin, 2012

Nutrition and L and K-enteroendocrine cells, 2011

Alterations of incretins functions in T2DM

Alterations in the activity of incretins cannot be considered as a cause of diabetes, but a consequence of chronic hyperglycemia that exacerbates the functional damage in the beta-cells.

GIP incretinic effect is almost completely lost in T2DM. This is due not to a reduction of intenstinal secretion, which is rather normal or augmented in basal and post-prandial conditions, but to a desensitization of GIP receptor and to a reduction of the number of receptors expressed on the beta-cells membrane. This events are caused by mechanism of receptor internalization, down-regulation, and uncoupling from G proteins. In particular, a surely important role is played by residues of serine 426-427, that regulate the rate of receptor internalization, and serine 406-411, important for receptor desensitization. So it is clear that hyperglycemia, through activation of certain kinase proteins, leads to an increase of this serine residues phosphorylation, with consequent appearance of a GIP-resistance phenotype.

GLP-1 levels result to be normal or augmented in diabetic patients, but there is a reduction of receptor expression. Receptor internalization seems to be the most important mechanism, and it is mediated by phosphorylation of specific amino acids residues in the C-terminal segment of the receptor. However, GLP-1 response in T2DM is not completely lost, but moderately reduced (approximately for 30%).
Recent studies have highlighted a close association between SNP (single nucleotide polymorphism) of TCF7L2 gene and T2DM. This gene encode for a protein involved in Wnt-signalling pathway which, as stated above, plays an important role inside the physiological functions of incretins.
In the beta-cells of a patient suffering from T2DM and in the same cells of an healthy person whose TCF7L2 gene has been silenced through siRNA (short-interfering RNA) a reduction of TCF7L2 protein is detectable, associated to an increase of the corresponding mRNA, a decrease of incretin receptors expression, a strong suppression of insulin secretion and an increase of apoptosis; over-expression of the same gene has opposite effects.
Studies on pancreatic beta-cells have been designed with the purpose to identify a possible involvement of GLP-1R/GIP-R signalling in the cellular dysfunctions following decrease of protein TCF7L2 concentration. The results have highlighted a down-regulation of incretin receptors following TCF7L2 gene silencing.
Reduction of TCF7L2 protein determines an increase of apoptotic process through an alteration of PKB functionality. As stated above, incretins carry out their protective effects through phosphorylation and activation of PKB. In TCF7L2 depleted cells phosphorylation of PKB is drastically reduced, due to reduced expressions of incretin receptors; this in turn determines a diminished phosphorylation of FoxO1 which consequently, instead of transferring in the cytoplasm, remains inside the nucleus leading to an inhibition of PDX-1 and to an increase of TCF7L2 gene transcription with accumulation of the corresponding mRNA. Why the increase of transcription is associated to a diminished mRNA translation and consequently to a reduction of protein levels remains to be cleared; it is thought that a role might be played by some defects in the post-translational regulation that lead to the synthesis of an abnormal protein which is eventually degraded.

Reduction of TCF7L2 protein levels determines also a beta-cell dysfunction, and this is demonstrated by the fact that siRNA-mediated gene silencing causes, as previously stated, a remarkable reduction in insulin secretion. However, in the same cells the administering of agents capable of increasing cAMP concentration in an incretin-independent way determines a normal enhancement of insulin release, proving that cAMP response in this cells is preserved. So, what is the mechanism by which a reduction of protein TCF7L2 leads to an altered functionality? Firstly, TCF7L2 gene silencing negatively affects the concentration of exocytotic proteins such as syntaxin-1 and munc18-1, with consequent alterations of the interactions between insulin-containing granules and cellular membrane. But this mechanism alone in surely not sufficient. Recent studies have demonstrated that TCF7L2 protein is in some way capable of influencing distribution of voltage-dependent Ca2+ channels across the cellular membrane: so, its reduction causes a reduced expression of this channels and eventually an alteration of insulin release.

Down-regulation of pancreatic transcription factors and incretin receptors in type 2 diabetes, 2013

Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function, 2009

Mechanisms by which common variants in the TCF7L2 gene increase risk of type 2 diabetes, 2007

Type 2 Diabetes Susceptibility Gene TCF7L2 and Its Role in β-Cell Function, 2009

Extrapancreatic effects of incretins

GIP receptors are present in the central nervous system, gastrointestinal tract, adipose tissue and bone.

In the central nervous system GIP seems to play a role in the proliferation of neural progenitor cells, especially in the hippocampal dentate gyrus, suggesting a role of GIP in neurogenesis. Several studies on mice have shown a possible role of GIP in the modification of behaviour and regulation of locomotor activity and exploration, but other studies are requested in order to assess the same effects in men.

In the GI tract GIP determines an inhibition of gastric acid secretion and a reduction of gastrointestinal motility, but this effects in men are obtained at a supraphysiological plasma concentration.

In adipose tissue GIP inhibits glucagon-induced lipolysis and enhances the activity of lipoprotein lipase; furthermore, it promotes glucose and fatty acids uptake and their transformation in triglycerides. So, an involvement of this hormone in the onset of obesity is very likely, as demonstrated by the fact that GIP-antagonist administration is capable of inducing a reduction of total body weight, adipocytes mass and triglycerides storage in muscle and liver.

In bone GIP has an anabolic effect and prevents loss of bone mass; this effects are obtained thanks to an increase of intracellular concentration of cAMP and calcium, an augmentation of type I collagen deposition and an enhancement of alkaline phosphatase activity.

GLP-1 receptors are present in the central nervous system, GI tract, muscle, adipose tissue, liver and cardiovascular system.

In the central nervous system GLP-1 is an important regulator of caloric intake, appetite and body weight. GLP-1 receptors are present in the nodose ganglion of the vagus nerve, in the brainstem and in the hypothalamus, and their stimulation induces a reduction of appetite and caloric intake; GLP-1 is therefore a key actor in the GI tract-brainstem-hypothalamus axis, which is very important in the regulation of dietary behaviour.
Recent studies have put into light a role of central GLP-1, produced by neurons in the nucleus of the solitary tract in the brainstem, in the regulation of total body mass and tissue-specific glucose homeostasis; in particular, it seems that central GLP-1 is able to facilitate hepatic uptake of glucose at the expense of the muscle one.

In the gastrointestinal tract physiological concentration of GLP-1 induces a reduction of gastric acid secretion and intestinal motility, with a neuro-mediated mechanism.

In human skeletal muscle GLP-1, through activation of PI-3K/PKB and MAPK transduction pathways, stimulates an increase of glucose uptake and catabolism, and glycogen synthesis.

In adipose tissue GLP-1 determines enhanced insulin-stimulated glucose uptake and lipolysis, while in hepatocytes it reduces gluconeogenesis and stimulates glycogen synthesis.

In the cardiovascular system GLP-1 affects arterial pressure and heart rate through a neuroendocrine and autonomic control mediated by the binding of GLP-1 to its receptors on the vagus nerves terminations; in addition, GLP-1 also induces an improvement of endothelial dysfunction, with a mechanism based on a TNF-alpha induced reduction of tissue plasminogen activator inhibitor expression.
In addition, it has been demonstrated that GLP-1 administration in patients with heart failure is able to improve global left ventricular functionality and metabolic control, and also it seems to have a protective effect against ischemic damage.

The Role of Incretins in Glucose Homeostasis and Diabetes Treatment, 2008

GIP receptor antagonism reverses obesity, insulin resistance, and associated metabolic disturbances induced in mice by prolonged consumption of high-fat diet, 2007

Incretins and therapy

Two new types of drug classes have been recently started to be utilized in the clinical practice to treat T2DM, besides the classic hypoglycaemic and insulinotropic drugs such as metformin and sulfonylureas: they are the GLP-1R agonists and the dipeptidyl-peptidase-4 inhibitors.

GLP-1R agonists.

• Exenatide: it is a protein made up of 39 amino acids, with a 53% homology with GLP-1, isolated from the salivary glands of Heloderma Suspectum, a species of lizard. It has an half-life superior to that of GLP-1 (from 3.3 to 4 hours), it is resistant to the action of DPP-4 (this is due to the presence of a Glycine-8 in place of an Alanine-8) and it interacts with GLP-1R with great affinity; administration is subcutaneous and it is eliminated by the kidneys through glomerular filtration. This drug determines a reduction of post-prandial hyperglycaemia, through three different mechanism: an increase in insulin secretion and a concomitant reduction of glucagon secretion in the pancreas, an increase of glucose uptake in the liver, and a reduction of gastric emptying rate. The most important collateral effects, reported by 35-45% of the patients, are nausea, vomiting and diarrhea, all of which tend to reduce with time. Randomized clinical trials have been carried out to test the efficacy of this drug, and the results have shown that subcutaneous administration of exenatide in association with metformin/sulfonylureas reduces post-prandial hyperglycemia more efficaciously in respect to what happens in the placebo-treated control group. Because the subcutaneous administration two times a day is uncomfortable for patients, new forms of the drugs have been tested, and the most encouraging is exenatide LAR (long-acting release), that can be administered just one time a week inside polymeric-based microspheres. A recent study ( DURATION-1 ) have successfully demonstrated a better glycemic control in the patients treated with this form of the drug, with a greater reduction of glycated haemoglobin in the absence of an increased risk of hypoglycaemia. Overall, there were six DURATION studies that all had similar outcomes, showing that exenatide once weekly resulted in significant improvements in glycemic control.
• Lixisenatide: it is a potent GLP-1R agonist; it is thought that it has an affinity for GLP-1R four times superior than that of the endogenous ligand. Presently it is still in the development phase, but preclinical studies have highlighted an important effect on the pancreatic beta-cells, such as an increased synthesis and secretion of insulin, a reduction of apoptosis, an increase of cellular mass, a reduced caloric intake and gastric emptying.
• Albiglutide: it is another GLP-1R agonist that consists of a DPP-4-resistant GLP-1 dimer fused to recombinant human albumin; it has an half-life of about 5 days, which is useful to the purpose of effecting a lesser number of administration (one time a week is sufficient); this drug determines a reduction of post-prandial hyperglycaemia in a dose-dependent manner, with headache and nausea as the most important side effects.

DPP-4 inhibitors.

• Sitagliptin: it is the only DPP-4 inhibitor that is currently in use in the clinical practice. It is a little non-peptidic molecule orally administered and rapidly absorbed in the gut; it has an half-life of 8 to 14 hours and it is eliminated by the kidneys. Inhibiting the action of DPP-4 the half-lives of GLP-1 and GIP are prolonged, and the final effect is a strengthening of incretins action. To achieve this purpose, a residue of insulin secretion might be present. The most important side effects are headache, nasopharyngitis, contact dermatitis, arthralgias and a little increase in the risk of contracting urinary infection; it has not been revealed an increase in the risk of undergoing hypoglycaemia or body weight modifications.
• Vildagliptin: it is a DPP-4 inhibitor currently undergoing study, already approved in Europe and some other countries all over the world; his effect on post-prandial hyperglycemia is efficient and well-tolerated.
• Algogliptin: it is a DPP-4 inhibitor currently in the planning phase, which has the important feature of being highly specific for the DPP-4 isoform, while the others inhibitors also act on the isoform 8 and 9. This is relevant because DPP-8 and 9 inhibition determines a weaker T lymphocytes activation, with a consequent immunosuppressive effect which could be avoided or at least reduced with the employment of this new drug.

In general, GLP-1R agonists are more effective in reducing glycated hemoglobin and post-prandial hyperglycemia, and have a more powerful protective effect on beta-cells compared with DPP-4 inhibitors. Both this classes of drugs seem to have a protective effect in the cardiovascular system, improving arterial pressure and lipid levels. Recent studies have suggested the possibility that incretinic therapy might increase the risk of acute pancreatitits and thyroid cancer, but further validations are necessary.

The important role of incretins in the physiopathology of T2DM has allowed the possibility to identify some surgical strategies to treat diabetes cases resistant to medical therapy, such as duodenal-jejunal bypass, gastric bypass and biliopancreatic diversion. This techniques have given proof of being effective in improving or even resolving the disease in a considerable number of patients. It is thought that this is due to an anatomical rearrangement of the GI tract: duodenal and proximal jejunum exclusion determines a faster arrival of the chyme in the distal jejunum, improving incretins release (especially GLP-1), and at the same time it is possible that it also causes a reduced secretion of other intestinal molecules involved in the onset of insulin-resistance. For example, duodenal-jejunal bypass is an operation that consists of a stomach-preserving bypass of a short segment of proximal small intestine, equivalent to the amount of intestine bypassed in a standard Roux-en-Y gastric bypass; patients who have undergone this operation have significantly increased postprandial GLP-1 and insulin secretion, compared with obese and lean controls.
The vision of diabetes as an intestinal disease surgically treatable probably represents the most innovative and revolutionary perspective from which analyze it, proving once again that the intestine has an important role in the physiopathology of this varied and complex disease.

Update on the Protective Molecular Pathways Improving Pancreatic Beta-Cell Dysfunction, 2013

Pathogenesis and management of postprandial hyperglycemia: role of incretin-based therapies, 2013

Diabetes is predominantly an intestinal disease, 2013