DPP-IV in plasma and endothelium
The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) play important roles in the regulation of blood glucose homeostasis. GLP-1 stimulates insulin secretion, decreases glucagon secretion and inhibits appetite and food intake. GIP also stimulates insulin secretion but increases glucagon secretion. The enzyme, Dipeptidyl peptidase-4 (DPP-4) was first described in 1966. Because of their penultimate N-terminal amino acid, it was soon suspected that the incretin hormones might be substrates for DPP-4 and the enzyme was subsequently found to play a pivotal role in the metabolism of the hormones in humans. DPP-4 is expressed in the capillary endothelium, the renal tubules and in the liver. In particular it is expressed in the endothelium of capillaries adjacent to the cells of the intestine secreting GIP and GLP-1. Since 1993 it has been known that both GLP-1 and GIP are N-terminally degraded to inactive metabolites (in terms of insulin secretion) by DPP-4.
DPP-4 has numerous additional substrates, but the functional implications of this are not clear. DPP-4 is also expressed in the membranes of lymphocytes where it is also known as CD 26, and has been thought to be involved in immune reactions. Some studies have suggested that it may play a role in oncology. However, in clinical studies long lasting inhibition of the catalytic activity of the enzyme has not been associated with adverse effects from the immune system or any other systems.
Dipeptidyl Peptidase-4 (DPP-4), also known as CD26, is a 766 amino acid peptidase and a member of the family of serine proteases. The enzyme was first described in 1966 when it was isolated from bacteria and eukaryotes and in 1992 the human DPP-4 cDNA was sequenced. The gene coding for DPP-4 is located on the human chromosome 2 and spans approximately 70 Kb.
DPP-4 exists in two forms, a soluble form in plasma and a membrane-bound form located on the luminal surface of endothelial cells in numerous tissues including the kidneys, the vascular endothelium and the hepatocytes. It is also expressed in the brush borders of the gut and the kidneys, where it serves to digest proteins and peptides for absorption.
The membrane-bound form of DPP-4 bulks out from the tissue membrane, and faces the extracellular space. The extracellular part is large and linked to the hydrophobic transmembrane segment of the tissue cells. Besides the extracellular part, DPP-4 also has a smaller cytoplasmic intracellular tail.
The soluble form of the enzyme lacks the intracellular part, and is apparently simply shed from the membranes into the plasma.
The catalytic function of the enzyme is related to extracellular parts of the protein consisting of two domains, one large beta-propeller and one smaller catalytic domain. The beta-propeller controls the access of the substrates to the active site and the catalytic domain is responsible for the actual cleavage.
In 1993 Sjostrom et al. showed that DPP-4 is expressed in the pancreatic islets of Langerhans in pigs  and recent studies show that DPP-4 is also present and active in human islet cells. In one study the activity of the enzyme was reported to depend on the presence or absence of diabetes, with type 2 diabetic patients having decreased DPP-4 activity compared to non-diabetic individuals.
A large number of peptides with roles in nutrition are potential substrates for DPP-4, but the list also includes peptides influencing the central nervous system and the immune system. The enzyme works by selectively cleaving the N-terminal dipeptide from the substrate peptide, which in some cases might change the function of the peptide. A lot of the substrates have been identified by in vitro incubations, but this does not mean they are important in vivo. Peptides with proline or alanine in the second position in the N-terminal-end are substrates to DPP-4.
The activity of the DPP-4 enzyme is measured by a simple assay depending on cleavage of a chromogenic substrate. It is also possible to measure the concentration of the DPP-4 protein. However, it should be pointed out that when plasma DPP-4 activity is assessed ex vivo (i.e. in plasma samples) it is often not corrected for the inherent dilution of the sample in the assay. Hence, the true DPP-4 activity in vivo may be higher than the measured values suggest.
Some studies have found the activity of DPP-4 to be lower in women than in men and to decrease with age. Furthermore the activity tends to fluctuate with pathological conditions. It is however difficult to compare the DPP-4 activity between studies as the methods for measuring DPP-4 activity are not uniform.
DPP-4 cleaves peptides between the amino acids 2 and 3 from the N-terminal end . The best known of its many substrates are the incretin hormones: Glucose-dependent insulinotropic polypeptide (GIP) and Glucagon-like peptide-1 (GLP-1) – secreted from the gastrointestinal tract in response to ingestion of nutrients. These incretin hormones have a powerful effect on the secretion of insulin and glucagon from the pancreas and are thus important for the regulation of glucose levels . The incretin hormones are rapidly degraded by DPP-4 to inactive metabolites. DPP-4 is estimated to degrade around 75% of newly secreted GLP-1 even before it reaches the hepatic portal vein; hepatic DPP-4 activity degrades another 50% so that only about 10% reaches the systemic circulation in the intact form . These findings prompted studies of DPP-4 inhibitors to assess their ability to protect endogenous incretin hormones from degradation, and this was first demonstrated in 1998. These findings spurred the development of the DPP-4 inhibitors for clinical use, the first of which, sitagliptin, was introduced to the market in 2006.
The notion of the incretins as important substrates which explain the mechanism of actions of the DPP-4 inhibitors in the treatment of diabetes has however been questioned during the past two years . The focus has shifted somewhat to substrates such as Stromal cell-derived factor 1 (SDF-1α), Polypeptide YY (PYY) and Gastrin-releasing peptide (GRP), which may have potential new effects resulting from DPP-4 activity and inhibition.
The discovery of GLP-1 and GIP, as important substrates to the DPP-4 enzyme, led to development of the DPP-4 inhibitor treatment. The inhibitors prevent the degradation of the incretin hormones increasing the levels of intact incretin hormones, resulting in increased insulinotropic and glucagonostatic effects, which will further influence the blood glucose levels, but recent studies have suggested that DPP-4 inhibitor treatment may also have direct effects on islet function by inhibiting DPP-4 produced by the pancreatic islets.
Currently, there are 5 different inhibitors at the market. First on the market was sitagliptin. Later vildagliptin, saxagliptin, alogliptin, and linagliptin were introduced. They all have the same endpoint – raising the circulating amount of especially GLP-1, leading towards a better glucose control within the treated patients.
The widespread use of the DPP-4 inhibitors has lead to renewed interest in this enzyme, not only regarding the treatment of diabetes, but also in cardiology, immunology and oncology where the enzyme and the inhibitors might have potential. The question of additional substrates deserves further research and studies of the actual mechanism of action of the individual inhibitors will be pursued further.
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