Incretins & Gastrointestinal Hormones

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The digestion and absorption of nutrients subsequent to food intake is associated with increased secretion of multiple gut peptides that are synthesized by specialized enteroendocrine cells located in the epithelium of the stomach, the distal ileum and the large intestine, and in pancreatic islets [1].

A number of peptide hormones are encoded by the single proglucagon gene. In the pancreas, proglucagon is cleaved to glucagon, glicentin-related pancreatic peptide (GRPP) and a major proglucagon fragment. In the intestinal L-cells, the molecule is processed to GLP-1 (glucagon-like peptide-1), GLP-2 (glucagon-like peptide-2), and glicentin, which is further broke down to oxyntomodulin [2].

Two hormones, glucagon-like peptide-1 (GLP-1), and glucose dependent insulinotropic polypeptide (GIP) are responsible for the incretin effect [3]. Incretin increases the magnitude of meal-stimulated insulin secretion from islet cells in a glucose-dependent manner. Incretin action facilitates the uptake of glucose by muscle tissue and the liver while simultaneously suppressing glucagon secretion by the cells of the islets, leading to reduced endogenous production of glucose from hepatic sources. Preclinical studies indicate that both GLP-1 and GIP increase levels of cAMP leading to expansion of β cell mass and resistance to β cell apoptosis [4, 5].

GLP-1 is a 30 amino acid peptide hormone produced in the intestinal epithelial endocrine L-cells. In addition to stimulate insulin secretion and to inhibit glucagon secretion, GLP-1 also inhibits gastrointestinal motility and secretion and thus acts as an enterogastrone and part of the “ileal brake” mechanism. GLP-1 also appears to be a physiological regulator of appetite and food intake. Decreased secretion of GLP-1 may contribute to the development of obesity, and exaggerated secretion may be responsible for postprandial reactive hypoglycemia [2, 6].

GIP is a 42 amino acid peptide hormone synthesized in and secreted from K cells in the intestinal epithelium. Unlike GLP-1, which exerts multiple non-incretin activities in the regulation of blood glucose, the primary action of GIP is the stimulation of glucose-dependent insulin secretion.

GIP receptors are expressed on adipocytes. Experimental data derived from studies of the GIP receptor knockout mice strongly implicates a role for the GIP receptor in the regulation of body weight. Emerging evidence suggests that gastric bypass surgery rapidly cures diabetes in grossly obese subjects, at least in part as the result of surgical bypass of GIP-secreting K-cells in the upper small intestine. GIP may in addition to its insulinotropic effects, promote fat storage and obesity, either by direct insulin-mimetic effects in adipose tissue or via enhancement of resistin-mediated stimulated lipoprotein lipase activity. Thus, blocking these effects pharmacologically could be a strategy for treatment of obesity [3, 7].

Both, GLP-1 and GIP, are inactivated by the enzyme dipeptidyl peptidase-4 (DPP-4) [8].

In circulation, the first two N-terminal amino acids of active form of GLP-1, GLP-1 (7-36) amide and GLP-1 (7-37), are extremely rapidly cleaved by DPP-4, which leads to the formation of the major circulating forms of GLP-1 in peripheral plasma, GLP-1 (9-36) amide and GLP-1 (9-37).

In fact, inhibitors of DPP-4 have been approved as therapeutic agents for the treatment of type 2 diabetes (e.g. sitagliptin). While the measurement of total GLP-1, i.e. the sum of the intact hormone and its metabolites, reveals the total L-cell secretion, the level of the intact GLP-1 reflects the endocrine actions of the peptide. The biologically active form of GIP (amino acids 1-42) is also rapidly inactivated to the biologically inactive GIP (3-42). GIP infused into human subjects is rapidly degraded, with a t1/2 of ~7 min [9, 10].

In addition to GLP-1, distal-intestinal L cells secrete postprandially oxyntomodulin and peptide tyrosine-tyrosine (PYY). These two hormones are considered to be satiety signals decreasing food intake, and body weight [11, 12].

Oxyntomodulin, a 37 amino-acid peptide (the first 29 amino-acids contain the glucagon structure), delays gastric emptying and decreases gastric acid secretion. In rodents, exogenous administration of oxyntomodulin decreases food intake while increasing energy expenditure, and chronic injections reduce body-weight gain. Although the mechanisms are not entirely understood, most probably these effects are mediated via GLP-1 receptor, since oxyntomodulin does not alter feeding in GLP1 receptor-deficient mice, and the GLP1-receptor antagonist, exendin9–39, blocks oxyntomodulin-induced anorexia. Oxyntomodulin is a candidate substrate for DPP-4. The midsection of oxyntomodulin may also be a target for degradative enzymes such as the ectopeptidases and neutral endopeptidase 24.11 (NEP) [13].

PYY, the other gut hormone, increases ileal absorption and inhibits gastric emptying along with inhibition of gallbladder and pancreatic secretion [14]. Both isoforms of the PYY, PYY (1-36), and that cleaved by DPP-4, PYY (3-36), have anorexigenic effects. However the truncated form is more potent. In adults, PYY (3-36) accounts for 37% and 54% of total PYY immunoreactivity in basal and postprandial plasma, respectively. The anorectic effects of PYY (3-36) appear to be mediated by the Y2 receptor, thus Y2 receptor agonists are considered in the treatment
of obesity.

Ghrelin, discovered as a natural ligand for the growth hormone (GH) secretagogue receptor is produced predominantly by the enteroendocrine cells in the oxyntic glands of the stomach. In addition to potently stimulating secretion of GH from the pituitary, ghrelin, which increases preprandially and drop rapidly after meals, acts as an orexigenic peptide increasing appetite, food intake, body weight, and adipogenesis [15].

During its synthesis, ghrelin is acylated on 3-serine with an ester-linked fatty acid group. The acylation is essential for ghrelin’s activity at the GH secretagogue receptor, but is readily cleaved by endogenous esterase activity [16]. Both active acyl-ghrelin and des-acyl ghrelin are found in the circulation. Recent studies suggest that des-acyl ghrelin has multiple biological activities, too. Since some of these actions of des-acyl ghrelin oppose those of acyl-ghrelin, the ratio of acyl- to desacyl ghrelin may determine the overall physiological response. In the circulation, ghrelin, but not des-acyl ghrelin, may be predominantly bound to carrier proteins. Ghrelin has specific lipoprotein interactions not seen with des-acyl ghrelin, and antisera to ghrelin differ in their ability to detect these bound forms [16, 17].

Pre-Analytical Considerations

Plasma levels of most gut hormones rise shortly after a meal and fall rapidly thereafter, mainly because they are eliminated by the kidneys, but also because they are enzymatically inactivated.

Testing for incretins, and other gut hormones, presents numerous challenges because of their instability. The degradation and modification of gut hormones caused by proteolytic enzymes, especially DPP-4 and NEP, occur not only in vivo, but also during and after blood collection. Therefore, proper sample collection and meticulous pre-analytical and analytical sample handling are crucial for successful quantification of those biomarkers.

The best practice for minimizing the preanalytical variability associated with blood collection, processing and storage is to collect samples directly into tubes containing protease inhibitors. Mixing blood with the protease inhibitors during collection instantly protects plasma proteins, which enhances the analyte recovery and long-term preservation.

To measure the active forms of GLP-1 and GIP, blood must be immediately preserved with DPP-4 inhibitor. Tubes containing DPP-4 inhibitor suitable for the measurements of active forms of incretins are commercially available (BD Diagnostics P700 tubes).

BD is introducing P800 tubes that contain a protease inhibitor cocktail in addition to DPP-4 inhibitor. P800 tubes are recommended not only for active forms of incretins, but also for other gut hormones. The addition of a protease inhibitor cocktail to inhibit proteases that may act on the peptides during long-term storage (even in the frozen state) is important.

As an alternative to using collection tubes containing the appropriate inhibitors, blood sample may be spiked with a protease inhibitor cocktail including DPP-4 inhibitor within 30 seconds of blood collection. An appropriate cocktail should contain in addition to a DPP-4 inhibitor (e.g. Diprotin A), a broad spectrum of inhibitors including EDTA, inhibiting metalloproteinases, serine protease inhibitors (e.g. aprotinin and/or AEBSF) and cysteine protease inhibitors (e.g. E-64). Sample preservatives must validated and be compatible with the assays used.

Additional pre-analytical steps should be considered when samples are obtained for quantification of the active acylated ghrelin. The simplest and most reliable procedure that stabilizes the labile side chain is acidification. After blood is collected into tubes containing protease inhibitors (e.g. BD P100), the separated plasma should be spiked with acid (e.g. 5N HCl) to pH 4. Instead of acidification, esterase inhibitor, may be considered. As of this writing, none of the commercially available collection tubes fully protect the acylated form of ghrelin.

In addition to applying appropriate protease inhibitors, blood samples should be kept on ice at all times and processed in a refrigerated centrifuge, aliquoted immediately, and stored frozen at -70 or below.

Minimizing time at ambient temperature during testing helps maintain sample integrity. Immediate snap freeze on dry-ice after use permits the same aliquot of sample to be used at a later time.

It is important to recognize that the sample stability tolerance usually applies to total time. The users need to take under consideration the cumulative effect of all handling and storage. For example, samples that went through a number of freeze thaw cycles verified during assay validation as being safe for analyte integrity (i.e. % mean difference was not higher than 10% compared to baseline values) cannot be stored for the same time at -70?C or lower as samples that have never thawed at all before use. Similarly, samples that were not processed immediately after blood collection, or were spun in the centrifuge without refrigeration will not tolerate a short storage at 4?C as well as samples handled appropriately. It is important to minimize the exposure of the samples to sub-optimal conditions at every step of sample handling or storage.

Analytical Considerations

Appropriate sample handling is key to successful laboratory testing. Another significant challenge in the measurement of incretins and gut hormones is the performance of the available assays. PBI’s experience indicates that often commercial assays are inadequate for clinical studies. However, modification of manufacturer’s recommendation and careful user-oversight can achieve acceptable assay performance.

The major concerns with commercial assays comprise assay sensitivity, matrix effect, and among-lot reagent consistency.

Often normal values of gut hormones, particularly levels of incretins, fall below the lower limit of assay quantification. Proper curve fitting such as a weighted 5-parameter logistics curve-fit, and introduction of additional standards at the low end of the standard curve can improve the sensitivity. Whenever feasible, standard material should be prepared in matrix corresponding to patient sample matrix including appropriate preservatives. In case of incretins, standards should be prepared in a broad spectrum of protease and DPP-4 inhibitors-protected human EDTA plasma spiked with appropriate synthesized or recombinant peptides. Similarly, control samples prepared in human plasma assure more accurate monitoring of short-term and long-term assay performance.

HORMONE	SOURCE	ACTIONS	BIOACTIVE FORMS	INACTIVATING ENZYME	T1/2 OF ACTIVE BIOFORM (IN VIVO)
GLP-1	Distal ileal L cells	Insulinotropic, decreases glucagon secretion, regulates gastro-intestinal motility and appetite	7-36 amide, 7-37	DPP-4	1 ½ to 2 min
GIP	Duodenal K cells	Insulinotropic, increases glucagon secretion, stimulates β-cells growth	1-42	DPP-4	~7 min
PYY	Colorectal L cells	Decreases food intake, inhibits gastric acid secretion and gastric emptying	3-36*	DPP-4
Glucagon	α-cells of the islets of Langerhans	Increases blood glucose, stimulates the release of insulin	1-29	DPP-4, NEP	~6 to 7 min
Oxyntomodulin	Colorectal L cells	Decreases food intake, inhibits gastric acid secretion, no effect on gastric emptying	1-37	DPP-4, NEP	~6 to 7 min

* Both isoforms are bioactive, however PYY (3-36) is more potent than PYY (1-36)

References

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