Dulaglutide for the treatment of type 2 diabetes.

INTRODUCTION: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are injectable agents used for the treatment of hyperglycemia in type 2 diabetes. The interest for this pharmacological class is rising with the development of once weekly compounds and the demonstration of a potential reduction in cardiorenal outcomes. Areas covered: The paper describes the main pharmacokinetic/pharmacodynamic characteristics of dulaglutide, a new once-weekly GLP-1 RA. Dulaglutide was extensively investigated in the phase-3 AWARD program, which demonstrated its safety and efficacy when compared to placebo or active glucose-lowering agents in patients treated with diet alone, metformin or sulfonylurea monotherapy, oral dual therapies and basal insulin. In both Caucasian and Japanese patients, comparative trials showed better glucose control with dulaglutide, with a minimal risk of hypoglycemia and weight loss, but at the expense of an increased dropout rate due to side effects, mostly transient gastrointestinal disturbances. Dulaglutide proved its non-inferiority versus liraglutide and the safety and tolerance profile is similar to that of other GLP-1 RAs. Expert opinion: The once-weekly formulation and the combined positive effects on both glucose control and weight improves patient satisfaction despite nausea. Dulaglutide must prove its capacity to reduce cardiovascular and diabetic complications in the ongoing prospective REWIND trial.

Increased intracellular content of enteroglucagon in proximal colon is related to intestinal adaptation to germ-free status.

Changes in the frequency of endocrine cells are evidence of intestinal adaptation to germ-free (GF) status. Not only the distribution of these cells along the intestine, but also the differences in intracellular content of these regulatory peptides may be explored to explain functional and structural aspects of GF intestinal adaptation. Focusing on the endocrine L-cells, we analyzed the intracellular content of enteroglucagon (EG) and peptide YY (PYY) throughout the intestine of the 14 GF and 14 conventional (CV) mice by using immunohistochemistry and the supra-optimal dilution technique. The percentage of EG-immunoreactive cells, but not of PYY-immunoreactive cells stained at supra-optimal dilution was significantly higher in the proximal colon of GF mice than in the CV counterparts (P < 0.05). Since the content of co-stored PYY did not differ between GF and CV mice, the higher content of EG was compatible with a selective cellular response. Moreover, in the cecum of GF mice, the density of EG-immunoreactive cells was significantly higher than that of PYY-immunoreactive cells (P < 0.05). These results are consistent with preferential production of EG by L-cells at the expense of PYY in the proximal colon and in the enlarged cecum of GF mice. In addition, they may reflect the dynamics of the GF intestinal epithelium and/or be correlated with the higher serum levels of these peptides. The role of endocrine cells needs to be better studied in human and other experimental adaptative conditions in order to elucidate the regulatory mechanisms of intestinal functions.

Distribution of enteroglucagon- and peptide YY-immunoreactive cells in the intestinal mucosa of germ-free and conventional mice.

There are evidences that microflora modulates endocrine cells in the gastrointestinal tract. In the present study we investigated the distribution of EG- and PYY-immunoreactive cells throughout the intestine of adult male NMRI conventional and germ-free mice. EG-immunoreactive cells were significantly more frequent in the proximal and middle colon than in the remainder of the intestine in both groups. In germ-free animals, these cells were more frequent in the cecum and less frequent in the distal ileum compared to conventional mice. PYY-immunoreactive cells were more frequent in the distal colon than in the remainder of the intestine in both groups, but they were significantly more frequent in the middle and distal colon of germ-free animals than in that of conventional counterparts. The number of EG-immunoreactive cells was 4.5-fold higher than the number of PYY-immunoreactive cells in the cecum of germ-free mice. The present results indicate the existence of an inverse gradient of EG- and PYY-immunoreactive cells along the colon, which is not significantly changed in the absence of a microflora. PYY production seems to be more significant in the distal colon. The cecum and the proximal portion of the colon are probably the regions of greatest functional importance for EG production, which is related to the microflora and probably to fermentation products, whether or not the effect of this peptide is trophic or antitrophic.

Immunocytochemical detection of islet hormones in the digestive system of Protopterus annectens.

The presence, distribution, and interrelationships of the four typical pancreatic islet hormones were investigated in the digestive system of Protopterus annectens by single and double immunohistochemical methods. Insulin-, glucagon-, and somatostatin-immunoreactive (IR) elements were detected in both the pancreas and the gut. Pancreatic polypeptide (PP)-IR endocrine cells were always present in the gut, but were only present in the pancreas of a few specimens. Some of the latter cells also seemed to react with glucagon antiserum. In the pancreas the immunopositive cells were organized into islets of different sizes, and their organizations were studied by the double immunohistochemical techniques. In the few large islets insulin-IR cells were present in the central zone, glucagon- and PP-IR cells at the periphery, and somatostatin-IR cells intermingled with both the peripheral and the central endocrine cells. In the smaller islets, the number and the staining intensity of glucagon- and PP-IR endocrine cells varied markedly. In the gut, insulin-, somatostatin-, and PP-IR cells were of the open type; glucagon-containing cells were very few and had no luminal contact. They were differently distributed along the intestinal epithelium. Somatostatin-IR nerve fibers and somatostatin-IR neuron cell bodies were also observed in the intestinal wall. The organization of pancreatic endocrine cells in P. annectens is similar to that observed in the majority of teleosts even if a different topographical association can be found. Furthermore, islets of different sizes seem to display a different metabolic turnover, and the detection of pancreatic PP-immunoreactivity varied according to the specimens utilized. In the intestinal portion insulin-IR cells, in addition to PP-, glucagon- and somatostatin-IR cells are present: this suggests that intestinal insulin-like immunoreactivity may be more widespread than previously supposed.

The role of gut-glucagon-like immunoreactants in the control of gastrointestinal epithelial cell renewal.

In vitro and in vivo studies have provided considerable information on the possible physiologic function of circulating gastrointestinal hormones as well as locally acting regulatory peptides in the multifactorial control of adaptive gastrointestinal epithelial cell proliferation and cell renewal. It has been suggested by circumstantial evidences that enteroglucagon (EG; G-GLI I) may act as a trophic factor on the intestinal mucosa which may account for adaptive changes of the small intestine following various stimuli. However, we have shown that there are experimental conditions (germ-free rats after conventionalisation; jejunal self-filling blind loops) in which intestinal hyperplasia does not correspond to an increase in the concentrations of enteroglucagon in plasma or intestinal mucosa. Furthermore, despite a continuous immunoneutralisation of circulating endogenous enteroglucagon by monoclonal antibodies there was an adaptive, hyperplastic response of the ileal remnants after a 70% proximal small bowel resection which was of the same magnitude as in the control group but was even greater considering the increased number of mitoses per crypt. In order to gain additional insight into the putative role of enteroglucagon as an enterotrophic regulatory peptide, an in vitro model was used to investigate the effect of highly purified rat G-GLI I on the proliferative response of primary small intestinal epithelial cells of fetal rats. Whereas there was a well known growth-promoting action of EGF, the proliferation of rat fetal intestinal epithelial cells was inhibited by the addition of purified G-GLI I. These results indicate that enteroglucagon does not act as an enterotrophic factor but provide the first direct evidence consistent with an antitrophic role of enteroglucagon in the small intestine.

Effect of monoclonal antibodies to enteroglucagon on ileal adaptation after proximal small bowel resection.

On the basis of circumstantial clinical and experimental evidence, it has been suggested that enteroglucagon (EG) may act as an enterotrophic factor. This study was undertaken to evaluate the effects of long term in vivo immunoneutralisation of EG, using monoclonal antibodies to EG, on the hyperplastic ileal response after small bowel resection. Nineteen rats had a 70% proximal resection. A group of 10 rats was given iv 0.5 ml of undiluted hybridoma ascites immediately after the operation and on the 7th day postoperatively. Furthermore 0.025 ml/day of the same hybridoma ascitic fluid was continuously delivered ip for 14 days via mini-osmotic pumps. The hybridoma ascites was prepared from the clone 23.6B4 synthesising a monoclonal antibody directed toward the N-terminal to central region of the glucagon molecule which showed a marked crossreaction with EG. A control group of 9 rats was given a corresponding amount of antibody-free plasmacytoma ascites (Ag 8.653) by the same technique. Seven and 14 days postoperatively there was a plasma anti-EG-antibody excess with an excess binding capacity of 84.9 glucagon eq nM and 88.5 glucagon eq nM respectively. The three dimensional architecture and the proliferative activity of the ileal remnant were evaluated two weeks postoperatively. Despite a continuous immunoneutralisation of circulating endogenous EG by monoclonal antibodies, the adaptive response of the ileal remnants was of the same magnitude as that seen in the control group. These data do not support the hypothesis that EG is a circulating enterotrophic regulatory peptide.

Production and characterization of N-terminally and C-terminally directed monoclonal antibodies against pancreatic glucagon.

Hybridoma technology has been successfully applied to the production of monoclonal antibodies against a variety of small soluble peptides. We report herein for the first time on the development of monoclonal antibodies to pancreatic glucagon. Twenty-three stable positive hybridomas were detected by radioimmunoassay from five separate fusions and cloned by the limiting dilution method. Four selected monoclonal antibodies were all of the IgG 2a subclass type kappa and bound to protein A. One monoclonal antibody (23.8B6) was shown to be directed toward the C-terminal region and another (23.6B4) toward the N-terminal to central region of the glucagon molecule. These antibodies did not cross-react with any of the other peptides tested. Two further monoclonal antibodies (23.4A1, 22.3A6) reacted with the C-terminal third of the glucagon molecule and showed a cross-reaction with the structurally related gastric inhibitory polypeptide of 0.7% and 9.1%, respectively. All but the C-terminal monoclonal antibody 23.8B6 showed a marked cross-reaction with ileal extracts. The N-terminally directed monoclonal antibody 23.6B4 was of sufficient avidity for use in the radioimmunoassay of pancreatic glucagon and gut glucagon-like immunoreactivity in tissue extracts, being sensitive to changes of pancreatic glucagon of 2.0 fmol/tube at a final titer of culture supernatant of 1:1.4 X 10(5). In gel permeation chromatography of intestinal extracts, two major peaks were detectable (Kav 0.27 and 0.54). The present findings show that monoclonal antibodies provide sensitive tools for detecting pancreatic glucagon and gut glucagon-like immunoreactivity. They will be valuable immunoreactants for the development of immunoradiometric assays as well as for large-scale immunoaffinity purification of gut glucagon-like immunoreactivity.

Molecular forms of human enteroglucagon in tissue and plasma: plasma responses to nutrient stimuli in health and in disorders of the upper gastrointestinal tract.

A means of estimating human enteroglucagon (glucagon-like immunoreactivity of intestinal origin) in tissues and plasma is described, based on the subtraction of RIA values obtained with the C-terminal-directed glucagon antiserum RCS5 from the total glucagon-like immunoreactivity determined with the N-terminal- to midmolecule-directed glucagon antiserum R59. Gel filtration on Sephadex G-50 of human plasma and extracts of normal human intestine separated the R59 immunoreactivity into three peaks: a small peak of void volume material, a major peak coeluting with porcine glicentin, and a smaller peak coeluting with pancreatic glucagon. No RCS5 immunoreactivity was detected in the human gut, except for a small amount constituting less than 2% of the total glucagon-like immunoreactivity in the ileum and rectum only. In extracts of human pancreas, the chromatographic profiles obtained with RCS5 and R59 assays differed from the intestinal patterns, but were identical to each other, giving no evidence of a significant amount of pancreatic R59 immunoreactivity that was not also reactive with RCS5. Chromatography of plasmas from healthy subjects and patients with dumping syndrome, active coeliac disease, and tropical sprue showed that only the second major peak of R59 immunoreactivity reflected the basal or postnutrient increases in the plasma enteroglucagon concentration. In patients with exaggerated enteroglucagon release, the rise was again found to be entirely due to an increase in this peak of immunoreactivity. This major molecular form of human enteroglucagon, similar in size to porcine glicentin, is, thus, the form most likely to be of physiological and pathophysiological significance.

Exaggerated response of plasma glucagon-like immunoreactivity (GLI) to oral glucose in patients with reactive hypoglycemia.

In order to study the pathogenesis of reactive hypoglycemia, the responses of plasma glucose, IRI, glucagon immunoreactivity (GI) and total glucagon-like immunoreactivity (GLI) to 100 g oral glucose load were investigated in twenty-six patients of normal weight with reactive hypoglycemia. Of these patients, nineteen exhibited a diabetic OGTT curve. The findings in these patients were compared to normal control subjects (N = 20) and to disease-matched patients controls (N = 43). The psychological status was assessed by Cornell Medical Index Health Questionnaire in most of the subjects, who also received an x ray examination of the upper gastrointestinal tract. In addition, IVGTT was performed in the hypoglycemic patients. No apparent difference in plasma IRI response to oral glucose was observed between the hypoglycemic patients and their respective controls. Plasma total GLI concentrations were significantly increased during OGTT in both hypoglycemic groups. Following an oral glucose load, plasma GI levels were suppressed in the hypoglycemic groups to an extent similar to that in the control despite an apparent fall in their plasma glucose levels to the hypoglycemic range in the former. Radiological alterations in the upper gastrointestinal tract; deformity of the duodenal cap, gastric and/or duodenal ulcer, were found more frequently in the hypoglycemic groups. However, no characteristic change in personality was noticed in the patients. During IVGTT, neither plasma glucose nor total GLI level of the hypoglycemics differed from that of each control. The pathogenic factors responsible for reactive hypoglycemia will be discussed.

Effect of acetylcholine on the secretion of gut glucagon immunoreactivity and gut glucagon-like immunoreactivity in pancreatectomized dogs.

Effect of the infusion of acetylcholine on the secretion of gut glucagon immunoreactivity (gut GI) that was measured using C-terminal specific glucagon antiserum after pancreatectomy, and gut glucagon-like immunoreactivity (gut GLI) that was obtained by subtracting GI from total glucagon-like immunoreactivity (total GLI) which was measured using non-specific glucagon antiserum, was investigated in sixteen pancreatectomized dogs untreated with insulin, in order to demonstrate whether the secretion of gut GI and gut GLI is influenced by the parasympathetic nervous system. During the infusion of acetylcholine at a rate of 10 microM/kg/min, gut GI in the femoral venous blood showed a significant increase from the basal value of 181 +/- 22 pg/ml to a maximum of 569 +/- 107 pg/ml at 30 min (p less than 0.01), and "true gut GI secretion increment" in the portal venous blood showed a maximum significant increase of 916 +/- 144% at 30 min from the basal value (p less than 0.001). However, gut GLI showed no significant change. One shot administration of atropine at a rate of 15 micrograms/kg could significantly inhibit the stimulatory effect of acetylcholine on gut GI (p less than 0.05--0.001). It is concluded that the parasympathetic nervous system might play an important role in the control mechanism of the release of gut GI, but not of gut GLI in pancreatectomized dogs untreated with insulin.

Half life of gastrointestinal glucagon-like immunoreactive materials.

The composition, half life and hyperglycemic action of the porcine gastrointestinal glucagon-like immunoreactive materials were examined. Glucagon immunoreactivity (GI) measured using specific antiglucagon serum was more abundunt in the extract from the gastric fundus than in the one from the small intestine. When the extract from the gastric fundus was injected in dogs, the half life (T1/2) of total glucagon-like immunoreactivity (total GLI) measured using nonspecific antiglucagon serum was 9.5 +/- 1.1 min (mean +/- SEM), which was longer than that of crystalline pancreatic glucagon, 3.4 +/- 0.2 min, but shorter than that of the extract from the small intestine, 15.9 +/- 1.3 min. On the other hand, T1/2 for GI from the gastric fundus was 5.1 +/- 0.9 min, which was not significantly different from that of crystalline pancreatic glucagon. Blood sugar levels were significantly increased from the basal by 25 +/- 4 mg/100 ml at 10 min and 19 +/- 4 mg/100 ml at 15 min following an injection of the extract from the gastric fundus, but such a change in blood sugar levels was not demonstrated when the extract from the small intestine was injected. These results suggest that GI of the gastric fundus is close to pancreatic glucagon in respect of its metabolism and hyperglycemic activity.

Lack of gastrointestinal glucagon response to hypoglycaemia in depancreatized dogs.

Fasting (24 h) normal dogs and depancreatized dogs were injected intravenously with highly purified porcine insulin (Actrapid) in the doses of 0.2 U/kg and 0.5 U/kg, respectively. Blood glucose decreased from 152 +/- 41 (SEM) mg/100 ml to 39 +/- 7 mg/100 ml in the depancreatized dog and from 95 +/- 3 mg/100 ml to 42 +/- 4 mg/100 ml in the normal animal. Using a specific antiserum for "pancreatic" glucagon, the circulating level of glucagon immunoreactivity did not rise from the basal value of 247 +/- 31 pg/ml in the depancreatized group whereas it rose significantly from 223 +/- 24 pg/ml to 321 +/- 41 pg/ml in the normal group. In contrast intravenous infusion of 7 g of arginine increased "pancreatic" glucagon immunoreactivity in both groups. Thus, extrapancreatic glucagon of the pancreatic type does not respond to hypoglycaemia but to arginine infusion.