A new report in Nature Medicine (Finan et al. 2014) describes a synthetic peptide agonist capable of binding three different hormone receptors that are key regulators of metabolism. The triagonist peptide reduced body weight, improved glycemic control, reversed hepatic steatosis and maintained lean body mass in several rodent models of human type 2 diabetes.
Hormonal Control of Metabolism
The peptide engineered by these investigators was designed to act as an agonist mimicking the action of three different hormones. The first, glucagon-like peptide-1 (GLP1), is an incretin made by intestinal L cells and is a potent regulator of blood glucose. GLP1 stimulates insulin secretion, inhibits glucagon secretion, and decreases food intake by improving satiety. It also stimulates insulin-producing β-cell proliferation and differentiation and inhibits apoptosis. The second, glucagon (GCG), is made by the alpha cells of the pancreas. GCG stimulates hepatocytes to convert glycogen to glucose and therefore increases glucose in the blood. The third, glucose-dependent insulinotrophic polypeptide (a.k.a. gastric inhibitory peptide, GIP), is also an incretin. GIP is made by the K cells of the intestine and stimulates insulin secretion as well as fatty acid metabolism through lipoprotein kinase activity in adipocytes. The report by Finan et al. demonstrates these three components work together to improve insulin sensitivity and metabolic processing of glucose.
In prior studies, these researchers showed significantly improved glycemic control using two different biagonist peptides. One peptide had GLP1 and GCG activity and a second had GLP1 and GIP activity. These observations led to the hypothesis that a peptide could be engineered to have activity for all three pathways. Ultimately, a triagonist was synthesized with triple human receptor activity as well as activity in mice, rats, and monkeys. The structure of the molecule contained a single binding moiety capable of recognition by all three receptors without cross-reaction with other G-protein coupled receptors.
Target Validation In Vitro and In Vivo
Validation of the triagonist activity was performed by measuring production of cAMP from cell lines that each express one of the appropriate receptors and associated metabolic pathways. The researchers used the mouse pancreatic β-cell line MIN6 which has high expression of Glp-1r, mouse 3T3-L1 adipocytes that express Gipr, and rat hepatocytes with high Gcgr expression. The triagonist was found to bind each receptor with high affinity and activate the respective metabolic pathways. The peptide was also screened against >70 other receptors to determine if it contained cross reactivity, and none was identified. These results showed the triagonist had high specificity and functional stimulation of all three intended receptor pathways.
The in vivo validation was performed in part using the well-established C57BL/6J (000664) mouse model of diet induced obesity (B6 DIO) and pre-type 2 diabetes (Figure 1). B6 DIO mice are obese, have moderately elevated fed blood glucose, and are glucose intolerant (slowed ability to clear blood glucose when challenged). B6 DIO mice treated with the triagonist showed improved glucose tolerance (faster rate of blood glucose clearance after challenge) that could be blocked by pre-treatment of the mice with a GLPR antagonist. The specificity of the triagonist for GIPR was tested by placing C57BL/6J mice with a knockout mutation of Glp on a high fat diet (B6 Glp -/- DIO). The B6 Glp -/- DIO mice treated with the triagonist showed improved glucose tolerance that was blocked by pre-treatment with a GIPR antagonist. These results demonstrated the triagonist ability to improve glucose tolerance through GIPR independent of the GLPR pathway.
Validation of GCGR antagonism was performed using a different model: C57BL/6J (000664) mice treated with streptozotocin (B6 STZ) have hyperglycemia in the absence of metabolic and autoimmune abnormalities. STZ has a relatively specific toxicity for the insulin-producing pancreatic β cells, thus diminishing insulin production. B6 STZ mice treated with the triagonist show transient super-elevated blood glucose levels during glucose challenge in the glucose tolerance test that was inhibited by pre-treatment with a GCGR antagonist. This experiment demonstrated the ability of the triagonist peptide to stimulate the glucagon pathway even in the presence of systemic hyperglycemia.
Pre-Clinical Validation of Treatment for Type 2 Diabetes
Following target validation, the triagonist was tested in comparison to the coagonist (GLP1 and GIP specificity) in the B6 DIO mice for correction of type 2 diabetes phenotypes. Mice treated with the coagonist showed a 15.7% reduction in body weight and those treated with the triagonist showed a 26.6% reduction. The loss of body weight was due to elimination of fat stores specifically, because lean body mass in both treatment groups was maintained. In addition, both treatment groups showed a similar reduction in food intake (improved satiety), indicating the greater reduction in body weight with the triagonist was not simply due to a greater reduction in food intake. Both treatment groups also showed a similar reduction in blood glucose and improved glucose tolerance. In contrast, the triagonist group had lower serum insulin and improved HOMA-IR score (ratio of insulin release to blood glucose) than the coagonist group, indicating the triagonist stimulated greater insulin sensitivity. The triagonist also showed more significant reduction in total cholesterol and reduced hepatosteatosis. Taken together, these results show the triagonist enhanced overall energy expenditure and corrected a number of type 2 diabetes phenotypes.
Further pre-clinical validation of the triagonist was performed in the B6 db/db mouse model of type 2 diabetes and obesity, strain B6.BKS(D)-Leprdb/J (000697) (Figure 2). A spontaneous deletion of the leptin receptor in these mice leads to obesity, transient hyperglycemia, and glucose intolerance. B6 db/db mice were treated with vehicle, the coagonist (GLP1 and GIP specificity) or the triagonist. The coagonist showed a reduction in body weight, but the triagonist completely prevented the progressive obesity observed in untreated mice. Interestingly, there was no difference in food intake between vehicle and either agonist treatment group. Both agonists protected the mice from fasting hyperglycemia, but the triagonist showed a greater improvement in glucose tolerance than the coagonist. The triagonst also showed reduced alpha cell infiltration of islets, indicating alleviation of pancreatic architectural changes associated with type 2 diabetes.
Triple Agonist Therapy for Obesity and Diabetes
In conclusion, Finan et al. demonstrate the synthesis of an engineered small peptide with high specificity for and activation of three key molecular pathways that together significantly improve energy expenditure. The molecule showed efficacy in the improvement of multiple obesity and diabetes phenotypes commonly associated with type 2 diabetes. Importantly, the triagonist showed in vivo activity in two different mouse models of human disease as well as in diabetic Zucker rats (not discussed here), demonstrating cross species specificity. Further studies examining safety and toxicity will hopefully allow this exciting new treatment to move on to human clinical trials.