Investigating function and evolution of genes and proteins involved in metabolic control in mammals

Abstract

The duck-billed platypus and the short-beaked echidna represent the most basal lineage of living mammals and therefore provide important information about mammalian evolution. Monotremes have also undergone remarkable anatomical, physiological and genetic changes. One of the most radical changes involves the monotreme digestive system and genes associated with metabolic control. I have investigated several genes that are vital for metabolic control (specifically genes in the ghrelin and incretin pathways) as well as the histology of the monotreme pancreas. Given the glandless gut in monotremes, I first sought to investigate genes in the appetite regulating ghrelin pathway. Surprisingly, I discovered that genes encoding ghrelin and ghrelin O-acyl transferase (GOAT) are missing in the platypus and echidna genome, whilst, its receptor, growth hormone secretagogue receptor 1a (GHS-R 1a) is present. This is the first report suggesting the loss of ghrelin in a mammal. The conservation of the ghrelin receptor gene despite the lack of the ghrelin and GOAT genes in platypus suggests that another ligand maybe acting via this receptor in monotremes (Chapter 2). Ghrelin is expressed in human pancreatic ε-cells. Hence the lack of ghrelin in platypus led us to investigate in more detail the structure of the monotreme pancreas, another key organ in both metabolic control and digestion (Chapter 3). Generally, the monotreme pancreata share the basic characteristics of other mammalian pancreata, including both endocrine islets and exocrine acini. I performed immunohistochemical analysis to reveal the detailed architecture of the platypus and echidna endocrine islets of Langerhans. The unique phenotypes of the PP-lobe, smaller size of islets, and the abundance of α-cells indicate the monotreme pancreata have more resemblance to that of birds and marsupials than eutherian species. One of the key functions of the pancreas is to release insulin upon food intake. Glucagon-like peptide 1 (GLP-1), a hormone released from the small intestine upon food intake, triggers insulin release via the GLP-1 receptor (GLP-1R) in β-cells of the pancreas. In human, GLP-1 is rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4) and thereby has a very short serum half-life (<2 min). Searching for longacting GLP-1 analogs to improve insulin sensitivity has been a key strategy in Type 2 diabetes (T2D) treatment. We identified and characterised Glp-1, Glp-1r and Dpp-4 and found both Glp-1 and Dpp-4 are expressed in gut and pancreas as expected, and interestingly also in venom. Importantly, evolutionary changes in monotreme GLP-1 sequences led us to predict that it would be resistant to enzymatic degradation. Extensive biochemical analysis of monotreme GLP-1 revealed that this variant is in fact resistant to DDP-4 degradation, however can be degraded by other enzymes (trypsinlike) in their own sera. Moreover, we demonstrated that monotreme GLP-1s can bind and activate both pGLP-1R and hGLP-1R with similar potency and stimulate insulin release in isolated mouse islets (Chapter 4). Together this work provides fundamental new insights into the molecular and anatomical characteristics of the monotreme digestive system, the evolution of metabolic control and potential novel avenues for diabetes treatment based on monotreme GLP-1.