Jejunal control of glucose homeostasis in the human body

Abstract: Background: The prevalence of obesity and type-2 diabetes mellitus (T2DM) have risen dramatically over the last decades, and are major threats to the human health. Obesity, and also T2DM, is treated with lifestyle modifications, medications or by bariatric surgery, with varying success. Recent findings demonstrate that the improved glucose homeostasis following bariatric surgery is partially weight-loss independent. Improvements in glucose homeostasis and incretin secretion profiles are seen already before the patients leave the operating hospital after Roux-en-Y gastric bypass (RYGB) surgery. Therefore, it has been speculated that this improvement is induced by the anatomical reconfiguration of the gut. The exact mechanism for this has been elusive. The overall aim of this thesis was to investigate how jejunum, and the expression of different proteins in the jejunum, helps regulate glucose homeostasis in the human body. Methods: In Paper I, global proteomics was used on jejunal mucosa biopsies in patients undergoing RYGB surgery, and biopsies retrieved from the Roux limb 6-8 months after surgery to search for major regulations in the proteome. The biological functions of the proteomics findings were further studied in vivo in mice and in vitro in murine primary jejunal enteroendocrine cells (EECs). In Paper II and III, the effect of two weeks of iso-caloric high-fat diet (HFD) and high-carbohydrate diet (HCD) were assessed in healthy and normal weight volunteers in a cross-over design. For Paper II, a mixed meal test (MMT) with sequential blood sampling was performed at end of each dietary period to examine the glucose homeostasis. Metabolomics was also used to explore the effect of each dietary period on metabolite profiles. For Paper III, jejunal mucosa biopsies were retrieved following each dietary period, and the protein expression was assessed with western blot while functional characteristics of glucose transport were evaluated with Ussing chambers. The findings from the biopsy material were further studied in vitro in Caco-2 cells. In Paper IV, in vitro cultures of murine GLUTag cells and differentiated human jejunal enteroid monolayers were used to study the effect of the ketone body β-hydroxybutyrate (βHB) on glucose-induced GLP-1 secretion. Results: From global proteomics analysis, it was shown that the ketogenesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) is drastically down-regulated in jejunum after RYGB. Furthermore, prolonged HFD in mice increased the jejunal expression of the same enzyme. In the diet study, MMT resulted in similar glucose and insulin profiles, while the secreted levels of GLP-1 were higher after HFD. The HFD also increased the levels of several metabolites, i.e. valine, leucine and creatine, that has previously been shown to be indicators of early insulin resistance. Evaluation of the jejunal mucosa revealed that HFD decreased the expression of sodium-glucose linked transporter 1 (SGLT1) and the acetylation of histone 3 at lysine 9, while the expression of HMGCS2 was increased. In vitro studies in Caco-2 cells stipulate a sirtuin dependent regulation of SGLT1 expression, induced by jejunal ketogenesis. The ketone body βHB had significant inhibitory effect on glucose-induced GLP-1 secretion in murine primary jejunal EECs, in GLUTag cells and in differentiated human jejunal enteroid monolayers. Protein expression analysis displayed an increased phosphorylation of kinase Akt as well as expression of kinase ERK1/2 after addition of βHB to GLUTag cells. Conclusions: The results from this doctoral thesis display an interesting role of intestinal ketogenesis. The rate-limiting enzyme of ketogenesis HMGCS2, and therefore intestinal ketogenesis, is induced by a prolonged fat-dominated diet, and is almost completely abolished following RYGB surgery. The results also display a capacity of the jejunal ketogenesis to influence glucose homeostasis both by inhibition of GLP-1 secretion from EECs and by regulation of jejunal glucose transporters.