Biology subjects

Rutter, G. A.

Publications and source records attributed to Rutter, G. A..

42 records · Page 3Linked to original sources

bioRxiv2019

Differences in signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide

Background and purposeAmino acid substitutions at the N-termini of glucagon-like peptide-1 receptor agonist (GLP-1RA) peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here we aimed to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1RAs exendin-4 and lixisenatide lead to similar phenomena. We also sought to establish the impact of the C-terminus on signal bias resulting from modifications elsewhere in the molecule. Experimental approachExendin-4, lixisenatide, and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand, and their biased derivatives, were assessed in mice. Key resultsLixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was 5-fold less potent for cAMP signalling. Both peptides were rapidly endocytosed, but the GLP-1R recycled more slowly to the plasma membrane after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice. N-terminal substitutions to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. Conclusion and implicationsChanges to the C-terminus of exendin-4 affect signalling potency and GLP-1R trafficking via mechanisms unrelated to GLP-1R occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.

pharmacology and toxicology↗

bioRxiv2019

β-cell dedifferentiation is associated with epithelial-mesenchymal transition triggered by miR-7-mediated repression of mSwi/Snf complex

{beta}-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. However, little is known on the genetic and epigenetic changes linked with the dedifferentiation of {beta}-cells. We now report that {beta}-cell dedifferentiation is associated with epithelial to mesenchymal transition (EMT) triggered by miR-7-mediated repression of Smarca4/Brg1 expression, a catalytic subunit of the mSwi/Snf chromatin remodeling complexes essential for {beta}-cell transcription factors ({beta}-TFs) activity. miR-7-mediated repression of Brg1 expression in diabetes causes an overall compaction of chromatin structure preventing {beta}-TFs from accessing and transactivating genes maintaining the functional and epithelial identity of {beta}-cells. Concomitantly, loss of {beta}-cell identity impairs the ability of {beta}-TFs Pdx1, Nkx6-1, Neurod1 to repress non-{beta}-cell genes enriched selectively in mesenchymal cells leading to EMT, change in islet microenvironment, and fibrosis. Remarkably, anti-EMT agents normalized glucose tolerance of diabetic mice, thus revealing mesenchymal reprogramming of {beta}-cells as a novel therapeutic target in diabetes. This study sheds light on the genetic signature of dedifferentiated {beta}-cells and highlights how loss of mSwi/Snf activity in diabetes initiating a step-wise remodeling of epigenetic landscapes of {beta}-cells leading to the induction of an EMT process reminiscent of a response to tissue injury.

genomics↗

bioRxiv2019

The mitochondrial Ca2+ uniporter MCU is required for normal glucose-stimulated insulin secretion in vitro and in vivo

Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic {beta}-cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in {beta}-cells has not previously been examined in vivo. Here, we inactivated the pore-forming subunit MCUa (MCU) selectively in the {beta}-cell in mice using Ins1Cre-mediated recombination. Glucose-stimulated mitochondrial Ca2+ accumulation, ATP production and insulin secretion were strongly (p<0.05 and p<0.01) inhibited in MCU null animals ({beta}MCU-KO) in vitro. Interestingly, cytosolic Ca2+ concentrations increased (p<0.001) whereas mitochondrial membrane depolarisation improved in {beta}MCU-KO animals. Male {beta}MCU-KO mice displayed impaired in vivo insulin secretion at 5 (p<0.001) but not 15 min. post intraperitoneal (IP) injection of glucose while the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p<0.05) in young {beta}MCU-KO (<12 weeks), but not older animals. We conclude that MCU is crucial for mitochondrial Ca2+ uptake in pancreatic {beta}-cells and is required for normal GSIS. The apparent compensatory mechanisms which maintain glucose tolerance in {beta}MCU-KO mice remain to be established.

physiology↗

bioRxiv2019

Vertical sleeve gastrectomy lowers kidney SGLT2 expression in the mouse

BackgroundBariatric surgery has been established to improve insulin sensitivity and glucose clearance, but also increases insulin and glucagon secretion. Each of the above effects have also been observed following treatment with sodium glucose co-transporter 2 (SGLT2) inhibitors.\n\nAimTo determine whether there is an effect of bariatric surgery (Vertical Sleeve Gastrectomy; VSG) on renal SGLT2 expression in mice.\n\nMethodsEighteen lean mice underwent VSG (n=8) or sham (n=9) surgery. Glucose tolerance tests with or without treatment with the SGLT2 inhibitor dapagliflozin were performed four weeks post operatively, in order to assess if pharmacological SGLT2 inhibition has the same euglycemic effects after bariatric surgery. Kidneys were harvested from fed mice and SGLT2 expression was analysed using Quantitative reverse-transcription PCR and immunofluorescence.\n\nResultsVSG mice displayed significantly improved glucose tolerance (AUC=103{+/-}6.8; AUC=66.6{+/-}2.9 in control and VSG mice, respectively; p<0.001), despite an absence of significant weight loss when compared to sham operated mice (p=0.37, Mann-Whitney test). Treatment of sham-operated mice with dapagliflozin (10 mg/kg) improved glucose tolerance. In contrast, dapagliflozin did not further improve glucose tolerance in VSG-operated mice. Moreover, qRT-PCR and immunofluorescence analysis on mouse kidneys demonstrated a significant lowering of SGLT2 expression at both the mRNA (n=7, p<0.0001) and protein (n=5, p=0.0007) levels four weeks after VSG.\n\nConclusionsVertical sleeve gastrectomy in lean animals causes a significant inhibition of SGLT2 expression in the kidney cortex. These findings are in line with our previous results on the effects of Duodenal Jejunal Bypass in lean rats, and point towards a physiologically-relevant gut-kidney axis. SGLT2 inhibition may thus be an important mechanism through which bariatric surgery improves glucose tolerance in man.

physiology↗

bioRxiv2018

Agonist binding affinity determines palmitoylation of the glucagon-like peptide-1 receptor and its functional interaction with plasma membrane nanodomains in pancreatic beta cells

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes and obesity, is known to undergo palmitoylation by covalent ligation of an acyl chain to cysteine 438 in its carboxyl-terminal tail. Work with other GPCRs indicates that palmitoylation can be dynamically regulated to allow receptors to partition into plasma membrane nanodomains that act as signaling hotspots. Here, we demonstrate that the palmitoylated state of the GLP-1R is increased by agonist binding, leading to its segregation and clustering into plasma membrane signaling nanodomains before undergoing internalization in a clathrin-dependent manner. Both GLP-1R signaling and trafficking are modulated by strategies targeting nanodomain segregation and cluster formation, including depletion of cholesterol or expression of a palmitoylation-defective GLP-1R mutant. Differences in receptor binding affinity exhibited by biased GLP-1R agonists, and modulation of binding kinetics with the positive allosteric modulator BETP, influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

cell biology↗

bioRxiv2018

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion

A rare loss-of-function variant p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8) enriched in Western Finland protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, especially compared with individuals matched for the genotype of a common T2D risk variant in SLC30A8, p.Arg325. In genome-edited human IPS-derived {beta}-like cells, we establish that the p.Arg138* variant results in reduced SLC30A8 expression due to haploinsufficiency. In human {beta}-cells loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function, which was also seen in isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aiming at maintaining insulin secretion capacity in T2D.

genetics↗