👤 Yusman Manchanda

Internalisation of G protein-coupled receptors (GPCRs) can contribute to altered cellular responses by directing signalling from non-canonical locations, such as endosomes. If signalling processes are locally constrained, active receptors in different subcellular locations could produce different downstream effects. This phenomenon may be relevant to the optimal targeting of the glucagon-like peptide-1 receptor (GLP-1R), a type 2 diabetes and obesity target GPCR for which several ligands with varying internalisation tendency have been discovered. To investigate, we compared the signalling localisation effects of two prototypical GLP-1RAs with opposite signal bias and effects on GLP-1R trafficking: exendin-asp3 (ExD3), a full agonist that drives rapid internalisation, and exendin-phe1 (ExF1), which shows much slower internalisation. After using bioorthogonal labelling and fluorescent agonist conjugates to verify the divergent trafficking patterns of ExF1 and ExD3 in β-cell lines and primary pancreatic islets, we used live cell biosensors to monitor signalling at different subcellular locations. This revealed that cAMP/PKA/ERK signalling in β-cells is in fact distributed widely across the cell over short- (<5 min) and medium-term (up to 60 min) stimulation at pharmacological (>10 pM) concentrations, with no major differences in signal localisation that could be linked to internalised versus cell surface-bound GLP-1R. Moreover, washout experiments highlighted that, whilst fast-internalising ExD3 shows much greater accumulation and binding to GLP-1R in endosomes than slow-internalising ExF1, it is a rather inefficient driver of both cAMP production in β-cells and insulin secretion from perfused rat pancreata. These data provide a greater understanding of the cellular effects of biased GLP-1R agonism. Show less

The use of incretin analogues has emerged as an effective approach to achieve both enhanced insulin secretion and weight loss in Type 2 diabetes (T2D) patients. Agonists which bind and stimulate multiple receptors have shown particular promise. However, off-target effects remain a complication of using these agents, and modified versions with optimised pharmacological profiles and/or biased signalling are sought. Ligand synthesis was achieved using standard solid-phase techniques. Assessments of GLP-1R-binding kinetics, G protein recruitment and receptor internalisation were performed using biochemical and imaging approaches. Insulin secretion was measured in purified mouse and human islets, and drug efficacy was assessed in hyperglycaemic db/db mice. We describe the synthesis and properties of a molecule which binds to both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors (GLP-1R and GIPR) to enhance insulin secretion. HISHS-2001 shows increased affinity at the GLP-1R, as well as a tendency towards reduced internalisation and recycling at this receptor versus FDA-approved dual GLP-1R/GIPR agonist tirzepatide. HISHS-2001 also displayed significantly greater bias towards cAMP generation versus β-arrestin 2 recruitment compared to tirzepatide. In contrast, G HISHS-2001 represents a novel dual receptor agonist with a promising pharmacological profile and actions. Future clinical studies will be needed to assess the safety and efficacy of this molecule in humans. Show less

The use of incretin analogues has emerged in recent years as an effective approach to achieve both enhanced insulin secretion and weight loss in type 2 diabetes (T2D) patients. Agonists which bind and stimulate multiple receptors have shown particular promise. However, off target effects, including nausea and diarrhoea, remain a complication of using these agents, and modified versions with optimized pharmacological profiles and/or biased signaling at the cognate receptors are increasingly sought. Here, we describe the synthesis and properties of a molecule which binds to both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors (GLP-1R and GIPR) to enhance insulin secretion. HISHS-2001 shows increased affinity at the GLP-1R, as well as a tendency towards reduced internalization and recycling at this receptor Show less

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology, following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking, and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal vs plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function. Show less

Various population studies have reported the association of rare S2 allele of apolipoprotein C3 (APOC3) SstI polymorphism with hypertriglyceridemia (HTG) and coronary artery disease (CAD). We were the first to report an association of S2 allele with high triglyceride (TG) levels in healthy volunteers from Northern India. Since HTG is suggested to be a predominant risk factor for CAD among Indians, we have elucidated the relationship of APOC3 SstI polymorphism with the lipid profile and CAD. A total of 158 patients with > or = 70% stenosis in one or more coronary artery (angiographically proven CAD patients), 35 subjects with < 70% stenosis (NCAD) and 151 normal controls (free of heart disease) from Northern plains of India were recruited in the study. DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Lipid profile was estimated by enzymatic kit. We found a strong association of S2 allele with high TG levels, which was more significant in patients. Prevalence of S2 allele in normal controls and CAD patients were comparable, despite the fact that mean TG level was significantly higher in patients. A greater insight into this observation revealed that the prevalence of high TG, if not coupled with other risk factors (like high total cholesterol, low HDL), was comparable in patients and controls. Thus, our study reveals that rare S2 allele may be employed as a susceptibility marker for high TG. However, high TG or S2 allele alone may not contribute to the etiology of CAD. Show less

Several studies including a small case-control (hypertriglyceridemic/normotriglyceridemic individuals) study by us revealed close association between rare S2 allele ofAPOC3 Sstl polymorphism and hypertriglyceridemia. With the understanding that Asian Indians are highly vulnerable to the adverse effects of hypertriglyceridemia, we extended the investigation and studied the frequency distribution of this polymorphism in 216 healthy volunteers from Northern plains of India. We found that more than 50% of the study population had one or two S2 allele. This may suggest that a larger fraction of this population is genetically predisposed to hypertriglyceridemia. Show less

A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India. DNA samples were analyzed by polymerase chain reaction (PCR) followed by SstI digestion. Digested PCR products were run on 3% agarose gel and visualized by ethidium bromide staining. Rare S2 allele was highly prevalent in our study population (0.313) as compared to the Caucasians (0.00-0.11). The genotypic distribution was in agreement with Hardy-Weinberg equilibrium. S2 allele was almost two times more prevalent in the HTG group (N = 34) as compared to NTG group (N = 105) (p = 0.001). Multiple logistic regression revealed S1S2 individuals had age-adjusted odds ratio of 2.43 (95%CI = 0.99-6.01, p = 0.054) and S2S2 had 9.9 (95%CI = 2.66-37.29, p = 0.0006) for developing HTG in comparison to S1S1 genotype. Our study shows a significant association between rare S2 allele and HTG in Asian Indians. Show less