Epigenetic dysregulation may influence disease progression. Here we explore whether epigenetic alterations in human pancreatic islets impact insulin secretion and type 2 diabetes (T2D). In islets, 5,584 DNA methylation sites exhibit alterations in T2D cases versus controls and are associated with HbA1c in individuals not diagnosed with T2D. T2D-associated methylation changes are found in enhancers and regions bound by β-cell-specific transcription factors and associated with reduced expression of e.g. CABLES1, FOXP1, GABRA2, GLR1A, RHOT1, and TBC1D4. We find RHOT1 (MIRO1) to be a key regulator of insulin secretion in human islets. Rhot1-deficiency in β-cells leads to reduced insulin secretion, ATP/ADP ratio, mitochondrial mass, Ca²⁺, and respiration. Regulators of mitochondrial dynamics and metabolites, including L-proline, glycine, GABA, and carnitines, are altered in Rhot1-deficient β-cells. Islets from diabetic GK rats present Rhot1-deficiency. Finally, RHOT1methylation in blood is associated with future T2D. Together, individuals with T2D exhibit epigenetic alterations linked to mitochondrial dysfunction in pancreatic islets.

Glucagon has long been defined by its glucogenic action and as a result α-cells have been characterised based largely on their interaction with glucose. Recent findings have challenged this preconception, bringing to the fore the significant role glucagon plays in amino acid breakdown and underlining the importance of amino acids in glucagon secretion. The challenge that remains is defining the mechanism that underlie these effects - understanding which amino acids are most important, how they act on the α-cell and how their actions integrate with other fuels such as glucose and fatty acids. This review will describe the current relationship between amino acids and glucagon and how we can use this knowledge to redefine the α-cell.

Aim: MicroRNAs (miRNAs) regulate β‐cell function, and β‐cell mitochondria and insulin secretion are perturbed in diabetes. We aimed to identify key miRNAs regulating β‐cell mitochondrial metabolism and novel β‐cell miRNA‐mitochondrial pathways.Methods: TargetScan (http://www.targetscan.org/) was used to predict if 16 miRNAs implicated in β‐cell function target 27 cis‐eGenes implicated in mitochondrial activity. The expression of candidate miRNAs and insulin secretion after 24 and 1 h pre‐incubation in 2.8, 11.1‐ and 16.7‐mM glucose was measured in clonal INS‐1 832/13 β‐cells. MiR‐29 silenced INS‐1 832/13 cells were assessed for insulin secretion (glucose, pyruvate, and K⁺), target cis‐eGene expression (Ndufv3 and Ndufa10 components of mitochondrial complex I (CI)), OXPHOS (CI‐V) protein expression, and mitochondrial OXPHOS respiration/activity. The expression of differentially expressed miR‐29 miRNAs was evaluated in Goto‐Kakizaki (GK) rat, db/db mouse and type 2 diabetic (T2D) human islets, as well as NMRI mouse islets cultured under glucolipotoxic conditions.Results: MiR‐29, miR‐15 and miR‐124 were predicted to regulate ~20 cis‐eGenes, while miR‐29 alone was predicted to regulate ≥12 of these in rat and human species. MiR‐29 expression and insulin secretion were reduced in INS‐1 832/13 cells after 24 h in elevated glucose. MiR‐29 knockdown increased all tested insulin secretory responses, Nudfv3, Ndufa10, complex I and II expression, and cellular mitochondrial OXPHOS. MiR‐29 expression was reduced in db/db islets but increased in GK rat and T2D human islets.Conclusion: We conclude miR‐29 is a key miRNA in regulating β‐cell mitochondrial metabolism and insulin secretion via underlying miR‐29‐OXPHOS complex pathways. Furthermore, we infer reduced miR‐29 expression compensatorily enhances insulin secretion under glucotoxicity.

Aims/hypothesis: Roux-en-Y gastric bypass surgery (RYGB) frequently results in remission of type 2 diabetes as well as exaggerated secretion of glucagon-like peptide-1 (GLP-1). Here, we assessed RYGB-induced transcriptomic alterations in the small intestine and investigated how they were related to the regulation of GLP-1 production and secretion in vitro and in vivo. Methods: Human jejunal samples taken perisurgically and 1 year post RYGB (n=13) were analysed by RNA-seq. Guided by bioinformatics analysis we targeted four genes involved in cholesterol biosynthesis, which we confirmed to be expressed in human L cells, for potential involvement in GLP-1 regulation using siRNAs in GLUTag and STC-1 cells. Gene expression analyses, GLP-1 secretion measurements, intracellular calcium imaging and RNA-seq were performed in vitro. OGTTs were performed in C57BL/6j and iScd1^−/− mice and immunohistochemistry and gene expression analyses were performed ex vivo. Results: Gene Ontology (GO) analysis identified cholesterol biosynthesis as being most affected by RYGB. Silencing or chemical inhibition of stearoyl-CoA desaturase 1 (SCD1), a key enzyme in the synthesis of monounsaturated fatty acids, was found to reduce Gcg expression and secretion of GLP-1 by GLUTag and STC-1 cells. Scd1 knockdown also reduced intracellular Ca²⁺ signalling and membrane depolarisation. Furthermore, Scd1 mRNA expression was found to be regulated by NEFAs but not glucose. RNA-seq of SCD1 inhibitor-treated GLUTag cells identified altered expression of genes implicated in ATP generation and glycolysis. Finally, gene expression and immunohistochemical analysis of the jejunum of the intestine-specific Scd1 knockout mouse model, iScd1^−/−, revealed a twofold higher L cell density and a twofold increase in Gcg mRNA expression. Conclusions/interpretation: RYGB caused robust alterations in the jejunal transcriptome, with genes involved in cholesterol biosynthesis being most affected. Our data highlight SCD as an RYGB-regulated L cell constituent that regulates the production and secretion of GLP-1. Graphical

Biological tissue consists of populations of cells exhibiting different responses to pharmacological stimuli. To probe the heterogeneity of cell function, we propose a multiplexed approach based on real‐time imaging of the secondary messenger levels within each cell of the tissue, followed by extraction of the changes of single‐cell fluorescence over time. By utilizing a piecewise baseline correction, we were able to quantify the effects of multiple pharmacological stimuli added and removed sequentially to pancreatic islets of Langerhans, thereby performing a deep functional profiling for each cell within the islet. Cluster analysis based on the functional profile demonstrated dose‐dependent changes in statistical inter‐relationships between islet cell populations. We therefore believe that the functional cytometric approach can be used for routine quantitative profiling of the tissue for drug screening or pathological testing.

Aims: Survodutide is a novel long‐acting dual glucagon (GCGR)/glucagon‐like peptide‐1 (GLP‐1R) receptor agonist currently in phase‐3 clinical development for the treatment of people living with obesity and metabolic dysfunction‐associated steatohepatitis. Emerging evidence suggests impaired renal GCGR function in chronic kidney disease (CKD), including diabetic kidney disease (DKD). This study aimed to characterize the effects of survodutide in a translational mouse model of advanced DKD, induced by adeno‐associated virus‐mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) diabetic db/db mice. Materials and Methods: Female obese and diabetic db/db C57BKS mice received a single intravenous dose of ReninAAV 1 week prior to UNx. Three weeks post‐UNx, db/db UNx‐ReninAAV mice were administered (s.c., q.d.) vehicle or survodutide (7.5 nmol/kg) for 10 weeks. Endpoints included blood/urine biochemistry, histological markers of glomerulosclerosis, tubular injury and inflammation, as well as quantitative 3D whole‐pancreas and whole‐kidney imaging. Results: Compared to vehicle controls, survodutide reduced body weight, hyperglycemia, and HbA1c levels concurrent with improved hyperinsulinemia and β‐cell islet volume distribution. Notably, survodutide promoted substantial benefits on advanced albuminuria, severe glomerulosclerosis, kidney hypertrophy and inflammation. 3D whole‐kidney imaging revealed that survodutide significantly reduced kidney and glomerular hypertrophy while increasing glomerular density and improving glomerular sphericity. Survodutide reduced liver weight and plasma amino acid levels, including alanine, confirming hepatic glucagon receptor activation. Conclusions: Survodutide markedly improved metabolic and renal outcomes in the db/db UNx‐ReninAAV mouse model of advanced DKD, supporting further investigation into therapeutic options of survodutide in chronic kidney diseases.