We have investigated the effects of prolonged exposure (24 h) to pro-inflammatory cytokines on β-cell metabolism and insulin secretion using clonal BRIN-BD11 β cells. Addition of IL-1β, tumour necrosis factor-α and IFN-γ (at concentrations that did not induce apoptosis) inhibited chronic (24 h) and acute stimulated levels of insulin release (by 59 and 93% respectively), increased cellular glucose and alanine consumption, and also elevated lactate and glutamate release. However, ATP levels and cellular triacylglycerol were decreased while glutathione was increased. We conclude that sub-lethal concentrations of pro-inflammatory cytokines appear to shift β-cell metabolism away from a key role in energy generation and stimulus–secretion coupling and towards a catabolic state which may be related to cell defence.

Bone is permanently remodeled by a complex network of local, hormonal, and neuronal factors that affect osteoclast and osteoblast biology. Among these factors, a role for gastrointestinal hormones has been proposed based on the evidence that bone resorption dramatically falls after a meal. Glucagon-like peptide-1 (GLP1) is one of these gut hormones, and despite several reports suggesting an anabolic effect of GLP1, or its stable analogs, on bone mass, little is known about the effects of GLP1/GLP1 receptor on bone strength. In this study, we investigated by three-point bending, quantitative X-ray microradiography, microcomputed tomography, qBEI, and FTIRI bone strength and bone quality in male Glp1r knockout (Glp1r KO) mice when compared with control WT animals. Animals with a deletion of Glp1r presented with a significant reduction in ultimate load, yield load, stiffness, and total absorbed and post-yield energies when compared with WT animals. Furthermore, cortical thickness and bone outer diameter were significantly decreased in deficient animals. The mineral quantity and quality were not significantly different between Glp1r KO and WT animals. On the other hand, the maturity of the collagen matrix was significantly reduced in deficient animals and associated with lowered material properties. Taken together, these data support a positive effect of GLP1R on bone strength and quality.

This study examined the effects of glucagon-like peptide-1 (GLP-1) on insulin secretion alone and in combination with sulphonylureas or nateglinide, with particular attention to K_ATP channel-independent insulin secretion. In depolarised cells, GLP-1 significantly augmented glucose-induced K_ATP channel-independent insulin secretion in a glucose concentration-dependent manner. GLP-1 similarly augmented the K_ATP channel-independent insulin-releasing effects of tolbutamide, glibenclamide or nateglinide. Downregulation of protein kinase A (PKA)- or protein kinase C (PKC)-signalling pathways in culture revealed that the K_ATP channel-independent effects of sulphonylureas or nateglinide were critically dependent upon intact PKA and PKC signalling. In contrast, GLP-1 exhibited a reduced but still significant insulin-releasing effect following PKA and PKC downregulation, indicating that GLP-1 can modulate K_ATP channel-independent insulin secretion by protein kinase-dependent and -independent mechanisms. The synergistic insulin-releasing effects of combinatorial GLP-1 and sulphonylurea/nateglinide were lost following PKA- or PKC-desensitisation, despite GLP-1 retaining an insulin-releasing effect, demonstrating that GLP-1 can induce insulin release under conditions where sulphonylureas and nateglinide are no longer effective. Our results provide new insights into the mechanisms of action of GLP-1, and further highlight the promise of GLP-1 or similarly acting analogues alone or in combination with sulphonylureas or meglitinide drugs in type 2 diabetes therapy.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid hormone secreted from intestinal K-cells, which exhibits a number of actions including stimulation of insulin release. A truncated form, GIP(1–30), has recently been demonstrated in intestine and islet α-cells. To evaluate the potential physiological significance of this naturally occurring form of GIP, the present study has examined and compared the bioactivity of enzymatically stabilised forms, [d-Ala²]GIP(1–30) and [d-Ala²]GIP(1–42), in high-fat fed mice. Twice-daily injection of GIP peptides for 42 days had no significant effect on food intake or body weight. However, non-fasting glucose levels were significantly lowered, and insulin levels were elevated in both treatment groups compared to saline controls. The glycaemic response to i.p. glucose was correspondingly improved (P<0.05) in [d-Ala²]GIP(1–30)- and [d-Ala²]GIP(1–42)-treated mice. Furthermore, glucose-stimulated plasma insulin levels were significantly elevated in both treatment groups compared to control mice. Insulin sensitivity was not significantly different between any of the groups. Similarly, plasma lipid profile, O₂ consumption, CO₂ production, respiratory exchange ratio, and energy expenditure were not altered by 42 days twice-daily treatment with [d-Ala²]GIP(1–30) or [d-Ala²]GIP(1–42). In contrast, ambulatory activity was significantly (P<0.05) elevated during the light phase in both GIP treatment groups compared to saline controls. The results reveal that sustained GIP receptor activation exerts a spectrum of beneficial metabolic effects in high-fat fed mice. However, no differences were discernable between the biological actions of the enzyme-resistant analogues of the naturally occurring forms, GIP(1–30) and GIP(1–42).

Cholecystokinin (CCK) is a hormone that has important physiological effects on energy balance. This study has used a stable CCK₁ receptor agonist, (pGlu-Gln)-CCK-8, to evaluate the metabolic effects of prolonged administration in normal mice. Twice-daily injection of (pGlu-Gln)-CCK-8 for 28 days resulted in significantly lowered body weights (P<0.05) on days 24 and 28, which was associated with decreased accumulated calorie intake (P<0.01) from day 12 onward. Nonfasting plasma glucose was significantly reduced (P<0.05) on day 28, while plasma insulin concentrations were increased (P<0.05). After 28 days, glucose tolerance and glucose-mediated insulin secretion were not significantly different in (pGlu-Gln)-CCK-8-treated mice. However, following a 15-min refeeding period in 18-h fasted mice, glucose levels were significantly (P<0.05) decreased by (pGlu-Gln)-CCK-8 despite similar food intake and nutrient-induced insulin levels. Insulin sensitivity in (pGlu-Gln)-CCK-8-treated mice was significantly (P<0.01) improved compared with controls. Accumulation of triacylglycerol in liver was reduced (P<0.01) but there were no differences in circulating cholesterol and triacylglycerol concentrations, as well as triacylglycerol content of pancreatic, muscle, and adipose tissue in (pGlu-Gln)-CCK-8 mice. These data highlight the beneficial metabolic effects of prolonged (pGlu-Gln)-CCK-8 administration and confirm a lack of detrimental effects.

Evidence for involvement of toll-like receptors (TLRs) (e.g. TLR4 and TLR2, whose agonists include lipopolysaccharides (LPS) and saturated fatty acids) in altered patterns of signalling in adipose, liver and muscle from animal models of insulin resistance and obesity has been published. We have now extended this area of research and have determined the effects of LPS on cell viability, insulin secretion, insulin signalling and metabolism in a clonal β-cell line. BRIN-BD11 β-cells were treated for 24 h with increasing concentrations of LPS. Chronic (24 h) and acute (20 min) insulin secretion, insulin content and parameters of cell metabolism and insulin signalling were determined. Incubation of BRIN-BD11 cells for 24 h in the presence of increasing concentrations of the TLR4 ligand LPS significantly decreased chronic (24 h) insulin secretion from 1.09±0.19 to 0.76±0.18 μg insulin/mg protein in the presence of 100 ng/ml LPS (P<0.05). There was no change in acute (20 min) stimulated insulin secretion or insulin content. Cell metabolism was not changed. Insulin receptor-β (IRβ) expression levels were increased significantly from 1±0.52 to 8.6±1.83 units (P<0.01), whereas calcineurin activity and Akt phosphorylation were significantly (P<0.01 and P<0.05 respectively) reduced in response to 24 h incubation in the presence of LPS. There was no change in IR substrate-1 protein expression or phosphorylation after 24 h. Further incubation for 24 h in the absence of LPS resulted in the recovery of chronic insulin secretion. The negative β-cell effects of LPS may contribute to hyperglycaemia in vivo.

We have investigated the effects of prolonged exposure (24 h) to the amino acid l-glutamine, on gene and protein expression using clonal BRIN-BD11 β-cells. Expression profiling of BRIN-BD11 cells was performed using oligonucleotide microarray analysis. Culture for 24 h with 10 mM l-glutamine compared with 1 mM resulted in substantial changes in gene expression with 148 genes upregulated more than 1.8-fold, and 18 downregulated more than 1.8-fold, including many genes involved in cellular signaling, metabolism, gene regulation, and the insulin-secretory response. Subsequent functional experiments confirmed that l-glutamine increased the activity of the Ca²⁺ regulated phosphatase calcineurin and the transcription factor Pdx1. Additionally, we demonstrated that β-cell-derived l-glutamate was released into the extracellular medium at high rates. As calcineurin is a regulator of the glutamate N-methyl-d-aspartate (NMDA) receptor activity, we investigated the action of NMDA on nutrient-induced insulin secretion, and demonstrated suppressed insulin release. These observations indicate important long-term effects of l-glutamine in regulating β-cell gene expression, signaling, and secretory function.

Recent approval of the dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist, tirzepatide, for the management of type 2 diabetes mellitus (T2DM) has reinvigorated interest in exploitation of GIP receptor (GIPR) pathways as a means of metabolic disease management. However, debate has long surrounded the use of the GIPR as a therapeutic target and whether agonism or antagonism is of most benefit in management of obesity/diabetes. This controversy appears to be partly resolved by the success of tirzepatide. However, emerging studies indicate that prolonged GIPR agonism may desensitise the GIPR to essentially induce receptor antagonism, with this phenomenon suggested to be more pronounced in the human than rodent setting. Thus, deliberation continues to rage in relation to benefits of GIPR agonism vs antagonism. That said, as with GIPR agonism, it is clear that the metabolic advantages of sustained GIPR antagonism in obesity and obesity-driven forms of diabetes can be enhanced by concurrent GLP-1 receptor (GLP-1R) activation. This narrative review discusses various approaches of pharmacological GIPR antagonism including small molecule, peptide, monoclonal antibody and peptide-antibody conjugates, indicating stage of development and significance to the field. Taken together, there is little doubt that interesting times lie ahead for GIPR agonism and antagonism, either alone or when combined with GLP-1R agonists, as a therapeutic intervention for the management of obesity and associated metabolic disease.

This study assessed the metabolic and neuroprotective actions of the sodium glucose cotransporter-2 inhibitor dapagliflozin in combination with the GLP-1 agonist liraglutide in dietary-induced diabetic mice. Mice administered low-dose streptozotocin (STZ) on a high-fat diet received dapagliflozin, liraglutide, dapagliflozin-plus-liraglutide (DAPA-Lira) or vehicle once-daily over 28 days. Energy intake, body weight, glucose and insulin concentrations were measured at regular intervals. Glucose tolerance, insulin sensitivity, hormone and biochemical analysis, dual-energy X-ray absorptiometry densitometry, novel object recognition, islet and brain histology were examined. Once-daily administration of DAPA-Lira resulted in significant decreases in body weight, fat mass, glucose and insulin concentrations, despite no change in energy intake. Similar beneficial metabolic improvements were observed regarding glucose tolerance, insulin sensitivity, HOMA-IR, HOMA-β, HbA1c and triglycerides. Plasma glucagon, GLP-1 and IL-6 levels were increased and corticosterone concentrations decreased. DAPA-Lira treatment decreased alpha cell area and increased insulin content compared to dapagliflozin monotherapy. Recognition memory was significantly improved in all treatment groups. Brain histology demonstrated increased staining for doublecortin (number of immature neurons) in dentate gyrus and synaptophysin (synaptic density) in stratum oriens and stratum pyramidale. These data demonstrate that combination therapy of dapagliflozin and liraglutide exerts beneficial metabolic and neuroprotective effects in diet-induced diabetic mice. Our results highlight important personalised approach in utilising liraglutide in combination with dapagliflozin, instead of either agent alone, for further clinical evaluation in treatment of diabetes and associated neurodegenerative disorders.

The insulin-releasing effects, cellular mechanisms of action and anti-hyperglycaemic activity of 10 analogues of esculentin-2CHa lacking the cyclic C-terminal domain (CKISKQC) were evaluated. Analogues of the truncated peptide, esculentin-2CHa(1–30), were designed for plasma enzyme resistance and increased biological activity. Effects of those analogues on insulin release, cell membrane integrity, membrane potential, intracellular Ca²⁺ and cAMP levels were determined using clonal BRIN-BD11 cells. Their acute effects on glucose tolerance were investigated using NIH Swiss mice. d-Amino acid substitutions at positions 7(Arg), 15(Lys) and 23(Lys) and fatty acid (l-octanoate) attachment to Lys at position 15 of esculentin-2CHa(1–30) conveyed resistance to plasma enzyme degradation whilst preserving insulin-releasing activity. Analogues, [d-Arg⁷,d-Lys¹⁵,d-Lys²³]-esculentin-2CHa(1–30) and Lys¹⁵-octanoate-esculentin-2CHa(1–30), exhibiting most promising profiles and with confirmed effects on both human insulin-secreting cells and primary mouse islets were selected for further analysis. Using chemical inhibition of adenylate cyclase, protein kinase C or phospholipase C pathways, involvement of PLC/PKC-mediated insulin secretion was confirmed similar to that of CCK-8. Diazoxide, verapamil and Ca²⁺ omission inhibited insulin secretion induced by the esculentin-2CHa(1–30) analogues suggesting an action on K_ATP and Ca²⁺ channels also. Consistent with this, the analogues depolarised the plasma membrane and increased intracellular Ca²⁺. Evaluation with fluorescent-labelled esculentin-2CHa(1–30) indicated membrane action, with internalisation; however, patch-clamp experiments suggested that depolarisation was not due to the direct inhibition of K_ATP channels. Acute administration of either analogue to NIH Swiss mice improved glucose tolerance and enhanced insulin release similar to that observed with GLP-1. These data suggest that multi-acting analogues of esculentin-2CHa(1–30) may prove useful for glycaemic control in obesity-diabetes.