David M. Smith

Improved glucose control and reduced body fat mass in free fatty acid receptor 2-deficient mice fed a high-fat diet

Free fatty acid receptor 2 (Ffar2), also known as GPR43, is activated by short-chain fatty acids (SCFA) and expressed in intestine, adipocytes, and immune cells, suggesting involvement in lipid and immune regulation. In the present study, Ffar2-deficient mice (Ffar2-KO) were given a high-fat diet (HFD) or chow diet and studied with respect to lipid and energy metabolism. On a HFD, Ffar2-KO mice had lower body fat mass and increased lean body mass. The changed body composition was accompanied by improved glucose control and lower HOMA index, indicating improved insulin sensitivity in Ffar2-KO mice. Moreover, the Ffar2-KO mice had higher energy expenditure accompanied by higher core body temperature and increased food intake. The liver weight and content of triglycerides as well as plasma levels of cholesterol were lower in the Ffar2-KO mice fed a HFD. A histological examination unveiled decreased lipid interspersed in brown adipose tissue of the Ffar2-KO mice. Interestingly, no significant differences in white adipose tissue (WAT) cell size were observed, but significantly lower macrophage content was detected in WAT from HFD-fed Ffar2-KO compared with wild-type mice. In conclusion, Ffar2 deficiency protects from HFD-induced obesity and dyslipidemia at least partly via increased energy expenditure.

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)‐stimulated leptin secretion by activating Gαi. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gαi signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild‐type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild‐type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gαi signalling mediated by GPR43 in SCFA‐stimulated leptin secretion.

Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes

Summary Hormone-secreting cells within pancreatic islets of Langerhans play important roles in metabolic homeostasis and disease. However, their transcriptional characterization is still incomplete. Here, we sequenced the transcriptomes of thousands of human islet cells from healthy and type 2 diabetic donors. We could define specific genetic programs for each individual endocrine and exocrine cell type, even for rare δ, γ, ε, and stellate cells, and revealed subpopulations of α, β, and acinar cells. Intriguingly, δ cells expressed several important receptors, indicating an unrecognized importance of these cells in integrating paracrine and systemic metabolic signals. Genes previously associated with obesity or diabetes were found to correlate with BMI. Finally, comparing healthy and T2D transcriptomes in a cell-type resolved manner uncovered candidates for future functional studies. Altogether, our analyses demonstrate the utility of the generated single-cell gene expression resource.

11β-Hydroxysteroid Dehydrogenase Type 1 Regulates Glucocorticoid-Induced Insulin Resistance in Skeletal Muscle

OBJECTIVE Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11β-HSD1 inhibitors improve insulin sensitivity. RESEARCH DESIGN AND METHODS Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11β-HSD1 inhibition upon insulin signaling and action. RESULTS Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer307 insulin receptor substrate (IRS)-1. 11β-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11β-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer307 IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11β-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer307 IRS1 decreased and pThr308 Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression. CONCLUSIONS Prereceptor facilitation of glucocorticoid action via 11β-HSD1 increases pSer307 IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11β-HSD1 inhibition decreases pSer307 IRS1, increases pThr308 Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.

CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22–52, CGRP8–37 and BIBN4096BS

Adrenomedullin (AM) has two known receptors formed by the calcitonin receptor‐like receptor (CL) and receptor activity‐modifying protein (RAMP) 2 or 3: We report the effects of the antagonist fragments of human AM and CGRP (AM22–52 and CGRP8–37) in inhibiting AM at human (h), rat (r) and mixed species CL/RAMP2 and CL/RAMP3 receptors transiently expressed in Cos 7 cells or endogenously expressed as rCL/rRAMP2 complexes by Rat 2 and L6 cells. AM22–52 (10 μM) antagonised AM at all CL/RAMP2 complexes (apparent pA2 values: 7.34±0.14 (hCL/hRAMP2), 7.28±0.06 (Rat 2), 7.00±0.05 (L6), 6.25±0.17 (rCL/hRAMP2)). CGRP8–37 (10 μM) resembled AM22–52 except on the rCL/hRAMP2 complex, where it did not antagonise AM (apparent pA2 values: 7.04±0.13 (hCL/hRAMP2), 6.72±0.06 (Rat2), 7.03±0.12 (L6)). On CL/RAMP3 receptors, 10 μM CGRP8–37 was an effective antagonist at all combinations (apparent pA2 values: 6.96±0.08 (hCL/hRAMP3), 6.18±0.18 (rCL/rRAMP3), 6.48±0.20 (rCL/hRAMP3)). However, 10 μM AM22–52 only antagonised AM at the hCL/hRAMP3 receptor (apparent pA2 6.73±0.14). BIBN4096BS (10 μM) did not antagonise AM at any of the receptors. Where investigated (all‐rat and rat/human combinations), the agonist potency order on the CL/RAMP3 receptor was AM∼βCGRP>αCGRP. rRAMP3 showed three apparent polymorphisms, none of which altered its coding sequence. This study shows that on CL/RAMP complexes, AM22–52 has significant selectivity for the CL/RAMP2 combination over the CL/RAMP3 combination. On the mixed species receptor, CGRP8–37 showed the opposite selectivity. Thus, depending on the species, it is possible to discriminate pharmacologically between CL/RAMP2 and CL/RAMP3 AM receptors.

Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line.

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Glucose-dependent modulation of insulin secretion and intracellular calcium ions by GKA50, a glucokinase activator.

Because glucokinase is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of novel glucokinase activator compound 50 (GKA50) on islet function. Insulin secretion was determined by enzyme-linked immunosorbent assay, and microfluorimetry with fura-2 was used to examine intracellular Ca2+ homeostasis ([Ca2+]i) in isolated mouse, rat, and human islets of Langerhans and in the MIN6 insulin-secreting mouse cell line. In rodent islets and MIN6 cells, 1 μmol/l GKA50 was found to stimulate insulin secretion and raise [Ca2+]i in the presence of glucose (2–10 mmol/l). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1 μmol/l) caused a leftward shift in the glucose-concentration response profiles, and the half-maximal effective concentration (EC50) values for glucose were shifted by 3 mmol/l in rat islets and ∼10 mmol/l in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of glucose-stimulated insulin secretion. In the absence of glucose, GKA50 failed to elevate [Ca2+]i (1 μmol/l GKA50) or to stimulate insulin release (30 nmol/l–10 μmol/l GKA50). At 5 mmol/l glucose, the EC50 for GKA50 in MIN6 cells was ∼0.3 μmol/l. Inhibition of glucokinase with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca2+]i but not the actions of tolbutamide. Finally, the ATP-sensitive K+ channel agonist diazoxide (200 μmol/l) inhibited GKA50-induced insulin release and its elevation of [Ca2+]i. We show that GKA50 is a glucose-like activator of β-cell metabolism in rodent and human islets and a Ca2+-dependent modulator of insulin secretion.

The pharmacology of adrenomedullin receptors and their relationship to CGRP receptors.

Adrenomedullin (AM) has two specific receptors formed by the calcitonin-receptor-like receptor (CL) and receptor activity-modifying protein (RAMP) 2 or 3. These are known as AM1 and AM2 receptors, respectively. In addition, AM has appreciable affinity for the CGRP1 receptor, composed of CL and RAMP1. The AM1 receptor has a high degree of selectivity for AM over CGRP and other peptides, and AM22–52 is an effective antagonist at this receptor. By contrast, the AM2 receptor shows less specificity for AM, having appreciable affinity for βCGRP. Here, CGRP8–37 is either equipotent or more effective as an antagonist than AM22–52, depending on the species from which the receptor components are derived. Thus, under the appropriate circumstances it seems that βCGRP might be able to activate both CGRP1 and AM2 receptors and AM could activate both AM1 and AM2 receptors as well as CGRP1 receptors. Current peptide antagonists are not sufficiently selective to discriminate between these three receptors. The CGRP-selectivity of RAMP1 and RAMP3 may be conferred by a putative disulfide bond from the N-terminus to the middle of the extracellular domain of these molecules. This is not present in RAMP2.

Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G‐protein coupled receptor (GPCR) calcitonin receptor‐like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5′‐RACE and cloned the proximal 5′‐flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA‐boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp‐1, Pit‐1, glucocorticoid receptor, and hypoxia‐inducible factor‐1 α (HIF‐1α) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5′‐flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site‐directed mutagenesis of the consensus hypoxia‐response element (HRE) in the 5′‐flanking region abolished such a response. We also demonstrated by semi‐quantitative RT‐PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up‐regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.

GPR120 (FFAR4) is preferentially expressed in pancreatic delta cells and regulates somatostatin secretion from murine islets of Langerhans

Aims/hypothesisThe NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/β-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas.MethodsA KO/KI mouse was generated in which exon 1 of the Gpr120 gene (also known as Ffar4) was replaced in frame by LacZ, thereby allowing for regulated expression of β-galactosidase under the control of the endogenous GPR120 promoter. The distribution of GPR120 was inferred from expression studies detecting β-galactosidase activity and protein production. Islet hormone secretion was measured from isolated mouse islets treated with selective GPR120 agonists.Resultsβ-galactosidase activity was detected as a surrogate for GPR120 expression exclusively in a small population of islet endocrine cells located peripherally within the islet mantle. Immunofluorescence analysis revealed co-localisation with somatostatin suggesting that GPR120 is preferentially produced in islet delta cells. In confirmation of this, glucose-induced somatostatin secretion was inhibited by a range of selective GPR120 agonists. This response was lost in GPR120-knockout mice.Conclusions/interpretationThe results imply that GPR120 is selectively present within the delta cells of murine islets and that it regulates somatostatin secretion.