Fiona M. Gribble

An SCN9A channelopathy causes congenital inability to experience pain.

The complete inability to sense pain in an otherwise healthy individual is a very rare phenotype. In three consanguineous families from northern Pakistan, we mapped the condition as an autosomal-recessive trait to chromosome 2q24.3. This region contains the gene SCN9A, encoding the α-subunit of the voltage-gated sodium channel, Nav1.7, which is strongly expressed in nociceptive neurons. Sequence analysis of SCN9A in affected individuals revealed three distinct homozygous nonsense mutations (S459X, I767X and W897X). We show that these mutations cause loss of function of Nav1.7 by co-expression of wild-type or mutant human Nav1.7 with sodium channel β1 and β2 subunits in HEK293 cells. In cells expressing mutant Nav1.7, the currents were no greater than background. Our data suggest that SCN9A is an essential and non-redundant requirement for nociception in humans. These findings should stimulate the search for novel analgesics that selectively target this sodium channel subunit.

Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein–Coupled Receptor FFAR2

Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of “indigestible” prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1–secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.

Glucose Sensing in L Cells: A Primary Cell Study

Summary Glucagon-like peptide-1 (GLP-1) is an enteric hormone that stimulates insulin secretion and improves glycaemia in type 2 diabetes. Although GLP-1-based treatments are clinically available, alternative strategies to increase endogenous GLP-1 release from L cells are hampered by our limited physiological understanding of this cell type. By generating transgenic mice with L cell-specific expression of a fluorescent protein, we studied the characteristics of primary L cells by electrophysiology, fluorescence calcium imaging, and expression analysis and show that single L cells are electrically excitable and glucose responsive. Sensitivity to tolbutamide and low-millimolar concentrations of glucose and α-methylglucopyranoside, assessed in single L cells and by hormone secretion from primary cultures, suggested that GLP-1 release is regulated by the activity of sodium glucose cotransporter 1 and ATP-sensitive K+ channels, consistent with their high expression levels in purified L cells by quantitative RT-PCR. These and other pathways identified using this approach will provide exciting opportunities for future physiological and therapeutic exploration.

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Correlating structure and function in ATP-sensitive K+ channels

ATP-sensitive K+ channels couple cell metabolism to electrical activity in nerve, muscle and endocrine cells, and play important roles in these tissues under both physiological and pathological conditions. The KATP channel is an octameric complex of two unrelated types of subunit: a pore-forming subunit (Kir6.2) and a regulatory subunit, the sulphonylurea receptor (SUR). This review focuses on the regulation of KATP channel activity by nucleotides and cell metabolism and considers which regulatory mechanisms are intrinsic to Kir6.2 and which are conferred by association with SUR.

Genetic cause of hyperglycaemia and response to treatment in diabetes

BACKGROUND Type 2 diabetes shows evidence of underlying heterogeneity. No studies have assessed whether different causes for diabetes change the response to oral hypoglycaemic therapy. In a few cases, patients with diabetes caused by mutations in the hepatocyte nuclear factor 1alpha (HNF-1alpha) gene have been described as sensitive to the hypoglycaemic effects of sulphonylureas. We aimed to see whether the glycaemic response to the sulphonylurea gliclazide and the biguanide metformin differed in HNF-1alpha diabetes and type 2 diabetes, and to investigate the mechanism for differences in sulphonylurea sensitivity. METHODS We did a randomised crossover trial of glicazide and metformin in 36 patients, either with diabetes caused by HNF-1alpha mutations or type 2 diabetes, who were matched for body-mass index and fasting plasma glucose. The primary outcome was reduction in fasting plasma glucose. Analysis was by intention to treat. We assessed possible mechanisms for sulphonylurea sensitivity through insulin sensitivity, insulin secretory response to glucose and tolbutamide, and tolbutamide clearance. FINDINGS Patients with HNF-1alpha diabetes had a 5.2-fold greater response to gliclazide than to metformin (fasting plasma glucose reduction 4.7 vs 0.9 mmol/L, p=0.0007) and 3.9-fold greater response to gliclazide than those with type 2 diabetes (p=0.002). Patients with HNF-1alpha diabetes had a strong insulin secretory response to intravenous tolbutamide despite a small response to intravenous glucose, and were more insulin sensitive than those with type 2 diabetes. Sulphonylurea metabolism was similar in both patient groups. INTERPRETATION The cause of hyperglycaemia changes the response to hypoglycaemic drugs; HNF-1alpha diabetes has marked sulphonylurea sensitivity. This pharmacogenetic effect is consistent with models of HNF-1alpha deficiency, which show that the beta-cell defect is upstream of the sulphonylurea receptor. Definition of the genetic basis of hyperglycaemia has implications for patient management.

The essential role of the Walker A motifs of SUR1 in K‐ATP channel activation by Mg‐ADP and diazoxide

The ATP‐sensitive K‐channel (K‐ATP channel) plays a key role in insulin secretion from pancreatic β‐cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg‐ADP, which inhibit and potentiate channel activity, respectively. The β‐cell K‐ATP channel consists of a pore‐forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (WA) motifs of the first (K719A) and second (K1384M) nucleotide‐binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the WA lysine of NBD1 (but not NBD2) is essential for activation of K‐ATP currents by diazoxide. The potentiatory effects of Mg‐ADP required the presence of the WA lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild‐type currents. Metabolic inhibition led to activation of wild‐type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K‐ATP channel activity.

Na+-d-glucose Cotransporter SGLT1 is Pivotal for Intestinal Glucose Absorption and Glucose-Dependent Incretin Secretion

To clarify the physiological role of Na+-d-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1−/− mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1−/− mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1−/− mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose–free diet. In wild-type mice, passage of d-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1−/− mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

OBJECTIVE Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS ZnT8−/− mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8−/− islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn2+ transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.

Molecular determinants of KATP channel inhibition by ATP

ATP‐sensitive K+ (KATP) channels are both inhibited and activated by intracellular nucleotides, such as ATP and ADP. The inhibitory effects of nucleotides are mediated via the pore‐forming subunit, Kir6.2, whereas the potentiatory effects are conferred by the sulfonylurea receptor subunit, SUR. The stimulatory action of Mg‐nucleotides complicates analysis of nucleotide inhibition of Kir6.2/SUR1 channels. We therefore used a truncated isoform of Kir6.2, that expresses ATP‐sensitive channels in the absence of SUR1, to explore the mechanism of nucleotide inhibition. We found that Kir6.2 is highly selective for ATP, and that both the adenine moiety and the β‐phosphate contribute to specificity. We also identified several mutations that significantly reduce ATP inhibition. These are located in two distinct regions of Kir6.2: the N‐terminus preceding, and the C‐terminus immediately following, the transmembrane domains. Some mutations in the C‐terminus also markedly increased the channel open probability, which may account for the decrease in apparent ATP sensitivity. Other mutations did not affect the single‐channel kinetics, and may reduce ATP inhibition by interfering with ATP binding and/or the link between ATP binding and pore closure. Our results also implicate the proximal C‐terminus in KATP channel gating.