Brian T. Layden

Short chain fatty acids and their receptors: new metabolic targets.

Fatty acids are carboxylic acids with aliphatic tails of different lengths, where short chain fatty acids (SCFAs) typically refer to carboxylic acids with aliphatic tails less than 6 carbons. In humans, SCFAs are derived in large part from fermentation of carbohydrates and proteins in the colon. By this process, the host is able to salvage energy from foods that cannot be processed normally in the upper parts of the gastrointestinal tract. In humans, SCFAs are a minor nutrient source, especially for people on Western diets. Intriguingly, recent studies, as highlighted here, have described multiple beneficial roles of SCFAs in the regulation of metabolism. Further interest in SCFAs has emerged due to the association of gut flora composition with obesity and other metabolic states. The recent identification of receptors specifically activated by SCFAs has further increased interest in this area. These receptors, free fatty acid receptor-2 and -3 (FFAR2 and FFAR3), are expressed not only in the gut epithelium where SCFAs are produced, but also at multiple other sites considered to be metabolically important, such as adipose tissue and pancreatic islets. Because of these relatively recent findings, studies examining the role of these receptors, FFAR2 and FFAR3, and their ligands, SCFAs, in metabolism are emerging. This review provides a critical analysis of SCFAs, their recently identified receptors, and their connection to metabolism.

An acetate-specific GPCR, FFAR2, regulates insulin secretion

G protein-coupled receptors have been well described to contribute to the regulation of glucose-stimulated insulin secretion (GSIS). The short-chain fatty acid-sensing G protein-coupled receptor, free fatty acid receptor 2 (FFAR2), is expressed in pancreatic β-cells, and in rodents, its expression is altered during insulin resistance. Thus, we explored the role of FFAR2 in regulating GSIS. First, assessing the phenotype of wild-type and Ffar2(-/-) mice in vivo, we observed no differences with regard to glucose homeostasis on normal or high-fat diet, with a marginally significant defect in insulin secretion in Ffar2(-/-) mice during hyperglycemic clamps. In ex vivo insulin secretion studies, we observed diminished GSIS from Ffar2(-/-) islets relative to wild-type islets under high-glucose conditions. Further, in the presence of acetate, the primary endogenous ligand for FFAR2, we observed FFAR2-dependent potentiation of GSIS, whereas FFAR2-specific agonists resulted in either potentiation or inhibition of GSIS, which we found to result from selective signaling through either Gαq/11 or Gαi/o, respectively. Lastly, in ex vivo insulin secretion studies of human islets, we observed that acetate and FFAR2 agonists elicited different signaling properties at human FFAR2 than at mouse FFAR2. Taken together, our studies reveal that FFAR2 signaling occurs by divergent G protein pathways that can selectively potentiate or inhibit GSIS in mouse islets. Further, we have identified important differences in the response of mouse and human FFAR2 to selective agonists, and we suggest that these differences warrant consideration in the continued investigation of FFAR2 as a novel type 2 diabetes target.

New Insights Into Gestational Glucose Metabolism: Lessons Learned From 21st Century Approaches

Pregnancy presents a unique physiological challenge that requires changes coordinated by placentally and non–placentally derived hormones to prepare the mother for the metabolic stress presented by fetal development and to ensure appropriate nutrient allocation between mother and fetus. Of particular importance is the maintenance of normal glucose metabolism during pregnancy. Here, we describe physiological changes in glucose metabolism during pregnancy and highlight new insights into these adaptations that have emerged over the past decade using novel methodologies, specifically genome-wide association studies (GWAS) and metabolomics. While GWAS have identified some novel associations with metabolic traits during pregnancy, the majority of the findings overlap with those observed in nonpregnant populations and individuals with type 2 diabetes (T2D). Metabolomics studies have provided new insight into key metabolites involved in gestational diabetes mellitus (GDM). Both of these approaches have suggested that a strong link exists between GDM and T2D. Most recently, a role of the gut microbiome in pregnancy has been observed, with changes in the microbiome during the third trimester having metabolic consequences for the mother. In this Perspectives in Diabetes article, we highlight how these new data have broadened our understanding of gestational metabolism, and emphasize the importance of future studies to elucidate differences between GDM and T2D.

Identification of HKDC1 and BACE2 as Genes Influencing Glycemic Traits During Pregnancy Through Genome-Wide Association Studies

Maternal metabolism during pregnancy impacts the developing fetus, affecting offspring birth weight and adiposity. This has important implications for metabolic health later in life (e.g., offspring of mothers with pre-existing or gestational diabetes mellitus have an increased risk of metabolic disorders in childhood). To identify genetic loci associated with measures of maternal metabolism obtained during an oral glucose tolerance test at ∼28 weeks’ gestation, we performed a genome-wide association study of 4,437 pregnant mothers of European (n = 1,367), Thai (n = 1,178), Afro-Caribbean (n = 1,075), and Hispanic (n = 817) ancestry, along with replication of top signals in three additional European ancestry cohorts. In addition to identifying associations with genes previously implicated with measures of glucose metabolism in nonpregnant populations, we identified two novel genome-wide significant associations: 2-h plasma glucose and HKDC1, and fasting C-peptide and BACE2. These results suggest that the genetic architecture underlying glucose metabolism may differ, in part, in pregnancy.

Regulation of Pancreatic Islet Gene Expression in Mouse Islets by Pregnancy

Pancreatic β cells adapt to pregnancy-induced insulin resistance by unclear mechanisms. This study sought to identify genes involved in β cell adaptation during pregnancy. To examine changes in global RNA expression during pregnancy, murine islets were isolated at a time point of increased β cell proliferation (E13.5), and RNA levels were determined by two different assays (global gene expression array and G-protein-coupled receptor (GPCR) array). Follow-up studies confirmed the findings for select genes. Differential expression of 110 genes was identified and follow-up studies confirmed the changes in select genes at both the RNA and protein level. Surfactant protein D (SP-D) mRNA and protein levels exhibited large increases, which were confirmed in murine islets. Cytokine-induced expression of SP-D in islets was also demonstrated, suggesting a possible role as an anti-inflammatory molecule. Complementing these studies, an expression array was performed to define pregnancy-induced changes in expression of GPCRs that are known to impact islet cell function and proliferation. This assay, the results of which were confirmed using real-time reverse transcription-PCR assays, demonstrated that free fatty acid receptor 2 and cholecystokinin receptor A mRNA levels were increased at E13.5. This study has identified multiple novel targets that may be important for the adaptation of islets to pregnancy.

Maternal short-chain fatty acids are associated with metabolic parameters in mothers and newborns

During the course of pregnancy, dynamic remodeling of the gut microbiota occurs and contributes to maternal metabolic changes through an undefined mechanism. Because short chain fatty acids (SCFAs) are a major product of gut microbiome fermentation, we investigated whether serum SCFA levels during pregnancy are related to key metabolic parameters in mothers and newborns. In this prospective study, 20 pregnant women without gestational diabetes were evaluated at 36-38 weeks of gestation, and their newborns were assessed after parturition. In this cohort, which included normal (n = 10) and obese (n = 10) subjects based on prepregnancy body mass index, serum levels of SCFAs (acetate, propionate, and butyrate), maternal adipokines, maternal glucose, and C-peptide were measured at 36-38 weeks of gestation. Maternal weight gain and newborn anthropometrics were also determined. Data were analyzed using linear regression to test for associations, adjusting for prepregnancy obesity. In this cohort, serum acetate levels were associated with maternal weight gain and maternal adiponectin levels. In addition, serum propionate correlated negatively with maternal leptin levels, newborn length, and body weight. Taken together, this study observed that novel relationships exist among maternal SCFA levels and multiple interrelated maternal/newborn metabolic parameters.

G Protein Coupled Receptors in Embryonic Stem Cells: A Role for Gs-Alpha Signaling

Background Identification of receptor mediated signaling pathways in embryonic stem (ES) cells is needed to facilitate strategies for cell replacement using ES cells. One large receptor family, largely uninvestigated in ES cells, is G protein coupled receptors (GPCRs). An important role for these receptors in embryonic development has been described, but little is known about GPCR expression in ES cells. Methodology/Principal Findings We have examined the expression profile of 343 different GPCRs in mouse ES cells demonstrating for the first time that a large number of GPCRs are expressed in undifferentiated and differentiating ES cells, and in many cases at high levels. To begin to define a role for GPCR signaling in ES cells, the impact of activating Gs-alpha, one of the major alpha subunits that couples to GPCRs, was investigated. Gs-alpha activation resulted in larger embryoid bodies (EBs), due, in part, to increased cell proliferation and prevented the time-related decline in expression of transcription factors important for maintaining ES cell pluripotency. Significance/Conclusions These studies suggest that Gs-alpha signaling contributes to ES cell proliferation and pluripotency and provide a framework for further investigation of GPCRs in ES cells.

Coordinated regulatory variation associated with gestational hyperglycaemia regulates expression of the novel hexokinase HKDC1

Maternal glucose levels during pregnancy impact the developing fetus, affecting metabolic health early and later in life. Both genetic and environmental factors influence maternal metabolism, but little is known about the genetic mechanisms that alter glucose metabolism during pregnancy. Here we report that haplotypes previously associated with gestational hyperglycemia in the third trimester disrupt regulatory element activity and reduce expression of the nearby HKDC1 gene. We further find that experimentally reducing or increasing HKDC1 expression reduces or increases hexokinase activity, respectively, in multiple cellular models; and that purified HKDC1 protein has hexokinase activity in vitro. Together, these results suggest a novel mechanism of gestational glucose regulation in which the effects of genetic variants in multiple regulatory elements alter glucose homeostasis by coordinately reducing expression of the novel hexokinase HKDC1.

FFAR3 modulates insulin secretion and global gene expression in mouse islets.

The short chain fatty acid (SCFA) receptor (free fatty acid receptor-3; FFAR3) is expressed in pancreatic β cells; however, its role in insulin secretion is not clearly defined. Here, we examined the role of FFAR3 in insulin secretion. Using islets from global knockout FFAR3 (Ffar3−/−) mice, we explored the role of FFAR3 and ligand-induced FFAR3 signaling on glucose stimulated insulin secretion. RNA sequencing was also performed to gain greater insight into the impact of FFAR3 deletion on the islet transcriptome. First exploring insulin secretion, it was determined that Ffar3−/− islets secrete more insulin in a glucose-dependent manner as compared to wildtype (WT) islets. Next, exploring its primary endogenous ligand, propionate, and a specific agonist for FFAR3, signaling by FFAR3 inhibited glucose-dependent insulin secretion, which occurred through a Gαi/o pathway. To help understand these results, transcriptome analyses by RNA-sequencing of Ffar3−/− and WT islets observed multiple genes with well-known roles in islet biology to be altered by genetic knockout of FFAR3. Our data shows that FFAR3 signaling mediates glucose stimulated insulin secretion through Gαi/o sensitive pathway. Future studies are needed to more rigorously define the role of FFAR3 by in vivo approaches.

SCFA Receptors in Pancreatic β Cells: Novel Diabetes Targets?

Nutrient sensing receptors are key metabolic mediators of responses to dietary and endogenously derived nutrients. These receptors are largely G-protein-coupled receptors (GPCRs) and many are gaining significant interest as drug targets with a potential therapeutic role in metabolic diseases. A distinct subclass of nutrient sensing GPCRs, two short chain fatty acid (SCFA) receptors (FFA2 and FFA3) are uniquely responsive to gut microbiota derived nutrients (such as acetate, propionate, and butyrate). Pharmacological, molecular, and genetic studies have investigated their role in organismal glucose metabolism and recently in pancreatic β cell biology. Here, we summarize the present knowledge on the role of these receptors as metabolic sensors in β cell function and physiology, revealing new therapeutic opportunities for type 2 diabetes.