Kashan Ahmed

MyomiR-133 regulates brown fat differentiation through Prdm16

Brown adipose tissue (BAT) uses the chemical energy of lipids and glucose to produce heat, a function that can be induced by cold exposure or diet. A key regulator of BAT is the gene encoding PR domain containing 16 (Prdm16), whose expression can drive differentiation of myogenic and white fat precursors to brown adipocytes. Here we show that after cold exposure, the muscle-enriched miRNA-133 is markedly downregulated in BAT and subcutaneous white adipose tissue (SAT) as a result of decreased expression of its transcriptional regulator Mef2. miR-133 directly targets and negatively regulates PRDM16, and inhibition of miR-133 or Mef2 promotes differentiation of precursors from BAT and SAT to mature brown adipocytes, thereby leading to increased mitochondrial activity. Forced expression of miR-133 in brown adipogenic conditions prevents the differentiation to brown adipocytes in both BAT and SAT precursors. Our results point to Mef2 and miR-133 as central upstream regulators of Prdm16 and hence of brown adipogenesis in response to cold exposure in BAT and SAT.

Nicotinic acid– and monomethyl fumarate–induced flushing involves GPR109A expressed by keratinocytes and COX-2–dependent prostanoid formation in mice

The antidyslipidemic drug nicotinic acid and the antipsoriatic drug monomethyl fumarate induce cutaneous flushing through activation of G protein-coupled receptor 109A (GPR109A). Flushing is a troublesome side effect of nicotinic acid, but may be a direct reflection of the wanted effects of monomethyl fumarate. Here we analyzed the mechanisms underlying GPR109A-mediated flushing and show that both Langerhans cells and keratinocytes express GPR109A in mice. Using cell ablation approaches and transgenic cell type-specific GPR109A expression in Gpr109a-/- mice, we have provided evidence that the early phase of flushing depends on GPR109A expressed on Langerhans cells, whereas the late phase is mediated by GPR109A expressed on keratinocytes. Interestingly, the first phase of flushing was blocked by a selective cyclooxygenase-1 (COX-1) inhibitor, and the late phase was sensitive to a selective COX-2 inhibitor. Both monomethyl fumarate and nicotinic acid induced PGE2 formation in isolated keratinocytes through activation of GPR109A and COX-2. Thus, the early and late phases of the GPR109A-mediated cutaneous flushing reaction involve different epidermal cell types and prostanoid-forming enzymes. These data will help to guide new efficient approaches to mitigate nicotinic acid-induced flushing and may help to exploit the potential antipsoriatic effects of GPR109A agonists in the skin.

Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

An Autocrine Lactate Loop Mediates Insulin-Dependent Inhibition of Lipolysis through GPR81

Lactate is an important metabolic intermediate released by skeletal muscle and other organs including the adipose tissue, which converts glucose into lactate under the influence of insulin. Here we show that lactate activates the G protein-coupled receptor GPR81, which is expressed in adipocytes and mediates antilipolytic effects through G(i)-dependent inhibition of adenylyl cyclase. Using GPR81-deficient mice, we demonstrate that the receptor is not involved in the regulation of lipolysis during intensive exercise. However, insulin-induced inhibition of lipolysis and insulin-induced decrease in adipocyte cAMP levels were strongly reduced in mice lacking GPR81, although insulin-dependent release of lactate by adipocytes was comparable between wild-type and GPR81-deficient mice. Thus, lactate and its receptor GPR81 unexpectedly function in an autocrine and paracrine loop to mediate insulin-induced antilipolytic effects. These data show that lactate can directly modulate metabolic processes in a hormone-like manner, and they reveal a new mechanism underlying the antilipolytic effects of insulin.

GPR109A, GPR109B and GPR81, a family of hydroxy-carboxylic acid receptors

G-protein-coupled receptors (GPCRs) are the most versatile receptor family as they have the ability to respond to chemically diverse ligands. Despite intensive efforts during the past two decades, there are still more than 100 orphan GPCRs for which endogenous ligands are unknown. Recently, GPR109A, GPR109B and GPR81, which form a GPCR subfamily, have been deorphanized. The physiological ligands of these receptors are the ketone body 3-hydroxy-butyrate, the metabolite 2-hydroxy-propanoate (lactate) as well as the beta-oxidation intermediate 3-hydroxy-octanoate. Thus, this receptor subfamily is activated by hydroxy-carboxylic acid ligands which are intermediates of energy metabolism. All three receptors are predominantly expressed in adipocytes and mediate antilipolytic effects. In this article, we propose that the hydroxy-carboxylic acid structure of their endogenous ligands is the defining property of this receptor subfamily and that hydroxy-carboxylic acid receptors function as metabolic sensors which fine-tune the regulation of metabolic pathways.

Loss of FFA2 and FFA3 increases insulin secretion and improves glucose tolerance in type 2 diabetes

Type 2 diabetes is a major health problem worldwide, and one of its key features is the inability of elevated glucose to stimulate the release of sufficient amounts of insulin from pancreatic beta cells to maintain normal blood glucose levels. New therapeutic strategies to improve beta cell function are therefore believed to be beneficial. Here we demonstrate that the short-chain fatty acid receptors FFA2 (encoded by FFAR2) and FFA3 (encoded by FFAR3) are expressed in mouse and human pancreatic beta cells and mediate an inhibition of insulin secretion by coupling to Gi-type G proteins. We also provide evidence that mice with dietary-induced obesity and type 2 diabetes, as compared to non-obese control mice, have increased local formation by pancreatic islets of acetate, an endogenous agonist of FFA2 and FFA3, as well as increased systemic levels. This elevation may contribute to the insufficient capacity of beta cells to respond to hyperglycemia in obese states. Indeed, we found that genetic deletion of both receptors, either on the whole-body level or specifically in pancreatic beta cells, leads to greater insulin secretion and a profound improvement of glucose tolerance when mice are on a high-fat diet compared to controls. On the other hand, deletion of Ffar2 and Ffar3 in intestinal cells did not alter glucose tolerance in diabetic animals, suggesting these receptors act in a cell-autonomous manner in beta cells to regulate insulin secretion. In summary, under diabetic conditions elevated acetate acts on FFA2 and FFA3 to inhibit proper glucose-stimulated insulin secretion, and we expect antagonists of FFA2 and FFA3 to improve insulin secretion in type 2 diabetes.

Thyrocyte-specific Gq/G11 deficiency impairs thyroid function and prevents goiter development

The function of the adult thyroid is regulated by thyroid-stimulating hormone (TSH), which acts through a G protein-coupled receptor. Overactivation of the TSH receptor results in hyperthyroidism and goiter. The Gs-mediated stimulation of adenylyl cyclase-dependent cAMP formation has been regarded as the principal intracellular signaling mechanism mediating the action of TSH. Here we show that the Gq/G11-mediated signaling pathway plays an unexpected and essential role in the regulation of thyroid function. Mice lacking the alpha subunits of Gq and G11 specifically in thyroid epithelial cells showed severely reduced iodine organification and thyroid hormone secretion in response to TSH, and many developed hypothyroidism within months after birth. In addition, thyrocyte-specific Galphaq/Galpha11-deficient mice lacked the normal proliferative thyroid response to TSH or goitrogenic diet, indicating an essential role of this pathway in the adaptive growth of the thyroid gland. Our data suggest that Gq/G11 and their downstream effectors are promising targets to interfere with increased thyroid function and growth.

The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes

Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell–specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200–induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.

Deorphanization of GPR109B as a receptor for the β-oxidation intermediate 3-OH-octanoic acid and its role in the regulation of lipolysis

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the β-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to Gi-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC50 values of about 4 and 8 μm, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased β-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in β-oxidation rates.

Biological and Pharmacological Roles of HCA Receptors

The hydroxy-carboxylic acid (HCA) receptors HCA(1), HCA(2), and HCA(3) were previously known as GPR81, GPR109A, and GPR109B, respectively, or as the nicotinic acid receptor family. They form a cluster of G protein-coupled receptors with high sequence homology. Recently, intermediates of energy metabolism, all HCAs, have been reported as endogenous ligands for each of these receptors. The HCA receptors are predominantly expressed on adipocytes and mediate the inhibition of lipolysis by coupling to G(i)-type proteins. HCA(1) is activated by lactate, HCA(2) by the ketone body 3-hydroxy-butyrate, and HCA(3) by hydroxylated β-oxidation intermediates, especially 3-hydroxy-octanoic acid. Both HCA(2) and HCA(3) are part of a negative feedback loop which keeps the release of fat stores in check under starvation conditions, whereas HCA(1) plays a role in the antilipolytic (fat-conserving) effect of insulin. HCA(2) was first discovered as the molecular target of the antidyslipidemic drug nicotinic acid (or niacin). Many synthetic agonists have since been designed for HCA(2) and HCA(3), but the development of a new, improved HCA-targeted drug has not been successful so far, despite a number of clinical studies. Recently, it has been shown that the major side effect of nicotinic acid, skin flushing, is mediated by HCA(2) receptors on keratinocytes, as well as on Langerhans cells in the skin. In this chapter, we summarize the latest developments in the field of HCA receptor research, with emphasis on (patho)physiology, receptor pharmacology, major ligand classes, and the therapeutic potential of HCA ligands.