Loss of the nutrient receptor Tas1R3 reduces atherosclerotic plaque accumulation and hepatic steatosis in ApoE-/- mice.

The taste receptor type I (Tas1R) family consists of three G protein-coupled receptors (T1R1, T1R2, and T1R3) that form heterodimers recognizing sweet compounds (T1R2/T1R3) or amino acids (T1R1/T1R3). These receptors are nutrient sensors that facilitate appropriate physiological responses with nutrient availability. However, their contribution to the development of pathologies associated with overnutrition (e.g., atherosclerosis) is unclear. The aim of the present study was to determine if T1R3 deletion would reduce atherosclerotic plaque development in mice. We generated atherosclerotic mice with whole-body deletion of T1R3 by crossing T1R3-/- mice with ApoE-/- mice. T1R3+/+ ApoE-/- and T1R3-/- ApoE-/- mice were maintained on an atherogenic high-fat diet for 8 weeks. Weight gain and food consumption were measured during the 8-week diet. Atherosclerotic lesion development and size were assessed by en face analysis of intact aortas and microscopic analysis of aortic roots. Our results indicate that T1R3 deletion in male and female ApoE-/- mice reduces aortic atherosclerotic plaque accumulation. Hepatic triglyceride accumulation, which was measured by quantification of oil red O staining, was also reduced in T1R3-/- mice. While the ablation of T1R3 reduced the final body weight of both males and females by approximately 12%, serum lipids, insulin, and glucose were either unchanged or slightly reduced. Immunoblot analysis of the phosphorylation of p70S6K, an effector of mTORC1, suggests T1R3 ablation reduces mTORC1 activity by approximately 50% in the male livers. Collectively, these findings suggest that the whole-body deletion of T1R3 reduces atherosclerosis and hepatic steatosis in a manner largely independent of the measured effects on whole-body glucose and lipid homeostasis.

Loss of the nutrient sensor TAS1R3 leads to reduced bone resorption.

The taste receptor type 1 (TAS1R) family of heterotrimeric G protein-coupled receptors participates in monitoring energy and nutrient status. TAS1R member 3 (TAS1R3) is a bi-functional protein that recognizes amino acids such as L-glycine and L-glutamate or sweet molecules such as sucrose and fructose when dimerized with TAS1R member 1 (TAS1R1) or TAS1R member 2 (TAS1R2), respectively. It was recently reported that deletion of TAS1R3 expression in Tas1R3 mutant mice leads to increased cortical bone mass but the underlying cellular mechanism leading to this phenotype remains unclear. Here, we independently corroborate the increased thickness of cortical bone in femurs of 20-week-old male Tas1R3 mutant mice and confirm that Tas1R3 is expressed in the bone environment. Tas1R3 is expressed in undifferentiated bone marrow stromal cells (BMSCs) in vitro and its expression is maintained during BMP2-induced osteogenic differentiation. However, levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) are unchanged in the serum of 20-week-old Tas1R3 mutant mice as compared to controls. In contrast, levels of the bone resorption marker collagen type I C-telopeptide are reduced greater than 60% in Tas1R3 mutant mice. Consistent with this, Tas1R3 and its putative signaling partner Tas1R2 are expressed in primary osteoclasts and their expression levels positively correlate with differentiation status. Collectively, these findings suggest that high bone mass in Tas1R3 mutant mice is due to uncoupled bone remodeling with reduced osteoclast function and provide rationale for future experiments examining the cell-type-dependent role for TAS1R family members in nutrient sensing in postnatal bone remodeling.

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells.

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic ß-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic ß-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

G protein-coupled receptors and the regulation of autophagy.

Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. Although autophagy is generally accepted to be regulated in a cell-autonomous fashion, recent studies suggest that nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets.

Muscarinic control of MIN6 pancreatic ß cells is enhanced by impaired amino acid signaling.

We have shown recently that the class C G protein-coupled receptor T1R1/T1R3 taste receptor complex is an early amino acid sensor in MIN6 pancreatic ß cells. Amino acids are unable to activate ERK1/2 in ß cells in which T1R3 has been depleted. The muscarinic receptor agonist carbachol activated ERK1/2 better in T1R3-depleted cells than in control cells. Ligands that activate certain G protein-coupled receptors in pancreatic ß cells potentiate glucose-stimulated insulin secretion. Among these is the M3 muscarinic acetylcholine receptor, the major muscarinic receptor in ß cells. We found that expression of M3 receptors increased in T1R3-depleted MIN6 cells and that calcium responses were altered. To determine whether these changes were related to impaired amino acid signaling, we compared responses in cells exposed to reduced amino acid concentrations. M3 receptor expression was increased, and some, but not all, changes in calcium signaling were mimicked. These findings suggest that M3 acetylcholine receptors are increased in ß cells as a mechanism to compensate for amino acid deficiency.

Minireview: Nutrient sensing by G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.

Off-target effects of MEK inhibitors.

The mitogen-activated protein kinases (MAPKs) ERK1/2 regulate numerous cellular processes, including gene transcription, proliferation, and differentiation. The only known substrates of the MAP2Ks MEK1/2 are ERK1/2; thus, MEK inhibitors PD98059, U0126, and PD0325901 have been important tools in determining the functions of ERK1/2. By using these inhibitors and genetically manipulating MEK, we found that ERK1/2 activation is neither sufficient nor necessary for regulated secretion of insulin from pancreatic ß cells or secretion of epinephrine from chromaffin cells. We show that both PD98059 and U0126 reduce agonist-induced entry of calcium into cells in a manner independent of their ability to inhibit ERK1/2. Caution should be used when interpreting results from experiments using these compounds.

Amino acid regulation of autophagy through the GPCR TAS1R1-TAS1R3.

Cells require the ability to rapidly detect decreases in concentrations of free amino acids so that homeostatic mechanisms, including autophagy, can be engaged to replenish amino acids. Amino acids are transported into cells where it is generally accepted that they are detected by an intracellular sensor. We now show that the cell surface G protein coupled receptor (GPCR) TAS1R1-TAS1R3 (T1R1-T1R3) can sense extracellular amino acids, activate MTORC1, and inhibit autophagy. This receptor is expressed in most tissues and fasted TAS1R3 (-/-) mice have increased autophagy in the heart, skeletal muscle and liver.

The G protein-coupled taste receptor T1R1/T1R3 regulates mTORC1 and autophagy.

Cells continually assess their energy and nutrient state to maintain growth and survival and engage necessary homeostatic mechanisms. Cell-autonomous responses to the fed state require the surveillance of the availability of amino acids and other nutrients. The mammalian target of rapamycin complex 1 (mTORC1) integrates information on nutrient and amino acid availability to support protein synthesis and cell growth. We identify the G protein-coupled receptor (GPCR) T1R1/T1R3 as a direct sensor of the fed state and amino acid availability. Knocking down this receptor, which is found in most tissues, reduces the ability of amino acids to signal to mTORC1. Interfering with this receptor alters localization of mTORC1, downregulates expression of pathway inhibitors, upregulates key amino acid transporters, blocks translation initiation, and induces autophagy. These findings reveal a mechanism for communicating amino acid availability through a GPCR to mTORC1 in mammals.

RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly.

The study of macroautophagy in mammalian cells has described induction, vesicle nucleation, and membrane elongation complexes as key signaling intermediates driving autophagosome biogenesis. How these components are recruited to nascent autophagosomes is poorly understood, and although much is known about signaling mechanisms that restrain autophagy, the nature of positive inductive signals that can promote autophagy remain cryptic. We find that the Ras-like small G protein, RalB, is localized to nascent autophagosomes and is activated on nutrient deprivation. RalB and its effector Exo84 are required for nutrient starvation-induced autophagocytosis, and RalB activation is sufficient to promote autophagosome formation. Through direct binding to Exo84, RalB induces the assembly of catalytically active ULK1 and Beclin1-VPS34 complexes on the exocyst, which are required for isolation membrane formation and maturation. Thus, RalB signaling is a primary adaptive response to nutrient limitation that directly engages autophagocytosis through mobilization of the core vesicle nucleation machinery.

MAP-ping unconventional protein-DNA interactions.

Control of gene expression depends on a myriad of protein-DNA interactions, and the number of proteins involved just got larger. In this issue, Hu et al. (2009) identify hundreds of human proteins that bind to DNA, including many surprises such as the protein kinase ERK2 (MAPK1) that now appears to control gene expression directly.

The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD.

The human Rad9 checkpoint protein is a subunit of the heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex that plays a role as a damage sensor in the DNA damage checkpoint response. Rad9 has been found to interact with several other proteins outside the context of the 9-1-1 complex with no obvious checkpoint functions. During our studies on the 9-1-1 complex, we found that Rad9 immunoprecipitates contained a 240 kDa protein that was identified as carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Further investigations revealed that only free Rad9, but not Rad9 within the 9-1-1 complex, bound to CAD. The rate-limiting step in de novo pyrimidine nucleotide synthesis is catalyzed by the carbamoyl phosphate synthetase II (CPSase) domain of CAD. We find that Rad9 binds to the CPSase domain, and, moreover, this binding results in a 2-fold stimulation of the CPSase activity of CAD. Similar results were also obtained with an N-terminal Rad9 fragment. These findings suggest that Rad9 may play a role in ribonucleotide biosynthesis.

Sodium arsenite inhibits and reverses expression of adipogenic and fat cell-specific genes during in vitro adipogenesis.

Arsenic causes cancer in humans, but its mechanism of action is unique among known carcinogenic agents. As a naturally occurring component of sediments and ground water, human exposure to arsenic is inevitable, necessitating the establishment of exposure limits. Because cancer is characterized as an imbalance between cell growth and differentiation, it has been hypothesized that arsenic exerts its carcinogenic effect, in part, by perturbing the balance between these antagonistic processes. Previous work in this laboratory has demonstrated that sodium arsenite prevents adipocytic differentiation of C3H 10T1/2 cells, leading to the hypothesis that the underlying mechanism involves downregulation of genes associated with adipogenesis. In support of this hypothesis, it was found that mRNA levels of peroxisome proliferative-activated receptor gamma (PPAR gamma), CCAAT-enhancer binding protein alpha (C/EBP alpha), and adipocyte-selective, fatty acid-binding protein (aP2) are decreased in arsenic-treated cells; arsenic-induced phenotypic reversion of differentiated adipocytes correlates with reduced aP2 expression. Arsenic also blocks upregulation of p21(Cip1/Waf1), a factor whose expression is tightly regulated during adipogenesis. The differentiating effect of pioglitazone, which induces adipogenesis by activating PPAR gamma, is inhibited by arsenic, suggesting that arsenic interferes with adipogenic signaling at or below the level of PPAR gamma. Because C/EBP alpha is important in the expression of certain keratinocyte-specific genes, the negative effect of arsenic on C/EBP alpha might also contribute to the development of skin cancer. PPAR gamma, C/EBP alpha, and p21(Cip1/Waf1) are important in numerous normal and pathological processes, including carcinogenesis, leading us to postulate that perturbation of these factors by arsenic might contribute to the carcinogenic effect of this metalloid.