Maternal gut microbiota in pregnancy influences offspring metabolic phenotype in mice.

Antibiotics and dietary habits can affect the gut microbial community, thus influencing disease susceptibility. Although the effect of microbiota on the postnatal environment has been well documented, much less is known regarding the impact of gut microbiota at the embryonic stage. Here we show that maternal microbiota shapes the metabolic system of offspring in mice. During pregnancy, short-chain fatty acids produced by the maternal microbiota dictate the differentiation of neural, intestinal, and pancreatic cells through embryonic GPR41 and GPR43. This developmental process helps maintain postnatal energy homeostasis, as evidenced by the fact that offspring from germ-free mothers are highly susceptible to metabolic syndrome, even when reared under conventional conditions. Thus, our findings elaborate on a link between the maternal gut environment and the developmental origin of metabolic syndrome.

Ketone body receptor GPR43 regulates lipid metabolism under ketogenic conditions.

Ketone bodies, including ß-hydroxybutyrate and acetoacetate, are important alternative energy sources during energy shortage. ß-Hydroxybutyrate also acts as a signaling molecule via specific G protein-coupled receptors (GPCRs); however, the specific associated GPCRs and physiological functions of acetoacetate remain unknown. Here we identified acetoacetate as an endogenous agonist for short-chain fatty acid (SCFA) receptor GPR43 by ligand screening in a heterologous expression system. Under ketogenic conditions, such as starvation and low-carbohydrate diets, plasma acetoacetate levels increased markedly, whereas plasma and cecal SCFA levels decreased dramatically, along with an altered gut microbiota composition. In addition, Gpr43-deficient mice showed reduced weight loss and suppressed plasma lipoprotein lipase activity during fasting and eucaloric ketogenic diet feeding. Moreover, Gpr43-deficient mice exhibited minimal weight decrease after intermittent fasting. These observations provide insight into the role of ketone bodies in energy metabolism under shifts in nutrition and may contribute to the development of preventive medicine via diet and foods.

Receptor tyrosine kinase activation induces free fatty acid 4 receptor phosphorylation, ß-arrestin interaction, and internalization.

FFA4 (Free Fatty Acid receptor 4, previously known as GPR120) is a G protein-coupled receptor that acts as a sensor of long-chain fatty acids, modulates metabolism, and whose dysfunction participates in endocrine disturbances. FFA4 is known to be phosphorylated and internalized in response to agonists and protein kinase C activation. In this paper report the modulation of this fatty acid receptor by activation of receptor tyrosine kinases. Cell-activation with growth factors (insulin, epidermal growth factor, insulin-like growth factor-I, and platelet-derived growth factor) increases FFA4 phosphorylation in a time- and concentration-dependent fashion. This effect was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase, suggesting the involvement of these kinases in it. FFA4 phosphorylation did not alter agonist-induced FFA4 calcium signaling, but was associated with decreased ERK 1/2 phosphorylation. In addition, insulin, insulin-like growth factor-I, epidermal growth factor, and to a lesser extent, platelet-derived growth factor, induce receptor internalization. This action of insulin, insulin-like growth factor I, and epidermal growth factor was blocked by inhibitors of protein kinase C and phosphoinositide 3-kinase. Additionally, cell treatment with these growth factors induced FFA4-ß-arrestin coimmunoprecipitation. Our results evidenced cross-talk between receptor tyrosine kinases and FFA4 and suggest roles of protein kinase C and phosphoinositide 3-kinase in such a functional interaction.

Complex formation between the vasopressin 1b receptor, ß-arrestin-2, and the µ-opioid receptor underlies morphine tolerance.

Chronic morphine exposure upregulates adenylate cyclase signaling and reduces analgesic efficacy, a condition known as opioid tolerance. Nonopioid neurotransmitters can enhance morphine tolerance, but the mechanism for this is poorly understood. We show that morphine tolerance was delayed in mice lacking vasopressin 1b receptors (V1bRs) or after administration of V1bR antagonist into the rostral ventromedial medulla, where transcripts for V1bRs and µ-opioid receptors are co-localized. Vasopressin increased morphine-binding affinity in cells expressing both V1bR and µ-opioid receptors. Complex formation among V1bR, ß-arrestin-2, and µ-opioid receptor resulted in vasopressin-mediated upregulation of ERK phosphorylation and adenylate cyclase sensitization. A leucine-rich segment in the V1bR C-terminus was necessary for the association with ß-arrestin-2. Deletion of this leucine-rich segment increased morphine analgesia and reduced vasopressin-mediated adenylate cyclase sensitization. These findings indicate that inhibition of µ-opioid-receptor-associated V1bR provides an approach for enhancing morphine analgesia without increasing analgesic tolerance.

The short chain fatty acid receptor GPR43 regulates inflammatory signals in adipose tissue M2-type macrophages.

The regulation of inflammatory responses within adipose tissue by various types of immune cells is closely related to tissue homeostasis and progression of metabolic disorders such as obesity and type 2 diabetes. G-protein-coupled receptor 43 (GPR43), which is activated by short-chain fatty acids (SCFAs), is known to be most abundantly expressed in white adipose tissue and to modulate metabolic processes. Although GPR43 is also expressed in a wide variety of immune cells, whether and how GPR43 in adipose tissue immune cells regulates the inflammatory responses and metabolic homeostasis remains unknown. In this study, we investigated the role of GPR43 in adipose tissue macrophages by using Gpr43-deficient mice and transgenic mice with adipose-tissue-specific overexpression of GPR43. We found that GPR43 activation by SCFA resulted in induction of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) in anti-inflammatory M2-type macrophages within adipose tissue. By contrast, this effect was not noted in inflammatory M1-type macrophages, suggesting that GPR43 plays distinct functions depending on macrophage types. Local TNF-α signaling derived from steady-state adipose tissue is associated with proper tissue remodeling as well as suppression of fat accumulation. Thus, GPR43-involving mechanism that we have identified supports maintenance of adipose tissue homeostasis and increase in metabolic activity. This newly identified facet of GPR43 in macrophages may have clinical implications for immune-metabolism related episodes.

Agonists and protein kinase C-activation induce phosphorylation and internalization of FFA1 receptors.

FFA1 (previously known as GPR40) is a free fatty acid receptor involved in the regulation of inflammatory processes and insulin secretion. The cellular actions resulting from FFA1 activation have received considerable attention. However, little is known on the regulation of the receptor function. In the present work, using cells transfected with this receptor, docosahexaenoic acid and α-linolenic acid increased intracellular calcium concentration and ERK 1/2 phosphorylation. It was also observed that FFA1 is a phosphoprotein whose phosphorylation state was increased (2- to 3-fold) by agonists (i.e., free fatty acids) and also by phorbol myristate acetate. Agonist- and phorbol ester-mediated FFA1 phosphorylation was markedly reduced by inhibitors of protein kinase C. Receptor stimulation by free fatty acids and protein kinase C activation also induced receptor internalization as evidenced by confocal microscopy. In summary, our data show that FFA1 is a phosphoprotein whose phosphorylation state is modulated by agonists and protein kinase C activation; such covalent modification is associated with receptor internalization.

The Effects of a Selective CK2 Inhibitor on Anti-glomerular Basement Membrane Glomerulonephritis in Rats.

Protein kinase CK2 ("casein kinase II") is a protein serine/threonine kinase that plays critical roles in biological processes such as cell growth, cell cycle progression, and apoptosis. So far, we have identified that one catalytic isozyme of CK2, CK2α, is over-expressed in the kidney during the progression of glomerulonephritis (GN). Moreover, we have shown that in vivo inhibition of CK2 by administration of CK2 inhibitors was effective in the treatment of experimental GN. Hence the development of potent CK2 inhibitors should be considered in therapeutic strategies for GN. In the present study we identified compound 13, a pyrazine derivative, as a potent CK2 inhibitor. By performing enzyme kinetics analysis in vitro, we characterized the inhibition of compound 13 toward each CK2 catalytic isozyme. Furthermore, in vivo, we demonstrated that compound 13 is effective in attenuating proteinuria, decreasing the enhanced level of blood urea nitrogen and serum creatinine, and ameliorating glomerular crescent formation in an experimental GN rat model. On the other hand, cellular apoptosis was detected in the rat testis following administration of compound 13. This study provides clues for new strategies for developing applicable compounds into CK2-targeted GN treatments.

Effects of a Tricaprylin Emulsion on Anti-glomerular Basement Membrane Glomerulonephritis in Rats: In Vivo and in Silico Studies.

Glomerulonephritis (GN) is a set of pathological conditions that result in the destruction of glomeruli and loss of renal function, commonly leading to the development of end-stage renal disease. Current pharmacotherapy is limited to immunosuppressive therapy. In the present study, we found a novel antinephritic effect of a tricaprylin emulsion in the anti-glomerular basement membrane (anti-GBM) GN rat model. We evaluated the treatment in vivo by comparing administration of the emulsion with administration of a casein kinase II (CK2) inhibitor in this rat model, and performed a gene ontology-based microarray analysis to reveal in silico the detailed mechanism of action. Our results showed that administration of the tricaprylin emulsion, or even tricaprylin alone, significantly ameliorated the anti-GBM antibody-induced renal dysfunction in these rats. We believe that tricaprylin is the key active antinephritic component of the emulsion and might be a promising drug for the effective treatment of nephritis. Moreover, with respect to microarray analysis, we developed a generally applicable and rapid method to compare gene expression profile data for multiple models of nephritis and clinical samples from a public domain microarray database.

Identification of protein kinase CK2 inhibitors using solvent dipole ordering virtual screening.

Novel protein kinase CK2 inhibitors were identified using the solvent dipole ordering virtual screening method. A total of 26 compounds categorized in 15 distinct scaffold classes inhibited greater than 50% of enzyme activity at 50 µM, and eight exhibited IC50 values less than 10 µM. Most of the identified compounds are lead-like and dissimilar to known inhibitors. The crystal structures of two of the CK2 complexes revealed the high accuracy of the predicted binding modes.

The oral lipid sensor GPR120 is not indispensable for the orosensory detection of dietary lipids in mice.

Implication of the long-chain fatty acid (LCFA) receptor GPR120, also termed free fatty acid receptor 4, in the taste-guided preference for lipids is a matter of debate. To further unravel the role of GPR120 in the "taste of fat", the present study was conducted on GPR120-null mice and their wild-type littermates. Using a combination of morphological [i.e., immunohistochemical staining of circumvallate papillae (CVP)], behavioral (i.e., two-bottle preference tests, licking tests and conditioned taste aversion) and functional studies [i.e., calcium imaging in freshly isolated taste bud cells (TBCs)], we show that absence of GPR120 in the oral cavity was not associated with changes in i) gross anatomy of CVP, ii) LCFA-mediated increases in intracellular calcium levels ([Ca(2+)]i), iii) preference for oily and LCFA solutions and iv) conditioned avoidance of LCFA solutions. In contrast, the rise in [Ca(2+)]i triggered by grifolic acid, a specific GPR120 agonist, was dramatically curtailed when the GPR120 gene was lacking. Taken together, these data demonstrate that activation of lingual GPR120 and preference for fat are not connected, suggesting that GPR120 expressed in TBCs is not absolutely required for oral fat detection in mice.

The SCFA Receptor GPR43 and Energy Metabolism.

Free fatty acids (FFAs) are essential nutrients and act as signaling molecules in various cellular processes via binding with FFA receptors. Of these receptors, GPR43 is activated by short-chain fatty acids (SCFAs; e.g., acetate, propionate, and butyrate). During feeding, SCFAs are produced by microbial fermentation of dietary fiber in the gut, and these SCFAs become important energy sources for the host. The gut microbiota affects nutrient acquisition and energy regulation of the host and can influence the development of obesity, insulin resistance, and diabetes. Recently, GPR43 has been reported to regulate host energy homeostasis in the gastrointestinal tract and adipose tissues. Hence, GPR43 is also thought to be a potential drug target for metabolic disorders, such as obesity and diabetes. In this review, we summarize the identification, structure, and activities of GPR43, with a focus on host energy regulation, and present an essential overview of our current understanding of its physiological roles in host energy regulation that is mediated by gut microbiota. We also discuss the potential for GPR43 as a therapeutic target.

Regulation of Energy Homeostasis by GPR41.

Imbalances in energy regulation lead to metabolic disorders such as obesity and diabetes. Diet plays an essential role in the maintenance of body energy homeostasis by acting not only as energy source but also as a signaling modality. Excess energy increases energy expenditure, leading to a consumption of it. In addition to glucose, mammals utilize short-chain fatty acids (SCFAs), which are produced by colonic bacterial fermentation of dietary fiber, as a metabolic fuel. The roles of SCFAs in energy regulation have remained unclear, although the roles of glucose are well-studied. Recently, a G-protein-coupled receptor deorphanizing strategy successfully identified GPR41 (also called free fatty acid receptor 3 or FFAR3) as a receptor for SCFAs. GPR41 is expressed in adipose tissue, gut, and the peripheral nervous system, and it is involved in SCFA-dependent energy regulation. In this mini-review, we focus on the role of GPR41 in host energy regulation.

Role of free fatty acid receptors in the regulation of energy metabolism.

Free fatty acids (FFAs) are energy-generating nutrients that act as signaling molecules in various cellular processes. Several orphan G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified and play important physiological roles in various diseases. FFA ligands are obtained from food sources and metabolites produced during digestion and lipase degradation of triglyceride stores. FFARs can be grouped according to ligand profiles, depending on the length of carbon chains of the FFAs. Medium- and long-chain FFAs activate FFA1/GPR40 and FFA4/GPR120. Short-chain FFAs activate FFA2/GPR43 and FFA3/GPR41. However, only medium-chain FFAs, and not long-chain FFAs, activate GPR84 receptor. A number of pharmacological and physiological studies have shown that these receptors are expressed in various tissues and are primarily involved in energy metabolism. Because an impairment of these processes is a part of the pathology of obesity and type 2 diabetes, FFARs are considered as key therapeutic targets. Here, we reviewed recently published studies on the physiological functions of these receptors, primarily focusing on energy homeostasis.

Free fatty acids and protein kinase C activation induce GPR120 (free fatty acid receptor 4) phosphorylation.

GPR120, free fatty acid receptor 4, is a recently deorphanized G protein-coupled receptor that seems to play cardinal roles in the regulation of metabolism and in the pathophysiology of inflammatory and metabolic disorders. In the present work a GPR120-Venus fusion protein was expressed in HEK293 Flp-In T-REx cells and its function (increase in intracellular calcium) and phosphorylation were studied. It was observed that the fusion protein migrated in sodium dodecyl sulfate-polyacrylamide gels as a band with a mass of ≈70-75kDa, although other bands of higher apparent weight (>130kDa) were also detected. Cell stimulation with docosahexaenoic acid or α-linolenic acid induced concentration-dependent increases in intracellular calcium and GPR120 phosphorylation. Activation of protein kinase C with phorbol esters also induced a marked receptor phosphorylation but did not alter the ability of 1µM docosahexaenoic acid to increase the intracellular calcium concentration. Phorbol ester-induced GPR120 phosphorylation, but not that induced with docosahexaenoic acid, was blocked by protein kinase C inhibitors (bis-indolyl-maleimide I and Gö 6976) suggesting that conventional kinase isoforms mediate this action. The absence of effect of protein kinase C inhibitors on agonist-induced GPR120 phosphorylation indicates that this kinase does not play a major role in agonist-induced receptor phosphorylation. Docosahexaenoic acid action was associated with marked GPR120 internalization whereas that induced with phorbol esters was smaller at early times.

Mice genetically deficient in vasopressin V1a and V1b receptors are resistant to jet lag.

Jet-lag symptoms arise from temporal misalignment between the internal circadian clock and external solar time. We found that circadian rhythms of behavior (locomotor activity), clock gene expression, and body temperature immediately reentrained to phase-shifted light-dark cycles in mice lacking vasopressin receptors V1a and V1b (V1a(-/-)V1b(-/-)). Nevertheless, the behavior of V1a(-/-)V1b(-/-) mice was still coupled to the internal clock, which oscillated normally under standard conditions. Experiments with suprachiasmatic nucleus (SCN) slices in culture suggested that interneuronal communication mediated by V1a and V1b confers on the SCN an intrinsic resistance to external perturbation. Pharmacological blockade of V1a and V1b in the SCN of wild-type mice resulted in accelerated recovery from jet lag, which highlights the potential of vasopressin signaling as a therapeutic target for management of circadian rhythm misalignment, such as jet lag and shift work.

The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43.

The gut microbiota affects nutrient acquisition and energy regulation of the host, and can influence the development of obesity, insulin resistance, and diabetes. During feeding, gut microbes produce short-chain fatty acids, which are important energy sources for the host. Here we show that the short-chain fatty acid receptor GPR43 links the metabolic activity of the gut microbiota with host body energy homoeostasis. We demonstrate that GPR43-deficient mice are obese on a normal diet, whereas mice overexpressing GPR43 specifically in adipose tissue remain lean even when fed a high-fat diet. Raised under germ-free conditions or after treatment with antibiotics, both types of mice have a normal phenotype. We further show that short-chain fatty acid-mediated activation of GPR43 suppresses insulin signalling in adipocytes, which inhibits fat accumulation in adipose tissue and promotes the metabolism of unincorporated lipids and glucose in other tissues. These findings establish GPR43 as a sensor for excessive dietary energy, thereby controlling body energy utilization while maintaining metabolic homoeostasis.

Voluntary locomotion linked with cerebral activation is mediated by vasopressin V1a receptors in free-moving mice.

We previously reported that cerebral activation suppressed baroreflex control of heart rate (HR) at the onset of voluntary locomotion. In the present study, we examined whether vasopressin V1a receptors in the brain were involved in these responses by using free-moving V1a receptor knockout (KO, n = 8), wild-type mice locally infused with a V1a receptor antagonist into the nucleus tractus solitarii (BLK, n = 8) and control mice (CNT, n = 8). Baroreflex sensitivity (HR/MAP) was determined from HR response (HR) to a spontaneous change in mean arterial pressure (MAP) every 4 s during the total resting period, which was ∼8.7 h, of the 12 h measuring period in the three groups. HR/MAP was determined during the periods when the cross-correlation function (R(t)) between HR and MAP was significant (P < 0.05). Cerebral activity was determined from the power density ratio of to δ wave band (/δ) on the electroencephalogram every 4 s. Spontaneous changes in /δ were significantly correlated with R(t) during 62 ± 3% of the total resting period in CNT (P < 0.05), but only 38 ± 4% in KO and 47 ± 2% in BLK (vs. CNT, both P < 0.001). When R(t) and HR/MAP were divided into six bins according to the level of /δ, both were positively correlated with /δ in CNT (both P < 0.001), while neither was correlated in KO or BLK (all P > 0.05). Moreover, the probability that mice started to move after an increase in /δ was 24 ± 4% in KO and 24 ± 6% in BLK, markedly lower than 61 ± 5% in CNT (both P < 0.001), with no suppression of the baroreflex control of HR. Thus, central V1a receptors might play an important role in suppressing baroreflex control of HR during cerebral activation at the onset of voluntary locomotion.

Diversity-oriented synthesis of pyrazolo[4,3-b]indoles by gold-catalysed three-component annulation: application to the development of a new class of CK2 inhibitors.

Pyrazolo[4,3-b]indole derivatives have been designed as novel CK2 inhibitor compounds based on the binding mode analysis of a previously reported phenylpyrazole-type CK2 inhibitor. A series of pyrazolo[4,3-b]indoles and related dihydropyrazolo[4,3-b]indoles were efficiently prepared from simple starting materials using a gold-catalysed three-component annulation reaction as a key step. Several of the newly synthesized compounds displayed high levels of inhibitory activity, indicating that the pyrazolo[4,3-b]indole core represents a promising scaffold for the development of potent CK2 inhibitors.

FFA1-selective agonistic activity based on docking simulation using FFA1 and GPR120 homology models.

BACKGROUND AND PURPOSE The free fatty acid FFA1 receptor and GPR120 are GPCRs whose endogenous ligands are medium- and long-chain FFAs, and they are important in regulating insulin and GLP-1 secretion respectively. Given that the ligands of FFA1 receptor and GPR120 have similar properties, selective pharmacological tools are required to study their functions further. EXPERIMENTAL APPROACH We used a docking simulation approach using homology models for each receptor. Biological activity was assessed by phosphorylation of ERK and elevation of intracellular calcium ([Ca(2+) ]i ) in cells transfected with FFA1 receptor or GPR120. Insulin secretion from murine pancreatic beta cells (MIN6) was also measured. KEY RESULTS Calculated hydrogen bonding energies between a series of synthetic carboxylic acid compounds and the homology models of the FFA1 receptor and GPR120, using docking simulations, correlated well with the effects of the compounds on ERK phosphorylation in transfected cells (R(2) = 0.65 for FFA1 receptor and 0.76 for GPR120). NCG75, the compound with the highest predicted selectivity for FFA1 receptors from this structure-activity relationship analysis, activated ERK and increased [Ca(2+) ]i as potently as the known FFA1 receptor-selective agonist, Compound 1. Site-directed mutagenesis analysis based on the docking simulation showed that different amino acid residues were important for the recognition and activation by FFA1 receptor agonists. Moreover, NCG75 strongly induced ERK and [Ca(2+) ]i responses, and promoted insulin secretion from MIN6 cells, which express endogenous FFA1 receptors. CONCLUSION AND IMPLICATIONS A docking simulation approach using FFA1 receptor and GPR120 homology models could be useful in predicting FFA1 receptor-selective agonists.