Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity.

Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others activates G protein-coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector, which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the ß3-adrenergic receptor (ADRB3) in a CCAAT/enhancer binding protein (C/EBP)-α- and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, depletion of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.

Green Tea Polyphenol EGCG Upregulates Tollip Expression by Suppressing Elf-1 Expression.

TLR signaling is critical to innate immune system regulation; however, aberrant TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, and tumor metastasis. Moreover, a recent study found that TLR-4 signaling pathway inhibition might be a target for the suppression of chronic inflammatory disorders. In this article, we show that the green tea polyphenol epigallocatechin-3-O-gallate (EGCG) increases the expression of Toll interacting protein, a strong inhibitor of TLR4 signaling, by suppressing the expression of E74-like ETS transcription factor 1 (Elf-1). A mechanistic study revealed that EGCG suppressed Elf-1 expression via protein phosphatase 2A/cyclic GMP (cGMP)-dependent mechanisms. We also confirmed that orally administered EGCG and a cGMP inducer upregulated Toll interacting protein expression, increased intracellular levels of cGMP in macrophages, and suppressed Elf-1 expression. These data support EGCG and a cGMP inducer as potential candidate suppressors of TLR4 signaling.

Detailed localization of regulator of G protein signaling 2 messenger ribonucleic acid and protein in the rat brain.

Regulator of G protein signaling (RGS) proteins are a recently identified family of proteins which dampen G protein-coupled receptor-mediated signaling by accelerating the intrinsic GTPase activity of Galpha subunits of heterotrimeric G proteins. More than 20 different RGSs have been identified and at least 10 are expressed in the CNS. The present study describes in detail the localization in the rat brain of one member of this family, RGS2. The distribution of RGS2 mRNA and protein has been studied in parallel by performing in situ hybridization and immunoautoradiography on adjacent rat brain sections. Our localization study reveals that RGS2 mRNA and protein are widely expressed in the brain. Protein and mRNA are mostly colocalized such as in neocortex, piriform cortex, caudate-putamen, septum, hippocampus, locus coeruleus. Some mismatches were also observed such as presence of mRNA but not protein in the paraventricular nucleus, the substantia nigra pars compacta and the red nucleus, suggesting that RGS2 protein is present in neuronal projections. Previous reports describing an induction of RGS2 mRNA in the rat striatum after psychostimulants (amphetamine, cocaine) led us to focus on the distribution of RGS2 in the basal ganglia circuitry. The absence of RGS2 mRNA and protein in the globus pallidus suggests that RGS2 would play its regulatory role more in the direct (striatonigral) than in the indirect (striatopallidal) striatal output pathway. In addition, to delineate the implication of RGS2 in pre- and/or postsynaptic functions in the basal ganglia, we performed lesions of the nigrostriatal pathway by 6-hydroxydopamine (6-OHDA) and striatal quinolinic acid lesions. The 6-OHDA lesion did not modify RGS2 mRNA or protein levels in the caudate-putamen whereas the intrastriatal quinolinic acid infusion caused a marked reduction of RGS2 mRNA and protein in the lesioned zone. These data indicate that RGS2 is predominantly expressed in intrinsic striatal neurons. Moreover, the absence of detectable change in RGS2 expression after injections of 6-OHDA suggests also that RGS2 is not primarily involved in the hypersensitization of postsynaptic dopamine receptors observed after lesion of the nigrostriatal pathway.

Green fluorescent protein in the study of neuronal signaling pathways.

In recent years, techniques have been established for transiently co-transfecting cells with cDNA of the jellyfish green fluorescent protein (GFP), a reporter gene that encodes a non-toxic marker. This approach can be applied to primary neurons where it has become especially useful for the study of neuronal second messenger pathways. This unit describes procedures for transfecting neurons in primary culture: transfection with GFP DNA, including co-transfecting with separate GFP and gene-of-interest constructs, transfecting with a single construct containing the gene of interest fused to a GFP gene, and transfecting with a single construct containing separate gene-of-interest and GFP cassettes. Also included is a method for the rapid, large-scale preparation of a nearly homogeneous population of neurons from rat cerebellum. The Commentary provides several examples of how this approach can be applied to specific biological questions on neuronal signaling pathways.

Organization of the Drosophila genome in mutants with changes in second messenger metabolism and learning ability.

Mechanisms modifying the structural-functional organization of polytene chromosomes were studied in a Drosophila line in which the activating properties of calmodulin were altered and learning ability was increased, by treating mutants with homeopathic preparations which affect Ca2+ and F- ion metabolism. The results indicated a dominant role for Ca2+ ions and calmodulin in determining the chromocentric organization of the nucleus. F- ions, which stimulate the adenylate cyclase complex, were found not to have a role.

Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster.

A cDNA clone is described that encodes a novel G-protein-coupled dopamine receptor (DopR99B) expressed in Drosophila heads. The DopR99B receptor maps to 99B3-5, close to the position of the octopamine/tyramine receptor gene at 99A10-B1, suggesting that the two may be related through a gene duplication. Agonist stimulation of DopR99B receptors expressed in Xenopus oocytes increased intracellular Ca2+ levels monitored as changes in an endogenous inward Ca2+-dependent chloride current. In addition to initiating this intracellular Ca2+ signal, stimulation of DopR99B increased cAMP levels. The rank order of potency of agonists in stimulating the chloride current is: dopamine > norepinephrine > epinephrine > tyramine. Octopamine and 5-hydroxytryptamine are not active (< 100 microM). This pharmacological profile plus the second-messenger coupling pattern suggest that the DopR99B receptor is a D1-like dopamine receptor. However, the hydrophobic core region of the DopR99B receptor shows almost equal amino acid sequence identity (40-48%) with vertebrate serotonergic, alpha 1- and beta-adrenergic, and D1-like and D2-like dopaminergic receptors. Thus, this Drosophila receptor defines a novel structural class of dopamine receptors. Because DopR99B is the second dopamine receptor cloned from Drosophila, this work establishes dopamine receptor diversity in a system amenable to genetic dissection.

Cloning and functional characterization of extracellular Ca(2+)-sensing receptors from parathyroid and kidney.

Parathyroid cells recognize and respond to (i.e., "sense") minute perturbations in the extracellular ionized calcium concentration (Ca2+o), but the mechanisms underlying this process have remained obscure. Recently, we employed expression cloning in Xenopus laevis oocytes to isolate a cDNA coding for a Ca2+o-sensing receptor from bovine parathyroid. Like the native receptor, the cloned Ca2+o-sensing receptor stimulates phospholipase C (PLC) in a G-protein-dependent manner with a nearly identical pharmacological profile. Its deduced amino acid sequence confirms that it is a member of the superfamily of G-protein-coupled receptors (GPR). Transcripts for the receptor are expressed in parathyroid and other tissues that sense Ca2+o (viz., kidney and thyroidal C-cells) as well as those that have no known role in extracellular Ca2+ homeostasis, such as the brain. The availability of the cDNA clone for the Ca2+o-sensing receptor made it possible to test the hypothesis that mutations in the gene encoding the human homolog of the receptor cause inherited disorders of mineral ion metabolism. Familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are, in fact, caused by mutations that reduce the activity of the receptor when they are present in the heterozygous and homozygous states, respectively. In contrast, we have subsequently discovered a family in which a form of autosomal dominant hypocalcemia results from an activating mutation in the receptor gene. The Ca2+-sensing receptor, therefore, permits Ca2+o to play a "hormonelike" role as an extracellular first messenger in addition to its well described role as an important intracellular second messenger.

[The organizational characteristics of the Drosophila genome in mutants with altered second-messenger metabolism and with learning ability]. / Osobennosti organizatsii genoma drozofily u mutantov s izmenennym metabolizmom vtorichnykh posrednikov i sposobnosti k obucheniiu.

The data obtained on homeopathic correction of the Drosophila mutant strains with altered activation properties of calmodulin and increased ability for learning, suggest an important role of Ca ions calmodulin in formation of chromocentral organisation of the nucleus. No significant role of the F ions was revealed.

Molecular cloning and characterization of a murine LPS-inducible cDNA.

Murine macrophages respond to endotoxins by inducing a vast array of genes that play a major role in the host's response to infection and tumor growth. We have isolated and characterized a 1.8-kb cDNA, designated IRG2, from a cDNA library prepared from RNA isolated from the murine cell line, RAW 264.7, after bacterial LPS stimulation. The cDNA encodes a protein of 47 kDa that is the murine homologue of a small family of proteins described from IFN-induced human cells. The IRG2 message does not appear until 3 h after LPS exposure and its induction is dependent on new protein synthesis. IRG2 induction by LPS is slightly inhibited by the anti-inflammatory steroid, dexamethasone. Increasing cytosolic cAMP with either forskolin, dibutyryl cAMP, or 8-(4-chlorophenylthio)-cAMP caused marked inhibition of the LPS induction of IRG2. In contrast, activation of PKC with phorbol ester potentiated the LPS response. Removing extracellular Ca2+ with EGTA inhibited IRG2 induction; increasing intracellular calcium with the calcium ionophore A23187 led to enhanced levels of the IRG2 transcript. These data suggest that the induction of IRG2 occurs via a PKC pathway.