SUZ domain-containing proteins have multiple effects on nonsense-mediated decay target transcripts.

Many transcripts are targeted by nonsense-mediated decay (NMD), leading to their degradation and the inhibition of their translation. We found that the protein SUZ domain-containing protein 1 (SZRD1) interacts with the key NMD factor up-frameshift 1. When recruited to NMD-sensitive reporter gene transcripts, SZRD1 increased protein production, at least in part, by relieving translational inhibition. The conserved SUZ domain in SZRD1 was required for this effect. The SUZ domain is present in only three other human proteins besides SZRD1: R3H domain-containing protein 1 and 2 (R3HDM1, R3HDM2) and cAMP-regulated phosphoprotein 21 (ARPP21). We found that ARPP21, similarly to SZRD1, can increase protein production from NMD-sensitive reporter transcripts in an SUZ domain-dependent manner. This indicated that the SUZ domain-containing proteins could prevent translational inhibition of transcripts targeted by NMD. Consistent with the idea that SZRD1 mainly prevents translational inhibition, we did not observe a systematic decrease in the abundance of NMD targets when we knocked down SZRD1. Surprisingly, knockdown of SZRD1 in two different cell lines led to reduced levels of the NMD component UPF3B, which was accompanied by increased levels in a subset of NMD targets. This suggests that SZRD1 is required to maintain normal UPF3B levels and indicates that the effect of SZRD1 on NMD targets is not limited to a relief from translational inhibition. Overall, our study reveals that human SUZ domain-containing proteins play a complex role in regulating protein output from transcripts targeted by NMD.

Parkinson's disease protein PARK7 prevents metabolite and protein damage caused by a glycolytic metabolite.

Cells are continuously exposed to potentially dangerous compounds. Progressive accumulation of damage is suspected to contribute to neurodegenerative diseases and aging, but the molecular identity of the damage remains largely unknown. Here we report that PARK7, an enzyme mutated in hereditary Parkinson's disease, prevents damage of proteins and metabolites caused by a metabolite of glycolysis. We found that the glycolytic metabolite 1,3-bisphosphoglycerate (1,3-BPG) spontaneously forms a novel reactive intermediate that avidly reacts with amino groups. PARK7 acts by destroying this intermediate, thereby preventing the formation of proteins and metabolites with glycerate and phosphoglycerate modifications on amino groups. As a consequence, inactivation of PARK7 (or its orthologs) in human cell lines, mouse brain, and Drosophila melanogaster leads to the accumulation of these damaged compounds, most of which have not been described before. Our work demonstrates that PARK7 function represents a highly conserved strategy to prevent damage in cells that metabolize carbohydrates. This represents a fundamental link between metabolism and a type of cellular damage that might contribute to the development of Parkinson's disease.

Brief O2 uploading during continuous hypothermic machine perfusion is simple yet effective oxygenation method to improve initial kidney function in a porcine autotransplant model.

With oxygenation proposed as a resuscitative measure during hypothermic models of preservation, the aim of this study was to evaluate the optimal start time of oxygenation during continuous hypothermic machine perfusion (HMP). In this porcine ischemia-reperfusion autotransplant model, the left kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia prior to 22 hours of HMP and autotransplantation. Kidneys were randomized to receive 2 hours of oxygenation during HMP either at the start (n = 6), or end of the perfusion (n = 5) and outcomes were compared to standard, nonoxygenated HMP (n = 6) and continuous oxygenated HMP (n = 8). The brief initial and continuous oxygenated HMP groups were associated with superior graft recovery compared to either standard, nonoxygenated HMP or kidneys oxygenated at the end of HMP. This correlated with significant metabolic differences in perfusate (eg, lactate, succinate, flavin mononucleotide) and tissues (eg, succinate, adenosine triphosphate, hypoxia-inducible factor-1α, nuclear factor erythroid 2-related factor 2) suggesting superior mitochondrial preservation with initial oxygenation. Brief initial O2 uploading during HMP at procurement site might be an easy and effective preservation strategy to maintain aerobic metabolism, protect mitochondria, and achieve an improved early renal graft function compared with standard HMP or oxygen supply shortly at the end of HMP preservation.

The synthesis of branched-chain fatty acids is limited by enzymatic decarboxylation of ethyl- and methylmalonyl-CoA.

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methyl-branches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl-CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethyl-branched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function.

Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

A conserved phosphatase destroys toxic glycolytic side products in mammals and yeast.

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, 'metabolite repair enzymes' eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.

ISPD produces CDP-ribitol used by FKTN and FKRP to transfer ribitol phosphate onto α-dystroglycan.

Mutations in genes required for the glycosylation of α-dystroglycan lead to muscle and brain diseases known as dystroglycanopathies. However, the precise structure and biogenesis of the assembled glycan are not completely understood. Here we report that three enzymes mutated in dystroglycanopathies can collaborate to attach ribitol phosphate onto α-dystroglycan. Specifically, we demonstrate that isoprenoid synthase domain-containing protein (ISPD) synthesizes CDP-ribitol, present in muscle, and that both recombinant fukutin (FKTN) and fukutin-related protein (FKRP) can transfer a ribitol phosphate group from CDP-ribitol to α-dystroglycan. We also show that ISPD and FKTN are essential for the incorporation of ribitol into α-dystroglycan in HEK293 cells. Glycosylation of α-dystroglycan in fibroblasts from patients with hypomorphic ISPD mutations is reduced. We observe that in some cases glycosylation can be partially restored by addition of ribitol to the culture medium, suggesting that dietary supplementation with ribitol should be evaluated as a therapy for patients with ISPD mutations.

SRA regulates adipogenesis by modulating p38/JNK phosphorylation and stimulating insulin receptor gene expression and downstream signaling.

The Steroid Receptor RNA Activator (SRA) enhances adipogenesis and increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. To assess the mechanism, we differentiated ST2 mesenchymal precursor cells that did or did not overexpress SRA into adipocytes using combinations of methylisobutylxanthine, dexamethasone and insulin. These studies showed that SRA overexpression promotes full adipogenesis in part by stimulation of insulin/insulin-like growth factor-1 (IGF-1) signaling. SRA overexpression inhibited phosphorylation of p38 mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) in the early differentiation of ST2 cells. Conversely, knockdown of endogenous SRA in 3T3-L1 cells increased phosphorylation of JNK. Knockdown of SRA in mature 3T3-L1 adipocytes reduced insulin receptor (IR) mRNA and protein levels, which led to decreased autophosphorylation of IRß and decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt. This likely reflects a stimulatory role of SRA on IR transcription, as transfection studies showed that SRA increased expression of an IR promoter-luciferase reporter construct.

Identification of TP53-induced glycolysis and apoptosis regulator (TIGAR) as the phosphoglycolate-independent 2,3-bisphosphoglycerate phosphatase.

The p53-induced protein TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] is considered to be a F26BPase (fructose-2,6-bisphosphatase) with an important role in cancer cell metabolism. The reported catalytic efficiency of TIGAR as an F26BPase is several orders of magnitude lower than that of the F26BPase component of liver or muscle PFK2 (phosphofructokinase 2), suggesting that F26BP (fructose 2,6-bisphosphate) might not be the physiological substrate of TIGAR. We therefore set out to re-evaluate the biochemical function of TIGAR. Phosphatase activity of recombinant human TIGAR protein was tested on a series of physiological phosphate esters. The best substrate was 23BPG (2,3-bisphosphoglycerate), followed by 2PG (2-phosphoglycerate), 2-phosphoglycolate and PEP (phosphoenolpyruvate). In contrast the catalytic efficiency for F26BP was approximately 400-fold lower than that for 23BPG. Using genetic and shRNA-based cell culture models, we show that loss of TIGAR consistently leads to an up to 5-fold increase in the levels of 23BPG. Increases in F26BP levels were also observed, albeit in a more limited and cell-type dependent manner. The results of the present study challenge the concept that TIGAR acts primarily on F26BP. This has significant implications for our understanding of the metabolic changes downstream of p53 as well as for cancer cell metabolism in general. It also suggests that 23BPG might play an unrecognized function in metabolic control.

Secreted frizzled-related protein 5 suppresses adipocyte mitochondrial metabolism through WNT inhibition.

Preadipocytes secrete several WNT family proteins that act through autocrine/paracrine mechanisms to inhibit adipogenesis. The activity of WNT ligands is often decreased by secreted frizzled-related proteins (SFRPs). Sfrp5 is strongly induced during adipocyte differentiation and increases in adipocytes during obesity, presumably to counteract WNT signaling. We tested the hypothesis that obesity-induced Sfrp5 expression promotes the development of new adipocytes by inhibiting endogenous suppressors of adipogenesis. As predicted, mice that lack functional SFRP5 were resistant to diet-induced obesity. However, counter to our hypothesis, we found that adipose tissue of SFRP5-deficient mice had similar numbers of adipocytes, but a reduction in large adipocytes. Transplantation of adipose tissue from SFRP5-deficient mice into leptin receptor-deficient mice indicated that the effects of SFRP5 deficiency are tissue-autonomous. Mitochondrial gene expression was increased in adipose tissue and cultured adipocytes from SFRP5-deficient mice. In adipocytes, lack of SFRP5 stimulated oxidative capacity through increased mitochondrial activity, which was mediated in part by PGC1α and mitochondrial transcription factor A. WNT3a also increased oxygen consumption and the expression of mitochondrial genes. Thus, our findings support a model of adipogenesis in which SFRP5 inhibits WNT signaling to suppress oxidative metabolism and stimulate adipocyte growth during obesity.

Multiple roles for the non-coding RNA SRA in regulation of adipogenesis and insulin sensitivity.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARγ coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA, Steroid receptor RNA Activator (SRA), associates with PPARγ and coactivates PPARγ-dependent reporter gene expression. Overexpression of SRA in ST2 mesenchymal precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes involved in the cell cycle, and insulin and TNFα signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARγ. SRA in adipocytes increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the expression of adipocyte-related inflammatory genes and TNFα-induced phosphorylation of c-Jun NH(2)-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways.

Expression of miR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation.

The regulation of synthesis, degradation, and distribution of lipids is crucial for homeostasis of organisms and cells. The sterol regulatory element-binding protein (SREBP) transcription factor family is post-translationally activated in situations of reduced lipid abundance and activates numerous genes involved in cholesterol, fatty acid, and phospholipid synthesis. In this study, we provide evidence that the primary transcript of SREBP2 contains an intronic miRNA (miR-33) that reduces cellular cholesterol export via inhibition of translation of the cholesterol export pump ABCA1. Notably, miR-33 also inhibits translation of several transcripts encoding proteins involved in fatty acid ß-oxidation including CPT1A, HADHB, and CROT, thereby reducing fatty acid degradation. The genetic locus encoding SREBP2 and miR-33 therefore contains a protein that increases lipid synthesis and a miRNA that prevents export and degradation of newly synthesized lipids. These results add an additional layer of complexity to our understanding of lipid homeostasis and might open possibilities for future therapeutic intervention.

Roles for miRNA-378/378* in adipocyte gene expression and lipogenesis.

In this study, we explored the roles of microRNAs in adipocyte differentiation and metabolism. We first knocked down Argonaute2 (Ago2), a key enzyme in the processing of micro-RNAs (miRNAs), to investigate a potential role for miRNAs in adipocyte differentiation and/or metabolism. Although we did not observe dramatic differences in adipogenesis between Ago2 knock-down and control 3T3-L1 cells, incorporation of [(14)C]glucose or acetate into triacylglycerol, and steady-state levels of triacyglycerol were all reduced, suggesting a role for miRNAs in adipocyte metabolism. To study roles of specific miRNAs in adipocyte biology, we screened for miRNAs that are differentially expressed between preadipocytes and adipocytes for the 3T3-L1 and ST2 cell lines. Distinct subsets of miRNAs decline or increase during adipocyte conversion, whereas most miRNAs are not regulated. One locus encoding two miRNAs, 378/378*, contained within the intron of PGC-1beta is highly induced during adipogenesis. When overexpressed in ST2 mesenchymal precursor cells, miRNA378/378* increases the size of lipid droplets and incorporation of [(14)C]acetate into triacylglycerol. Although protein and mRNA expression levels of C/EBPalpha, C/EBPbeta, C/EBPdelta, and PPARgamma1 are unchanged, microarray and quantitative RT-PCR analyses indicate that a set of lipogenic genes are upregulated, perhaps due to increased expression of PPARgamma2. Knock-down of miRNA378 and/or miRNA378* decreases accumulation of triacylglycerol. Interestingly, we made the unexpected finding that miRNA378/378* specifically increases transcriptional activity of C/EBPalpha and C/EBPbeta on adipocyte gene promoters.

On the role of FOX transcription factors in adipocyte differentiation and insulin-stimulated glucose uptake.

In this study, we explore the effects of several FOX and mutant FOX transcription factors on adipocyte determination, differentiation, and metabolism. In addition to Foxc2 and Foxo1, we report that Foxf2, Foxp1, and Foxa1 are other members of the Fox family that show regulated expression during adipogenesis. Although enforced expression of FOXC2 inhibits adipogenesis, Foxf2 slightly enhances the rate of differentiation. Constitutively active FOXC2-VP16 inhibits adipogenesis through multiple mechanisms. FOXC2-VP16 impairs the transient induction of C/EBPbeta during adipogenesis and induces expression of the transcriptional repressor Hey1 as well as the activator of Wnt/beta-catenin signaling, Wnt10b. The constitutive transcriptional repressor, FOXC2-Eng, enhances adipogenesis of preadipocytes and multipotent mesenchymal precursors and determines NIH-3T3 and C2C12 cells to the adipocyte lineage. Although PPARgamma ligand or C/EBPalpha are not necessary for stimulation of adipogenesis by FOXC2-Eng, at least low levels of PPARgamma protein are absolutely required. Finally, expression of FOXC2-Eng in adipocytes increases insulin-stimulated glucose uptake, further expanding the profound and pleiotropic effects of FOX transcription factors on adipocyte biology.

Dax-1 and steroid receptor RNA activator (SRA) function as transcriptional coactivators for steroidogenic factor 1 in steroidogenesis.

The nuclear receptor steroidogenic factor 1 (SF-1) is essential for adrenal development and steroidogenesis. The atypical orphan nuclear receptor Dax-1 binds to SF-1 and represses SF-1 target genes. Paradoxically, however, loss-of-function mutations of Dax-1 also cause adrenal hypoplasia, suggesting that Dax-1 may function as an SF-1 coactivator under some circumstances. Indeed, we found that Dax-1 can function as a dosage-dependent SF-1 coactivator. Both SF-1 and Dax-1 bind to steroid receptor RNA activator (SRA), a coactivator that functions as an RNA. The coactivator TIF2 also associates with Dax-1 and synergistically coactivates SF-1 target gene transcription. A naturally occurring Dax-1 mutation inhibits this transactivation, and the mutant Dax-1-TIF2 complex mislocalizes in living cells. Coactivation by Dax-1 is abolished by SRA knockdown. The expression of the steroidogenic gene products steroidogenic acute regulatory protein (StAR) and melanocortin 2 receptor is reduced in adrenal Y1 cells following the knockdown of endogenous SRA. Similarly, the knockdown of endogenous Dax-1 downregulates the expression of the steroidogenic gene products CYP11A1 and StAR in both H295R adrenal and MA-10 Leydig cells. These findings reveal novel functions of SRA and Dax-1 in steroidogenesis and adrenal biology.

Hyperphagia and obesity in female mice lacking tissue inhibitor of metalloproteinase-1.

Certain matrix metalloproteinases and their regulators, the tissue inhibitors of metalloproteinases (TIMPs), are involved in development and remodeling of adipose tissue. In studying Timp1() mice, which have a null mutation in Timp1 (Timp1(-/-)), we observed that females exhibit increased body weight by 3 months of age due to increased total body lipid and adipose tissue. Whereas Timp1(-/-) mice have increased size and number of adipocytes, they also display increased food intake despite hyperleptinemia, suggesting that alterations in hypothalamic leptin action or responsiveness may underlie their weight gain. Indeed, leptin promotes the expression of Timp1 mRNA in the hypothalamus, and leptin signaling via signal transducer and activator of transcription-3 mediates the expression of hypothalamic Timp1. Furthermore, Timp1(-/-) mice demonstrate increased food intake and altered expression of certain hypothalamic neuropeptide genes prior to elevated weight gain. Thus, whereas previous data suggested roles for matrix metalloproteinases and TIMPs in the regulation of adipose tissue, these data reveal that Timp1 mRNA is induced by leptin in the hypothalamus and that expression and action of Timp1 contributes to the regulation of feeding and energy balance.

The microRNA miR-8 is a conserved negative regulator of Wnt signaling.

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

Fat-specific protein 27 regulates storage of triacylglycerol.

FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins. In this study, we investigated the function of another member of this family, FSP27 (Cidec), in apoptosis and adipocyte metabolism. Although overexpression of FSP27 is sufficient to increase apoptosis of 293T and 3T3-L1 cells, more physiological levels of expression stimulate spontaneous lipid accumulation in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased beta-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L1 adipocytes. Stable knockdown of FSP27 during adipogenesis of 3T3-L1 cells substantially decreases lipid droplet size, increases mitochondrial and lipid droplet number, and modestly increases glucose uptake and lipolysis. Expression of FSP27 in subcutaneous adipose tissue of a human diabetes cohort decreases with total fat mass but is not associated with measures of insulin resistance (e.g. homeostasis model assessment). Together, these data indicate that FSP27 binds to lipid droplets and regulates their enlargement.

Resistance to diet-induced obesity and improved insulin sensitivity in mice with a regulator of G protein signaling-insensitive G184S Gnai2 allele.

OBJECTIVE: Guanine nucleotide binding protein (G protein)-mediated signaling plays major roles in endocrine/metabolic function. Regulators of G protein signaling (RGSs, or RGS proteins) are responsible for the subsecond turn off of G protein signaling and are inhibitors of signal transduction in vitro, but the physiological function of RGS proteins remains poorly defined in part because of functional redundancy. RESEARCH DESIGN AND METHODS: We explore the role of RGS proteins and G alpha(i2) in the physiologic regulation of body weight and glucose homeostasis by studying genomic "knock-in" mice expressing RGS-insensitive G alpha(i2) with a G184S mutation that blocks RGS protein binding and GTPase acceleration. RESULTS: Homozygous G alpha(i2)(G184S) knock-in mice show slightly reduced adiposity. On a high-fat diet, male G alpha(i2)(G184S) mice are resistant to weight gain, have decreased body fat, and are protected from insulin resistance. This appears to be a result of increased energy expenditure. Both male and female G alpha(i2)(G184S) mice on a high-fat diet also exhibit enhanced insulin sensitivity and increased glucose tolerance despite females having similar weight gain and adiposity compared with wild-type female mice. CONCLUSIONS: RGS proteins and G alpha(i2) signaling play important roles in the control of insulin sensitivity and glucose metabolism. Identification of the specific RGS proteins involved might permit their consideration as potential therapeutic targets for obesity-related insulin resistance and type 2 diabetes.

Wnt10b increases postnatal bone formation by enhancing osteoblast differentiation.

UNLABELLED: Overexpression of Wnt10b from the osteocalcin promoter in transgenic mice increases postnatal bone mass. Increases in osteoblast perimeter, mineralizing surface, and bone formation rate without detectable changes in pre-osteoblast proliferation, osteoblast apoptosis, or osteoclast number and activity suggest that, in this animal model, Wnt10b primarily increases bone mass by stimulating osteoblastogenesis. INTRODUCTION: Wnt signaling regulates many aspects of development including postnatal accrual of bone. Potential mechanisms for how Wnt signaling increases bone mass include regulation of osteoblast and/or osteoclast number and activity. To help differentiate between these possibilities, we studied mice in which Wnt10b is expressed specifically in osteoblast lineage cells or in mice devoid of Wnt10b. MATERIALS AND METHODS: Transgenic mice, in which mouse Wnt10b is expressed from the human osteocalcin promoter (Oc-Wnt10b), were generated in C57BL/6 mice. Transgene expression was evaluated by RNase protection assay. Quantitative assessment of bone variables was done by radiography, muCT, and static and dynamic histomorphometry. Mechanisms of bone homeostasis were evaluated with assays for BrdU, TUNEL, and TRACP5b activity, as well as serum levels of C-terminal telopeptide of type I collagen (CTX). The endogenous role of Wnt10b in bone was assessed by dynamic histomorphometry in Wnt10b(-/-) mice. RESULTS: Oc-Wnt10b mice have increased mandibular bone and impaired eruption of incisors during postnatal development. Analyses of femoral distal metaphyses show significantly higher BMD, bone volume fraction, and trabecular number. Increased bone formation is caused by increases in number of osteoblasts per bone surface, rate of mineral apposition, and percent mineralizing surface. Although number of osteoclasts per bone surface is not altered, Oc-Wnt10b mice have increased total osteoclast activity because of higher bone mass. In Wnt10b(-/-) mice, changes in mineralizing variables and osteoblast perimeter in femoral distal metaphyses were not observed; however, bone formation rate is reduced because of decreased total bone volume and trabecular number. CONCLUSIONS: High bone mass in Oc-Wnt10b mice is primarily caused by increased osteoblastogenesis, with a minor contribution from elevated mineralizing activity of osteoblasts.