The orphan nuclear receptor small heterodimer partner is required for thiazolidinedione effects in leptin-deficient mice

Background

Small heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp^−/−) double mutant mice.

Results

Both ob/ob and double mutant ob/ob;Shp^−/− mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp^−/− mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp^−/− liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp^−/− mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp^−/− mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.

Conclusions

Our results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.     

Corresponding increase in long-chain acyl-CoA and acylcarnitine after exercise in muscle from VLCAD mice

Long-chain acylcarnitines accumulate in long-chain fatty acid oxidation defects, especially during periods of increased energy demand from fat. To test whether this increase in long-chain acylcarnitines in very long-chain acyl-CoA dehydrogenase (VLCAD(-/-)) knock-out mice correlates with acyl-CoA content, we subjected wild-type (WT) and VLCAD(-/-) mice to forced treadmill running and analyzed muscle long-chain acyl-CoA and acylcarnitine with tandem mass spectrometry (MS/MS) in the same tissues. After exercise, long-chain acyl-CoA displayed a significant increase in muscle from VLCAD(-/-) mice [C16:0-CoA, C18:2-CoA and C18:1-CoA in sedentary VLCAD(-/-): 5.95 +/- 0.33, 4.48 +/- 0.51, and 7.70 +/- 0.30 nmol x g(-1) wet weight, respectively; in exercised VLCAD(-/-): 8.71 +/- 0.42, 9.03 +/- 0.93, and 14.82 +/- 1.20 nmol x g(-1) wet weight, respectively (P < 0.05)]. Increase in acyl-CoA in VLCAD-deficient muscle was paralleled by a significant increase in the corresponding chain length acylcarnitine. Exercise resulted in significant lowering of the free carnitine pool in VLCAD(-/-) muscle. This is the first study demonstrating that acylcarnitines and acyl-CoA directly correlate and concomitantly increase after exercise in VLCAD-deficient muscle.     

Glucocorticoids and cyclic AMP selectively increase hepatic lipin-1 expression, and insulin acts antagonistically

Glucocorticoids (GCs) increase hepatic phosphatidate phosphatase (PAP1) activity. This is important in enhancing the liver's capacity for storing fatty acids as triacylglycerols (TAGs) that can be used subsequently for beta-oxidation or VLDL secretion. PAP1 catalyzes the conversion of phosphatidate to diacylglycerol, a key substrate for TAG and phospholipid biosynthesis. PAP1 enzymes in liver include lipin-1A and -1B (alternatively spliced isoforms) and two distinct gene products, lipin-2 and lipin-3. We determined the mechanisms by which the composite PAP1 activity is regulated using rat and mouse hepatocytes. Levels of lipin-1A and -1B mRNA were increased by dexamethasone (dex; a synthetic GC), and this resulted in increased lipin-1 synthesis, protein levels, and PAP1 activity. The stimulatory effect of dex on lipin-1 expression was enhanced by glucagon or cAMP and antagonized by insulin. Lipin-2 and lipin-3 mRNA were not increased by dex/cAMP, indicating that increased PAP1 activity is attributable specifically to enhanced lipin-1 expression. This work provides the first evidence for the differential regulation of lipin activities. Selective lipin-1 expression explains the GC and cAMP effects on increased hepatic PAP1 activity, which occurs in hepatic steatosis during starvation, diabetes, stress, and ethanol consumption.     

In situ assay of fatty acid β-oxidation by metabolite profiling following permeabilization of cell membranes

Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO.     

Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p has previously been implicated in peroxisome proliferation based on morphological observations of PEX11 mutant cells. Pex11p-deficient cells fail to increase peroxisome number in response to growth on fatty acids and instead accumulate a few giant peroxisomes. We report that mutants deficient in genes required for medium-chain fatty acid (MCFA) beta-oxidation display the same phenotype as Pex11p-deficient cells. Upon closer inspection, we found that Pex11p is required for MCFA beta-oxidation. Disruption of the PEX11 gene results in impaired formation of MCFA-CoA esters as measured in intact cells, whereas their formation is normal in cell lysates. The sole S. cerevisiae MCFA-CoA synthetase (Faa2p) remains properly localized to the inner leaflet of the peroxisomal membrane in PEX11 mutant cells. Therefore, the in vivo latency of MCFA activation observed in Pex11p-deficient cells suggests that Pex11p provides Faa2p with substrate. When PEX11 mutant cells are shifted from glucose to oleate-containing medium, we observed an immediate deficiency in beta-oxidation of MCFAs whereas giant peroxisomes and a failure to increase peroxisome abundance only became apparent much later. Our observations suggest that the MCFA oxidation pathway regulates the level of a signaling molecule that modulates the number of peroxisomal structures in a cell.     

Bioinformatic profiling of the transcriptional response of adult rat cardiomyocytes to distinct fatty acids

Diabetes mellitus, obesity, and dyslipidemia increase risk for cardiovascular disease, and expose the heart to high plasma fatty acid (FA) levels. Recent studies suggest that distinct FA species are cardiotoxic (e.g., palmitate), while others are cardioprotective (e.g., oleate), although the molecular mechanisms mediating these observations are unclear. The purpose of the present study was to investigate the differential effects of distinct FA species (varying carbon length and degree of saturation) on adult rat cardiomyocyte (ARC) gene expression. ARCs were initially challenged with 0.4 mM octanoate (8:0), palmitate (16:0), stearate (18:0), oleate (18:1), or linoleate (18:2) for 24 h. Microarray analysis revealed differential regulation of gene expression by the distinct FAs; the order regarding the number of genes whose expression was influenced by a specific FA was octanoate (1,188) > stearate (740) > palmitate (590) > oleate (83) > linoleate (65). In general, cardioprotective FAs (e.g., oleate) increased expression of genes promoting FA oxidation to a greater extent than cardiotoxic FAs (e.g., palmitate), whereas the latter induced markers of endoplasmic reticulum and oxidative stress. Subsequent RT-PCR analysis revealed distinct time- and concentration-dependent effects of these FA species, in a gene-specific manner. For example, stearate- and palmitate-mediated ucp3 induction tended to be transient (i.e., initial high induction, followed by subsequent repression), whereas oleate-mediated induction was sustained. These findings may provide insight into why diets high in unsaturated FAs (e.g., oleate) are cardioprotective, whereas diets rich in saturated FAs (e.g., palmitate) are not.     

Temperature dependence of O2 consumption; opposite effects of leptin and etomoxir on respiratory quotient in mice

Objectives : The aims were to compare the temperature dependence of the metabolic rate in young ob/ob mice with that in mature ob/ob and db/db mice and to examine the effect on the metabolic substrate preference of leptin and etomoxir in ob/ob, C57BL/6J (wild‐type), and db/db mice. Research Methods and Procedures : In vivo oxygen consumption and carbon dioxide production were continuously measured by indirect calorimetry, and body temperature and total locomotor activity were measured by an implanted transponder. Leptin, etomoxir, or vehicle was administered intraperitoneally. Results : The temperature dependence of the metabolic rate of mature ob/ob and db/db mice were similar to that in wild‐type mice. In young 6‐week‐old ob/ob mice, the metabolic rate was almost doubled at 15 °C. Leptin (2 × 3 mg/kg) decreased the respiratory quotient (RQ) and carbon dioxide production but did not alter oxygen consumption, body temperature, or locomotor activity in ob/ob and C57BL/6J mice and had no effect in the db/db mice. Etomoxir (2 × 30 mg/kg) enhanced RQ and decreased oxygen consumption, carbon dioxide production, and body temperature in ob/ob, C57BL/6J, and db/db mice. Total locomotor activity was reduced in ob/ob and C57BL/6J mice. Discussion : In young ob/ob mice, the temperature sensitivity was enhanced compared with mature mice. Leptin and etomoxir had opposite effects on metabolic substrate preference. Leptin and lowered environmental temperature increased the relative fat oxidation as indicated by decreased RQ, possibly through activation of the sympathetic nervous system.     

Mitochondrial oxidative metabolism during respiratory infection in riboflavin deficient mice

Studies in children and mice have shown that respiratory infection alters riboflavin metabolism, resulting in increased urinary loss of this vitamin. This could be due to mobilization of riboflavin from the liver to blood because liver Flavin adenine dinucleotide (FAD) levels were lowered in the mice during infection. To understand the functional implications of lowered hepatic FAD levels during respiratory infection, flavoprotein functions such as oxidative phosphorylation and β-oxidation of the liver mitochondria were examined during infection in mice. Weanling mice were fed either riboflavin-restricted or control diet for 18 days and then injected with a sublethal dose of Klebsiella pneumoniae. During infection, the state 3 respiratory rate with palmitoyl-L-carnitine and glutamate were significantly lowered (27–29%) in the riboflavin-restricted group, whereas in the control group 10% reduction was observed with palmitoyl-L-carnitine as substrate. A 22% reduction in the respiratory control ratio with palmitoyl-L-carnitine as substrate was observed during infection in the riboflavin-restricted group. The β-oxidation of palmitoyl-L-carnitine was significantly lowered (29%) in the riboflavin-restricted infected group. The results of the study suggest that the effects of infection on vital physiologic functions were more pronounced in the riboflavin-restricted mice than in the control mice. © Elsevier Science Inc. 1999     

Arylacetamide deacetylase attenuates fatty-acid-induced triacylglycerol accumulation in rat hepatoma cells

Mobilization of hepatic triacylglycerol stores provides substrates for mitochondrial β-oxidation and assembly of VLDLs; however, the identity of lipolytic enzymes involved in the regulation of this process remains largely unknown. Arylacetamide deacetylase (AADA) shares homology with hormone-sensitive lipase and therefore could potentially participate in hepatic lipid metabolism, including the regulation of hepatic triacylglycerol levels. We have established McArdle-RH7777 (rat hepatoma) cell lines stably expressing mouse AADA cDNA and performed metabolic labeling as well as lipid mass analyses. Expression of AADA cDNA in McArdle-RH7777 cells significantly reduced intracellular triacylglycerol levels and apolipoprotein B secretion and increased fatty acid oxidation.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice

Muscle fibers attach to laminin in the basal lamina using two distinct mechanisms: the dystrophin glycoprotein complex and the alpha 7 beta 1 integrin. Defects in these linkage systems result in Duchenne muscular dystrophy (DMD), alpha 2 laminin congenital muscular dystrophy, sarcoglycan-related muscular dystrophy, and alpha 7 integrin congenital muscular dystrophy. Therefore, the molecular continuity between the extracellular matrix and cell cytoskeleton is essential for the structural and functional integrity of skeletal muscle. To test whether the alpha 7 beta 1 integrin can compensate for the absence of dystrophin, we expressed the rat alpha 7 chain in mdx/utr(-/-) mice that lack both dystrophin and utrophin. These mice develop a severe muscular dystrophy highly akin to that in DMD, and they also die prematurely. Using the muscle creatine kinase promoter, expression of the alpha 7BX2 integrin chain was increased 2.0-2.3-fold in mdx/utr(-/-) mice. Concomitant with the increase in the alpha 7 chain, its heterodimeric partner, beta 1D, was also increased in the transgenic animals. Transgenic expression of the alpha 7BX2 chain in the mdx/utr(-/-) mice extended their longevity by threefold, reduced kyphosis and the development of muscle disease, and maintained mobility and the structure of the neuromuscular junction. Thus, bolstering alpha 7 beta 1 integrin-mediated association of muscle cells with the extracellular matrix alleviates many of the symptoms of disease observed in mdx/utr(-/-) mice and compensates for the absence of the dystrophin- and utrophin-mediated linkage systems. This suggests that enhanced expression of the alpha 7 beta 1 integrin may provide a novel approach to treat DMD and other muscle diseases that arise due to defects in the dystrophin glycoprotein complex. A video that contrasts kyphosis, gait, joint contractures, and mobility in mdx/utr(-/-) and alpha 7BX2-mdx/utr(-/-) mice can be accessed at http://www.jcb.org/cgi/content/full/152/6/1207.     

Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members

The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.     

Ultrastructural localization of integrin subunits beta4 and alpha3 within the migrating epithelial tongue of in vivo human wounds

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.     

The role of β-sheets in the structure and assembly of keratins

X-ray diffraction, infrared and electron microscope studies of avian and reptilian keratins, and of stretched wool and hair, have played a central role in the development of models for the β-conformation in proteins. Both α- and β-keratins contain sequences that are predicted to adopt a β-conformation and these are believed to play an important part in the assembly of the filaments and in determining their mechanical properties. Interactions between the small β-sheets in keratins provide a simple mechanism through which shape and chemical complementarity can mediate the assembly of molecules into highly specific structures. Interacting β-sheets in crystalline proteins are often related to one another by diad symmetry and the data available on feather keratin suggest that the filament is assembled from dimers in which the β-sheets are related by a perpendicular diad. The most detailed model currently available is for feather and reptilian keratin but the presence of related β-structural forms in mammalian keratins is also noted.     

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.