Liver fatty acid-binding protein (L-Fabp

Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. Here, we showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5′-monophosphate-activated protein kinase)-dependent autophagy regression of hepatic steatosis in vivo, and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that, by inhibiting glucose transport, trehalose triggers beneficial cellular autophagy.

No abstract

Osteocalcin (OC) is a calcium binding protein expressed in mature osteoblasts undergoing mineralization. The OC gene has been identified as a target for transcriptional suppression by Msx2, a homeodomain transcription factor that controls ossification in calvarial bone of the developing skull. We have initiated systematic structure-function analyses of Msx2, using OC promoter suppression (luciferase reporter) in MC3T3-E1 calvarial osteoblasts as an assay. Msx2 variants were epitope ("FLAG")-tagged for monitoring Msx2 protein expression by Western blot analysis. Functional analyses of N- and C-terminally truncated molecules identify Msx2 residues 97-208 as the core suppressor domain. Internal deletion analyses indicate that suppressor function is dependent upon structural features encoded by residues 132-148--upstream of the homeodomain and overlapping the homeodomain N-terminal extension--but not upon residues in the three homeodomain helices. Mutations that enhance DNA binding activity do not proportionally enhance Msx2 suppressor function; moreover, a Msx2 point mutant Msx2(T147A) that completely lacks DNA binding activity is indistinguishable from wild-type Msx2 in its ability to suppress the OC promoter, demonstrating that direct interaction with DNA is not required for Msx2 suppressor function. This suggests that Msx2 suppresses transcription via protein-protein interactions with components of the basal transcriptional machinery, either alone or in concert with co-regulators. Using interaction "Far Western" blotting assays, we systematically tested for protein-protein interactions between Msx2 and components of the basal transcriptional machinery known to mediate transcriptional activation (TBP, TFIIB, and TFIIF). Msx2 binds both components of TFIIF (RAP74, RAP30), but not TFIIB or TBP. Msx2(55-208) encompasses core suppressor domain residues and binds TFIIF; in this context, deletion of the seventeen amino acid residues 132-148 that are required for core suppressor function abrogates interactions with TFIIF components. Co-expression of RAP74 in MC3T3-E1 cells partially reverses (>50%) suppression of OC promoter activity by Msx2, while co-expression of TFIIB or RAP30 has no effect. Thus the core suppressor domain of Msx2 participates in functionally important interactions with RAP74 that regulate OC promoter activity in calvarial osteoblasts.

Microsomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifeninducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an ϳ80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion. The mobilization and secretion of triglyceride (TG)3 -rich lipoproteins from mammalian enterocytes and hepatocytes are critically dependent on the integrated function of at least two dominant genes. These include the microsomal triglyceride transfer protein (MTTP), a resident endoplasmic reticulum protein that facilitates the transfer of neutral lipid to a large hydrophobic acceptor protein, apolipoprotein B (apoB) (1). ApoB in turn functions as a requisite structural component of the surface of TG-rich lipoproteins and plays a vital role in plasma lipoprotein metabolism (2). Their physiological importance is exemplified through the phenotypes associated with abetalipoproteinemia and homozygous familial hypobetalipoproteinemia, where structural defects in either the MTTP or APOB gene, respectively, lead to a syndrome of hypocholesterolemia (including low levels of high density lipoprotein (HDL)), mild fat malabsorption with fasting lipid accumulation in the small intestine along with hepatic steatosis (3, 4).Exploration of the mechanisms underlying these phenotypes has been advanced through studies in murine genetic models in which either the Apob or Mttp gene was deleted. Germ line deletion of either the Apob or Mttp gene, however, resulted in embryonic lethality (5-7), the result of defective lipid delivery to the developing embryo, necessitating alternative approaches to the s...

Osteocalcin (OC) is a small calcium binding protein expressed in bones and teeth undergoing mineralization. OC expression in calvarial osteoblasts and odontoblasts is regulated in part via protein-protein interactions between the homeodomain repressor, Msx2, and components of the cell type-specific basal OC promoter. Recent work suggests that homeodomain proteins form heterodimers that confer transcriptional regulation. Since the homeodomain proteins Dlx5 and Msx2 are both expressed by primary rat calvarial osteoblasts, we examined whether Msx2 and Dlx5 functionally interact to regulate the OC promoter. In both primary rat calvarial and MC3T3E1 mouse calvarial osteoblasts, transient expression of Dlx5 only mildly augments basal OC promoter (luciferase reporter) activity, while Msx2 suppresses transcriptional activity by ca. 80%. However, Dlx5 completely reverses Msx2 repression of the OC promoter. Structure-function analyses using far-Western blot and transient cotransfection assays reveal that (i) Msx2 and Dlx5 can form dimers, (ii) Dlx5 residues 127-143 are necessary for dimerization and to reverse Msx2-dependent OC repression, and (iii) intrinsic DNA binding activity of Dlx5 is not required for OC regulation. Msx2 inhibits the DNA binding activity of a third complex, the OC fibroblast growth factor response element binding protein (OCFREB), that supports activity of the basal OC promoter. Dlx5 completely abrogates Msx2 suppression of OCFREB DNA binding activity, and residues required for Dlx5 transcriptional de-repression in vivo are also required for reversing inhibition of OCFREB binding in vitro. Finally, Dlx5 reverses Msx2 inhibition of OC promoter activation by FGF2/forskolin. Thus, Dlx5 regulates the expression of the OC promoter in calvarial osteoblasts in part by de-repression, antagonizing Msx2 repression of transcription factors that support basal OC promoter activity.

Liver regeneration is impaired following partial hepatectomy (PH) in mice with genetic obesity and hepatic steatosis and also in wild-type mice fed a high-fat diet. These findings contrast with other data showing that liver regeneration is impaired in mice in which hepatic lipid accumulation is suppressed by either pharmacologic leptin administration or by disrupted glucocorticoid signaling. These latter findings suggest that hepatic steatosis may actually be required for normal liver regeneration. We have reexamined this relationship using several murine models of altered hepatic lipid metabolism. Liver fatty acid (FA) binding protein knockout mice manifested reduced hepatic triglyceride (TG) content compared to controls, with no effect on liver regeneration or hepatocyte proliferation. Examination of early adipogenic messenger RNAs revealed comparable induction in liver from both genotypes despite reduced hepatic steatosis. Following PH, hepatic TG was reduced in intestine-specific microsomal TG transfer protein deleter mice, which fail to absorb dietary fat, increased in peroxisome proliferator activated receptor alpha knockout mice, which exhibit defective FA oxidation, and unchanged (from wild-type mice) in liver-specific FA synthase knockout mice in which endogenous hepatic FA synthesis is impaired. Hepatic TG increased in the regenerating liver in all models, even in animals in which lipid accumulation is genetically constrained. However, in no model-and over a >90-fold range of hepatic TG content-was liver regeneration significantly impaired following PH. T he mammalian liver has a remarkable capacity to regenerate. For example, following experimental partial hepatectomy (PH), with removal of 70% of the liver, hepatocytes begin within hours to respond to signals that initiate and sustain a complex yet well-integrated process of proliferation. 1,2 Growth factor-dependent priming of liver regeneration promotes the reentry of quiescent hepatocytes into the cell cycle, while progression through the restriction point in late growth phase 1 (G 1 ) is maintained by other growth factors, cyclins, and their respective kinases, perpetuating a proliferative drive that over several days restores hepatocyte mass (reviewed in Fausto et al. 2 ). However, despite intensive study over the last several years, intersections between these molecular signaling cascades and the multiple metabolic pathways that may influence liver regeneration remain incompletely characterized.Integral to the successful initiation and completion of liver regeneration is that the remaining cells within the liver acquire sufficient energy substrate to support the metabolic demands of rapid proliferation. It has long been recognized that the regenerating liver generates signals (still poorly understood) that couple fatty acid (FA) release from peripheral adipose stores to augmented he- PPAR, peroxisome proliferator-activated receptor; TG, triglyceride; WT, wild-type. From the

Msx2 is a homeodomain transcriptional repressor that exerts tissue-specific actions during craniofacial skeletal and neural development. To identify coregulatory molecules that participate in transcriptional repression by Msx2, we applied a Farwestern expression cloning strategy to identify transcripts encoding proteins that bind Msx2. A lambdagt11 expression library from mouse brain was screened with radiolabeled GST-Msx2 fusion protein encompassing the core suppressor domain of Msx2. A cDNA was isolated that encodes a novel protein fragment that binds radiolabeled Msx2. Homeoprotein binding activity was confirmed by Farwestern analysis of the T7-epitope-tagged recombinant protein fragment, and interactions in vitro require Msx2 residues necessary for transcriptional suppression in vivo. On the basis of biochemical analyses, this novel protein was named MINT, an acronym for Msx2-interacting nuclear target protein. The original clone is part of a 12.6 kb transcript expressed at high levels in testis and at lower levels in calvarial osteoblasts and brain. Multiple clones isolated from a mouse testis library were sequenced to construct a MINT cDNA contig of 11 kb. Starting from an initiator Met in good Kozak context, a large nascent polypeptide of 3576 amino acids is predicted, in contiguous open reading frame with the Msx2 interaction domain residues 2070-2394. Protein sequence analysis reveals that MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature approximately 110 kDa (N-terminal) and approximately 250 kDa (C-terminal) MINT protein fragments accumulate in chromatin and nuclear matrix fractions, cosegregating with Msx2 and topoisomerase II. In gel shift assays, the MINT RRM domain selectively binds T- and G-rich DNA sequences; this includes a large G/T-rich inverted repeat element present in the proximal rat osteocalcin (OC) promoter, overlapping three cognates that support OC expression in osteoblasts. MINT and OC mRNAs are reciprocally regulated during differentiation of MC3T3E1 calvarial osteoblasts. Consistent with its proposed role as a nuclear transcriptional factor, transient expression of MINT(1-812) suppresses the FGF/forskolin-activated OC promoter, does not significantly regulate CMV promoter activity, but markedly upregulates the HSV thymidine kinase promoter in MC3T3E1 cells. In toto, these data indicate that the novel nuclear protein MINT binds the homeoprotein Msx2 and coregulates OC during craniofacial development. Msx2 and MINT both target an information-dense, osteoblast-specific regulatory region of the OC proximal promoter, nucleotides -141 to -111. The N-terminal MINT RRM domain represents an authentic dsDNA binding module for this novel vertebrate nuclear matrix protein. Acting as a scaffold protein, MINT potentially exerts both positive and negative regulatory actions by organizing transcriptional complexes in the nuclear matrix.