The low-density lipoprotein receptor–related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

Summary: We generated two complementary systems for Cre-mediated recombination of target genes in the mouse digestive epithelium and tested them with a Crereporter mouse strain. Cre was expressed under the control of a 9 kb regulatory region of the murine villin gene (vil-Cre). Genetic recombination was initiated at embryonic day (E) 9 in the visceral endoderm, and by E12.5 in the entire intestinal epithelium, but not in other tissues. Cre expression was maintained throughout adulthood. Furthermore, transgenic mice bearing a tamoxifen-dependent Cre recombinase (vil-Cre-ER T2 ) expressed under the control of the villin promoter were created to perform targeted spatiotemporally controlled somatic recombination. After tamoxifen treatment, recombination was detectable throughout the digestive epithelium. The recombined locus persisted for 60 days after tamoxifen administration, despite rapid intestinal cell renewal, indicating that epithelial progenitor cells had been targeted. The villin-Cre and villin-Cre-ER T2 mice provide valuable tools for studies of cell lineage allocation and gene function in the developing and adult intestine. genesis 39: 186 -193, 2004.

Obesity is a disorder of energy balance. Hormone-sensitive lipase (HSL) mediates the hydrolysis of triacylglycerol, the major form of stored energy in the body. Perilipin (encoded by the gene Plin), an adipocyte protein, has been postulated to modulate HSL activity. We show here that targeted disruption of Plin results in healthy mice that have constitutively activated fat-cell HSL. Plin -/- mice consume more food than control mice, but have normal body weight. They are much leaner and more muscular than controls, have 62% smaller white adipocytes, show elevated basal lipolysis that is resistant to beta-adrenergic agonist stimulation, and are cold-sensitive except when fed. They are also resistant to diet-induced obesity. Breeding the Plin -/- alleles into Leprdb/db mice reverses the obesity by ncreasing the metabolic rate of the mice. Our results demonstrate a role for perilipin in reining in basal HSL activity and regulating lipolysis and energy balance; thus, agents that inactivate perilipin may prove useful as anti-obesity medications.

SUMMARY Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1, and an inducible podocyte-specific knock-in mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at Serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics.

and collect data. BHC helped generate Tug1-transgenic mice and edit the manuscript. PAO helped edit the manuscript, and FRD oversaw experiments, prepared the manuscript, and provided guidance on overall project design.

Adipose differentiation-related protein (ADFP; also known as ADRP or adipophilin), is a lipid droplet (LD) protein found in most cells and tissues. ADFP expression is strongly induced in cells with increased lipid load. We have inactivated the Adfp gene in mice to better understand its role in lipid accumulation. The Adfpdeficient mice have unaltered adipose differentiation or lipolysis in vitro or in vivo. Importantly, they display a 60% reduction in hepatic triglyceride (TG) and are resistant to diet-induced fatty liver. To determine the mechanism for the reduced hepatic TG content, we measured hepatic lipogenesis, very-low-density lipoprotein (VLDL) secretion, and lipid uptake and utilization, all of which parameters were shown to be similar between mutant and wild-type mice. The finding of similar VLDL output in the presence of a reduction in total TG in the Adfp-deficient liver is explained by the retention of TG in the microsomes where VLDL is assembled. Given that lipid droplets are thought to form from the outer leaflet of the microsomal membrane, the reduction of TG in the cytosol with concomitant accumulation of TG in the microsome of Adfp ؊/؊ cells suggests that ADFP may facilitate the formation of new LDs. In the absence of ADFP, impairment of LD formation is associated with the accumulation of microsomal TG but a reduction in TG in other subcellular compartments.Adipose differentiation-related protein (ADFP) was first isolated by differential hybridization screening of 1246 cells during their differentiation to adipocytes (29). Its mRNA is induced 100 fold during the process. Using 3T3L1 cells, Brasaemle et al. (3) showed that Adfp gene expression is induced early, at day 1 of adipocyte differentiation, and that mRNA levels are maintained throughout differentiation. In contrast, ADFP protein levels, initially upregulated, gradually go down after day 4 (3), suggesting that these levels are subject to significant translational or posttranslational regulation. At the same time, upregulation of perilipin (PLIN), another lipid droplet (LD) protein, is observed at day 4 of differentiation; it has been postulated that perilipin and ADFP might compete for LD localization during the differentiation of 3T3L1 cells (3, 36). ADFP protein is localized to the surface of the LD, though it also has been detected in the LD core by freeze fracture electron microscopy (49, 50).ADFP shares sequence homology with other LD proteins including perilipin and Tip47, collectively known as PAT domain-containing proteins (36, 43), as well as with another LD protein, S3-12 (53). S3-12 shares a 11-amino-acid repeat motif with ADFP, in addition to another region of homology to both ADFP and Tip47 at its carboxyl terminus (6,24,36). With the exception of perilipin, which is expressed only in fat and steroidogenic tissues, the other LD proteins are detected in a variety of cells and tissues (22). Perilipin is phosphorylated by protein kinase A during lipolysis, resulting in an altered conformation to allow hormone-sensitive lipase ...

Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation. MicroRNAs (miRNAs)2 comprise a broad class of small noncoding RNAs that negatively regulate gene expression by basepairing to partially complementary sites in the 3Ј-untranslated regions (UTR) of specific target mRNAs (1, 2). An emerging body of evidence suggests that miRNAs serve as important therapeutic targets in a wide range of complex human diseases, including cancer and cardiovascular diseases, by targeting multiple transcripts (3-6). Recent studies have also revealed the involvement of miRNAs in diabetic nephropathy (DN) (7-9). However, despite the growing evidence for the regulatory effects of miRNAs in DN, limited information is available on the consequences of modulating miRNAs expression in vivo.We hypothesized that an unbiased global miRNA expression profiling might reveal novel miRNAs, which may play critical regulatory roles in the pathogenesis of DN. Accordingly, by using an integrated in vitro and in vivo comparative miRNA expression profiling, we identified up-regulated miR-29c as a signature miRNA in the diabetic environment.Previously published work suggested that down-regulation of miR-29c resulted in cardiac fibrosis (10, 11). In contrast, herein we identified miR-29c as a signature miRNA in the diabetic milieu whose expression was increased in hyperglycemic conditions both in vitro and in vivo. Thus, our objective was to explore the role of increased miR-29c expression in DN.We found t...

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