We have used a mRNA differential display technique to identify new genes involved in the reprogramming of gene expression during the adipose conversion of mouse 3T3 preadipocyte cell lines. We report here on the identification and cloning of a novel adipose-specific cDNA encoding a predicted membrane protein of 413 amino acids. The level of the corresponding 3.2-kilobase mRNA is tremendously increased during 3T3-L1 and 3T3-F442A differentiation into adipocytes. A single, very abundant 3.2-kilobase transcript is also found in inguinal and epididymal white adipose tissues and in interscapular brown adipose tissue but not in any other tissues examined. Its expression in adipose tissue is under tight nutritional regulation. The level of this novel 3.2-kilobase transcript becomes virtually nondetectable during fasting but is dramatically increased when fasted mice are refed a high carbohydrate diet. Based on its adipose specificity and dietary regulation, the novel gene product has been designated adiponutrin. The expression of adiponutrin mRNA is also 50-fold elevated in genetically obese fa/fa rats, indicating a link between adiponutrin and obesity. Western blot and confocal imagery analyses with epitope-tagged protein transiently expressed in 3T3-L1 adipocytes, and COS cells show that adiponutrin strictly localizes to membranes and is absent from the cytosol. Sequence analysis reveals homologies with several other members of related eukaryotic proteins, including a human paralog, which has been recently described in vesicular transport mechanisms. This leads us to suggest that adiponutrin could be involved in vesicular targeting and protein transport restricted to the adipocyte function.

Adiponutrin expression in mouse adipose tissue is dependent upon changes in nutritional status. 8-week-old Swiss mice were divided into three experimental groups and were either allowed free access to food (Fed) or subjected to 19.5 h of fast (Fast) or 12 h of fast followed by 7.5 h of refeeding (Fast/Refed). The animals were sacrificed at the end of 19.5 h, and 10 µg of total RNA extracted from epididymal fat pads was used for Northern blot analysis for adiponutrin, ADD1, and FAS mRNAs. Mean values of insulinemia and glycemia corresponding to each nutritional status are indicated at the top. alpha-Enolase mRNA, probed with the 3'-untranslated region of the murine mRNA (accession number ACX52379) was used as invariant control. 18 S ribosomal RNA from ethydium bromide-stained gels is shown. Baulande S. , et al. J. Biol. Chem., Vol. 276, Issue 36, 33336-33344, September 7, 2001

Desnutrin mRNA levels in various adult mouse tissues and cells. A, 10 µg of total RNA from various mouse tissues was analyzed by Northern blotting and hybridized with radiolabeled desnutrin and adiponutrin cDNA probes. Sk, skeletal; Ing, inguinal; Gon, gonadal; Ren, renal. B, shown is desnutrin mRNA expression in gonadal WAT from 12-h fasted wild-type (WT), db/db, and ob/ob mice. C, 5 µg of total RNA from cells of the stromal vascular fraction (SVF) or adipocytes isolated from mouse inguinal adipose tissue (left panels) or 10 µg of total RNA from 3T3-L1 cells at the indicated days of differentiation (right panels) was examined by Northern blot analysis for the expression of desnutrin and various adipocyte markers. Prol. Pre., proliferating preadipocytes; Conf. pre., confluent preadipocytes; FAS, fatty-acid synthase; aFABP, adipocyte fatty acid-binding protein.
Villena JA, et al. J Biol Chem. 2004 Nov 5;279(45):47066-75. Epub 2004 Aug 27.

Desnutrin mRNA levels during adipocyte differentiation of 3T3-L1 cells. Two-day post-confluent 3T3-L1 preadipocytes (day 0) were induced to differentiate by treatment with 1 µM dexamethasone and 0.5 mM MIX for 2 days and then maintained in differentiation medium for an additional 5 days. Ten µg of total RNA prepared from cells collected at the indicated time points was examined for the expression of desnutrin and other adipocyte markers by Northern blot analysis. aFABP, adipocyte fatty acid-binding protein; FAS, fatty-acid synthase; PPAR, peroxisome proliferator-activated receptor-gamma.
Villena JA, et al. J Biol Chem. 2004 Nov 5;279(45):47066-75. Epub 2004 Aug 27.

Nutritional regulation of desnutrin mRNA levels. A, expression of desnutrin mRNA in WAT from mice fasted for 24 h (F) or fasted and then refed for 12 h (R) as assessed by Northern blot analysis; B, time course analysis of desnutrin and adiponutrin mRNA expression levels in WAT during fasting. Mice were fasted for 12, 24, or 48 h, and total RNA was extracted for examination of desnutrin mRNA levels by Northern blot analysis. Desnutrin mRNA levels in WAT were compared with those in mice that were fasted for 48 h and subsequently fed for 12 h. FAS, fatty-acid synthase.

Subcellular localization of desnutrin-EGFP fusion protein. A, COS-7 cells were transfected with the desnutrin-EGFP expression vector, and localization of the fusion protein was assessed by confocal microscopy. B, COS-7 cells were transfected with an HA-tagged desnutrin expression vector, and nuclear (Nuc.), mitochondrial (Mit.), microsomal (Mic.), and cytosolic (Cyt.) fractions were prepared as described under "Experimental Procedures." Five µg of protein from each fraction was subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and analyzed for the presence of HA-desnutrin using anti-HA antibody. As a positive control for the immunodetection, 10 µg of whole cell lysate was used.