3',5'-cyclic adenosine monophosphate-response sequences of the uncoupling protein gene are sequentially recruited during darglitazone-induced brown adipocyte differentiation.

Uncoupling protein-1 (UCP) is uniquely expressed in brown adipose tissue (BAT) and is essential to the thermogenic function of this tissue. The UCP gene is under the control of norepinephrine (NE) via cAMP. However, the precise delineation of the cAMP response sequences and mechanisms whereby cAMP stimulate the gene have remained elusive. A BAT tumor cell line, HIB-1B, can be differentiated into UCP-expressing brown adipocytes. We report here that when these cells are differentiated with a standard differentiation protocol including insulin, T3, hydrocortisone, IBMX, and indomethacin (standard differentiation, StD), cAMP stimulation of the rat UCP gene is largely mediated by an upstream 90-bp sequence -2,399/-2,490 (R90) with a lesser contribution of a downstream sequence -57/+114 (dnCRS). This latter is functional also in non-BAT cells, whereas the cAMP response sequence contained in R90 (upCRS) is BAT-specific. Thiazolidinediones (TZD) are a new group of drugs known to increase sensitivity to insulin and, more recently, to induce adipocyte differentiation (adipogenesis) via PPARgamma. A TZD, darglitazone (darg), can rapidly induce differentiation of HIB-1B cells, as judged by the expression of the adipocyte lipid binding protein (aP2), lipoprotein lipase (LPL), uncoupling protein (UCP) and beta3-adrenergic receptors. UCP messenger RNA (mRNA) responsive to NE is evidenced as early as one day after exposure to darg. While UCP-CAT vectors (+114/-3673 bp of rat UCP gene) are barely responsive to NE in HIB-1B preadipocytes, both darg and StD markedly enhance NE responsiveness of such constructs. However, by 3 days of exposure to darg, the responses were less vigorous than in StD cells (4- to 10-fold vs. 20- to 50-fold), and the deletion of R90 did not affect the response to NE in darg-differentiated cells, whereas this deletion caused a 75% reduction in StD cells. Prolongation of darg exposure to 5-7 days resulted in greater response of UCP mRNA to NE and 50-80% inhibition of the response of UCP-CAT vectors by the deletion of R90. Thus, darg-induced differentiation of HIB-1B cells suggests that the NE-dependent expression of the UCP gene takes place in a step-wise manner: first, the gene is "enabled," as no UCP mRNA is detected in HIB-1B preadipocytes; thereafter and transiently, the response of the gene to NE is sustained by dnCRS; finally, as differentiation progresses, a cell-specific and more powerful cis-acting sequence, upCRS, is recruited, accounting in the fully differentiated cell for most of the response to NE. These results also suggest that TZDs might increase energy expenditure by inducing terminal differentiation of BAT, and that these drugs may be useful in the differential cloning of the factors involved in the recruitment of the BAT specific cAMP response sequence.

Thiazolidinediones stimulate uncoupling protein-2 expression in cell lines representing white and brown adipose tissues and skeletal muscle.

Thiazolidinediones (TZD) are PPAR gamma ligands that sensitize tissues to insulin. A cDNA encoding a mitochondrial protein likely to act as uncoupler (uncoupling protein 2, UCP2) has been recently cloned. Since TZD have been reported to increase energy expenditure in animals, we have examined the effects of these drugs on the expression of UCP2 mRNA in cell lines representing white (3T3-L1 and 3T3-F442A) and brown (HIB-1B) adipose tissues and skeletal muscle (L6). Northern blots probed with a mouse UCP2 full-length cDNA showed a mRNA of 1.6 kb both in tissues and the aforementioned cells lines. Within 4 h of exposing these cells to 30 microM darglitazone, there was an increase in UCP2 mRNA which reached a plateau of 5-10 times the basal in about 8 h. In all cells TZDs (darglitazone, troglitazone) were more active than the predominantly PPAR alpha ligands WY-14,613 and clofibrate, or the non-selective ligand linoleic acid. These results indicate that TZDs can stimulate the expression of UCP2 gene probably via PPAR gamma and hence have the potential to increase energy expenditure in adult humans, in whom UCP2 is expressed ubiquitously.

Uncoupling protein-2 and -3 messenger ribonucleic acids in adipose tissue and skeletal muscle of healthy males: variability, factors affecting expression, and relation to measures of metabolic rate.

Mitochondrial uncoupling protein-2 and -3 (UCP2 and UCP3) may be involved in the modulation of resting metabolic rate and energy balance. To investigate their variability, the influence of this on the variability of energy expenditure, and potential regulatory factors of the expression of the corresponding genes, we measured their messenger ribonucleic acids (mRNAs) in muscle and white adipose tissue of lean, healthy men and correlated the abundance of these mRNAs (attomoles per microg total RNA) with measures of resting metabolic rate, hormone levels (thyroid hormones, insulin, glucagon, leptin, and catecholamines), and fuels potentially involved in energy balance regulation. We also investigated whether the thiazolidinedione, troglitazone, stimulates UCP2 and UCP3 mRNA levels to follow up on the observation that this antidiabetic drug increases the levels of expression in cultured cells. We found UCP2 and UCP3 mRNA levels to be highly variable and poorly correlated with measures of energy expenditure and with most factors affecting energy balance. Only nocturnal urinary norepinephrine excretion could explain a significant fraction of the variability in both UCP2 and UCP3 expression in muscle, but not adipose tissue. Thyroid hormone and norepinephrine excretion were found to contribute to the variability of resting metabolic rate, but this could not be explained by an effect on UCP mRNAs. Troglitazone affected neither the expression of UCPs nor the hormones or the measures of metabolic rate investigated. In conclusion, our results show that the expression of UCP2 and UCP3 genes is quite variable in healthy males and that this variability does not explain that in resting energy expenditure, and suggest that sympathetic activity is an important potential regulator of the expression of these proteins in skeletal muscle. However, the data do not support the concept that regulation of the expression of these genes is the most important level of control of UCP3 and UCP2 functions, and other levels of control have to be invoked.

Glucocorticoids inhibit the transcriptional response of the uncoupling protein-1 gene to adrenergic stimulation in a brown adipose cell line.

Glucocorticoids may inhibit brown adipose tissue (BAT) thermogenesis acting at a central level as well as reducing the responses of the tissue to adrenergic stimulation in vivo. This latter effect is not well understood. We investigated whether or not glucocorticoids directly reduce the expression of the key molecule for BAT thermogenesis, uncoupling protein-1 (UCP1), and if so, to what extent and by what mechanisms. We used HIB-1B brown adipose cells obtained from a hibernoma. The response of UCP1 mRNA to adrenergic stimulation in these cells is qualitatively and quantitatively similar to that seen in vivo. Dexamethasone and other glucocorticoids, given simultaneously with NE, nearly abolish the ensuing UCP1 mRNA accumulation. This effect was negated by the glucocorticoid receptor antagonist RU-486. Significant inhibition is seen within the physiological range of concentrations, with ID(50)s for dexamethasone and corticosterone of 1 and 75 nM, respectively. Within the time span of the experiments, glucocorticoids did not reduce the strength of the NE signal nor did they necessitate ongoing protein synthesis or reduce the stability of mature UCP1 mRNA, but they significantly inhibited the stimulation of transcription by NE in a run-on in vitro transcription assay. These observations indicate that glucocorticoids are powerful inhibitors of the UCP1 gene response to adrenergic stimulation acting at transcriptional level, and provide further evidence for a global inhibitory effect of glucocorticoids on BAT thermogenesis.

A comparison between parallel hairline incisions and perpendicular incisions when performing a face lift.

Our profession has not decided if it is better to incise parallel or perpendicular (at an angle) to hair follicles when performing a brow lift or a face lift. Thirty patients had one side incised parallel and the other side incised perpendicular to hair follicles. Neither the patients nor the examiners knew how each side was incised. By comparing the invisibility, the nonlinearity, the absence of hypopigmentation, and the abundance of hair into and in front of the scar, we obtained statistical proof that if we incised perpendicular to hair follicles (to preserve the deep follicles in the proximal flap), scars were better in 95 percent of patients.

Anatomy, pathophysiology, and prevention of senile enophthalmia and associated herniated lower eyelid fat pads.

We describe in detail the anatomy and function of the "Lockwood suspensory ligament" and the interrelated function of the orbital contents responsible for the intraorbital position of the eyeball and fat. With age, or because of genetic disposition, the eyeball descends, reducing the space between it and the floor of the orbit. This will inevitably cause forward projection of the extraconical orbital fat, creating herniated fat pads and resulting in enophthalmia. Based on the volume of the bony orbit and its contents, it is likely that relocating, rather then removing, herniated fat pads will greatly improve and prevent the enophthalmia of aging and give the globe a position and a projection of youth. Based on the results of surgery using the capsulopalpebral flap, it is likely that a descended Lockwood suspensory ligament, rather than a weakened orbital septum, is the cause of herniated fat pads and enophthalmia. We feel neither a weakened orbital septum nor an overabundance of orbital fat nor a shallow orbit is responsible for either of these conditions. We give a detailed description of how to raise the globe, preserve and relocate herniated fat pads, and manage and prevent enophthalmia and obtain a beautiful, youthful looking eye.

Breast augmentation: compression--a very important factor in preventing capsular contracture.

The incidence of capsular contracture is reduced when smooth-surfaced, saline-filled implants are placed subpectorally, avoiding contamination and the presence of blood. In addition, since the authors began using compression, they have not had one single capsular contracture requiring surgery. The authors' technique, their preoperative and postoperative regimens, an explanation of how they use compression, and the scientific and mathematical explanation of why compression could be the most important factor in preventing capsular compression are described. Of more than 1700 augmentations performed in our private clinic, only 830 could be included in the study. The patients were followed clinically for 1 year, after which time they completed a questionnaire, the results of which showed the authors that capsular contracture had not developed.

The aging eye: pathophysiology and management.

As humans go through the normal process of aging, there is drooping of many of the deep structures of the eyes. For generations, and continuing today, aesthetic plastic surgeons are removing superficial structures (i.e., skin and fat) as the primary management of drooping upper and lower eyelids. However, the dissatisfaction of patients with the results of these conventional approaches (Fig. 1) has led to further research and the development of innovative techniques that give a more youthful and harmonious appearance to the face.

Why I no longer use short incisions.

According to the author, excision is the only effective method of eliminating chalasis, and a good-quality scar is the well-camouflaged key to superior results. He describes a method of using long hairline incisions that avoids temporal hair loss and forehead widening and improves the appearance of the scar.

Treatment of dynamic crow's feet while performing a blepharoplasty.

A classification of crow's feet and its pathophysiology are discussed. The author demonstrates a technique of removing a portion of the overactive orbicularis muscle, the underlying cause of crow's feet, while performing the blepharoplasty. The procedure is effective, safe, and simple.

[Improving of scars from temporal and frontal face lifts]. / Amélioration des cicatrices de lifting temporal et frontal.

In order to prevent a widening of the frontal and temporal area and to avoid reducing the presence of hair in these areas we practice "W" like incisions at the junction of the scalp and skin. We preserve the deep part of the hair follicules and eliminate tension as we suture the wound. We will demonstrate a technique which is simple and quick allowing us to obtain excellent scars and we discuss its physiology.

Needle dermabrasion.

In this article we describe a technique of needle dermabrasion (tattoo without pigment) used to improve achromic, hypertrophic, and unsightly scars. It is simple, safe (no complications), and it gives us consistently good results.

Biosynthesis of the fucose-containing xyloglucan nonasaccharide by pea microsomal membranes.

Pea microsomal membranes catalyze the transfer of [(14)C]fucose (Fuc) from GDP-[U-(14)C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [(14)C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc(4) Xyl(3) Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, alpha-linked in a terminal nonreducing position. By comparison, in incubations where GDP-[(14)C] fucose is absent and replaced by UDP-[(3)H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[(14)C]fucose and UDP-[(3)H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP-fucose.