Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, abdominal obesity, and insulin resistance. However, its etiology is unclear, and its management is often unsatisfactory or requires a diversified approach. Here, we describe a new rat PCOS model, the first to exhibit both ovarian and metabolic characteristics of the syndrome. Female rats received the nonaromatizable androgen dihydrotestosterone (DHT) or the aromatase inhibitor letrozole by continuous administration, beginning before puberty, to activate androgen receptors. Adult DHT rats had irregular cycles, polycystic ovaries characterized by cysts formed from atretic follicles, and a diminished granulosa layer. They also displayed metabolic features, including increased body weight, increased body fat, and enlarged mesenteric adipocytes, as well as elevated leptin levels and insulin resistance. All letrozole rats were anovulatory and developed polycystic ovaries with structural changes strikingly similar to those in human PCOS. Our findings suggest that the formation of a "hyperplastic" theca interna reflects the inclusion of luteinized granulosa cells in the cyst wall rather than true hyperplasia. We conclude that the letrozole model is suitable for studies of the ovarian features of human PCOS, while the DHT model is suitable for studies of both ovarian and metabolic features of the syndrome.

Enlarged adipocytes are associated with insulin resistance and are an independent predictor of type 2 diabetes. To understand the molecular link between these diseases and adipocyte hypertrophy, we developed a technique to separate human adipocytes from an adipose tissue sample into populations of small cells (mean 57.6+/-3.54 microm) and large cells (mean 100.1+/-3.94 microm). Microarray analysis of the cell populations separated from adipose tissue from three subjects identified 14 genes, of which five immune-related, with more than fourfold higher expression in large cells than small cells. Two of these genes were serum amyloid A (SAA) and transmembrane 4 L six family member 1 (TM4SF1). Real-time RT-PCR analysis of SAA and TM4SF1 expression in adipocytes from seven subjects revealed 19-fold and 22-fold higher expression in the large cells, respectively, and a correlation between adipocyte size and both SAA and TM4SF1 expression. The results were verified using immunohistochemistry. In comparison with 17 other human tissues and cell types by microarray, large adipocytes displayed by far the highest SAA and TM4SF1 expression. Thus, we have identified genes with markedly higher expression in large, compared with small, human adipocytes. These genes may link hypertrophic obesity to insulin resistance/type 2 diabetes.

In PCOS, adipose tissue has aberrant morphology/function. Increased waist-to-hip ratio indicates abdominal/visceral fat accumulation, but this is not supported by MRI. Enlarged adipocytes and reduced serum adiponectin, together with a large waistline, rather than androgen excess, may be central factors in the pathogenesis/maintenance of insulin resistance in PCOS.

Res. 2003;11:699-708. Objective: Accumulation of visceral fat is recognized as a predictor of obesity-related metabolic disturbances. Factors that are predominantly expressed in this depot could mediate the link between visceral obesity and associated diseases. Research Methods and Procedures: Paired subcutaneous and omental adipose tissue biopsies were obtained from 10 obese men. Gene expression was analyzed by DNA microarrays in triplicate and by real-time polymerase chain reaction. Serum C3 and C4 were analyzed by radial immunodiffusion assays in 91 subjects representing a cross section of the general population. Body composition was measured by computerized tomography. Results: Complement components C2, C3, C4, C7, and Factor B had higher expression in omental compared with subcutaneous adipose tissue (ϳ2-, 4-, 17-, 10-, and 7-fold, respectively). In addition, adipsin, which belongs to the alternative pathway, and the classical pathway components C1QB, C1R, and C1S were expressed in both depots. Analysis of tissue distribution showed high expression of C2, C3, and C4 in omental adipose tissue, and only liver had higher expression of these genes. Serum C3 levels correlated with both visceral and subcutaneous adipose tissue in both men (r ϭ 0.65 and p Ͻ 0.001 and r ϭ 0.52 and p Ͻ 0.001, respectively) and women (r ϭ 0.34 and p ϭ 0.023 and r ϭ 0.49 and p Ͻ 0.001, respectively), whereas C4 levels correlated with only visceral fat in men (r ϭ 0.36, p ϭ 0.015) and with both depots in women (visceral: r ϭ 0.58, p Ͻ 0.001; and subcutaneous: r ϭ 0.51, p Ͻ 0.001). Discussion: Recent studies show that the metabolic syndrome is associated with chronically elevated levels of several immune markers, some of which may have metabolic effects. The high expression of complement genes in intra-abdominal adipose tissue might suggest that the complement system is involved in the development of visceral adiposity and/or contributes to the metabolic complications associated with increased visceral fat mass.

Enlarged subcutaneous abdominal adipocytes have been shown to predict incidence of type 2 diabetes (T2D) in the Pima population of Arizona (USA). We investigated the role of subcutaneous abdominal adipocyte size (AAS), as well as femoral adipocyte size (FAS), as predictors of T2D in a populationbased Swedish cohort. In 1974 -1975, a sample of 1302 middle-aged women underwent a health examination, including anthropometry and evaluation of parental medical history. In addition, body composition (total body potassium and total body water), AAS and FAS (adipose tissue needle biopsy) were assessed in a subsample of 245 women. Incidence of T2D was followed until 2001, with 36 cases eligible for inclusion in this analysis. Women developing T2D had larger AAS at baseline vs. women remaining healthy (age/heredityadjusted hazard ratio for increase of AAS by 1 SD [AAS-HR] 1.91; P<0.001). Further adjustment for both body fat percentage and waist-to-height ratio (WHtR) indicated a robust association. For FAS, the corresponding associations were consistently weaker. WHtR retained a strong predictive association independent of AAS and FAS (WHtR-HR 2.6 and 2.7, respectively; P<0.001). To conclude, in addition to the amount and distribution of body fat in women, subcutaneous adipocyte size, particularly in the abdominal region, predicts incidence of T2D in later life.-Lönn, M., Mehlig, K., Bengtsson, C., Lissner, L. Adipocyte size predicts incidence of type 2 diabetes in women. FASEB J. 24, 326 -331 (2010). www.fasebj.org

Research Methods and Procedures:Using 52 human adipose tissue expression profiles (HU95), 10 putative reference genes with the lowest variation in expression levels were selected for further studies. Expression stability of these 10 novel and 5 previously established reference genes was evaluated by real-time reverse transcriptase-polymerase chain reaction analysis. For this purpose, 44 adipose tissue biopsies from 27 subjects were chosen to include a wide range of parameters such as sex, age, BMI, depot origin, biopsy procedure, and effects of nutrition. Results: LRP10 was identified as the gene with the least variation in expression levels. The frequently used reference genes RPLP0, 18S rRNA, PPIA, ACTB, and GAPD were ranked as 4, 6, 7, 8, and 10, respectively. Discussion: Our results suggest that LRP10 is a better choice as reference for expression studies of human adipose tissue compared with the most frequently used reference genes.

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, abdominal obesity, and insulin resistance. Pharmacotherapy is often unsatisfactory. This study evaluates the effects of low-frequency electro-acupuncture (EA) and physical exercise on metabolic disturbances and adipose tissue mRNA expression of selected genes in a rat PCOS model characterized by insulin resistance and adiposity. Dihydrotestosterone (inducing PCOS) or vehicle (control) was administrated continuously, beginning before puberty. At age 10 wk, PCOS rats were randomly divided into three groups; PCOS, PCOS EA, and PCOS exercise. PCOS EA rats received 2-Hz EA (evoking muscle twitches) three times/wk during 4-5 wk. PCOS exercise rats had free access to a running wheel for 4-5 wk. EA and exercise improved insulin sensitivity, measured by clamp, in PCOS rats. Exercise also reduced adiposity, visceral adipocyte size, and plasma leptin. EA increased plasma IGF-I. Real-time RT-PCR revealed increased expression of leptin and IL-6 and decreased expression of uncoupling protein 2 in visceral adipose tissue of PCOS rats compared with controls. EA restored the expression of leptin and uncoupling protein 2, whereas exercise normalized adipose tissue leptin and IL-6 expression in PCOS rats. Thus, EA and exercise ameliorate insulin resistance in rats with PCOS. This effect may involve regulation of adipose tissue metabolism and production because EA and exercise each partly restore divergent adipose tissue gene expression associated with insulin resistance, obesity, and inflammation. In contrast to exercise, EA improves insulin sensitivity and modulates adipose tissue gene expression without influencing adipose tissue mass and cellularity.

The supply of polyunsaturated fatty acids (PUFA) is important for optimal fetal and postnatal development. We have previously shown that leptin levels in suckling rats are reduced by maternal PUFA deficiency. In the present study, we evaluated the effect of maternal dietary intake of (n-3) and (n-6) PUFA on the leptin content in rat milk and serum leptin levels in suckling pups. For the last 10 days of gestation and throughout lactation, the rats were fed an isocaloric diet containing 7% linseed oil (n-3 diet), sunflower oil (n-6 diet), or soybean oil (n-6/n-3 diet). Body weight, body length, inguinal fat pad weight, and adipocyte size of the pups receiving the n-3 diet were significantly lower during the whole suckling period compared with n-6/n-3 fed pups. Body and fat pad weights of the n-6 fed pups were in between the other two groups at week one, but not different from the n-6/n-3 group at week 3. Feeding dams the n-3 diet resulted in decreased serum leptin levels in the suckling pups compared with pups in the n-6/n-3 group. The mean serum leptin levels of the n-6 pups were between the other two groups but not different from either group. There were no differences in the milk leptin content between the groups.These results show that the balance between the n-6 and n-3 PUFA in the maternal diet rather than amount of n-6 or n-3 PUFA per se could be important for adipose tissue growth and for maintaining adequate serum leptin levels in the offspring. -Korotkova, M., B. Gabrielsson, M. Lönn, L-Å. Hanson, and B. Strandvik. Leptin levels in rat offspring are modified by the ratio of linoleic to ␣ -linolenic acid in the maternal diet.