Effects of two types of energy restriction on methylation levels of adiponectin receptor 1 and leptin receptor overlapping transcript in a mouse mammary tumour virus-transforming growth factor-α breast cancer mouse model.

The role of adiponectin and leptin signalling pathways has been suggested to play important roles in the protective effects of energy restriction (ER) on mammary tumour (MT) development. To study the effects of ER on the methylation levels in adiponectin receptor 1 (AdipoR1) and leptin receptor overlapping transcript (Leprot) genes using the pyrosequencing method in mammary fat pad tissue, mouse mammary tumour virus-transforming growth factor-α (MMTV-TGF-α) female mice were randomly assigned to ad libitum (AL), chronic ER (CER, 15 % ER) or intermittent ER (3 weeks AL and 1 week 60 % ER in cyclic periods) groups at 10 weeks of age until 82 weeks of age. The methylation levels of AdipoR1 in the CER group were higher than those in the AL group at week 49/50 (P < 0·05), while the levels of methylation for AdipoR1 and Leprot genes were similar among the other groups. Also, the methylation levels at CpG2 and CpG3 regions of the promoter region of the AdipoR1 gene in the CER group were three times higher (P < 0·05), while CpG1 island of Leprot methylation was significantly lower compared with the other groups (P < 0·05). Adiponectin and leptin gene expression levels were consistent with the methylation levels. We also observed a change with ageing in methylation levels of these genes. These results indicate that different types of ER modify methylation levels of AdipoR1 and Leprot in different ways and CER had a more significant effect on methylation levels of both genes. Epigenetic regulation of these genes may play important roles in the preventive effects of ER against MT development and ageing processes.

Effect of obesity on breast cancer development.

In recent years, obesity has been identified as a risk factor for the development of breast cancer in postmenopausal women, and it has been associated with a poor outcome. Many factors appear to be important in the mechanism of this increased risk, including estrogen, estrogen receptors, and the adipokines leptin and adiponectin. Estrogen, a potent mitogen for mammary cells, has long been implicated in the development of mammary tumors. Because adipose-associated aromatase activity increases the conversion of androgen to estrogen, mammary adipose tissue is thought to be an important source of local estrogen production. Leptin, which increases in the circulation in proportion to body fat stores, has been demonstrated in vitro to promote breast cancer cell growth. Animal models have also identified leptin as an important factor for the development of mammary tumors. In contrast to leptin, serum adiponectin concentrations are inversely related to body fat stores, and the addition of adiponectin to human breast cancer cells reduces cell proliferation and enhances apoptosis. This review explores the relationship between these factors and the development of mammary cancer in humans and mouse models.

Sequential changes in the activities of lipoprotein lipase and lipogenic enzymes during tumor growth in rats.

The sequential changes in lipid metabolism during tumor growth were evaluated in inbred Lewis rats bearing a mammary adenocarcinoma (AC33). Serum lipids, insulin, glucagon, and liver and adipose tissue lipogenic enzymes were measured in tumor-bearing and control rats after 6, 12, 18, 24, and 32 days of tumor growth. Lipoprotein lipase (LPL) activity in heart, soleus muscle, and epididymal fat pads was also determined. On the sixth day, the activity of LPL was reduced in the adipose tissue and remained lower throughout the duration of the experiment. Serum triglycerides were elevated from the 12th day followed by an increase in free fatty acid levels from the 18th day of tumor growth. These changes were accompanied by a decrease in serum insulin levels in the tumor-bearing rats from Day 12. The presence of the tumor also decreased the activities of some of the lipogenic enzymes in liver and adipose tissue, but these changes occurred at the later time points. On the 24th day, a decrease in fat pad weights was found and characterized by a decrease in fat cell size but not in fat cell number. These results suggest that a defect in clearance, due to the decrease in the activity of adipose tissue LPL, may be responsible for the early development of hypertriglyceridemia during tumor growth. In this study, the alterations in the lipogenic enzymes and LPL cannot be attributed to reduced food intake but may be due to the direct or an indirect effect of the tumor on a hormone such as insulin.

Obesity is associated with a decreased leptin transport across the blood-brain barrier in rats.

Leptin exerts important effects on the regulation of food intake and energy expenditure by acting in the brain. Leptin is secreted by adipocytes into the bloodstream and must gain access to specific regions in the brain involved in regulating energy balance. Its action is mediated by interaction with a receptor that is mainly expressed in the hypothalamus but is also present in other cerebral areas. To reach these target areas, leptin most likely needs to cross the blood-brain barrier (BBB). In this study, we compared the permeability of leptin at the BBB in homozygous lean (FA/FA), high-fat diet-induced (HFD) obese rats (FA/FA rats on a highfat diet), and genetically obese fa/fa Zucker rats by quantifying the permeability coefficient surface area (PS) product after correction for the residual plasma volume (Vp) occupied by leptin in the vessel bed of different brain regions. The intravenous bolus injection technique was used in the cannulated brachial vein and artery using leptin radioiodinated with 2 isotopes of iodine (125I and 131I) to separately determine the PS and Vp values. The PS for leptin at the BBB in lean FA/FA rats ranged from 11.0 +/- 1.6 at the cortex to 14.8 +/- 1.4 x 10(-6) ml x g(-1) x ml(-1) at the posterior hypothalamus. The PS for leptin in HFD obese FA/FA and obese fa/fa rats ranged from 3.0- to 4.0-fold lower than in lean FA/FA rats. The Vp values were not significantly different among the 3 groups studied. SDS-PAGE analysis of the radioiodinated leptin after 60 min of uptake revealed intact protein in the 8 different brain regions. Plasma leptin levels were significantly higher in both obese rat groups compared with those in lean FA/FA rats. Leptin levels in cerebrospinal fluid were not significantly different among the 3 groups of rats. These findings strongly suggest that the leptin receptor (OB-R) in the BBB can be easily saturated. Saturation of the BBB OB-R in obese individuals would explain the defect in leptin transport into the brain described in this study.

Effects of carbohydrate restriction on renal injury in the obese Zucker rat.

The obese Zucker rat model of nonimmune-mediated, spontaneous focal glomerulosclerosis is ideally suited to study the influence of diet on the initiation and progression of glomerular injury. Young (6 wk) and old (33 wk) lean and obese female Zucker rats were fed a carbohydrate-restricted diet intermittently for 27 wk. Carbohydrate restriction resulted in lower body weight (460 +/- 16 versus 310 +/- 7 g, p less than 0.025), kidney weight (1.26 +/- 0.04 versus 1.07 +/- 0.05 g, p less than 0.025), and glomerular area (6930 +/- 290 versus 5780 +/- 230 micron2, p less than 0.025) in young obese Zucker rats compared to ad libitum-fed rats. Although urine-albumin excretion was substantially reduced by carbohydrate restriction in young obese Zucker rats (41.1 +/- 12.3 versus 6.9 +/- 2.9 mg/24 h, p less than 0.01), glomerular injury was not significantly altered. In old obese rats, carbohydrate restriction did not significantly reduce albuminuria or prevent the progression of glomerular injury. Thus, intermittent carbohydrate restriction failed to alter significantly either the initiation of glomerular injury in young, or the progression of nephron damage in old, obese Zucker rats.

Effect of long-term clofibric acid treatment on serum and tissue lipid and cholesterol levels in obese Zucker rats.

The long-term effects of clofibric acid (200 mg/kg body weight) injected subcutaneously from 6-36 weeks of age were assessed in obese, hyperlipemic Zucker rats. At 18 and 36 weeks of age, treated rats had significantly lower fasted serum cholesterol levels but triacylglycerol levels were not affected. Rats were killed at 36 weeks of age at which time there were no differences in body and kidney weights between control and clofibric acid-treated rats. Liver, spleen and heart weights were lowered by clofibric acid treatment. In liver there was an elevation of lipid/g due to treatment but there were no effects on cholesterol/g or on either total liver lipid or cholesterol levels. In the epididymal fat pad of clofibric acid-treated rats, there was a 21% elevation of cholesterol level on a per pad basis. In the other organs, there were no effects of treatment on lipid or cholesterol levels except for lowered total cholesterol in kidney. Several liver lipogenic enzymes were lowered by treatment but malic enzyme was two times higher.

Glucose metabolism in isolated adipocytes from lean and obese Zucker rats following treatment with dehydroepiandrosterone.

Previous work has demonstrated that chronic administration of dehydroepiandrosterone (DHEA) to obese Zucker rats reduces the severity of hyperinsulinemia that is usually present. There were also significant decreases in body weight, fat depot weight, and adipose tissue cellularity. It was hypothesized that the decreased serum insulin was a reflection of improved tissue responsiveness to insulin. The purpose of the present study was to evaluate this hypothesis by examining the insulin response in isolated adipocytes of DHEA-treated rats. Glucose incorporation into CO2, fatty acids, and glyceride-glycerol was measured in isolated parametrial and retroperitoneal adipocytes. Cells from control and DHEA-treated lean rats and control and DHEA-treated obese rats were used, as well as cells from a group of obese rats pair-fed to the DHEA-obese rats. Increased basal and insulin-stimulated rates of incorporation of glucose into CO2 and fatty acids were found in adipocytes from DHEA-lean rats compared to control, lean rats. In contrast, cells from DHEA-treated obese rats tended to incorporate less glucose into CO2 and fatty acids than either the control or pair-fed obese rats. These data indicate that the decrease in serum insulin levels seen in DHEA-treated obese rats is not due to an improvement of adipose tissue responsiveness.

Alterations in lipogenic enzymes and lipoprotein lipase activity during gram-negative sepsis in the rat.

The effects of sepsis on lipid metabolism have not been clearly defined. This study was designed to observe the changes in adipose tissue lipoprotein lipase (LPL) and fatty acid synthetase (FAS) after administration of Escherichia coli bacteria. Male Lewis rats, weighing 245 to 270 g, were assigned to two groups and fed a powdered chow diet for 14 days. On day 14, one group was inoculated with E coli. Twenty-four hours later, both groups were killed by decapitation. Serum triglyceride levels were significantly elevated in the E coli-treated rats. Adipose tissue LPL and FAS activity was significantly decreased by 50% in E coli-treated rats compared with the control rats. These results suggest that the elevated serum triglyceride levels associated with sepsis maybe caused by a decreased rate of clearance of lipids from the blood and an increased rate of hepatic lipid synthesis.

Studies on nuclear binding of dehydroepiandrosterone in hepatocytes.

Experiments were conducted to determine if dehydroepiandrosterone (DHEA) specifically binds to liver nuclei or interferes in the binding of other steroid hormones. Hepatocytes were isolated from obese, female Zucker rats that were treated with and without 0.6% DHEA in their diets for 2 weeks. The hepatocytes were incubated with either [3H]DHEA, [3H]estrone, or [3H]corticosterone. The nuclei isolated from the incubated cells from either control or DHEA-treated rats had no binding with [3H]DHEA, but had the expected binding with [3H]estrone and [3H]corticosterone. Furthermore, increasing concentrations of cold, unlabeled DHEA in the incubation media with [3H]estrone or [3H]corticosterone failed to have any effect on the binding of either of these steroid hormones to the nuclei. These results suggest that DHEA treatment does not exert its effects on cellular metabolism through a receptor-mediated mechanism like other steroid hormones or by interfering in the expression of steroid hormones such as estrone and corticosterone.

Liver, serum and adipose tissue fatty acid composition in suckling Zucker rats.

Young adult obese Zucker rats have altered tissue fatty acid (FA) composition. The present study was aimed at determining whether such changes were seen in either liver, serum or adipose tissue obtained from 17-day-old obese (fafa) rats in comparison to both homozygous (FaFa) and heterozygous (Fafa) lean rats. Body weights of obese pups (30.3 g) were significantly greater than those of homozygous lean rats (25.2 g) (P < 0.05). Liver weight and lipid content were similar in all groups. Inguinal fat pad weight and lipid content were greatest in obese pups (573 mg) followed by heterozygous lean pups (303 mg); homozygous lean pups (146 mg) had the lowest values. There were no differences among the groups in hepatic FA composition in either triacylglycerol (TG) or phospholipid fractions. Serum TG was similar among the groups, while serum phospholipid was greater (P < 0.05) in obese (269 mg/dL) than in homozygous lean pups (184 mg/dL); heterozygous lean pups had an intermediate value not significantly different from either homozygous group. On a percent basis, there were no differences in FA composition in either serum lipid fraction among the three groups. There were a number of significant differences in adipose tissue FA composition between the groups on a percent basis. The adipose tissue FA composition on a percent basis reflected that of maternal milk. The results indicate that suckling obese Zucker rats do not have tissue FA profiles that are characteristic of essential FA deficiency.

Effects of adiponectin on breast cancer cell growth and signaling.

Obesity is a risk factor for postmenopausal breast cancer. Adiponectin/Acrp30 is lower in obese individuals and may be negatively regulating breast cancer growth. Here we determined that five breast cancer cell lines, MDA-MB-231, MDA-MB-361, MCF-7, T47D, and SK-BR-3, expressed one or both of the Acrp30 receptors. In addition, we found that the addition of Acrp30 to MCF-7, T47D, and SK-BR-3 cell lines inhibited growth. Oestrogen receptor (ER) positive MCF-7 and T47D cells were inhibited at lower Acrp30 concentrations than ER-negative SK-BR-3 cells. Growth inhibition may be related to apoptosis since PARP cleavage was increased by Acrp30 in the ER-positive cell lines. To investigate the role of ER in the response of breast cancer cells to Acrp30, we established the MDA-ERalpha7 cell line by insertion of ER-alpha into ER-alpha-negative MDA-MB-231 cells. This line readily formed tumours in athymic mice and was responsive to oestradiol in vivo. In vitro, MDA-ERalpha7 cells were growth inhibited by globular Acrp30 while the parental cells were not. This inhibition appeared to be due to blockage of JNK2 signalling. These results provide information on how obesity may influence breast cancer cell proliferation and establish a new model to examine interactions between ER and Acrp30.

Response of adult lean and obese female Zucker rats to intermittent food restriction/refeeding.

Previously, it was found that lean and obese Zucker rats (9-15 wk of age) responded differently to the first of four cycles of food restriction/refeeding. In later cycles, they responded similarly. The present study was undertaken to determine if this finding was due to age, adaptation to the intervention or the obesity. Adult (35-wk-old) lean and obese rats were classified into four groups, ad libitum-fed lean and obese and food-restricted lean and obese. Food-restricted rats underwent four 3-wk periods when they were fed 50% of their ad libitum intake, each followed by a 3-wk period of ad libitum refeeding. Food-restricted rats lost and regained sufficient weight in each cycle to weigh a similar amount as their ad libitum-fed groups by the end of each refeeding period. In lean rats, there were no permanent effects of this intervention except for a 25% reduction in carbohydrate intake. Similar results were found in obese rats, although they did have significantly lower retroperitoneal fat pad weight and serum triacylglycerol levels than ad libitum-fed obese rats at the end of the experiment. These results indicate that lean and obese adult rats respond to each food restriction/refeeding cycle in a similar manner. Results in the earlier experiment would appear to be due both to age and genotype.

Consequences of restricted feeding/refeeding cycles in lean and obese female Zucker rats.

Lean and obese female Zucker rats underwent four, 6-wk cycles of restricted feeding/refeeding. Restricted-fed, lean (RL) rats lost and then regained sufficient weight in each cycle to weigh the same amount as ad libitum-fed, lean (AL) rats at the end of each refeeding period. After the final period of restricted intake, RL rats had significantly lower parametrial and retroperitoneal pad weights and fat cell sizes than AL rats, but their organ growth and lipogenic enzymes were not affected. After the final refeeding period, there were no differences between RL and AL rats except in cumulative food intake. During the first period of restricted intake restricted-fed, obese (RO) rats did not lose weight; however, they were unable to attain the body weight of ad libitum-fed, obese (AO) rats during the subsequent refeeding period. In later cycles, RO rats lost and regained weight, but always weighed significantly less than AO rats. Following the final restricted feeding and refeeding periods, fat pad weights and cell numbers were significantly lower in RO than AO rats, but fat cell size was not affected. Liver weight and lipogenic enzymes were lower in RO than in AO rats after the final period of restricted intake, but were similar to AO rats after the final refeeding period. Permanent effects on heart and kidney growth were found in RO rats. Obese rats appeared to respond differently than lean rats to this form of dietary intervention.

The antiobesity effect of dehydroepiandrosterone in rats.

Initial studies showed that dehydroepiandrosterone (DHEA) treatment in mice resulted in lower body weight gain. Subsequent studies have shown that DHEA treatment in rats has a similar effect. In adult rodents, weight loss is a consequence of DHEA treatment. In general, these effects are independent of changes in food intake and are accompanied by lower body fat. DHEA treatment has been shown in some circumstances to alter a number of serum factors including glucose, insulin, cholesterol, and triacylglycerol. Recent studies have focused on the effects of DHEA on liver metabolism. Studies have been undertaken to determine whether the antiobesity effect of DHEA is mediated by the previously described inhibition of glucose-6-phosphate dehydrogenase by this steroid. It appears that inhibition of glucose-6-phosphate dehydrogenase in liver is not the initial metabolic response to DHEA but may play a contributing role. Inhibition of glucose-6-phosphate dehydrogenase in adipose tissue may affect differentiation of fat cells. A number of other enzymes involved in lipid and carbohydrate metabolism have also been shown to be altered by DHEA treatment, and several futile cycles involving some of these enzymes have been proposed to play a role in DHEA's antiobesity action. In addition, mitochondrial protein content is elevated by DHEA treatment. There appear to be time-dependent changes due to DHEA treatment on hepatic mitochondrial state three rates of respiration. Studies continue to evaluate the role of alterations in mitochondrial metabolism in DHEA's antiobesity action.

Effect of dehydroepiandrosterone treatment on liver metabolism in rats.

1. Administration of the 17-ketosteroid, dehydroepiandrosterone (DHEA), to rats results in lowered body weight. 2. A number of changes are seen in livers of treated rats. 3. These include higher liver weights and DNA, RNA and/or protein content, but lowered lipid and glycogen levels. 4. Activities of a number of liver enzymes involved in lipid and carbohydrate metabolism are altered by treatment. 5. In addition, net mitochondrial respiration is elevated by DHEA treatment. 6. Some of these findings may explain DHEA's antiobesity effect.

Anti-obesity effect of two different levels of dehydroepiandrosterone in lean and obese middle-aged female Zucker rats.

Dehydroepiandrosterone has previously been shown to prevent weight gain in growing lean and obese mice and rats. In the present study, lean and obese female Zucker rats were treated with either 0.6 or 1.0 percent DHEA in the diet from 8 until 14 months of age. In lean rats, 0.6 percent DHEA prevented weight gain and 1.0 percent DHEA resulted in significant weight loss compared to initial body weight. Control lean rats had a significant weight gain. Both 0.6 and 1.0 percent DHEA obese rats lost weight over the experimental period while control obese rats gained weight. Food intake of DHEA-treated obese rats was lowered compared to control obese rats but was similar to that of all lean groups. DHEA lowered serum insulin levels in both lean and obese rats relative to control groups. Both 0.6 and 1.0 percent DHEA lean rats had elevated hepatic G6PD activity compared to control lean rats. DHEA obese rats had lowered G6PD activity compared to the control obese rats. Hepatic malic enzyme was elevated by DHEA treatment in both lean and obese Zucker rats. Adipose tissue weights were lowered substantially in DHEA treated lean and obese rats versus their control groups. These data indicate that DHEA treatment in adult rats has an anti-obesity effect.

Dehydroepiandrosterone and related steroids inhibit mitochondrial respiration in vitro.

1. Dehydroepiandrosterone (DHEA) inhibited mitochondrial respiratory rates in the presence of tricarboxylic acid cycle intermediates with the exception of succinate. 2. Several other steroids tested for inhibitory activity on mitochondrial state 3 rate showed inhibitory potencies ranging from 0-90%. 3. Mitochondria isolated from adrenals, heart, kidneys, brain and brown adipose tissue were also inhibited by DHEA. 4. The aspartate malate shuttle system was inhibited, but the alpha-glycerophosphate shuttle was totally insensitive to DHEA. 5. The high-amplitude swelling of mitochondria in hypotonic media was inhibited by DHEA and a few other steroids.