Animal and Dairy Science

Docosahexaenoic acid (DHA, C22:6), a (n-3) fatty acid in fish oil, has been shown to decrease body fat and fat accumulation in rodents. We investigated the direct effect of DHA on cell growth, differentiation, apoptosis, and lipolysis using 3T3-L1 adipocytes. Cells were treated with 25-200 mmol/L DHA containing 0.2 mmol/L a-tocopherol or bovine serum albumin vehicle as a control. Proliferation of preconfluent preadipocytes was not affected by the DHA treatment. When added to postconfluent preadipocytes, all concentrations of DHA inhibited differentiation-associated mitotic clonal expansion (P , 0.01). Postconfluent preadipocytes demonstrated apoptosis after 48 h with 100 mmol/L DHA and after 24 and 48 h with 200 mmol/L DHA (P , 0.01). Differentiation was examined by Oil Red O staining and glycerol-3-phosphate dehydrogenase

Resveratrol, a phytoalexin, has gained much attention recently due to its effects on sirtuins. While the anti-cancer properties of resveratrol have been extensively investigated, the anti-adipogenic and osteogenic effects of resveratrol are also gaining considerable interest. The finding that resveratrol supplementation mimics caloric restriction prompted researchers to study the effects of resveratrol on lipid metabolism. Mesenchymal stem cells are the precursors for both adipocytes and osteoblasts. In the aging population, differentiation to adipocytes dominates over the differentiation to osteoblasts in bone marrow, contributing to the increased tendency for fractures to occur in the elderly. Thus, an inverse relationship exists between adipocytes and osteoblasts in the bone marrow. Resveratrol acts on several molecular targets in adipocytes and osteoblasts leading to a decrease in adipocyte number and size and an increase in osteogenesis. Furthermore, resveratrol in combination with genistein and quercetin synergistically decreased adipogenesis in murine and human adipocytes. A recent in vivo study showed that phytochemicals including resveratrol in combination with vitamin D prevented weight gain and bone loss in a postmenopausal rat model. Therefore, combinations of resveratrol with other phytochemicals may lead to potential novel potent therapies for both obesity and osteoporosis.

This study examined if dietary supplementation with a combination of phytochemicals with vitamin D inhibits bone loss and decreases adiposity in ovariectomized female rats. Ovariectomized female rats were given control, vitamin D or vitamin D + resveratrol + quercetin + genistein for 8 week. The high dose combination treatment significantly reduced body weight gain (control: 80±5 g; vitamin D: 81.5±5 g; high: 62.0±3, p<0.05) and fat pads as a percentage of body weight (control: 7.5±0.2; vitamin D: 7.6±0.2; high: 6.4±0.2, p<0.05) compared to the control and vitamin D alone.

Higher levels of body fat are associated with increased risk for development of numerous adverse health conditions. Phytochemicals are potential agents to inhibit differentiation of preadipocytes, stimulate lipolysis, and induce apoptosis of existing adipocytes, thereby reducing adipose tissue mass. Resveratrol decreased adipogenesis and viability in maturing preadipocytes; these effects were mediated not only through down-regulating adipocyte specific transcription factors and enzymes but also by genes that modulate mitochondrial function. Additionally, resveratrol increased lipolysis and reduced lipogenesis in mature adipocytes. In addition, combining resveratrol with other natural products produced synergistic activities from actions on multiple molecular targets in the adipocyte life cycle. Treatment of mice with resveratrol alone was shown to improve resistance to weight gain caused by a high-fat diet. Moreover, dietary supplementation of aged ovariectomized rats with a combination of resveratrol and vitamin D, quercetin, and genistein not only decreased weight gain but also inhibited bone loss. Combining several phytochemicals, including resveratrol, or using them as templates for synthesizing new drugs, provides a large potential for using phytochemicals to target adipocyte adipogenesis, apoptosis, and lipolysis.

Evidence from both epidemiological studies and basic research suggests that obesity and osteoporosis are interrelated. Though there is an increase in the 10 prevalence of these disorders, a limited number of treatments are available, one of the reasons being the complexity of the pathways involved and difficulty in 11 identifying a single molecular target. Due to adverse effects of pharmaceuticals, intake of herbal drugs by patients without a physician's recommendation is 12 increasing globally. Lack of success with targeted monotherapy has encouraged scientists to determine whether combinations of phytochemicals that interfere 13 with numerous cell-signaling pathways can be a more effective approach to treat complex diseases. For example, evidence is emerging that specific combinations 14 of phytochemicals are far more effective than single compounds in decreasing adipogenesis and promoting bone formation. Since multiple pathways are 15 dysfunctional in obesity and osteoporosis, an ideal approach for preventing and treating these diseases may be to use a combination of phytochemicals to 16 address several targets simultaneously.

Both central and peripheral leptin administrations reduce body weight, food intake, and adiposity in ob/ob mice. In this study we compared effects of intracerebroventricular (ICV) and subcutaneous (SC) administration of leptin on bone metabolism in the appendicular and axial skeleton and adipose tissue gene expression and determined the effects of ICV leptin on bone marrow gene expression in ob/ob mice. In experiment 1, leptin (1.5 or 0.38 µg/d) or control was continuously injected ICV for 12 days. Gene expression analysis of femoral bone marrow stromal cells showed that expression of genes associated with osteogenesis was increased after ICV injection, whereas those associated with osteoclastogenesis, adipogenesis, and adipocyte lipid storage were decreased. In experiment 2, leptin was injected continuously ICV (0.0 or 1.5 µg/d) or SC (0.0 or 10 µg/d) for 12 days. In both experiments, regardless of mode of administration, leptin decreased body weight, food intake, and body fat and increased muscle mass, bone mineral density, bone mineral content, bone area, marrow adipocyte number, and mineral apposition rate. Serum insulin was decreased, whereas serum osteocalcin, insulin-like growth factor 1, osteoprotegerin, pyridinoline, and receptor activator of nuclear factor κB ligand concentrations were increased. In experiment 2, expression of genes in adipose tissue associated with apoptosis, lipid mobilization, insulin sensitivity, and thermogenesis was increased, whereas expression of genes associated with cell differentiation and maturation was decreased regardless of mode of administration. Thus ICV injection of leptin promotes expression of pro-osteogenic factors in bone marrow, leading to enhanced bone formation in ob/ob mice. © 2011 American Society for Bone and Mineral Research

Obesity is an increasing health problem all over the world. Phytochemicals are potential agents to inhibit differentiation of preadipocytes, stimulate lipolysis, and induce apoptosis of existing adipocytes, thereby reducing the amount of adipose tissue. Flavonoids and stilbenoids represent the most researched groups of phytochemicals with regards to their effect on adipogenesis, but there are also a number of in vitro and in vivo studies with phenolic acids, alkaloids, and vitamins, as well as other plant compounds. Although phytochemicals like epigallocatechin-3-gallate, genistein, and resveratrol reduce lipid accumulation and induce adipocyte apoptosis in vitro and reduce body weight and adipose tissues mass in animal models of diet-induced obesity, well-conducted clinical trials are lacking. Pharmacological doses are often used in vitro and when applied in physiological doses in animals or humans, the phytochemicals are often ineffective in affecting adipogenesis. However, by combining several phytochemicals or using them as templates for synthesizing new drugs, there is a large potential in targeting adipogenesis using phytochemicals. © 2010 International Union of Biochemistry and Molecular Biology, Inc.

The objectives of this experiment were to determine whether leptin causes adipocyte apoptosis in mice, whether peripheral administration is an effective means of studying leptin-induced adipocyte apoptosis, and whether high-fat feeding results in reduced responsiveness to leptin-induced adipocyte apoptosis. Continuous 13-day intraperitoneal infusion of 10 lg/day leptin significantly increased adipocyte apoptosis in the epididymal/parametrial fat pads of male and female mice, but only male mice developed reduced responsiveness to leptin-induced adipocyte apoptosis after high-fat (45% fat) feeding for 5 or 15 weeks. There was a positive correlation between serum leptin concentration and percent apoptotic adipocytes. These findings demonstrate that leptin administered peripherally is effective in inducing adipocyte apoptosis in mice, thus extending the possibility of studying this effect of leptin in a wider variety of animal models. In addition, high-fat feeding has a gender-specific effect on development of reduced responsiveness to leptin-induced adipocyte apoptosis.

Comparative proteomic analyses were performed in adipose tissue of leptin-deficient ob/ob mice treated with leptin or control buffer in order to identify the protein expression changes as the potential targets of leptin. Mice were treated with either phosphate-buffered saline (control) or 10 µg/day leptin for 14 days via subcutaneous osmotic minipumps. Total protein from white adipose tissue was extracted and labeled with different fluorescent cyanine dyes for analysis by two-dimensional difference gel electrophoresis (DIGE). Spots that were differentially expressed and appeared to have sufficient material for mass spectrometry analysis were picked and digested with trypsin and subjected to MALDI-TOF MS for protein identification. Twelve functional protein groups were found differentially expressed in adipose tissue of leptin-treated vs. control ob/ob mice, including molecular chaperones and redox proteins such as calreticulin (CALR), protein disulfide isomerase-associated 3 (PDIA3), prohibitin (PHB), and peroxiredoxin-6 (PRDX6); cytoskeleton proteins such as β actin, desmin, and α-tubulin; and some other proteins. The mRNA levels of CALR, PDIA3, and PHB were measured by real-time reverse transcription-PCR and found to be upregulated (P < 0.05), consistent with the fold change in protein expression level. Our findings suggest that leptin's effects on lipid metabolism and apoptosis may be mediated in part by alterations in expression of molecular chaperones and redox proteins for regulating endoplasmic reticulum stress and cytoskeleton proteins for regulating mitochondrial morphology.

Normal mice and leptin-deficient ob/ob mice were treated with leptin to study effects on osteogenesis and adipogenesis in bone marrow. Leptin treatment significantly decreased bone marrow adipocyte size and number in ob/ob mice while increasing bone formation, BMC, and BMD. The results suggest that, in leptin-sensitive animals, the reduction in marrow adipocytes has positive effects on bone formation.Introduction: Adipocytes, osteoblasts, and osteoclasts have leptin receptors, and leptin can also affect bone metabolism indirectly through its receptors in the hypothalamus. We examined the effects of leptin treatment on bone formation, BMD, and marrow adipocyte population in normal mice and leptin-deficient ob/ob mice.Materials and Methods: At the age of 15 weeks, mice were implanted with Alzet osmotic pumps for subcutaneous delivery of treatment solutions (saline, 2.5 μg leptin/day, or 10 μg leptin/day) for 14 days at a delivery rate of 0.25 μl/h. Bone formation was assessed using fluorochrome labels, cell populations were quantified using histomorphometry, and bone densitometry was measured using DXA. We also used a Luminex Beadlyte assay system to quantify cell survival markers in bone marrow samples.Results and Conclusions: Results indicate that both doses of leptin decreased the number of marrow adipocytes in ob/ob mice by >20% (p < 0.05) compared with PBS-treated ob/ob mice. The decrease in adipocyte number with leptin treatment is accompanied by an increase in concentration of the apoptosis marker caspase-3 in bone marrow adipocytes and hematopoietic cells. Both leptin doses also significantly (p < 0.05) increased the percentage of fluorochrome-labeled tibial endosteal surface by >30% compared with PBS-treated ob/ob mice. Leptin treatment increased whole body BMC by >30% in the ob/ob mice receiving the highest leptin dose. Leptin treatment provided no increase in bone formation, BMC, or BMD in normal, leptin-replete mice.

The melanocortin (MC) system in the brain is believed to be an important downstream effector of leptin signaling; interference with MC functioning results in severe obesity. Melanotan II (MTII), an MC3/4-receptor agonist, produces similar behavioral and metabolic outcomes to those observed after leptin treatments, which enhance apoptosis in specific fat depots. To determine whether MTII also mediates adipose apoptosis induced by leptin treatment, two groups of rats (n = 8) received MTII (2 mg/kg, ip) or saline (2 ml/kg) once daily for 4 days and had free access to food and water, and a third group was injected with saline and pair-fed (PF) to MTII treated rats. Food intake, water intake, body temperature, and body weight were measured daily. MTII reduced food and water intake and body weight gain ( P < .05) and decreased body temperature compared to PF and saline-treated control groups. Retroperitoneal white adipose tissue (WAT) mass and epididymal WAT mass were reduced 46.3% and 21.1%, respectively ( P < .05), after MTII, but not after PF, compared with the saline control rats. Both MTII-(25.0%) and PF (33.3%)-treated rats had decreased brown fat weight ( P < .05), whereas muscle mass remained unchanged. Free fatty acid concentrations in serum were not different between MTII and control groups, but increased by 56.4% in PF group. DNA fragmentation assay did not support a role for MTII as an apoptotic signal in any of the fat tissues tested. These results show that in addition to reducing food intake and inhibiting body weight gain, intraperitoneal administration of MTII reduces fat mass, most likely by accelerated lipid mobilization, but not by apoptosis. D

Growth hormone (GH) has an inhibitory effect on adipogenesis, and its effect is associated with insulin action in obesity. In this study, the relationship between GH effect on insulin sensitivity and adipocyte differentiation in vivo was investigated. Transgenic (TG) female mice expressing porcine GH had reduced body weights and weights of retroperitoneal and parametrial fat depots. Insulin treatment increased PPARγ and GLUT4 expression in adipose tissue of WT mice but had no effect in TG mice. Content of transcription factors, PPARγ and C/EBPα and β, was higher in adipose tissue of WT mice, and for C/EBPα and PPARγ, the difference occurred primarily in 24-, compared to 12-week-old, mice. Expression of preadipocyte factor-1 was higher in adipose tissue of TG mice, and expression of TNF-α and leptin was reduced in adipose tissue of 24-week-old TG mice. Our results suggest that increased expression of GH reduces adipogenesis by inducing adipocyte resistance to the adipogenic effect of insulin.

During the past 40 years, many of the known metabolites and hormones have been considered for possible roles in the control of feeding. In the development of this area of research, much attention was given to the relationship between obesity and diabetes (Mayer, 1955). This led to many experiments on the possible roles of insulin, glucose metabolism and feeding behaviours . The development of the concept of the glucostatic control of feeding generated interest in many phases of glucose homoeostasis and the insulin control system, especially as they are related to control of meal size and frequency. Research on the role of hypothalamic structures in energy balance regulation and feeding behaviour included many studies on the effects of both insulin and growth hormone (Bray & This brief review will be concerned primarily with current considerations for the roles of selected hormones in the control of feeding. Several peptides of the gastrointestinal (GI) tract and brain will be discussed, and the possible roles for the major pancreatic hormones, glucagon and insulin, will be reviewed.

McLAUGHLIN, C. L., C. A. BAILE, M. A. DELLA-FERA AND T. G. KASSER. Meal-stimulated increased concentrations of CCK in the hypothalamus of Zucker obese and lean rats. PHYSIOL BEHAV 35(2) 215-220, 1985.--CCK is a putative satiety peptide found to be active when administered peripherally and centrally. Concentrations of CCK have been measured in the brains of fed and fasted animals, but as yet no clear correlation with feeding has been found. In the present experiment rats were sacrificed after a 6-hr fast or 5 min after a meal. Areas of the hypothalamus were removed from these rats and assayed for CCK content. The relationship between obesity and CCK content in specific areas of the brain was also investigated by using Zucker obese and lean rats. In fed rats the CCK concentrations were higher than in fasted rats in the ventromedial hypothalamus (VMH) (56 vs. 42 pg/mg tissue, p<0.005), lateral hypothalamus (38 vs. 27 pg/mg, p<0.01) and supraoptic nucleus (48 vs. 39 pg/mg, p<0.01). In obese rats the concentrations were higher than in lean rats in the VMH (56 vs. 41 pg/mg, p<0.003), dorsal medial hypothalamus (37 vs. 30 pg/mg, p<0.04) and anterior hypothalamus (61 vs. 37 pg/mg, p<0.001). Average concentrations of CCK in all hypothalamic areas were higher in females than males (50 vs. 40 pg/mg, p<0.001), Thus, CCK concentrations in specific areas of the hypothalamus increased with feeding, supporting the potential role of CCK in the central nervous system as a satiety peptide. Further, although the concentrations of CCK in obese rats were higher than those in lean rats, the changes in CCK concentration with feeding were the same, showing that obesity is not a consequence of decreased concentrations or concentration changes of CCK in brain.

Genistein (G), an isoflavone, and guggulsterone (GS), the active substance in guggulipid, have been reported to possess therapeutic effects for obesity. In the present study, we investigated the effects of combinations of G plus GS on apoptosis and adipogenesis in 3T3-L1 cells. In mature adipocytes, G and GS individually caused apoptosis, but combination of G plus GS significantly increased apoptosis, more than either compound alone. Furthermore, G plus GS caused a greater increase in procaspase-3 cleavage, Bax expression, cytochrome c release, and proteolytic cleavage of PARP than either compound alone. In maturing preadipocytes G and GS each suppressed lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. These results suggest that combination of genistein and guggulsterone may exert anti-obesity effects by inhibiting adipogenesis and inducing apoptosis in adipocytes.

Certain flavonoids have been shown to have specific effects on biochemical and metabolic functions of adipocytes. In this study, we investigated the effects of combinations of resveratrol and quercetin on adipogenesis and apoptosis in 3T3-L1 cells. In maturing preadipocytes resveratrol and quercetin at 25 μM individually suppressed intracellular lipid accumulation by 9.4 ± 3.9% (p b 0.01) and 15.9 ± 2.5%, respectively, (p b 0.001). The combination of resveratrol and quercetin at the same dose, however, decreased lipid accumulation by 68.6 ± 0.7% (p b 0.001). In addition, combinations of resveratrol and quercetin at 25 μM significantly decreased the expression of peroxisome proliferators-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein (C/EBP)α, both of which act as key transcription factors. In mature adipocytes resveratrol and quercetin at 100 μM individually decreased viability by 18.1 ± 0.6% (p b 0.001) and 15.8 ± 1% (p b 0.001) and increased apoptosis (100 μM) by 120.5 ± 8.3% (p b 0.001) and 85.3 ± 10% (p b 0.001) at 48 h, respectively. Combinations of resveratrol and quercetin further decreased viability (73.5 ± 0.9%, p b 0.001) and increased apoptosis (310.3 ± 9.6%, p b 0.001) more than single compounds alone. The combination of resveratrol and quercetin at 100 μM increased release of cytochrome c from mitochondria to cytosol and decreased ERK 1/2 phosphorylation. Taken together, our data indicate that combinations of resveratrol and quercetin can exert potential anti-obesity effects by inhibiting differentiation of preadipocytes and inducing apoptosis of mature adipocytes.