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1.  Pharmacological concentrations of irisin increase cell proliferation without influencing markers of neurite outgrowth and synaptogenesis in mouse H19-7 hippocampal cell lines 
Irisin is a novel, myocyte secreted, hormone that has been proposed to mediate the beneficial effects of exercise on metabolism. Irisin is expressed, at lower levels, in human brains and knock-down of the precursor of irisin, FNDC5, decreases neural differentiation of mouse embryonic stem cells. No previous studies have evaluated whether irisin may directly regulate hippocampal neurogenesis in mouse hippocampal neuronal (HN) cells.
Hippocampal neurogenesis as well as irisin signaling were studied in vitro using mouse H19-7 HN cell lines.
We observed that cell proliferation is regulated by irisin in a dose-dependent manner in mouse H19-7 HN cells. Specifically, physiological concentrations of irisin, 5 to 10 nM, had no effect on cell proliferation when compared to control. By contrast, pharmacological concentrations of irisin, 50 to 100 nM, increased cell proliferation when compared to control. Similar to these results regarding irisin’s effects on cell proliferation, we also observed that only pharmacological concentrations of irisin increased STAT3, but not AMPK and/or ERK, activation. Finally, we observed that irisin did not activate either microtubule-associated protein 2, a specific neurite outgrowth marker, or Synapsin, a specific synaptogenesis marker in mouse H19-7 HN cells.
Our data suggest that irisin, in pharmacological concentrations, increases cell proliferation in mouse H19-7 HN cells via STAT3, but not AMPK and/or ERK, signaling pathways. By contrast, neither physiological nor pharmacological concentrations of irisin alter markers of hippocampal neurogenesis in mouse H19-7 HN cell lines.
PMCID: PMC4370428  PMID: 23664146
irisin; signaling; hippocampal neurogenesis
2.  Chemopreventive Effects of Korean Red Ginseng Extract on Rat Hepatocarcinogenesis 
Journal of Cancer  2015;6(1):1-8.
The objective of this study was to determine a chemopreventive activity of Korean red ginseng extract (KRG) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. After acclimatization for a week, Sprague-Dawley rats were randomized into five groups (n = 15) and fed either KRG (0.5, 1 or 2%) or control diets for 10 weeks. After two weeks of starting of experimental diets, the rats were initiated hepatocarcinogenesis by injection of DEN and were then subjected to two-thirds partial hepatectomy at five-week for developing the medium-term bioassay system. Both 0.5 and 1% KRG diets suppressed the area (55 and 60%; p= 0.0251 and 0.0144) and number (39 and 59%; p= 0.0433 and 0.0012) of glutathione S-transferase placental form (GST-P) positive foci when compared to the DEN-control group. The production of thiobarbituric acid reactive substances (TBARS) was significantly reduced in 0.5 and 1% KRG-treated rats. The supplementation of 1% KRG diet significantly elevated the levels of total glutathione (tGSH) and glutathione-related enzymes including cytosolic glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. It was also observed in cDNA microarray that the gene expressions (Cyp2c6, Cyp2e1, Cyp3a9, and Mgst1) involved in the xenobiotics metabolism via cytochrome P450 signaling pathway were down-regulated in the 1% KRG diet-treated group when compared to the DEN-control. The chemopreventive effects of KRG could be affected by 1) the decrease of lipid peroxidation, 2) the increase of tGSH content and GSH-dependent enzyme activities, and 3) the decrease of the gene expression profile involved in cytochrome P450 signaling pathway. These results suggest that KRG may prove to be a therapeutic agent against hepatocarcinogenesis.
PMCID: PMC4278909  PMID: 25553083
Korean red ginseng; rat; glutathione S-transferase placental form positive foci; hepatocarcinogenesis; antioxidant.
3.  Identification and Saturable Nature of Signaling Pathways Induced by Metreleptin in Humans: Comparative Evaluation of In Vivo, Ex Vivo, and In Vitro Administration 
Diabetes  2014;64(3):828-839.
Signaling pathways activated by leptin in metabolically important organs have largely been studied only in animal and/or cell culture studies. In this study, we examined whether leptin has similar effects in human peripheral tissues in vivo, ex vivo, and in vitro and whether the response would be different in lean and obese humans. For in vivo leptin signaling, metreleptin was administered and muscle, adipose tissue, and peripheral blood mononuclear cells were taken for analysis of signal activation. Experiments were also done ex vivo and with primary cultured cells in vitro. The signal activation was compared between male versus female and obese versus lean humans. Acute in vivo, ex vivo, and/or in vitro metreleptin administration similarly activated STAT3, AMPK, ERK1/2, Akt, mTOR, NF-κB, and/or IKKα/β without any differences between male versus female and obese versus lean subjects. All signaling pathways were saturable at ∼30–50 ng/mL, consistent with the clinical evidence showing no additional effect(s) in obese subjects who already have high levels of leptin. Our data provide novel information on downstream effectors of metreleptin action in humans that may have therapeutic implications.
PMCID: PMC4338590  PMID: 25249580
4.  Fibroblast Growth Factor 21 Levels in Young Healthy Females Display Day and Night Variations and Are Increased in Response to Short-Term Energy Deprivation Through a Leptin-Independent Pathway 
Diabetes Care  2013;36(4):935-942.
Fibroblast growth factor (FGF)-21 is an endocrine factor with potent metabolic effects. Its day–night patterns of secretion and/or its physiological response to energy deprivation and relationship to free fatty acids (FFAs) and/or leptin remain to be fully elucidated. We aim to elucidate day–night pattern of FGF-21 levels and its relationship to FFA, to assess whether energy deprivation alters its circulating patterns, and to examine whether leptin may mediate these changes.
Six healthy lean females were studied for 72 h in a cross-over interventional study under three different conditions: on isocaloric diet and in a fasting state with administration of either placebo or metreleptin in physiological replacement doses. Blood samples were obtained hourly from 8:00 a.m. on day 4 until 8:00 a.m. on day 5.
FGF-21 exhibited day–night variation pattern during the isocaloric fed state. Fasting significantly increased FGF-21 levels (P < 0.01) via a leptin-independent pathway. Day–night variation pattern in the fed state was lost on fasting. Leptin replacement in the hypoleptinemic state restored approximate entropy of FGF-21 time series but did not alter circulating levels. FGF-21 levels were closely cross-correlated with FFA levels in all three states.
A day–night variation in the levels of FGF-21 exists in young lean females in the fed state. Energy deprivation increases FGF-21 levels via a leptin-independent pathway. The interaction between FGF-21 and starvation-induced lipolysis, as indicated by its close cross-correlations with FFA in both fed state and energy deprivation, needs to be studied further.
PMCID: PMC3609498  PMID: 23193213
5.  Direct role of adiponectin and adiponectin receptors in endometrial cancer: in vitro and ex vivo studies in humans 
Molecular cancer therapeutics  2011;10(12):2234-2243.
Low adiponectin levels are an independent risk factor for, and mediate the effect of obesity on endometrial cancer in epidemiology studies. The direct or indirect mechanisms underlying these findings remain to be elucidated. We first examined the expression of adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) in normal human endometrium and in endometrial cancer tissues ex vivo. We then utilized KLE and RL95-2 human endometrial cancer cell lines in vitro to study relative expression of AdipoRs, to investigate the effect of adiponectin on activating intracellular signaling pathways, and to assess its potential to alter malignant properties. We report for the first time that the relative expression level of AdipoR1 is higher than AdipoR2 in human endometrial cancer tissue but the expression of AdipoRs is not statistically different from non-neoplastic tissues. We also demonstrate for the first time in endometrial cancer cell lines in vitro that adiponectin suppresses endometrial cancer proliferation acting through AdipoRs. Adiponectin also increases the expression of the adaptor molecule LKB1 which is required for adiponectin-mediated activation of AMPK/S6 axis and modulation of cell proliferation, colony formation, adhesion and invasion of KLE and RL95-2 cell lines. These novel mechanistic studies provide for the first time in vitro and ex vivo evidence for a causal role of adiponectin in endometrial cancer.
PMCID: PMC3237794  PMID: 21980131
adiponectin; adiponectin receptor; intracellular signaling; LKB1; AMPK; endometrial adenocarcinoma
6.  Leptin administration to overweight and obese subjects for six months increases free leptin concentrations but does not alter circulating hormones of the thyroid and IGF axes during weight loss induced by a mild hypocaloric diet 
Short-term energy deprivation reduces leptin concentrations and alters the levels of circulating hormones of the hypothalamic–pituitary–peripheral axis in lean subjects. Whether the reduction in leptin concentration during long-term weight loss in obese individuals is linked to the same neuroendocrine changes seen in lean, leptin sensitive subjects remains to be fully clarified.
Twenty-four overweight and obese adults (16 women and 8 men; BMI: 27.5–38.0 kg/m2) were prescribed a hypocaloric diet (−500 kcal/day) and were randomized to receive recombinant methionyl leptin (n=18, metreleptin, 10 mg/day self-injected s.c.) or placebo (n=6, same volume and time as metreleptin) for 6 months.
Metreleptin administration did not affect weight loss beyond that induced by hypocaloric diet alone (P for interaction = 0.341) but increased the serum concentrations of total leptin by 6–8–fold (P<0.001) and led to the generation of anti-leptin antibodies. Despite free leptin concentration (P for interaction = 0.041) increasing from 9±1 ng/ml at baseline to 43±15 and 36±12 ng/ml at 3 and 6 months, respectively, changes in circulating hormones of the thyroid and insulin-like growth factor axes at 3 and 6 months were not significantly different in the placebo- and metreleptin-treated groups.
Leptin does not likely mediate changes in neuroendocrine function in response to weight loss induced by a mild hypocaloric diet in overweight and obese subjects.
PMCID: PMC3159386  PMID: 21602313
caloric restriction; thyroid function; leptin resistance; leptin tolerance; obesity
7.  Efficacy of Metreleptin in Obese Patients With Type 2 Diabetes: Cellular and Molecular Pathways Underlying Leptin Tolerance 
Diabetes  2011;60(6):1647-1656.
Metreleptin has been efficacious in improving metabolic control in patients with lipodystrophy, but its efficacy has not been tested in obese patients with type 2 diabetes.
We studied the role of leptin in regulating the endocrine adaptation to long-term caloric deprivation and weight loss in obese diabetic subjects over 16 weeks in the context of a double-blinded, placebo–controlled, randomized trial. We then performed detailed interventional and mechanistic signaling studies in humans in vivo, ex vivo, and in vitro.
In obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA1c marginally (8.01 ± 0.93–7.96 ± 1.12, P = 0.03). Total leptin, leptin-binding protein, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ∼50 ng/mL. Consistent with clinical observations, all metreleptin signaling pathways studied in human adipose tissue and peripheral blood mononuclear cells were saturable at ∼50 ng/mL, with no major differences in timing or magnitude of leptin-activated STAT3 phosphorylation in tissues from male versus female or obese versus lean humans in vivo, ex vivo, or in vitro. We also observed for the first time that endoplasmic reticulum (ER) stress in human primary adipocytes inhibits leptin signaling.
In obese patients with diabetes, metreleptin administration did not alter body weight or circulating inflammatory markers but reduced HbA1c marginally. ER stress and the saturable nature of leptin signaling pathways play a key role in the development of leptin tolerance in obese patients with diabetes.
PMCID: PMC3114380  PMID: 21617185
8.  N-Docosahexaenoylethanolamide promotes development of hippocampal neurons 
The Biochemical journal  2011;435(2):327-336.
DHA (docosahexaenoic acid, C22:6,n−3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n−3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.
PMCID: PMC3169088  PMID: 21281269
docosahexaenoic acid (DHA); N-docosahexaenoylethanolamide (DEA); hippocampus; neurite growth; neuron; synaptogenesis
9.  Leptin and Amylin Act in an Additive Manner to Activate Overlapping Signaling Pathways in Peripheral Tissues 
Diabetes Care  2010;34(1):132-138.
Amylin interacts with leptin to alter metabolism. We evaluated, for the first time, amylin- and/or leptin-activated signaling pathways in human peripheral tissues (hPTs).
Leptin and amylin signaling studies were performed in vitro in human primary adipocytes (hPAs) and human peripheral blood mononuclear cells (hPBMCs) and ex vivo in human adipose tissue (hAT) from male versus female subjects, obese versus lean subjects, and subjects with subcutaneous versus omental adipose tissue.
The long form of leptin receptor was expressed in human tissues and cells studied in ex vivo and in vitro, respectively. Leptin and amylin alone and in combination activate signal transducer and activator of transcription 3 (STAT3), AMP-activated protein kinase, Akt, and extracellular signal-regulated kinase signaling pathways in hAT ex vivo and hPAs and hPBMCs in vitro; all phosphorylation events were saturable at leptin and amylin concentrations of ∼50 and ∼20 ng/ml, respectively. The effects of leptin and amylin on STAT3 phosphorylation in hPAs and hPBMCs in vitro were totally abolished under endoplasmic reticulum stress and/or in the presence of a STAT3 inhibitor. Results similar to those in the in vitro studies were observed in hAT studied ex vivo.
Leptin and amylin activate overlapping intracellular signaling pathways in humans and have additive, but not synergistic, effects in signaling pathways studied in hPTs in vitro and ex vivo.
PMCID: PMC3005478  PMID: 20870968
10.  Leptin in Human Physiology and Therapeutics 
Frontiers in neuroendocrinology  2010;31(3):377-393.
Leptin regulates energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. In this review, we describe the role of leptin in human physiology and review evidence from recent “proof of concept” clinical trials using recombinant human leptin in subjects with congenital leptin deficiency, hypoleptinemia associated with energy-deficient states, and hyperleptinemia associated with garden-variety obesity. Since most obese individuals are largely leptin-tolerant or -resistant, therapeutic uses of leptin are currently limited to patients with complete or partial leptin deficiency, including hypothalamic amenorrhea and lipoatrophy. Leptin administration in these energy-deficient states may help restore associated neuroendocrine, metabolic, and immune function and bone metabolism. Leptin treatment is currently available for individuals with congenital leptin deficiency and congenital lipoatrophy. The long-term efficacy and safety of leptin treatment in hypothalamic amenorrhea and acquired lipoatrophy are currently under investigation. Whether combination therapy with leptin and potential leptin sensitizers will prove effective in the treatment of garden-variety obesity and whether leptin may have a role in weight loss maintenance is being greatly anticipated.
PMCID: PMC2916735  PMID: 20600241
leptin; leptin deficiency; obesity; leptin resistance; energy homeostasis; insulin resistance; adipokines; amenorrhea; lipoatrophy
11.  Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function 
Journal of neurochemistry  2009;111(2):510-521.
Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.
PMCID: PMC2773444  PMID: 19682204
docosahexaenoic acid; hippocampal development; long-term potentiation; neurite growth; synaptic function; synaptogenesis
12.  Physico-chemical modifications of conjugated linoleic acid for ruminal protection and oxidative stability 
Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of octadecadienoic acid [linoleic acid (LA), 18:2n-6]. Although ruminant milk and meat products represent the largest natural source of CLA and therefore, their concentration in ruminant lipids are of interest to human health, chemical or physical modifications of CLA should be needed as a means to enhance oxidative stability, to improve post-ruminal bioavailability, and to increase the clinical application. In fact, CLA are rapidly decomposed to form furan fatty acids when its are oxidized in air, and the effectiveness of dietary supplements of CLA may be related to the extent that their metabolisms by rumen bacteria are avoided. For these reasons, many scientists have examined the effect of manufacturing and protection on the stability of CLA in ruminants and food products. In this review, physico-chemical modifications of CLA for ruminal protection such as calcium salt (Ca), formaldehyde protection (FP), lipid encapsulation (LE), and amide linkage (AL), and for oxidative stability such as green tea catechin (GTC), cyclodextrin (CD), arginine (Arg), amylase, and PEGylation are proposed.
PMCID: PMC2430566  PMID: 18513443

Results 1-12 (12)