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Article ; Online: Dietary pH Enhancement Improves Metabolic Outcomes in Diet-Induced Obese Male and Female Mice: Effects of Beef vs. Casein Proteins.

Menikdiwela, Kalhara R / Guimarães, João Pedro Tôrres / Scoggin, Shane / Gollahon, Lauren S / Moustaid-Moussa, Naima

2022 Volume 14, Issue 13

Abstract: 1) Consumption of diets that are caloric dense but not nutrient dense have been implicated in metabolic diseases, in part through low-grade metabolic acidosis. Mitigation strategies through dietary intervention to alleviate acidosis have not been ... ...

Abstract	(1) Consumption of diets that are caloric dense but not nutrient dense have been implicated in metabolic diseases, in part through low-grade metabolic acidosis. Mitigation strategies through dietary intervention to alleviate acidosis have not been previously reported. Our objective is to determine the effects of pH enhancement (with ammonia) in high fat diet-induced obese mice that were fed beef or casein as protein sources compared to low fat diet-fed mice. (2) Methods: B6 male and female mice were randomized (
MeSH term(s)	Ammonia ; Animals ; Body Weight ; Caseins/metabolism ; Caseins/pharmacology ; Cattle ; Diet, High-Fat/adverse effects ; Dietary Fats/metabolism ; Female ; Glucose ; Hydrogen-Ion Concentration ; Male ; Mice ; Mice, Obese ; Obesity/metabolism
Chemical Substances	Caseins ; Dietary Fats ; Ammonia (7664-41-7) ; Glucose (IY9XDZ35W2)
Language	English
Publishing date	2022-06-22
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2518386-2
ISSN	2072-6643 ; 2072-6643
ISSN (online)	2072-6643
ISSN	2072-6643
DOI	10.3390/nu14132583
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Article ; Online: Anti-Inflammatory Mechanisms of Curcumin and Its Metabolites in White Adipose Tissue and Cultured Adipocytes.

Islam, Tariful / Scoggin, Shane / Gong, Xiaoxia / Zabet-Moghaddam, Masoud / Kalupahana, Nishan S / Moustaid-Moussa, Naima

Nutrients

2023 Volume 16, Issue 1

Abstract: The plant-derived polyphenol curcumin alleviates the inflammatory and metabolic effects of obesity, in part, by reducing adipose tissue inflammation. We hypothesized that the benefits of curcumin supplementation on diet-induced obesity and systemic ... ...

Abstract	The plant-derived polyphenol curcumin alleviates the inflammatory and metabolic effects of obesity, in part, by reducing adipose tissue inflammation. We hypothesized that the benefits of curcumin supplementation on diet-induced obesity and systemic inflammation in mice occur through downregulation of white adipose tissue (WAT) inflammation. The hypothesis was tested in adipose tissue from high-fat diet-induced obese mice supplemented with or without curcumin and in 3T3-L1 adipocytes treated with or without curcumin. Male B6 mice were fed a high-fat diet (HFD, 45% kcal fat) with or without 0.4% (
MeSH term(s)	Animals ; Mice ; Curcumin/pharmacology ; Interleukin-6/genetics ; Lipopolysaccharides ; Proteomics ; Adipocytes ; Adipose Tissue, White ; Anti-Inflammatory Agents/pharmacology ; Inflammation/drug therapy ; TOR Serine-Threonine Kinases ; Obesity/drug therapy ; Glucuronides
Chemical Substances	curcumin glucuronide (BE1PK7RL4M) ; Curcumin (IT942ZTH98) ; Interleukin-6 ; Lipopolysaccharides ; Anti-Inflammatory Agents ; TOR Serine-Threonine Kinases (EC 2.7.11.1) ; Glucuronides
Language	English
Publishing date	2023-12-25
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2518386-2
ISSN	2072-6643 ; 2072-6643
ISSN (online)	2072-6643
ISSN	2072-6643
DOI	10.3390/nu16010070
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Article ; Online: Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.

Nejat, Shadi / Menikdiwela, Kalhara R / Efotte, Aliyah / Scoggin, Shane / Vandanmagsar, Bolormaa / Thornalley, Paul J / Dehbi, Mohammed / Moustaid-Moussa, Naima

Genes

2023 Volume 14, Issue 10

Abstract: Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, ... ...

Abstract	Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D.
MeSH term(s)	Animals ; Female ; Male ; Mice ; Body Weight/genetics ; CRISPR-Cas Systems/genetics ; Diabetes Mellitus, Type 2/genetics ; Diabetes Mellitus, Type 2/metabolism ; Diet, High-Fat/adverse effects ; Glucose/metabolism ; HSP40 Heat-Shock Proteins/genetics ; HSP40 Heat-Shock Proteins/metabolism ; Insulin/genetics ; Insulin/metabolism ; Insulin Resistance/genetics ; Mice, Knockout ; Obesity/genetics ; Obesity/metabolism ; Phenotype ; RNA, Messenger
Chemical Substances	Glucose (IY9XDZ35W2) ; HSP40 Heat-Shock Proteins ; Insulin ; RNA, Messenger ; Dnajb3 protein, mouse
Language	English
Publishing date	2023-09-24
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2527218-4
ISSN	2073-4425 ; 2073-4425
ISSN (online)	2073-4425
ISSN	2073-4425
DOI	10.3390/genes14101857
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Article ; Online: Temperature-Dependent Effects of Eicosapentaenoic Acid (EPA) on Browning of Subcutaneous Adipose Tissue in UCP1 Knockout Male Mice.

Zu, Yujiao / Pahlavani, Mandana / Ramalingam, Latha / Jayarathne, Shasika / Andrade, Jose / Scoggin, Shane / Festuccia, William T / Kalupahana, Nishan S / Moustaid-Moussa, Naima

International journal of molecular sciences

2023 Volume 24, Issue 10

Abstract: Uncoupling protein 1 (UCP1) plays a central role in thermogenic tissues by uncoupling cellular respiration to dissipate energy. Beige adipocytes, an inducible form of thermogenic cells in subcutaneous adipose tissue (SAT), have become a major focus in ... ...

Abstract	Uncoupling protein 1 (UCP1) plays a central role in thermogenic tissues by uncoupling cellular respiration to dissipate energy. Beige adipocytes, an inducible form of thermogenic cells in subcutaneous adipose tissue (SAT), have become a major focus in obesity research. We have previously shown that eicosapentaenoic acid (EPA) ameliorated high-fat diet (HFD)-induced obesity by activating brown fat in C57BL/6J (B6) mice at thermoneutrality (30 °C), independently of UCP1. Here, we investigated whether ambient temperature (22 °C) impacts EPA effects on SAT browning in wild-type (WT) and UCP1 knockout (KO) male mice and dissected underlying mechanisms using a cell model. We observed resistance to diet-induced obesity in UCP1 KO mice fed HFD at ambient temperature, with significantly higher expression of UCP1-independent thermogenic markers, compared to WT mice. These markers included the fibroblast growth factor 21 (FGF21) and sarco/endoplasmic reticulum Ca
MeSH term(s)	Male ; Animals ; Mice ; Temperature ; Uncoupling Protein 1/genetics ; Uncoupling Protein 1/metabolism ; Eicosapentaenoic Acid/pharmacology ; Eicosapentaenoic Acid/metabolism ; Mice, Knockout ; Mice, Inbred C57BL ; Adipose Tissue, Brown/metabolism ; Obesity/metabolism ; Subcutaneous Fat/metabolism ; Thermogenesis/genetics ; Adipose Tissue, White/metabolism
Chemical Substances	Uncoupling Protein 1 ; Eicosapentaenoic Acid (AAN7QOV9EA) ; Ucp1 protein, mouse
Language	English
Publishing date	2023-05-13
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms24108708
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Article: Dietary pH Enhancement Improves Metabolic Outcomes in Diet-Induced Obese Male and Female Mice: Effects of Beef vs. Casein Proteins

Menikdiwela, Kalhara R. / Guimarães, João Pedro Tôrres / Scoggin, Shane / Gollahon, Lauren S. / Moustaid-Moussa, Naima

Nutrients. 2022 June 22, v. 14, no. 13

2022

Abstract	(1) Consumption of diets that are caloric dense but not nutrient dense have been implicated in metabolic diseases, in part through low-grade metabolic acidosis. Mitigation strategies through dietary intervention to alleviate acidosis have not been previously reported. Our objective is to determine the effects of pH enhancement (with ammonia) in high fat diet-induced obese mice that were fed beef or casein as protein sources compared to low fat diet-fed mice. (2) Methods: B6 male and female mice were randomized (n = 10) into eight diets that differ in protein source, pH enhancement of the protein, and fat content, and fed for 13 weeks: low fat (11% fat) casein (LFC), LF casein pH-enhanced (LFCN), LF lean beef (LFB), LFBN, high fat (46%) casein (HFC), HFCN, HF beef (HFB), and HFBN. Body weights and composition, and glucose tolerance tests were conducted along with terminal serum analyses. Three-way ANOVA was performed. (3) Results: A significant effect of dietary fat (LF vs. HF) was observed across all variables in both sexes (final body weight, fat mass, glucose clearance, and serum leptin). Importantly, pH enhancement significantly reduced adiposity (males only) and final body weights (females only) and significantly improved glucose clearance in both sexes. Lastly, clear sex differences were observed across all variables. (4) Conclusions: Our findings demonstrate metabolic benefits of increasing dietary pH using ammonia, while high fat intake per se (not protein source) is the major contributor to metabolic dysfunctions. Additional research is warranted to determine mechanisms underlying the beneficial effects of pH enhancement, and interactions with dietary fat content and proteins.
Keywords	acidosis ; adiposity ; ammonia ; beef ; blood serum ; casein ; dietary fat ; fat intake ; females ; glucose ; glucose tolerance ; leptin ; lipid content ; males ; nutritional intervention ; pH ; protein sources
Language	English
Dates of publication	2022-0622
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2518386-2
ISSN	2072-6643
ISSN	2072-6643
DOI	10.3390/nu14132583
Database	NAL-Catalogue (AGRICOLA)

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Article: Uncoupling protein 1-independent effects of eicosapentaenoic acid in brown adipose tissue of diet-induced obese female mice

Miller, Emily K. / Pahlavani, Mandana / Ramalingam, Latha / Scoggin, Shane / Moustaid-Moussa, Naima

Journal of nutritional biochemistry. 2021 Dec., v. 98

2021

Abstract: Brown adipose tissue (BAT) plays a key role in energy expenditure through its thermogenic function, making its activation a popular target to reduce obesity. We recently reported that male mice housed at thermoneutrality with uncoupling protein 1 (UCP1) ... ...

Abstract	Brown adipose tissue (BAT) plays a key role in energy expenditure through its thermogenic function, making its activation a popular target to reduce obesity. We recently reported that male mice housed at thermoneutrality with uncoupling protein 1 (UCP1) deficiency had increased weight gain and glucose intolerance, but eicosapentaenoic acid (EPA) ameliorated these effects.Whether female mice respond similarly to lack of UCP1 and to EPA remains unknown. We hypothesize that the effects of EPA on BAT activation are independent of UCP1 expression. We used female wild type (WT) and UCP1 knockout (KO) mice housed at thermoneutrality (30°C) as an obesogenic environment and fed them high fat (HF) diets with or without EPA for up to 14 weeks. Body weight (BW), body composition, and insulin and glucose tolerance tests were performed during the feeding trial. At termination, serum and BAT were harvested for further analyses. Mice in the KO-EPA group had significantly lower BW than KO-HF mice. In addition, KO-HF mice displayed significantly impaired glucose tolerance compared to their WT-HF littermates. However, EPA significantly enhanced glucose clearance in the KO mice compared to KO-HF mice. Protein levels of the mitochondrial cytochrome C oxidase subunits I, II, and IV were significantly lower in KO mice compared to WT. Our findings support that ablation of UCP1 is detrimental to energy metabolism of female mice in thermoneutral conditions. However, unexpectedly, EPA's protective effects against diet-induced obesity and glucose intolerance in these mice were independent of UCP1.
Keywords	blood serum ; body composition ; brown adipose tissue ; eicosapentaenoic acid ; energy expenditure ; energy metabolism ; environmental factors ; females ; glucose ; glucose tolerance ; insulin ; males ; mitochondria ; obesity ; weight gain
Language	English
Dates of publication	2021-12
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1014929-6
ISSN	1873-4847 ; 0955-2863
ISSN (online)	1873-4847
ISSN	0955-2863
DOI	10.1016/j.jnutbio.2021.108819
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Impact of Long-Term Dietary High Fat and Eicosapentaenoic Acid on Behavior and Hypothalamic-Pituitary-Adrenal Axis Activity in Amyloidogenic APPswe/PSEN1dE9 Mice.

Harris, Breanna N / Yavari, Mahsa / Ramalingam, Latha / Mounce, P Logan / Alers Maldonado, Kaylee / Chavira, Angela C / Thomas, Sarah / Scoggin, Shane / Biltz, Caroline / Moustaid-Moussa, Naima

Neuroendocrinology

2024 Volume 114, Issue 6, Page(s) 553–576

Abstract: Introduction: Alzheimer's disease (AD) alters neurocognitive and emotional function and causes dysregulation of multiple homeostatic processes. The leading AD framework pins amyloid beta plaques and tau tangles as primary drivers of dysfunction. However, ...

Abstract	Introduction: Alzheimer's disease (AD) alters neurocognitive and emotional function and causes dysregulation of multiple homeostatic processes. The leading AD framework pins amyloid beta plaques and tau tangles as primary drivers of dysfunction. However, many additional variables, including diet, stress, sex, age, and pain tolerance, interact in ways that are not fully understood to impact the onset and progression of AD pathophysiology. We asked: (1) does high-fat diet, compared to low-fat diet, exacerbate AD pathophysiology and behavioral decline? And, (2) can supplementation with eicosapentaenoic (EPA)-enriched fish oil prevent high-fat-diet-induced changes? Methods: Male and female APPswePSdE9 mice, and their non-transgenic littermates, were randomly assigned to a diet condition (low-fat, high-fat, high-fat with EPA) and followed from 2 to 10 months of age. We assessed baseline corticosterone concentration during aging, pain tolerance, cognitive function, stress coping, and corticosterone response to a stressor. Results: Transgenic mice were consistently more active than non-transgenic mice but did not perform worse on either cognitive task, even though we recently reported that these same transgenic mice exhibited metabolic changes and had increased amyloid beta. Mice fed high-fat diet had higher baseline and post-stressor corticosterone, but diet did not impact cognition or pain tolerance. Sex had the biggest influence, as female mice were consistently more active and had higher corticosterone than males. Conclusion: Overall, diet, genotype, and sex did not have consistent impacts on outcomes. We found little support for predicted interactions and correlations, suggesting diet impacts metabolic function and amyloid beta levels, but these outcomes do not translate to changes in behaviors measured here.
Language	English
Publishing date	2024-02-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	123303-8
ISSN	1423-0194 ; 0028-3835
ISSN (online)	1423-0194
ISSN	0028-3835
DOI	10.1159/000536586
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Article ; Online: Uncoupling protein 1-independent effects of eicosapentaenoic acid in brown adipose tissue of diet-induced obese female mice.

Miller, Emily K / Pahlavani, Mandana / Ramalingam, Latha / Scoggin, Shane / Moustaid-Moussa, Naima

The Journal of nutritional biochemistry

2021 Volume 98, Page(s) 108819

Abstract	Brown adipose tissue (BAT) plays a key role in energy expenditure through its thermogenic function, making its activation a popular target to reduce obesity. We recently reported that male mice housed at thermoneutrality with uncoupling protein 1 (UCP1) deficiency had increased weight gain and glucose intolerance, but eicosapentaenoic acid (EPA) ameliorated these effects. Whether female mice respond similarly to lack of UCP1 and to EPA remains unknown. We hypothesize that the effects of EPA on BAT activation are independent of UCP1 expression. We used female wild type (WT) and UCP1 knockout (KO) mice housed at thermoneutrality (30°C) as an obesogenic environment and fed them high fat (HF) diets with or without EPA for up to 14 weeks. Body weight (BW), body composition, and insulin and glucose tolerance tests were performed during the feeding trial. At termination, serum and BAT were harvested for further analyses. Mice in the KO-EPA group had significantly lower BW than KO-HF mice. In addition, KO-HF mice displayed significantly impaired glucose tolerance compared to their WT-HF littermates. However, EPA significantly enhanced glucose clearance in the KO mice compared to KO-HF mice. Protein levels of the mitochondrial cytochrome C oxidase subunits I, II, and IV were significantly lower in KO mice compared to WT. Our findings support that ablation of UCP1 is detrimental to energy metabolism of female mice in thermoneutral conditions. However, unexpectedly, EPA's protective effects against diet-induced obesity and glucose intolerance in these mice were independent of UCP1.
MeSH term(s)	Adipose Tissue, Brown/metabolism ; Animals ; Body Weight/drug effects ; Diet, High-Fat/adverse effects ; Eicosapentaenoic Acid/pharmacology ; Energy Metabolism/drug effects ; Female ; Glucose Intolerance/metabolism ; Glucose Tolerance Test/methods ; Mice ; Mice, Knockout ; Mice, Obese ; Obesity/drug therapy ; Obesity/metabolism ; Temperature ; Thermogenesis/drug effects ; Uncoupling Protein 1/metabolism
Chemical Substances	Uncoupling Protein 1 ; Eicosapentaenoic Acid (AAN7QOV9EA)
Language	English
Publishing date	2021-07-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1014929-6
ISSN	1873-4847 ; 0955-2863
ISSN (online)	1873-4847
ISSN	0955-2863
DOI	10.1016/j.jnutbio.2021.108819
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Article: Discordant Dose-Dependent Metabolic Effects of Eicosapentanoic Acid in Diet-Induced Obese Mice

Pahlavani, Mandana / Ramalingam, Latha / Miller, Emily K / Davis, Hanna / Scoggin, Shane / Moustaid-Moussa, Naima

Nutrients. 2020 May 08, v. 12, no. 5

2020

Abstract: Obesity is a widespread epidemic that increases the risk for several metabolic diseases. Despite several beneficial health effects of eicosapentaenoic acid (C20:5n-3, EPA), previous studies have used very high doses of EPA. In this study, dose-dependent ... ...

Abstract	Obesity is a widespread epidemic that increases the risk for several metabolic diseases. Despite several beneficial health effects of eicosapentaenoic acid (C20:5n-3, EPA), previous studies have used very high doses of EPA. In this study, dose-dependent effects of EPA on metabolic outcomes were determined in diet-induced obese mice. We used B6 male mice, fed high-fat diet (HF, 45% kcal fat) or HF diet supplemented with 9, 18, and 36 g/kg of EPA-enriched fish oil for 14 weeks. We conducted metabolic phenotyping during the feeding period, and harvested tissues and blood at termination. Only mice fed 36 g/kg of EPA significantly (p < 0.05) lowered body weight, fat content and epididymal fat pad weight, compared to HF. Both 18 and 36 g/kg doses of EPA significantly increased glucose clearance and insulin sensitivity, compared to HF or 9 g/kg of EPA. Locomotor activity was significantly increased with both 18 and 36 g/kg doses of EPA. Interestingly, all doses of EPA compared to HF, significantly increased energy expenditure and oxygen consumption and significantly reduced serum insulin, leptin, and triglycerides levels. These results demonstrate weight- and adiposity-independent metabolic benefits of EPA, at doses comparable to those currently used to treat hypertriglyceridemia.
Keywords	blood serum ; dose response ; eicosapentaenoic acid ; energy expenditure ; epididymis ; fish oils ; glucose ; health effects assessments ; high fat diet ; hypertriglyceridemia ; insulin ; insulin resistance ; leptin ; lipid content ; locomotion ; males ; mice ; nutrients ; obesity ; oxygen consumption ; phenotype ; risk ; tissues ; triacylglycerols ; weight
Language	English
Dates of publication	2020-0508
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
Note	NAL-light
ZDB-ID	2518386-2
ISSN	2072-6643
ISSN	2072-6643
DOI	10.3390/nu12051342
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Article ; Online: AdipoGauge software for analysis of biological microscopic images.

Yosofvand, Mohammad / Liyanage, Sanka / Kalupahana, Nishan S / Scoggin, Shane / Moustaid-Moussa, Naima / Moussa, Hanna

Adipocyte

2020 Volume 9, Issue 1, Page(s) 360–373

Abstract: Obesity is a complex disease of global epidemic proportions. Adipose tissue expansion and chronic low-grade inflammation, locally and systemically, are hallmark features of obesity. Obesity is associated with several other chronic diseases, which are ... ...

Abstract	Obesity is a complex disease of global epidemic proportions. Adipose tissue expansion and chronic low-grade inflammation, locally and systemically, are hallmark features of obesity. Obesity is associated with several other chronic diseases, which are also characterized by inflammation. Determination of adipocyte size and macrophage content in adipose tissue is a critical step in assessing changes in this tissue with obesity. Here, we introduce a complete standalone software package,
MeSH term(s)	Adipocytes/pathology ; Adipose Tissue/pathology ; Animals ; Fluorescent Antibody Technique/methods ; Humans ; Image Processing, Computer-Assisted/methods ; Immunohistochemistry/methods ; Macrophages/pathology ; Mice ; Microscopy/methods ; Software
Language	English
Publishing date	2020-07-11
Publishing country	United States
Document type	Journal Article
ISSN	2162-397X
ISSN (online)	2162-397X
DOI	10.1080/21623945.2020.1787583
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