scholarly journals CRISPR-delivery particles targeting nuclear receptor–interacting protein 1 (Nrip1) in adipose cells to enhance energy expenditure

2018 ◽  
Vol 293 (44) ◽  
pp. 17291-17305 ◽  
Author(s):  
Yuefei Shen ◽  
Jessica L. Cohen ◽  
Sarah M. Nicoloro ◽  
Mark Kelly ◽  
Batuhan Yenilmez ◽  
...  
Keyword(s):  
Metabolic Disease ◽  
Energy Expenditure ◽  
Therapeutic Strategy ◽  
Gene Deletion ◽  
Cultured Cells ◽  
Uncoupling Protein ◽  
Acid Oxidation ◽  
Amphipathic Peptide ◽  
Cas9 Protein

RNA-guided, engineered nucleases derived from the prokaryotic adaptive immune system CRISPR-Cas represent a powerful platform for gene deletion and editing. When used as a therapeutic approach, direct delivery of Cas9 protein and single-guide RNA (sgRNA) could circumvent the safety issues associated with plasmid delivery and therefore represents an attractive tool for precision genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fatty acid oxidation, and secretion of bioactive factors through a “browning” process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed “CRISPR-delivery particles,” denoted CriPs, composed of nano-size complexes of Cas9 protein and sgRNA that are coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9–mediated gene deletion of ectopically expressed GFP by CriPs was achieved in multiple cell types, including a macrophage cell line, primary macrophages, and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP-expressing mice following intraperitoneal injection of CriPs containing Gfp-targeting sgRNA. Furthermore, disruption of a nuclear co-repressor of catabolism, the Nrip1 gene, in white adipocytes by CriPs enhanced adipocyte browning with a marked increase of uncoupling protein 1 (UCP1) expression. Of note, the CriP-mediated Nrip1 deletion did not produce detectable off-target effects. We conclude that CriPs offer an effective Cas9 and sgRNA delivery system for ablating targeted gene products in cultured cells and in vivo, providing a potential therapeutic strategy for metabolic disease.

scholarly journals CRISPR delivery particles for developing therapeutic strategies in metabolic disease

2018 ◽  
Author(s):  
Yuefei Shen ◽  
Jessica L. Cohen ◽  
Sarah M. Nicoloro ◽  
Mark Kelly ◽  
Batuhan Yenilmez ◽  
...  
Keyword(s):  
Metabolic Disease ◽  
Therapeutic Strategy ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Adaptive Immune System ◽  
Amphipathic Peptide ◽  
Cas9 Protein

ABSTRACTRNA-guided engineered nucleases derived from a prokaryotic adaptive immune system known as CRISPR-Cas represent a promising platform for gene deletion and editing. As a therapeutic approach, direct delivery of Cas9 protein and guide RNA could circumvent the safety problems associated with plasmid delivery and therefore represents an attractive tool for genome engineering. Gene deletion or editing in adipose tissue to enhance its energy expenditure, fat oxidation and secretion of bioactive factors through a “browning” process presents a potential therapeutic strategy to alleviate metabolic disease. Here, we developed novel CRISPR delivery particles, denoted CriPs, composed of nano-size complexes of Cas9 protein and single guide (sg)RNA, coated with an amphipathic peptide called Endo-Porter that mediates entry into cells. Efficient CRISPR-Cas9 mediated gene deletion of ectopically expressed Green fluorescent protein (GFP) by CriPs was achieved in multiple cell types including a macrophage cell line, primary macrophages and primary pre-adipocytes. Significant GFP loss was also observed in peritoneal exudate cells with minimum systemic toxicity in GFP expressing mice following intraperitoneal injection of CriPs containing sgRNA targeting Gfp. Furthermore, the disruption of the Nrip1 gene in white adipocytes by CriPs enhanced adipocyte “browning” with a marked increase of UCP1 expression. Deletion of Nrip1 by CriPs did not produce detectable off-target effects. Thus CriPs represent a novel CRISPR delivery system for Cas9 and sgRNA that is effective for ablating targeted gene products in cultured cells and in vivo, and provide a potential therapeutic strategy for metabolic disease.

scholarly journals Adipocyte-specific deletion of the oxygen-sensor PHD2 sustains elevated energy expenditure at thermoneutrality

2021 ◽  
Author(s):  
Mario Gomez Salazar ◽  
Iris Pruñonosa Cervera ◽  
Rongling Wang ◽  
Karen French ◽  
Ruben García-Martín ◽  
...  
Keyword(s):  
Metabolic Disease ◽  
Energy Expenditure ◽  
Therapeutic Strategy ◽  
Oxygen Sensor ◽  
Modern Human ◽  
Protein Levels ◽  
Serum Proteomics ◽  
Potential Biomarker ◽  
Brown Adipose ◽  
Increased Risk

AbstractEnhancing brown adipose tissue (BAT) function to combat metabolic disease is a promising therapeutic strategy. A major obstacle to this strategy is that a thermoneutral environment, relevant to most modern human living conditions, deactivates functional BAT. We showed that we can overcome the dormancy of BAT at thermoneutrality by inhibiting the main oxygen sensor HIF-prolyl hydroxylase, PHD2, specifically in adipocytes. Mice lacking adipocyte PHD2 (P2KOad) and housed at thermoneutrality maintained greater BAT mass, had detectable UCP1 protein expression in BAT and higher energy expenditure. Mouse brown adipocytes treated with the pan-PHD inhibitor, FG2216, exhibited higher Ucp1 mRNA and protein levels, effects that were abolished by antagonising the canonical PHD2 substrate, HIF-2a. Induction of UCP1 mRNA expression by FG2216, was also confirmed in human adipocytes isolated from obese individuals. Human serum proteomics analysis of 5457 participants in the deeply phenotyped Age, Gene and Environment Study revealed that serum PHD2 (aka EGLN1) associates with increased risk of metabolic disease. Our data suggest adipose–selective PHD2 inhibition as a novel therapeutic strategy for metabolic disease and identify serum PHD2 as a potential biomarker.

scholarly journals In vitro and in vivo induction of brown adipocyte uncoupling protein (thermogenin) by retinoic acid

1996 ◽  
Vol 317 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Pere PUIGSERVER ◽  
Francisca VÁZQUEZ ◽  
María L. BONET ◽  
Catalina PICÓ ◽  
Andreu PALOU
Keyword(s):  
Retinoic Acid ◽  
Cultured Cells ◽  
Uncoupling Protein ◽  
Primary Cultures ◽  
Brown Adipocyte ◽  
Brown Adipose ◽  
Adipocyte Cell ◽  
Differentiation Stage

The effects of retinoic acid (RA) isomers (all-trans-RA and 9-cis-RA) on the appearance of uncoupling protein (UCP; thermogenin), the only unequivocal molecular marker of the brown adipocyte differentiated phenotype, have been investigated in primary cultures of brown adipocytes, in the brown adipocyte cell line HIB 1B and directly in intact mice. The results obtained with cultured cells indicate that retinoids function as inducers of the appearance of UCP and, at the same time, partially inhibit brown adipocyte cell proliferation. The two RA isomers displayed similar effectiveness as UCP inducers, their effect being comparable with that triggered by noradrenaline, so far considered to be the main modulator of UCP gene expression. The effectiveness of retinoids as UCP inducers was dependent on the stage of brown adipocyte differentiation, being maximal in confluent primary cells and in the medium–late differentiation stage of HIB 1B cells. Corroborating the results obtained in vitro, we show that administration of all-trans-RA or 9-cis-RA to mice leads to an increase in their brown adipose tissue specific UCP content. 9-cis-RA treatment also prevented the loss of UCP on cold deacclimation. To our knowledge, this is the first report of a stimulatory effect of retinoid compounds on UCP induction in vivo.

scholarly journals Nipsnap1 – A Novel Thermogenic Regulator

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 1228-1228
Author(s):  
Yang Liu ◽  
Yue Qu ◽  
Chloe Cheng ◽  
Pei-Yin Tsai ◽  
Joeva Barrow
Keyword(s):  
Energy Expenditure ◽  
Cold Exposure ◽  
Proteomic Analysis ◽  
Uncoupling Protein ◽  
Treatment Options ◽  
Oxidative Capacity ◽  
Loss Of Function ◽  
Brown Adipocytes ◽  
Brown Adipose

Abstract Objectives We aim to discover molecular treatment options to combat obesity by studying a process known as non-shivering thermogenesis (NST). The major objective is to determine if Nipsnap1 can regulate NST in brown adipose tissue (BAT) and evaluate its potential for obesity treatment. Methods 1. Identification of Nipsnap1 and loss-of-function study•Unbiased proteomic analysis is performed on BAT isolated mitochondria samples from 6-week-old mice exposed to cold to activate NST (n = 3). •siRNA-mediated knockdown of Nipsnap1 is performed on primary brown adipocytes isolated from mice. Seahorse Bioanalyzer is used for Mitochondria Respiration Test. •Thermogenic tissue-specific Nipsnap1 knockout mice (N1-KO) are generated by crossing floxed Nipsnap1 (N1-Flox) transgenic mice with UCP1-Cre mice. 2. Role of Nipsnap1 in thermogenesis•Phenotypical studies in vivo are performed on 6-week-old N1-KO and N1-Flox mice (n = 8). Mice are acclimated in 30°C followed by cold exposure in 4°C for ten days. Rectal temperature is measured. •N1-KO mice metabolism level and locomotive movement are monitored by the Sable Promethion Metabolic Cage system (n = 8). Results Nipsnap1 displays potent thermogenic properties. By unbiased proteomic analysis, we identify Nipsnap1, which is highly induced when NST is activated and strongly correlates with the NST key protein Uncoupling Protein 1 (UCP1). Nipsnap1 ablation in primary brown adipocytes reveals significant reductions in thermogenic adipose function. siNipsnap1 causes complete ablation of UCP1 protein levels. Moreover, it causes a 50% reduction (P < 0.001) of mitochondrial oxidative capacity and a 40% reduction (P < 0.05) of glycolytic capacity in cells. Nipsnap1 KO mice have impaired NST and reduced energy expenditure in vivo. Under prolonged cold exposure (day 7–10), N1-KO mice exhibit significant defects (day 10, P < 0.05) to maintain body temperature by NST. We demonstrated that the N1-KO mice had a 10% (P < 0.01) reduction in energy expenditure during the active night period after prolonged cold (day 5–8) treatment compared to controls. Conclusions Nipsnap1 plays an essential role in regulating NST. Targeting Nipsnap1 to increase energy expenditure at the molecular level will provide new insights into developing a safe and effective method to combat obesity and metabolic disease. Funding Sources Agency: NIH; Institute: NIDDK.

Human skeletal muscle PPARα expression correlates with fat metabolism gene expression but not BMI or insulin sensitivity

2004 ◽  
Vol 286 (2) ◽  
pp. E168-E175 ◽  
Author(s):  
Junlong Zhang ◽  
D. I. W. Phillips ◽  
Chunli Wang ◽  
Christopher D. Byrne
Keyword(s):  
Skeletal Muscle ◽  
Lipid Metabolism ◽  
Insulin Sensitivity ◽  
Uncoupling Protein ◽  
Acid Oxidation ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Few Data

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator of fatty acid oxidation in skeletal muscle, but few data exist from humans in vivo. To investigate whether insulin sensitivity in skeletal muscle and body mass index (BMI) were associated with skeletal muscle expression of PPARα and with important genes regulating lipid metabolism in humans in vivo, we undertook hyperinsulinemic-euglycemic clamps and measured PPARα mRNA levels and mRNA levels of lipid regulating PPARα response genes in skeletal muscle biopsies. mRNA levels were measured in 16 men, using a novel highly sensitive and specific medium throughput quantitative competitive PCR that allows reproducible measurement of multiple candidate mRNAs simultaneously. mRNA levels of PPARα were positively correlated with mRNA levels of CD36 ( r = 0.77, P = 0.001), lipoprotein lipase ( r = 0.54, P = 0.024), muscle-type carnitine palmitoyltransferase-I ( r = 0.54, P = 0.024), uncoupling protein-2 ( r = 0.63, P = 0.008), and uncoupling protein-3 ( r = 0.53, P = 0.026), but not with measures of insulin sensitivity, BMI, or GLUT4, which plays an important role in insulin-mediated glucose uptake. Thus our data suggest that in humans skeletal muscle PPARα expression and genes regulating lipid metabolism are tightly linked, but there was no association between both insulin sensitivity and BMI with PPARα expression in skeletal muscle.

UCP3 and its putative function: consistencies and controversies

2001 ◽  
Vol 29 (6) ◽  
pp. 768-773 ◽  
Author(s):  
M.-E. Harper ◽  
R. M. Dent ◽  
V. Bezaire ◽  
A. Antoniou ◽  
A. Gauthier ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Uncoupling Protein ◽  
Expression Patterns ◽  
Physiological Role ◽  
In Vivo Studies ◽  
Proton Leak ◽  
Acid Oxidation ◽  
Mitochondrial Uncoupling

The physiological function of uncoupling protein 3 (UCP3) is as yet unknown. Based on its 57% homology to UCP1 whose physiologic function is uncoupling and thermogenesis, UCP3 was attributed with the function of mitochondrial uncoupling through proton-leak reactions. UCP3 is expressed selectively in muscle, a tissue in which it has been estimated that proton leak accounts for approx. 50% of resting energy metabolism. Genetic linkage, association and variant studies suggest a role for UCP3 in obesity and/or diabetes. Studies of the heterologous expression of UCP3 in yeast provide support for the idea that UCP3 can uncouple mitochondrial oxidative phosphorylation, but the physiological relevance of these results is questionable. In vitro studies of mitochondria from Ucp3− − mice provide support, but there are no changes in resting metabolic rate (RMR) of mice. In vivo studies demonstrate increased ATP synthesis, but estimates of substrate oxidation rate indicate no change. Mice that greatly overexpress Ucp3 in muscle have increased RMR. Inconsistent with the function of uncoupling are the observations that fasting results in increased expression of UCP3, but no change in muscle proton leak. Moreover, fasting decreases energy expenditure in muscle. Expression patterns for Ucp3 and lipid-metabolism genes support a physiological role in fatty acid oxidation. Overall, findings support a role for Ucp3 in fatty acid metabolism that may have implications for obesity and/or Type II diabetes.

scholarly journals Involvement of the vitamin D receptor in energy metabolism: regulation of uncoupling proteins

2009 ◽  
Vol 296 (4) ◽  
pp. E820-E828 ◽  
Author(s):  
Kari E. Wong ◽  
Frances L. Szeto ◽  
Wenshuo Zhang ◽  
Honggang Ye ◽  
Juan Kong ◽  
...  
Keyword(s):  
Vitamin D ◽  
Energy Metabolism ◽  
Energy Expenditure ◽  
Vitamin D Receptor ◽  
Fat Mass ◽  
Uncoupling Protein ◽  
Mutant Mice ◽  
Metabolism Regulation ◽  
Null Mutant Mice

Recent studies have established that vitamin D plays multiple biological roles beyond calcium metabolism; however, whether vitamin D is involved in energy metabolism is unknown. To address this question, we characterized the metabolic phenotypes of vitamin D receptor (VDR)-null mutant mice. Under a normocalcemic condition, VDR-null mice displayed less body fat mass and lower plasma triglyceride and cholesterol levels compared with wild-type (WT) mice; when placed on a high-fat diet, VDR-null mice showed a slower growth rate and accumulated less fat mass globally than WT mice, even though their food intake and intestinal lipid transport capacity were the same as WT mice. Consistent with the lower adipose mass, plasma leptin levels were lower and white adipocytes were histologically smaller in VDR-null mice than WT mice. The rate of fatty acid β-oxidation in the white adipose tissue was higher, and the expression of uncoupling protein (UCP) 1, UCP2 and UCP3 was markedly upregulated in VDR-null mice, suggesting a higher energy expenditure in the mutant mice. Experiments using primary brown fat culture confirmed that 1,25-dihydroxyvitamin D3 directly suppressed the expression of the UCPs. Consistently, the energy expenditure, oxygen consumption, and CO2 production in VDR-null mice were markedly higher than in WT mice. These data indicate that vitamin D is involved in energy metabolism and adipocyte biology in vivo in part through regulation of β-oxidation and UCP expression.

scholarly journals Exendin-4 increases oxygen consumption and thermogenic gene expression in muscle cells

2017 ◽  
Vol 58 (2) ◽  
pp. 79-90 ◽  
Author(s):  
Jin-Seung Choung ◽  
Young-Sun Lee ◽  
Hee-Sook Jun
Keyword(s):  
Gene Expression ◽  
Oxygen Consumption ◽  
Energy Metabolism ◽  
Energy Expenditure ◽  
Uncoupling Protein ◽  
Farnesoid X Receptor ◽  
C2c12 Cells ◽  
Peroxisome Proliferator ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP1) has many anti-diabetic actions and also increases energy expenditure in vivo. As skeletal muscle is a major organ controlling energy metabolism, we investigated whether GLP1 can affect energy metabolism in muscle. We found that treatment of differentiated C2C12 cells with exendin-4 (Ex-4), a GLP1 receptor agonist, reduced oleate:palmitate-induced lipid accumulation and triglyceride content compared with cells without Ex-4 treatment. When we examined the oxygen consumption rate (OCR), not only the basal OCR but also the OCR induced by oleate:palmitate addition was significantly increased in Ex-4-treated differentiated C2C12 cells, and this was inhibited by exendin-9, a GLP1 receptor antagonist. The expression of uncoupling protein 1 (UCP1), β3-adrenergic receptor, peroxisome proliferator-activator receptor a (PPARa) and farnesoid X receptor mRNA was significantly upregulated in Ex-4-treated differentiated C2C12 cells, and the upregulation of these mRNA was abolished by treatment with adenylate cyclase inhibitor (2′5′-dideoxyadenosine) or PKA inhibitor (H-89). As well, intramuscular injection of Ex-4 into diet-induced obese mice significantly increased the expression of UCP1, PPARa and p-AMPK in muscle. We suggest that exposure to GLP1 increases energy expenditure in muscle through the upregulation of fat oxidation and thermogenic gene expression, which may contribute to reducing obesity and insulin resistance.

Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance

2008 ◽  
Vol 295 (2) ◽  
pp. E356-E367 ◽  
Author(s):  
Vladimir Kus ◽  
Tomas Prazak ◽  
Petr Brauner ◽  
Michal Hensler ◽  
Ondrej Kuda ◽  
...  
Keyword(s):  
Energy Expenditure ◽  
Lipid Oxidation ◽  
Soleus Muscle ◽  
Uncoupling Protein ◽  
Total Content ◽  
Whole Body ◽  
Cold Sensitivity ◽  
Acid Oxidation ◽  
Nonshivering Thermogenesis ◽  
High Fat

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30°C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4°C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30°C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

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