CRISPR-enhanced human adipocyte browning as cell therapy for metabolic disease

AbstractObesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as “brown” and “brite/beige” adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.

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CRISPR-enhanced human adipocyte “browning” as cell therapy for metabolic disease

10.1101/2020.10.13.337923 ◽

2020 ◽

Author(s):

Emmanouela Tsagkaraki ◽

Sarah Nicoloro ◽

Tiffany De Souza ◽

Javier Solivan-Rivera ◽

Anand Desai ◽

...

Keyword(s):

Adipose Tissue ◽

Ex Vivo ◽

Uncoupling Protein ◽

Suppressor Gene ◽

Whole Body ◽

Long Term Stability ◽

Beige Adipocytes ◽

Liver Triglycerides ◽

Whole Body Metabolism ◽

Mouse Adipocytes

AbstractObesity and type 2 diabetes (T2D) are associated with poor tissue responses to insulin1,2, disturbances in glucose and lipid fluxes3–5 and comorbidities including steatohepatitis6 and cardiovascular disease7,8. Despite extensive efforts at prevention and treatment9,10, diabetes afflicts over 400 million people worldwide11. Whole body metabolism is regulated by adipose tissue depots12–14, which include both lipid-storing white adipocytes and less abundant “brown” and “brite/beige” adipocytes that express thermogenic uncoupling protein UCP1 and secrete factors favorable to metabolic health15–18. Application of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing19,20 to enhance “browning” of white adipose tissue is an attractive therapeutic approach to T2D. However, the problems of cell-selective delivery, immunogenicity of CRISPR reagents and long term stability of the modified adipocytes are formidable. To overcome these issues, we developed methods that deliver complexes of SpyCas9 protein and sgRNA ex vivo to disrupt the thermogenesis suppressor gene NRIP121,22 with near 100% efficiency in human or mouse adipocytes. NRIP1 gene disruption at discrete loci strongly ablated NRIP1 protein and upregulated expression of UCP1 and beneficial secreted factors, while residual Cas9 protein and sgRNA were rapidly degraded. Implantation of the CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreased adiposity and liver triglycerides while enhancing glucose tolerance compared to mice implanted with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic modification of human adipocytes without exposure of the recipient to immunogenic Cas9 or delivery vectors.

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Critical review of beige adipocyte thermogenic activation and contribution to whole-body energy expenditure

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2017-0042 ◽

2017 ◽

Vol 31 (2) ◽

Cited By ~ 8

Author(s):

Érique Castro ◽

Tiago E. Oliveira Silva ◽

William T. Festuccia

Keyword(s):

Energy Expenditure ◽

Metabolic Diseases ◽

Uncoupling Protein ◽

Whole Body ◽

Beige Adipocyte ◽

White Adipocytes ◽

Beige Adipocytes ◽

Whole Body Metabolism ◽

Beige Cells ◽

Body Energy

AbstractBeige (or brite, “brown in white”) adipocytes are uncoupling protein 1 (UCP1)-positive cells residing in white adipose depots that, depending on the conditions, behave either as classic white adipocytes, storing energy as lipids, or as brown adipocytes, dissipating energy from oxidative metabolism as heat through non-shivering thermogenesis. Because of their thermogenic potential and, therefore, possible usage to treat metabolic diseases such as obesity and type 2 diabetes, beige cells have attracted the attention of many scientists worldwide aiming to develop strategies to safely recruit and activate their thermogenic activity. Indeed, in recent years, a large variety of conditions, molecules (including nutrients) and signaling pathways were reported to promote the recruitment of beige adipocytes. Despite of those advances, the true contribution of beige adipocyte thermogenesis to whole-body energy expenditure is still not completely defined. Herein, we discuss some important aspects that should be considered when studying beige adipocyte biology and the contribution to energy balance and whole-body metabolism.

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Effects of the presence, absence, and overexpression of uncoupling protein-3 on adiposity and fuel metabolism in congenic mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00401.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. E1304-E1312 ◽

Cited By ~ 45

Author(s):

Sheila R. Costford ◽

Shehla N. Chaudhry ◽

Mahmoud Salkhordeh ◽

Mary-Ellen Harper

Keyword(s):

Adipose Tissue ◽

Glucose Tolerance ◽

Uncoupling Protein ◽

Liver Glycogen ◽

Whole Body ◽

Wild Type ◽

Insulin Tolerance ◽

Uncoupling Protein 3 ◽

Brown Adipose ◽

Inner Membrane Protein

Uncoupling protein-3 (UCP3) is a poorly understood mitochondrial inner membrane protein expressed predominantly in skeletal muscle. The aim of this study was to examine the effects of the absence or constitutive physiological overexpression of UCP3 on whole body energy metabolism, glucose tolerance, and muscle triglyceride content. Congenic male UCP3 knockout mice ( Ucp3 −/−), wild-type, and transgenic UCP3 overexpressing (UCP3Tg) mice were fed a 10% fat diet for 4 or 8 mo after they were weaned. UCP3Tg mice had lower body weights and were less metabolically efficient than wild-type or Ucp3 −/− mice, but they were not hyperphagic. UCP3Tg mice had smaller epididymal white adipose tissue and brown adipose tissue (BAT) depots; however, there were no differences in muscle weights. Glucose and insulin tolerance tests revealed that both UCP3Tg and Ucp3 −/− mice were protected from development of impaired glucose tolerance and were more sensitive to insulin. 2-Deoxy-d-[1-3H]glucose tracer studies showed increased uptake of glucose into BAT and increased storage of liver glycogen in Ucp3 −/− mice. Assessments of intramuscular triglyceride (IMTG) revealed decreases in quadriceps of UCP3Tg mice compared with wild-type and Ucp3 −/− mice. When challenged with a 45% fat diet, Ucp3 −/− mice showed increased accumulation of IMTG compared with wild-type mice, which in turn had greater IMTG than UCP3Tg mice. Results are consistent with a role for UCP3 in preventing accumulation of triglyceride in both adipose tissue and muscle.

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Identification of zinc finger protein Bcl6 as a novel regulator of early adipose commitment

Open Biology ◽

10.1098/rsob.160065 ◽

2016 ◽

Vol 6 (6) ◽

pp. 160065 ◽

Cited By ~ 12

Author(s):

Xiaoming Hu ◽

Yuanfei Zhou ◽

Yang Yang ◽

Jie Peng ◽

Tongxing Song ◽

...

Keyword(s):

Zinc Finger ◽

Zinc Finger Protein ◽

Ex Vivo ◽

Early Stage ◽

Adipogenic Differentiation ◽

B Cell Lymphoma ◽

Whole Body ◽

Loss Of Function ◽

Finger Protein ◽

Whole Body Metabolism

Adipose tissue is a key determinant of whole-body metabolism and energy homeostasis. Unravelling the transcriptional regulatory process during adipogenesis is therefore highly relevant from a biomedical perspective. In these studies, zinc finger protein B-cell lymphoma 6 (Bcl6) was demonstrated to have a role in early adipogenesis of mesenchymal stem cells. Bcl6 is enriched in preadipose versus non-preadipose fibroblasts and shows upregulated expression in the early stage of adipogenesis. Gain- and loss-of-function studies revealed that Bcl6 acts as a key regulator of adipose commitment and differentiation both in vitro and ex vivo . RNAi-mediated knockdown of Bcl6 in C3H10T1/2 cells greatly inhibited adipogenic potential, whereas Bcl6 overexpression enhanced adipogenic differentiation. This transcription factor also directly or indirectly targets and controls the expression of some early and late adipogenic regulators (i.e. Zfp423, Zfp467, KLF15, C/EBPδ, C/EBPα and PPARγ). We further identified that Bcl6 transactivated the signal transducers and activators of transcription 1 ( STAT1 ), which was determined as a required factor for adipogenesis. Moreover, overexpression of STAT1 rescued the impairment of adipogenic commitment and differentiation induced by Bcl6 knockdown in C3H10T1/2 cells, thereby confirming that STAT1 is a downstream direct target of Bcl6. This study identifies Bcl6 as a positive transcriptional regulator of early adipose commitment.

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Dietary Cocoa Flavanols Enhance Mitochondrial Function in Skeletal Muscle and Modify Whole-Body Metabolism in Healthy Mice

Nutrients ◽

10.3390/nu13103466 ◽

2021 ◽

Vol 13 (10) ◽

pp. 3466

Author(s):

Frédéric Nicolas Daussin ◽

Alexane Cuillerier ◽

Julianne Touron ◽

Samir Ben Said ◽

Bruno Melo ◽

...

Keyword(s):

Glucose Tolerance ◽

Mitochondrial Function ◽

Tolerance Test ◽

Whole Body ◽

Oral Glucose ◽

H2o2 Production ◽

Wild Type ◽

Nad Metabolism ◽

Whole Body Metabolism ◽

Cocoa Flavanols

Mitochondrial dysfunction is widely reported in various diseases and contributes to their pathogenesis. We assessed the effect of cocoa flavanols supplementation on mitochondrial function and whole metabolism, and we explored whether the mitochondrial deacetylase sirtuin-3 (Sirt3) is involved or not. We explored the effects of 15 days of CF supplementation in wild type and Sirt3-/- mice. Whole-body metabolism was assessed by indirect calorimetry, and an oral glucose tolerance test was performed to assess glucose metabolism. Mitochondrial respiratory function was assessed in permeabilised fibres and the pyridine nucleotides content (NAD+ and NADH) were quantified. In the wild type, CF supplementation significantly modified whole-body metabolism by promoting carbohydrate use and improved glucose tolerance. CF supplementation induced a significant increase of mitochondrial mass, while significant qualitative adaptation occurred to maintain H2O2 production and cellular oxidative stress. CF supplementation induced a significant increase in NAD+ and NADH content. All the effects mentioned above were blunted in Sirt3-/- mice. Collectively, CF supplementation boosted the NAD metabolism that stimulates sirtuins metabolism and improved mitochondrial function, which likely contributed to the observed whole-body metabolism adaptation, with a greater ability to use carbohydrates, at least partially through Sirt3.

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SUN-670 P300 and CBP Are Necessary for Skeletal Muscle Insulin-Stimulated Glucose Uptake

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1496 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Vitor Fernandes Martins ◽

Samuel LaBarge ◽

Kristoffer Svensson ◽

Jennifer M Cunliffe ◽

Dion Banoian ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Insulin Sensitivity ◽

Glucose Tolerance ◽

Glucose Uptake ◽

Ex Vivo ◽

Binding Protein ◽

Whole Body ◽

Oral Glucose ◽

Skeletal Muscle Insulin Sensitivity

Abstract Introduction: Akt is a critical mediator of insulin-stimulated glucose uptake in skeletal muscle. The acetyltransferases, E1A binding protein p300 (p300) and cAMP response element-binding protein binding protein (CBP) are phosphorylated and activated by Akt, and p300/CBP can acetylate and inactivate Akt, thus giving rise to a possible Akt-p300/CBP axis. Our objective was to determine the importance of p300 and CBP to skeletal muscle insulin sensitivity. Methods: We used Cre-LoxP methodology to generate mice with a tamoxifen-inducible, conditional knock out of Ep300 and/or Crebbp in skeletal muscle. At 13-15 weeks of age, the knockout was induced via oral gavage of tamoxifen and oral glucose tolerance, ex vivo skeletal muscle insulin sensitivity, and microarray and proteomics analysis were done. Results: Loss of both p300 and CBP in adult mouse skeletal muscle rapidly and severely impairs whole body glucose tolerance and skeletal muscle insulin sensitivity. Furthermore, giving back a single allele of either p300 or CBP rescues both phenotypes. Moreover, the severe insulin resistance in the p300/CBP double knockout mice is accompanied by significant changes in both mRNA and protein expression of transcript/protein networks critical for insulin signaling, GLUT4 trafficking, and metabolism. Lastly, in human skeletal muscle samples, p300 and CBP protein levels correlate significantly and negatively with markers of insulin resistance. Conclusions: p300 and CBP are jointly required for maintaining whole body glucose tolerance and insulin sensitivity in skeletal muscle.

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Screening of flavor compounds using Ucp1-luciferase reporter beige adipocytes identified 5-methylquinoxaline as a novel UCP1-inducing compoundsss

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab216 ◽

2021 ◽

Author(s):

Satoko Kawarasaki ◽

Kazuki Matsuo ◽

Hidetoshi Kuwata ◽

Lanxi Zhou ◽

Jungin Kwon ◽

...

Keyword(s):

High Throughput Screening ◽

Uncoupling Protein ◽

Mitochondrial Protein ◽

Whole Body ◽

Flavor Compounds ◽

Luciferase Reporter ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Beige Adipocytes ◽

Ucp1 Expression

Abstract Uncoupling protein 1 (UCP1) in brown or beige adipocytes is a mitochondrial protein that is expected to enhance whole-body energy expenditure. For the high-throughput screening of UCP1 transcriptional activity regulator, we established a murine inguinal white adipose tissue-derived Ucp1-luciferase reporter preadipocyte line. Using this reporter preadipocyte line, 654 flavor compounds were screened, and a novel Ucp1 expression-inducing compound, 5-methylquinoxaline, was identified. Adipocytes treated with 5-methylquinoxaline showed increased Ucp1 mRNA expression levels and enhanced oxygen consumption. 5-methylquinoxaline induced Ucp1 expression through peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), and 5-methylquinoxaline-induced PGC1α activation seemed to be partially regulated by its phosphorylation or deacetylation. Thus, our Ucp1-luciferase reporter preadipocyte line is a useful tool for screening of Ucp1 inductive compounds.

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Macrophage infiltration into obese adipose tissues suppresses the induction of UCP1 level in mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00028.2015 ◽

2016 ◽

Vol 310 (8) ◽

pp. E676-E687 ◽

Cited By ~ 50

Author(s):

Tomoya Sakamoto ◽

Takahiro Nitta ◽

Koji Maruno ◽

Yu-Sheng Yeh ◽

Hidetoshi Kuwata ◽

...

Keyword(s):

Energy Expenditure ◽

Cold Exposure ◽

Uncoupling Protein ◽

Mitochondrial Protein ◽

Whole Body ◽

Marker Genes ◽

Adipose Tissues ◽

Expression Levels ◽

Beige Adipocytes ◽

Thermogenic Adipocytes

Emergence of thermogenic adipocytes such as brown and beige adipocytes is critical for whole body energy metabolism. Promoting the emergence of these adipocytes, which increase energy expenditure, could be a viable strategy in treating obesity and its related diseases. However, little is known regarding the mechanisms that regulate the emergence of these adipocytes in obese adipose tissue. Here, we demonstrated that classically activated macrophages (M1 Mϕ) suppress the induction of thermogenic adipocytes in obese adipose tissues of mice. Cold exposure significantly induced the expression levels of uncoupling protein-1 (UCP1), which is a mitochondrial protein unique in thermogenic adipocytes, in C57BL/6 mice fed a normal diet. However, UCP1 induction was significantly suppressed in adipose tissues of C57BL/6 mice fed a high-fat diet, into which M1 Mϕ infiltrated. Depletion of M1 Mϕ using clodronate liposomes eliminated the suppressive effect and markedly reduced the mRNA level of tumor necrosis factor-α (TNFα) in the adipose tissues. Importantly, consistent with the observed changes in the expression levels of marker genes for thermogenic adipocytes, combination treatment of clodronate liposome and cold exposure resulted in metabolic benefits such as lowered body weight and blood glucose level in obese mice. Moreover, intraperitoneal injection of recombinant TNFα protein suppressed UCP1 induction in lean adipose tissues of mice. Collectively, our data indicate that infiltrated M1 Mϕ suppress the induction of thermogenic adipocytes in obese adipose tissues via TNFα. This report suggests that inflammation induced by infiltrated Mϕ could cause not only insulin resistance but also reduction of energy expenditure in adipose tissues.

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Skeletal muscle mitochondrial uncoupling drives endocrine cross-talk through the induction of FGF21 as a myokine

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00330.2013 ◽

2014 ◽

Vol 306 (5) ◽

pp. E469-E482 ◽

Cited By ~ 106

Author(s):

Susanne Keipert ◽

Mario Ost ◽

Kornelia Johann ◽

Francine Imber ◽

Martin Jastroch ◽

...

Keyword(s):

Skeletal Muscle ◽

Mitochondrial Function ◽

Uncoupling Protein ◽

Ectopic Expression ◽

Metabolic Phenotype ◽

Whole Body ◽

Brown Adipocyte ◽

Fibroblast Growth Factor 21 ◽

Mitochondrial Uncoupling ◽

Whole Body Metabolism

UCP1-Tg mice with ectopic expression of uncoupling protein 1 (UCP1) in skeletal muscle (SM) are a model of improved substrate metabolism and increased longevity. Analysis of myokine expression showed an induction of fibroblast growth factor 21 (FGF21) in SM, resulting in approximately fivefold elevated circulating FGF21 in UCP1-Tg mice. Despite a reduced muscle mass, UCP1-Tg mice showed no evidence for a myopathy or muscle autophagy deficiency but an activation of integrated stress response (ISR; eIF2α/ATF4) in SM. Targeting mitochondrial function in vitro by treating C2C12 myoblasts with the uncoupler FCCP resulted in a dose-dependent activation of ISR, which was associated with increased expression of FGF21, which was also observed by treatment with respiratory chain inhibitors antimycin A and myxothiazol. The cofactor required for FGF21 action, β-klotho, was expressed in white adipose tissue (WAT) of UCP1-Tg mice, which showed an increased browning of WAT similar to what occurred in altered adipocyte morphology, increased brown adipocyte markers (UCP1, CIDEA), lipolysis (HSL phosphorylation), and respiratory capacity. Importantly, treatment of primary white adipocytes with serum of transgenic mice resulted in increased UCP1 expression. Additionally, UCP1-Tg mice showed reduced body length through the suppressed IGF-I-GH axis and decreased bone mass. We conclude that the induction of FGF21 as a myokine is coupled to disturbance of mitochondrial function and ISR activation in SM. FGF21 released from SM has endocrine effects leading to increased browning of WAT and can explain the healthy metabolic phenotype of UCP1-Tg mice. These results confirm muscle as an important endocrine regulator of whole body metabolism.

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Breaking BAT: can browning create a better white?

Journal of Endocrinology ◽

10.1530/joe-15-0408 ◽

2015 ◽

Vol 228 (1) ◽

pp. R19-R29 ◽

Cited By ~ 24

Author(s):

Amy Warner ◽

Jens Mittag

Keyword(s):

Energy Expenditure ◽

Metabolic Diseases ◽

Uncoupling Protein ◽

Whole Body ◽

Direct Stimulation ◽

White Adipocytes ◽

Beige Adipocytes ◽

Beige Fat ◽

Underlying Mechanisms ◽

Body Energy

Obesity and its comorbidities are a growing problem worldwide. In consequence, several new strategies have been proposed to promote weight loss and improve insulin sensitivity. Recently, it has been demonstrated that certain populations of white adipocytes can be ‘browned’, i.e., recruited to a more brown-like adipocyte, capable of thermogenesis through increased expression of uncoupling protein 1. The list of browning agents that induce these so-called beige adipocytes is growing constantly. However, the underlying mechanisms are often poorly understood, with the possibility that some of these agents cause browning as a secondary effect. Moreover, it remains unclear whether beige adipocytes can contribute sufficiently to affect whole-body energy expenditure in a functionally significant manner. This review presents an overview of the different molecular pathways leading to the induction of beige fat, including direct stimulation and indirect actions on the CNS or the immune system. We discuss the available evidence on the capacity of beige adipocytes to influence whole-body energy expenditure in rodents, and lastly outline the potential problems of translating browning capacity into the potential treatment of human metabolic diseases.

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