OBJECTIVES: Nuclear receptor interacting protein 1 (NRIP1) suppresses energy expenditure via repression of nuclear receptors, and its depletion markedly elevates uncoupled respiration in mouse and human adipocytes. We tested whether NRIP1 deficient adipocytes implanted into obese mice would enhance whole body metabolism. Since β-adrenergic signaling through cAMP strongly promotes adipocyte thermogenesis, we tested whether the effects of NRIP1 knock-out (NRIP1KO) require the cAMP pathway. METHODS: NRIP1KO adipocytes were implanted in recipient high-fat diet (HFD) fed mice and metabolic cage studies conducted. The Nrip1 gene was disrupted by CRISPR in primary preadipocytes isolated from control vs adipose selective GsαKO (cAdGsαKO) mice prior to differentiation to adipocytes. Protein kinase A inhibitor was also used. RESULTS: Implanting NRIP1KO adipocytes into HFD fed mice enhanced whole-body glucose tolerance by increasing insulin sensitivity, reducing adiposity, and enhancing energy expenditure in the recipients. NRIP1 depletion in both control and GsαKO adipocytes was equally effective in upregulating uncoupling protein 1 (UCP1) and adipocyte beiging, while β-adrenergic signaling by CL 316,243 was abolished in GsαKO adipocytes. Combining NRIP1KO with CL 316,243 treatment synergistically increased Ucp1 gene expression and increased the adipocyte subpopulation responsive to beiging. Estrogen-related receptor α (ERRα) was dispensable for UCP1 upregulation by NRIPKO. CONCLUSIONS: The thermogenic effect of NRIP1 depletion in adipocytes causes systemic enhancement of energy expenditure when such adipocytes are implanted into obese mice. Furthermore, NRIP1KO acts independently but cooperatively with the cAMP pathway in mediating its effect on adipocyte beiging.

• MeSH
• lidé MeSH
• myši obézní MeSH
• myši MeSH
• nuclear receptor interacting protein 1 metabolismus   MeSH
• obezita metabolismus   MeSH
• signální transdukce * MeSH
• termogeneze genetika   MeSH
• tukové buňky * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Obesity 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.

• MeSH
• bílé tukové buňky metabolismus   MeSH
• buněčná diferenciace MeSH
• buněčné kultury metody   MeSH
• CRISPR-Cas systémy genetika   MeSH
• dieta s vysokým obsahem tuků škodlivé účinky   MeSH
• dospělé kmenové buňky fyziologie   MeSH
• editace genu metody   MeSH
• guide RNA, Kinetoplastida genetika   MeSH
• hnědé tukové buňky metabolismus   transplantace   MeSH
• lidé MeSH
• metabolismus lipidů genetika   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nuclear receptor interacting protein 1 genetika   metabolismus   MeSH
• obezita komplikace   metabolismus   terapie   MeSH
• podkožní tuk cytologie   MeSH
• porucha glukózové tolerance etiologie   metabolismus   terapie   MeSH
• termogeneze genetika   MeSH
• ztučnělá játra etiologie   metabolismus   prevence a kontrola   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

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.

• MeSH
• bílá tuková tkáň cytologie   metabolismus   MeSH
• buněčné linie MeSH
• CRISPR-Cas systémy MeSH
• editace genu MeSH
• energetický metabolismus * MeSH
• genový targeting metody   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• nuclear receptor interacting protein 1 genetika   metabolismus   MeSH
• plazmidy genetika   metabolismus   MeSH
• reportérové geny MeSH
• sekvence CRISPR MeSH
• tukové buňky metabolismus   MeSH
• uncoupling protein 1 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH