CRISPR/Cas9 has revolutionized the genome-editing field. So far, successful application in human adipose tissue has not been convincingly shown. We present a method for gene knockout using electroporation in preadipocytes from human adipose tissue that achieved at least 90% efficiency without any need for selection of edited cells or clonal isolation. We knocked out the FKBP5 and PPARG genes in preadipocytes and studied the resulting phenotypes. PPARG knockout prevented differentiation into adipocytes. Conversely, deletion of FKBP51, the protein coded by the FKBP5 gene, did not affect adipogenesis. Instead, it markedly modulated glucocorticoid effects on adipocyte glucose metabolism and, furthermore, we show some evidence of altered transcriptional activity of glucocorticoid receptors. This has potential implications for the development of insulin resistance and type 2 diabetes. The reported method is simple, easy to adapt, and enables the use of human primary preadipocytes instead of animal adipose cell models to assess the role of key genes and their products in adipose tissue development, metabolism and pathobiology. Adipose tissue is widely regarded as an endocrine organ that plays a central role in both obesity and insulin resistance 1. Dysregulation of adipose tissue transcriptional pathways may contribute to significant changes in energy balance, glucose and lipid metabolism, and adipokine secretion, which in turn can influence the whole-body metabolic homeostasis. Thus, identification of genes and functional assessment of their corresponding proteins involved in such pathways could help discover novel disease mechanisms that could be used for drug development. Different approaches such as pharmacological inhibition using receptor antagonists or neutralizing antibodies, as well as genetic manipulation using small interfering RNA-mediated knockdown 2 have been widely opted for studying the function of different gene products in human adipose cells. However, the specificity and stability of such approaches are critical factors and may sometimes limit their use. The recent advancements in clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology have given rise to a precise and highly efficient method for gene editing in cells, tissues and whole organisms 3. However, its application to human adipose tissue is scarce. To the best of our knowledge, only one study reported using this technique in primary human adipose cells, but the phenotypic results following a single nucleotide substitution in the fat mass and obesity-associated gene were equivocal 4. In another study, immortalized human brown preadipocytes were used to knockout genes using CRISPR/Cas9 5. Both of these studies have used an expression vector to deliver the CRISPR components into the cells. However, there are some potential complications associated with the use of plasmid DNA, as mentioned in the discussion.

Context Reduced estrogen levels in postmenopausal women predispose them to metabolic side effects, including insulin resistance, and type 2 diabetes, however, the cellular mechanisms are not well understood. Objective This work aimed to study the expression of estrogen receptors in adipose tissue from pre- and postmenopausal women and the effects of estradiol (E2) on glucose uptake of adipocytes. Methods Subcutaneous (SAT) and visceral adipose tissue (VAT) obtained from pre- and postmenopausal women (19-51 and 46-75 yo, respectively) were used to measure gene expression of ESR1 and ESR2. SAT tissue was incubated with E2 and glucose uptake and estrogen receptor levels were measured. Polymorphisms in ESR1 and ESR2 were addressed in public databases to identify SNPs associated with metabolic traits. Results ESR2 expression was lower in pre- vs postmenopausal women, corresponding to lower ESR1:ESR2 gene expression ratio in postmenopausal women. In premenopausal women the expression of ESR1 was higher in VAT than SAT. In both pre- and postmenopausal women, ESR2 expression was lower in VAT than SAT. In late, but not pre- or early postmenopausal women, E2 reduced glucose uptake, GLUT4 protein, and increased expression of ESR2. ESR1 polymorphisms were associated with weight, body-fat distribution, and total cholesterol, and ESR2 polymorphisms were associated with total cholesterol and triglyceride levels and with body-fat percentage. Conclusion E2 inhibits glucose utilization in human adipocytes in late postmenopausal women. Changes in glucose utilization may be explained by a lower ESR1:ESR2 ratio. This can have clinical implications on the timing of estrogen treatment in postmenopausal women.

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Oestrogen receptor 2 ( ESR2 ) expression has been shown to be higher in subcutaneous adipose tissue (SAT) from postmenopausal compared to premenopausal women. The functional significance of altered ESR2 expression is not fully known. This study investigates the role of ESR2 for adipose tissue lipid and glucose metabolism. SAT biopsies were obtained from 44 female subjects with or without T2D. Gene expression of ESR2 and markers of adipose function and metabolism was assessed. ESR2 knockdown was performed using CRISPR/Cas9 in preadipocytes isolated from SAT of females, and differentiation rate, lipid storage, and glucose uptake were measured. ESR2 expression was inversely correlated with measures of central obesity and expression of some fatty acid oxidation markers, and positively correlated with lipid storage and glucose transport markers. Differentiation was reduced in ESR2 knockdown preadipocytes. This corresponded to reduced expression of markers of differentiation and lipogenesis. Glucose uptake was reduced in knockdown adipocytes. Our results indicate that ESR2 deficiency in women is associated with visceral adiposity and impaired subcutaneous adipocyte differentiation as well as glucose and lipid utilization. High ESR2 expression, as seen after menopause, could be a contributing factor to SAT expansion. This may support a possible target to promote a healthy obesity phenotype.