The Role of MicroRNA-103/ 107 in Adipose Tissue and Natriuretic Peptide Receptor C- Mediated Targeting of White Adipocytes

Over the past decades acquired type-2 diabetes mellitus has developed to be the prevalent metabolic disorder. Type-2 diabetes mellitus is characterized by chronic hyperglycemia and reduced insulin sensitivity in the liver, muscle and adipose tissues. This increased insulin resistance leads to hepatic steatosis, dysfunctional and inflamed adipose tissue, dyslipidemia and eventually to pancreatic β- cell failure. While white adipose tissue is associated with energy storage in the form of triglycerides, brown adipose tissue utilizes fatty acids to generate heat via Uncoupling Protein 1 (UCP1). Active brown adipose tissue is associated with a lower degree of obesity and white adipose tissue can undergo browning, i.e. take on brown adipose tissue characteristics, under certain conditions; the strongest known stimulus is a drop in body temperature. MicroRNAs, short noncoding RNAs can, via binding to specific mRNA targets induce their degradation and hence alter the transcriptome and metabolism of the cell. Knockdown of microRNA- 103/ 107 (mir-103/ 107) with antagomirs, microRNA antisense nucleotide oligos coupled to cholesterol, has proven to increase insulin sensitivity by increasing the mir-103/ 107 target Cav-1 in the liver of obese mice. However, the microRNAs were also suspected to influence adipose tissue metabolism in a Cav-1 independent mechanism. This could be caused by an increase of adipocyte number due to enhanced adipocyte progenitor differentiation, by browning of white adipocytes or by improved brown adipose tissue function. Unraveling the exact mechanisms and identification of the target tissues of mir-103/ 107 knockdown will help to understand the microRNAs’ function and to utilize it in the therapy of diabetes mellitus and obesity. Here we show that highest mir-103/ 107 expression levels are indeed found in the visceral adipose tissue of diabetic obese mice and furthermore that also the whitened brown adipose tissue of these mice expresses mir-103/ 107 at higher levels. In vivo knockdown of mir-103/ 107, coupled with a cold stimulus or denervation of the brown adipose tissue, resulted in enhanced insulin sensitivity, but the phenotype could not be determined to be caused by an improvement of adipose tissue function or browning of white adipose tissue. Mir-103/ 107 is endogenously expressed during adipogenesis of primary adipocyte progenitors and gain-of-function as well as loss-of-function of mir-103/ 107 during adipogenesis resulted in blocking of differentiation ex vivo. Knockdown of mir-103/ 107 in ex vivo differentiated primary adipocytes resulted in upregulation of the mRNA levels of Ucp1 and other brown adipose tissue markers. However, targets identified in a screen of the transcriptome after mir-103/ 107 knockdown were found to be regulated on the mRNA level but not on the protein level. Furthermore functional studies of ex vivo differentiated primary adipocytes revealed neither increased glucose and fatty acid uptake nor enhanced mitochondrial respiration after knockdown of mir-103/ 107. Hence mir-103/ 107 did not influence adipocyte metabolism directly. However, as the highest mir-103/ 107 expression levels are found in the adipose tissue, the metabolic phenotype after mir-103/ 107 knockdown most likely could stem from crosstalk of the adipose tissue with other organs and tissues. To study the effects of mir-103/ 107 in the whole organism without side effects caused by artificial antisense oligos, three knockout mouse lines for mir-103-1, mir-103-2 and mir-107 were generated using CRISPR/ Cas9 in pronuclear zygote injections. These mouse lines provide a useful tool for the research on mir-103/ 107 function in the future. So far there are very limited options to directly target white adipocytes for delivery of bigger molecules like antisense oligos. Atrial Natriuretic Peptide (ANP) binds and is internalized by its clearance receptor NPRC. Treatment with the PPARy agonist pioglitazone induces downregulation of NPRC in adipose tissue, suggesting that NPRC expression levels positively correlate with white adipose tissue but not brown adipose tissue. This could make ANP or ANP derivatives ideal candidates for targeting specific tissues and hence for cell-type or tissue-specific delivery of ANP-coupled small molecules or antisense oligos. Here we show that ANP but not its derivative AP-811 are internalized by Nprc. Furthermore, Nprc mRNA expression levels are highest in white adipose tissue, the lung and atrium. In vivo uptake of fluorescently labeled ANP is limited to the atrium and the lung; due to limitations in detection the ANP-uptake in the adipose tissues could not be investigated. However, as seen by Nprc mRNA expression levels in the adipose tissues, only white primary adipocytes, i.e. adipocyte not expressing Ucp1, were able to internalize ANP. These findings suggest that ANP and ANP derivatives pose as a viable option for targeting of white adipocytes and NPRC-mediated delivery of therapeutic agents and should be investigated further