Increased circulating cell-free DNA in insulin resistanceMilena S. Bartels1; Lucas Scheffler1; Rima Chakaroun1,2; Arne Dietrich1,3; Matthias Blüher2; Michael Stumvoll2; Anke Tönjes2; Jana Breitfeld1; Peter Kovacs1

Cell-free DNA (cfDNA) is DNA which partially circulates outside of cells in bloodor urine. Based on its origin and size, the extracted cfDNA is commonlydistinguished into cfDNA resulting from apoptosis (~166 bp) and cfDNA resultingfrom necrosis (~10,000 bp).Higher death rates of adipocytes in obesity are accompanied by acceleratedcellular turnover and followed by active remodeling of adipose tissue (AT)

MATERIAL AND METHODS

1 Leipzig University Medical Center, IFB Adiposity Diseases, Germany; 2 Department of Medicine, University of Leipzig, Germany; 3 Department of Surgery, University of Leipzig, Germany

BACKGROUND & AIM

Extraction of cfDNA:- MagMax™ Cell-free DNA Isolation Kit (Applied Biosystems)- quantitative real-time reverse transcriptase PCR

(qRT-PCR) using SYBR Green methodology

Sorbs cohort:anthropometricparameters n = 851

gender(n, female, male) 527/324

age(years) 48 ± 16

body-massindex (kg/m²) 26.87 ± 4.94

i tt hi ti 0 87 ± 0 10cellular turnover and followed by active remodeling of adipose tissue (AT)resulting in an increased release of cfDNA into the circulation (1). It has recentlybeen shown that obesity-induced cfDNA release from adipocytes stimulateschronic AT inflammation (2).

Circulating cfDNA in the blood has been postulated to be a promising biomarkerin pathological conditions, such as cancer, fetal chromosomal aberrations orcardiovascular diseases (3, 4). In addition to the predictive function of cfDNA inprenatal and cancer diagnostics, there is emerging evidence that cfDNA may beinvolved in metabolic disease as well. It has been shown that AT dysfunction

if t d i i i d l t b li d t b li f ti i l di

cf DNA fragments:waist-to-hip-ratio 0.87 ± 0.10

bodyfat (%) 21.28 ± 9.27

glucosemetabolism

NGT/IGT/T2D (n) 638/102/88

fastingglucose (mmol/l) 5.54 ± 1.24

fastinginsulin (μU/mL) 5.80 ± 3.81

HOMA-B 59.92 ± 75.47

HOMA-IR 1.78 ± 1.45

Stumvoll index 0.09 ± 0.03

HbA1c (%) 5.48 ± 0.65

inflammatorymarkers

( / ) 4manifested in impaired glucose metabolism and metabolic functions includinginsulin resistance as well as tissue inflammation leads to the release of cfDNA (1,2).

CfDNA further activates the macrophages of the visceral AT via Toll-like-receptor9 (TLR9) (2, 5), which causes a low-grade inflammation of the adipose tissue,thus further promoting insulin resistance (6) and contributing to the vicious circle.

The aim was to test the relevance of circulating cfDNA in metabolic disease.

The subtraction (106 bp fragment quantity minus 384bp fragment quantity) corresponded to the quantity ofcfDNA fragments with a length between 106 bp and384 bp (which comes from apoptosis).

RESULTS

Statistics: Spearman correlation

base pairs

• two primer sets for beta-actin-gene (ACTB)• ACTB106 primer: DNA fragments > 106bp• ACTB384 primer: DNA fragments > 384 bp

C-reactiveprotein(mg/L) 2.39 ± 4.01

chemerin(ng/mL) 121.74 ± 42.45

progranulin(ng/mL) 110.33 ± 28.32

cfDNA was increased in patients withproinflammatory adipokines (P<0.005)• positive correlation with chemerin,

progranulin, C-reactive protein (CRP)

impaired glucose metabolism (all P<0.006)• positive correlation with fasting insulin,

HOMA B HOMA IR (homeostasis model

RESULTS

higher mean cfDNA concentrations in subjects with medically treated type 2 diabetes (T2D) or with impaired fasting glucose (IFG) or impaired glucose tolerance (IGT)

cfDNA levels comparable between women and men (P=0.203)age ranges from 18 to 88 years, with an average age of 48 years.subjects in upper cfDNA tertiles had significantly • higher fasting insulin (A)• higher HOMA-IR (B) (homeostasis model assessment;

index for insulin resistance) HOMA-B, HOMA-IR (homeostasis model assessment; index for insulin secretion and insulin resistance)

• negative correlation with Stumvoll index

index for insulin resistance) • lower Stumvoll index (C) (index for insulin sensitivity)• higher levels of proinflammatory markers

(C-reactive protein (D), chemerin (E), progranulin (F))

A B C D E F

REFERENCES

CONCLUSION

1. Haghiac et al., Increased death of adipose cells, a path to release cell-free DNA into systemic circulation of obese women. Obesity 2012

Our data suggest animportant role of cfDNA inthe pathoph siolog of

KONTAKTMilena S. Bartels

IFB Adipositas ErkrankungenLiebigstr. 19, Leipzig+49 178 6893352

milena.bartels@online.de

Obesity 20122. Nishimoto et al.; Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Sci Adv 20163. Ellinger et al.; Noncancerous PTGS2 DNA fragments of apoptotic origin in sera of prostate cancer patients qualify as diagnostic and prognostic indicators. Int J Cancer 20084. Le Conte et al.; Cell-free fetal DNA analysis in maternal plasma as a screening test for trisomy 21, 18 and 13 in twin pregnancies. Ultrasound Obstet Gynecol 20175. Hong et al.; TLR9 regulates adipose tissue inflammation and obesity-related metabolic disorders. Obesity (Silver Spring) 20156. Wensveen et al.; The "Big Bang" in obese fat: Events initiating obesity-induced adipose tissue inflammation. Eur J Immunol 2015

the pathophysiology ofmetabolic syndrome.

Published in July 2018, Bartels et al.,

Diabetes & Metabolism