
==== Front
J Endocrinol Invest
J Endocrinol Invest
Journal of Endocrinological Investigation
0391-4097
1720-8386
Springer International Publishing Cham

38460092
2323
10.1007/s40618-024-02323-4
Original Article
Adipokine secretion and lipolysis following gender-affirming treatment in transgender individuals
Subramanian N. 1
Wiik A. 2
Rullman E. 2
Melin M. 23
Lundberg T. R. 2
Flanagan J. 2
Holmberg M. 14
Dekanski A. 1
Dhejne C. 45
Arver S. 4
Gustafsson T. 2
Laurencikiene J. 1
http://orcid.org/0000-0003-4655-4837
Andersson D. P. daniel.p.andersson@ki.se

16
1 grid.24381.3c 0000 0000 9241 5705 Lipid Laboratory, Department of Medicine Huddinge (H7), Karolinska Institutet, C2:94, Karolinska University Hospital Huddinge, 141 86 Huddinge, Sweden
2 grid.24381.3c 0000 0000 9241 5705 Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, and Unit of Clinical Physiology, Karolinska University Hospital, Stockholm, Sweden
3 https://ror.org/00m8d6786 grid.24381.3c 0000 0000 9241 5705 Department of Cardiology, Heart and Vascular Center, Karolinska University Hospital, Stockholm, Sweden
4 https://ror.org/00m8d6786 grid.24381.3c 0000 0000 9241 5705 ANOVA, Andrology, Sexual Medicine and Transgender Medicine, Karolinska University Hospital, Stockholm, Sweden
5 https://ror.org/056d84691 grid.4714.6 0000 0004 1937 0626 Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden
6 https://ror.org/00m8d6786 grid.24381.3c 0000 0000 9241 5705 Department of Endocrinology, Karolinska University Hospital Huddinge, Stockholm, Sweden
9 3 2024
9 3 2024
2024
47 9 22492260
5 9 2023
28 1 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
Background

The organ-specific effects of gender-affirming sex hormone treatment (GAHT) in transgender women (TW) and transgender men (TM) are insufficiently explored. This study investigated the effects of GAHT on adipose tissue function.

Methods

In a single-center interventional prospective study, 32 adults undergoing GAHT, 15 TW and 17 TM, were examined with anthropometry and abdominal subcutaneous adipose tissue biopsies obtained before initiation of treatment, 1 month after endogenous sex hormone inhibition and three and 11 months after initiated GAHT. Fat cell size, basal/stimulated lipolysis and cytokine secretion in adipose tissue were analyzed.

Results

TW displayed an increase in complement component 3a and retinol-binding protein 4 (RBP4) secretion after sex hormone inhibition, which returned to baseline following estradiol treatment. No changes in lipolysis were seen in TW. TM showed downregulation of RBP4 after treatment, but no changes in basal lipolysis. In TM, the estrogen suppression led to higher noradrenaline stimulated (NA) lipolysis that was normalized following testosterone treatment. At 11 months, the ratio of NA/basal lipolysis was lower compared to baseline. There were no significant changes in fat cell size in either TW or TM.

Conclusion

In TW, gonadal hormone suppression results in transient changes in cytokines and in TM there are some changes in NA-stimulated lipolysis following testosterone treatment. However, despite the known metabolic effects of sex hormones, the overall effects of GAHT on adipose tissue function are small and likely have limited clinical relevance, but larger studies with longer follow-up are needed to confirm these findings.

Trial registration

ClinicalTrials.gov Identifier: NCT02518009, Retrospectively registered 7 August 2015.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40618-024-02323-4.

Keywords

Adipose tissue
Lipolysis
Transgender
Transmen
Transwomen
http://dx.doi.org/10.13039/501100004348 Stockholms Läns Landsting 963296 962029 954970 Andersson D. P. Center for Innovative Medicine at Karolinska Institutethttp://dx.doi.org/10.13039/501100004359 Vetenskapsrådet 2016-00694 Laurencikiene J. http://dx.doi.org/10.13039/501100009708 Novo Nordisk Fonden NNF210C0069989 Laurencikiene J. Karolinska InstituteOpen access funding provided by Karolinska Institute.

issue-copyright-statement© Italian Society of Endocrinology (SIE) 2024
==== Body
pmcIntroduction

Gender dysphoria is a condition characterized by distress from an incongruence between a person’s gender identity and their sex recorded at birth. There has been an increase of individuals seeking gender-affirming treatment [1, 2]. Possible treatments include psychological counseling as well as gender-affirming hormonal treatment (GAHT) and surgical procedures aimed at aligning the body with the gender identity. The hormonal treatment consists of downregulation of endogenous gonadal hormones and gender-affirming hormonal treatment.

There is ample evidence that sex hormones have strong systemic and local metabolic and immunomodulatory effects [3–6]. The effects of gender-affirming hormone treatment on clinical outcomes such as cardiovascular disease [7–9], mortality [10], body composition [11], incretin levels and insulin sensitivity [12] have been studied previously. However, very few studies have addressed organ-specific (e.g., adipose-specific) metabolic effects at the cellular level. The pernicious phenotypes of adipose tissue, including enlargement of fat cells and altered release of fatty acids (although basal lipolysis is increased and stimulated lipolysis is decreased) and release of inflammatory factors are associated with insulin resistance [13–15], dyslipidemia [13], metabolic disease [16] and weight gain [17] over time. Furthermore, several adipose tissue phenotypes, such as fat cell size, level of lipolysis and secretion of cytokines show sex dimorphism [13, 17, 18] and in vitro studies have shown that both estrogen and testosterone reduce stimulated lipolysis [6, 19]. An accumulation of visceral/upper body fat often seen in males confers an increased cardio-metabolic risk. In addition, fat cell size, which is negatively associated with insulin sensitivity [13], is regulated differently in males and females. Lean females have larger subcutaneous fat cells than lean males on average. On the other hand, in individuals with a larger amount of adipose tissue, the opposite is seen [20]. Furthermore, one of the important adipose tissue anti-inflammatory cytokines, IL-10, is upregulated only in obese females, suggesting a possible protective mechanism not present in males [21]. These findings indicate that sex hormones may play at least a partial role in sex-specific adipose tissue function.

Because sex dimorphism consists of several components, the direct role of hormones is difficult to determine in vivo. Cohorts undergoing gender-affirming treatment could act as a model for such studies. To our knowledge, there is only one previous study (19 transgender women [TW] and 17 transgender men [TM]) that have investigated the effects of gender-affirming treatment in adipose tissue [22]. However, neither the inflammatory status of adipose tissue nor the effects of gonadal downregulation were examined.

We hypothesized that hormone treatment may affect important adipose tissue features and in the current study, we aimed to comprehensively examine the effects of gender-affirming treatment on in vitro adipocyte lipolysis, fat cell size and secretion of 16 cytokines with known impact on adipose tissue metabolism.

Methods

Study design and participants

This study is part of a single-center interventional prospective study (GEnder Dysphoria Treatment in Sweden study, GETS, Clinical Trials Identifier NCT02518009) that has been described previously [23, 24]. The study, conducted between April 2015 and May 2021, recruited individuals who had been referred and accepted for gender-affirming hormonal treatment of gender dysphoria at the Andrology Sexual Medicine and Transgender Medicine unit (ANOVA), Karolinska University Hospital, Stockholm, Sweden. All inclusion and exclusion criteria can be found in a previous publication [23]. Inclusion criteria were age 20–40 years, individuals with gender dysphoria who had been accepted for hormonal treatment and who had requested as much hormonal feminization or masculinization as possible and were willing to participate in the study. Exclusion criteria included an already started hormonal therapy, ongoing infectious disease, treatment with warfarin or other anticoagulant, history of cardiovascular disease, type 1 diabetes, serious psychiatric or somatic disease, alcohol or drug abuse, and language difficulties. Those who identified themselves as non-binary and wanted partial hormonal treatment were also excluded from participation. All individuals were initially treated with an injection of a gonadotropin releasing hormone (GnRH) antagonist (degarelix 240 mg subcutaneously). This strategy results in an immediate reduction in gonadotropin secretion and bring sex hormone levels (estradiol and testosterone) to castrate levels within 24 h. In clinical practice, inhibition of endogenous sex hormone production is usually combined with gender-affirming hormonal treatment. However, initial GnRH antagonist treatment provides an opportunity to compare in vivo effects of an environment without the presence of androgens and estrogens in adipose tissue. The subsequent gender-affirming hormone treatment was started at 4 weeks, after post-castration assessments were made. TM were treated with testosterone injections (testosterone undecanoate 1000 mg intramuscularly) with the first two injections given with a 6-week interval and thereafter one injection every 10th week. Dose adjustments were made to maintain androgen levels within the normal adult male reference range. In TW, androgen suppression was maintained with a GnRH analogue (Triptorelin pamoate [Pamorelin®] 11.25 mg administered intramuscularly every third month). Estradiol was administered transdermally (gel or patches), orally, or in a few cases intramuscularly (estrogenpolyphosphate). The estradiol doses given were either 1–2 mg gel applied daily, 100–200 μg/24 h patches, delivered twice weekly, 4–8 mg orally, or 80 mg IM every 2–4 weeks. The different routes of delivery used in the study reflect clinical practice.

The subjects were examined following an overnight fasting at four time points; baseline, 4 weeks after initiated gonadal suppression of endogenous sex hormones but before gender-affirming hormone treatment and 3 and 11 months after initiated gender-affirming hormone treatment. In total, 34 subjects, 17 TW and 17 TM, were initially included in the study. Venous blood samples were obtained. An abdominal subcutaneous fat biopsy in the paraumbilical region was obtained by needle aspiration following local anesthesia with prilocaine 5 mg/ml (without adrenaline). No adipose tissue biopsies were obtained in two TW.

Fat cell size

Adipocytes were isolated from the adipose tissue biopsy after collagenase treatment [25]. By measuring the diameter of 100 isolated adipocytes using light microscopy, the mean fat cell volume was calculated using a formula as described previously [20].

Lipolysis

Lipolysis experiments were performed according to a standard protocol [26]. Isolated fat cells from collagenase treated adipose tissue biopsies were incubated for 2 h at 37 °C in a lipolysis buffer (Krebs Ringer Phosphate buffer containing bovine albumin (20 g/l), glucose (1 g/l) and ascorbic acid (0.1 g/l).) in the absence (basal lipolysis) or presence (stimulated lipolysis) of various concentrations of noradrenaline and isoprenaline. At the end of incubation, glycerol release into the medium was determined and related to number of incubated fat cells. During fat mobilization in adipocytes by lipolysis, a triglyceride molecule is broken down into one glycerol molecule and three fatty acid molecules. The release of glycerol from the fat cells is a more accurate measure of lipolysis compared to fatty acid release as some of the fatty acids may be re-esterified in the adipocyte. Glycerol, on the other hand, is not reused in adipocytes as they lack glycerol kinase. As there is no consensus on how to express lipolysis, we used both per lipid weight and per the number of fat cells as well as the ratio between stimulated/basal lipolysis.

Adipokine secretion and blood sample analyses

The selection of cytokines was based on previous studies where the cytokine had either shown a strong association to adipose tissue metabolism or to be modulated by sex hormones. These included inflammatory factors (interferon gamma (IFN-γ), interleukin (IL) 1 beta (IL-1β), IL-6, chemokine IL-8, macrophage migration inhibitory factor (MIF), tumor necrosis factor alfa (TNF-α), Plasminogen activator inhibitor-1 (PAI-1), retinol-binding protein 4 (RBP4), monocyte chemoattractant protein 1 (MCP-1) and IL-18), anti-inflammatory factors (IL-1 receptor antagonist (IL-1RA), IL-10 and adiponectin) as well as some factors with reported dual functions, including both pro-inflammatory and anti-inflammatory effects depending on the environment (complement component 3a (C3a), IL-2 and IL-17a). Supplementary table S1 depicts some of the studies used for selection but should by no means be regarded as a comprehensive list of all published data on these cytokines.

Explants of subcutaneous white adipose tissue were incubated as described previously [27]. In brief, WAT explants were incubated for 2 h at 37 °C in 1 mL/100 mg tissue in the lipolysis buffer (see above). Fifty µl of the total incubation volume were used to analyze the above-listed 16 cytokines in the adipose tissue of both TM and TW at all 4 time points by Luminex multiplex immunoassay (ProcartaPlex, ThermoFisher, Vienna, Austria) according to manufacturer´s instructions. Data were collected using Mapgix (Luminex xMAP™ Corporation, Austin, TX USA) and expressed as picograms per milliliter (pg/ml). The alignment between the observed and expected concentrations in standard curves, curve fit and the amount of counted beads (>50) were used to validate the assay for individual cytokines. Sensitivity and detection limits of this immunoassay are indicated in Supplementary Table S2.

All standard laboratory analyses on blood samples were performed at the ISO accredited Karolinska University Laboratory, Stockholm, Sweden. Plasma concentrations of total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were quantified using enzymatic photometric methods. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation when triglyceride levels were below 4.0 mmol/L. Estradiol and testosterone levels were determined through electrochemiluminescence immunoassays. All analyses were performed on Roche Cobas 8000 or 6000 series analyzers (Roche Diagnostics Scandinavia AB, Solna, Sweden), employing reagents from the same manufacturer. For cases presenting with low testosterone levels, quantification was further refined using liquid chromatography–tandem mass spectrometry (LC–MS/MS), incorporating internal deuterium-labeled isotopic standards for enhanced precision.

Statistics

Time effects, i.e., comparison of pre- and post-intervention data, were derived using mixed linear models with time as a fixed effect and subject/time as a random effect to account for the repeated measures design. The Linear and Nonlinear Mixed Effects Models (nlme) library in R version 3.5.5 was used for this purpose. All dependent variables were treated as independent hypothesis tests and the correction for multiple hypothesis tests was calculated using the Benjamini–Hochberg false discovery rate, where a false discovery rate of 1% was considered significant. Statistical analysis for secreted cytokines was performed only for individuals without any missing time points and, therefore, two-way ANOVA for repeated measures and multiple comparisons was used.

Results

Of the 17 TW and 17 TM included in the study, the measurements of anthropometry were available for 15 TW and 17 TM. Clinical characteristics of the individuals undergoing gender-affirming treatment and number of subjects for each datapoint are presented in Table 2. The mean age in the cohort was 26 ± 4 years (TW 26 ± 4 years and TM 25 ± 5 years, respectively). There were no significant changes in body weight, BMI, glucose, insulin or insulin resistance estimated by HOMA-IR in either group during the study period (Table 1). As expected, in both transmen and transwomen, endogenous sex hormone production was downregulated following 1 month of GnRH antagonist treatment (termed T0) and gender-affirming hormone treatment resulted in increased levels of sex hormones (see Table 1). Lipid levels for TW and TM are also shown in Table 2. In TW, there were no change in total cholesterol, TG or LDL (p = 0.13–0.98). HDL cholesterol increased after 3 months but returned to baseline after 11 months after gender-affirming treatment was started. In TM, total cholesterol was also unchanged, HDL decreased from 1.6 to 1.2 mmol/L (p = 0.0002) 11 months after initiation of gender-affirming treatment and there was a small decrease in TG after 3 months (Table 1).Table 1 Characterization of transwomen and transmen before initiated treatment (T00), after gonadal downregulation (T0), 3 months (T3) and 11 months (T11) after gender-affirming hormonal treatment was started

Time	T00	T0	T3	T11	T00 vs T0	T00 vs T3	T00 vs T11	
Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)	p-value	p-value	p-value	
Transwomen	
Age (years)	26 (24–27)							
Weight (kg)	67 (64–80)	65 (63–84)	68 (64–83)	73 (66–85)	0.94	0.38	0.07	
Height (cm)	180 (175–183)							
Body mass index (kg/m2)	21.7 (19.5–24.7)	20.4 (19.3–25.4)	21.4 (19.9–25.2)	21.9 (20.6–25.4)	0.81	0.48	0.06	
Glucose (mmol/L)	5.3 (4.9–5.6)	5.2 (5.1–5.4)	5.1 (4.9–5.3)	5.1 (5.0–5.3)	0.76	0.15	0.64	
Insulin (mU/L)	7.6 (6.1–9.6)	6.9 (5.4–11.0)	7.3 (4.6–9.9)	9.4 (5.7–12.0)	0.78	0.86	0.16	
HOMA-IR	1.86 (1.52–2.09)	1.56 (1.27–2.35)	1.61 (1.05–2.34)	2.06 (1.37–2.91)	0.76	0.52	0.31	
Cholesterol (mmol/L)	3.8 (3.4–4.4)	4.1 (3.5–4.5)	3.7 (3.0–4.7)	3.8 (3.5–4.2)	0.06	0.72	0.41	
HDL cholesterol (mmol/L)	1.3 (1.1–1.6)	1.5 (1.2–1.7)	1.4 (1.3–1.9)	1.4 (1.1–1.6)	0.83	0.05	0.98	
LDL cholesterol (mmol/L)	2.0 (1.7–2.8)	2.3 (1.7–2.9)	1.6 (1.3–1.6)	2.1 (1.6–2.4)	0.31	0.14	0.13	
Triglycerides (mmol/L)	0.7 (0.5–1.0)	0.7 (0.6–1.1)	0.6 (0.5–0.8)	0.7 (0.4–0.9)	0.14	0.81	0.90	
Estradiol (pmol/L)	77 (61–110)	16 (10–45)	232 (199–568)	442 (237–575)	0.28	<0.0001	<0.0001	
Testosterone (nmol/L)	19 (18–22)	0.5 (0.3–0.6)	0.4 (0.4–0.8)	0.6 (0.4–0.7)	<0.0001	<0.0001	<0.0001	
Sex hormone binding globulin (nmol/L)	41 (34–52)		70 (48–84)	57 (42–79)		0.004	0.008	
Transmen	
Age (years)	24 (22–29)							
Weight (kg)	59 (51–87)	60 (50–83)	65 (54–83)	67 (58–80)	0.67	0.77	0.93	
Height (cm)	167 (164–171)							
Body mass index (kg/m2)	20.7 (19.6–32.9)	20.7 (18.9–29.5)	22.1 (20.1–29.8)	22.9 (20.7–27.7)	0.54	0.90	0.95	
Glucose (mmol/L)	5.1 (4.9–5.3)	5.1 (4.9–5.5)	5.2 (5.0–5.6)	5.3 (5.1–5.5)	1.00	0.18	0.07	
Insulin (mU/L)	8.8 (6.3–13.5)	7.6 (5.7–13.5)	8.4 (6.8–10.5)	8.6 (6.4–11)	0.87	0.51	0.65	
HOMA-IR	2.00 (1.45–3.23)	1.81 (1.20–3.23)	1.94 (1.55–2.71)	2.09 (1.52–5.34)	0.80	0.62	0.95	
Cholesterol (mmol/L)	4.1 (3.6–4.6)	4.3 (4.0–4.6)	4.2 (3.6–4.6)	3.9 (3.3–4.4)	0.10	0.48	0.46	
HDL cholesterol (mmol/L)	1.6 (1.3–1.9)	1.8 (1.2–2.1)	1.4 (1.1–1.7)	1.3 (1.0–1.4)	0.14	0.14	0.0002	
LDL cholesterol (mmol/L)	1.9 (1.5–2.8)	2.2 (1.8–2.7)	2.2 (2.0–2.7)	2.1 (1.6–2.7)	0.15	0.89	0.98	
Triglycerides (mmol/L)	0.8 (0.6–1.0)	0.7 (0.6–0.9)	0.9 (0.6–1.2)	1.0 (0.7–1.2)	0.06	0.10	0.04	
Estradiol (pmol/L)	390 (218–603)	24 (13–40)	124 (76–187)	183 (143–248)	<0.0001	0.0002	<0.0001	
Testosterone (nmol/L)	1 (0.6–1.4)	0.9 (0.5–1.1)	21 (14–27)	21 (18–29)	0.95	<0.0001	<0.0001	
Sex hormone binding globulin (nmol/L)	67 (43–101)		35 (26–45)	24 (19–39)		0.002	0.0005	

Table 2 Fat cell volume and lipolysis in transwomen and transmen before initiated treatment (T00), after gonadal downregulation (T0), 3 months (T3) and 11 months (T11) after gender-affirming hormonal treatment was started

Time	T00	T0	T3	T11	T00 vs T0	T00 vs T3	T00 vs T12	
n	Median (IQR)	n	Median (IQR)	n	Median (IQR)	n	Median (IQR)	p-value	p-value	p-value	
Transwomen	
Fat cell volume (pL)	15	387 (209–561)	12	347 (271–534)	11	340 (222–424)	12	342 (284–517)	0.80	0.47	0.74	
Basal lipolys (µmol glycerol/g TG/2 h)	14	0.70 (0.57–0.88)	12	0.64 (0.54–0.82)	11	0.60 (0.43–0.98)	12	0.88 (0.43–2.10)	0.55	0.75	0.09	
Noradrenaline induced lipolysis (µmol glycerol/g TG/2 h)	14	2.67 (1.23–3.64)	12	2.98 (1.39–4.08)	11	2.36 (1.42–4.13)	12	3.00 (2.06–3.95)	0.69	0.90	0.16	
Isoprenaline induced lipolysis (µmol glycerol/g TG/2 h)	14	8.46 (3.51–9.40)	12	5.79 (3.47–11.11)	11	8.48 (4.08–9.87)	12	7.16 (6.35–8.85)	0.89	0.85	0.34	
Basal lipolysis (µmol glycerol/×107 cells/2 h)	14	2.72 (1.27–3.55)	12	2.03 (1.01–3.09)	11	1.56 (0.96–2.27)	12	3.21 (1.46–7.77)	0.32	0.47	0.19	
Noradrenaline stimulated lipolysis (µmol glycerol/×107 cells/2 h)	14	6.20 (4.79–13.10)	12	8.90 (5.23–14.25)	11	5.15 (4.50–13.38)	12	9.26 (6.66–21.94)	0.79	0.86	0.14	
Isoprenaline stimulated lipolysis (µmol glycerol/×107 cells/2 h)	14	18.09 (15.24–32.89)	12	19.26 (15.30–29.04)	11	19.33 (14.67–29.84)	12	22.38 (16.46–35.61)	0.76	0.84	0.72	
Noradrenaline/Basal lipolysis (ratio)	14	2.91 (1.85–4.41)	12	3.57 (2.27–6.62)	11	3.37 (2.70–5.68)	12	3.47 (2.31–4.59)	0.16	0.63	0.53	
Isoprenaline/Basal lipolysis (ratio)	14	11.70 (4.82–14.56)	12	10.22 (6.73–16.62)	11	14.37 (5.66–18.62)	12	7.68 (5.03–15.30)	0.86	0.59	0.71	
Transmen	
Fat cell volume (pL)	14	430 (299–576)	14	396 (308–519)	14	545 (349–680)	16	442 (300–609)	0.76	0.09	0.87	
Basal lipolys (µmol glycerol/g TG/2 h)	14	0.74 (0.34–1.02)	14	0.94 (0.36–1.20)	14	0.96 (0.52–1.30)	16	1.05 (0.79–1.48)	0.39	0.30	0.20	
Noradrenaline induced lipolysis (µmol glycerol/g TG/2 h)	14	2.77 (1.54–3.72)	14	3.81 (2.35–5.87)	14	2.72 (1.57–3.44)	16	2.71 (2.04–3.33)	0.04	0.59	0.57	
Isoprenaline induced lipolysis (µmol glycerol/g TG/2 h)	14	5.91 (5.14–7.38)	14	7.48 (6.04–8.77)	14	6.27 (5.43–7.95)	16	5.77 (5.09–9.12)	0.06	0.70	0.41	
Basal lipolysis (µmol glycerol/×107 cells/2 h)	14	2.35 (1.26–4.55)	14	2.94 (1.86–6.03)	14	4.30 (2.02–7.54)	16	3.09 (2.68–5.51)	0.48	0.05	0.15	
Noradrenaline stimulated lipolysis (µmol glycerol/×107 cells/2 h)	14	10.70 (4.68–16.60)	14	14.52 (6.80–24.28)	14	13.74 (6.66–22.35)	16	9.69 (5.93–15.81)	0.07	0.54	0.49	
Isoprenaline stimulated lipolysis (µmol glycerol/×107 cells/2 h)	14	23.73 (17.68–27.88)	14	31.12 (18.30–34.94)	14	26.85 (25.15–36.64)	16	23.19 (19.68–32.80)	0.11	0.10	0.42	
Noradrenaline/Basal lipolysis (ratio)	14	3.64 (2.38–5.45)	14	4.00 (3.08–7.65)	14	3.25 (1.54–4.97)	16	2.45 (1.63–3.26)	0.10	0.74	0.04	
Isoprenaline/Basal lipolysis (ratio)	14	8.90 (5.91–13.01)	14	9.06 (6.09–13.30)	14	7.87 (4.97–13.35)	16	6.19 (4.84–8.60)	0.92	0.95	0.15	
TG Triglycerides

Several factors secreted by adipose tissue are affected by hormone washout

Although there were no systemic changes in insulin sensitivity estimated with HOMA-IR in the treated patients, we further investigated if adipose tissue-secreted factors can be locally affected by sex hormone changes during gonadal downregulation and gender-affirming hormone treatment. Of the 16 cytokines that we selected, only 3 were affected by the treatment. A complement factor C3a was significantly increased by the gonadal downregulation in TW and then normalized following 11 months of estrogen treatment (Fig. 1a). A similar upregulation and normalization were observed for retinol binding protein 4 (RBP4) in TW (Fig. 1b), suggesting that removal of testosterone potentiates secretion of both these factors. Although IL-1RA showed a similar trend of regulation as RBP4 and C3a, the change was not significant (Fig. 1c). In TM, no changes were observed for C3a (Fig. 1d), but RBP4 was significantly downregulated by the hormonal intervention (comparing before (T00) and after 11 months (T11) after gender-affirming hormonal treatment was started) (Fig. 1e). Interestingly, IL-1RA was significantly higher after gonadal hormone downregulation in TM compared to after the 11 months treatment. IL-1RA showed a trend for upregulation after hormone washout (one month after removal of estrogens), but this effect was not significant (Fig. 1f). The levels of the thirteen cytokines that did not change significantly during gonadal downregulation or gender-affirming hormone treatment are displayed in Fig. 2.Fig. 1 Significantly regulated secreted adipokine. Adipokines C3a (a, d), RBP4 (b, e) and IL-1RA (c, f) were measured by Luminex multiplexing assay in TW (n = 6) and TM (n = 8). Measurements were performed before initiated treatment (T00), 1 month after initiated gonadal down-regulation (T0), 3 months (T3) and 11 months (T11) after gender-affirming hormonal treatment was started. The secretion is expressed as pg/ml. Differences between the groups were calculated using one-way ANOVA with multiple comparisons

Fig. 2 Secretion of the adipokines that are not affected by gender-affirming therapy. Adipokines were measured by Luminex multiplexing assay in TW (n = 6) and TM (n = 8). Measurements were performed before initiated treatment (T00), 1 month after initiated gonadal down-regulation (T0), 3 months (T3) and 11 months (T11) after gender-affirming hormonal treatment was started. The secretion is expressed as pg/ml. Differences between the groups were calculated using one-way ANOVA with multiple comparisons

No major changes in fat cell size and lipolysis

We further wanted to confirm whether the previously shown changes in adipocyte cell size and lipolysis following gender-affirming treatment [22] were also seen in our cohort. However, no changes in fat cell size were seen in TM or TW following gonadal downregulation or hormonal treatment in our study (Table 2 and Fig. 3a, b, p = 0.06–0.87). In addition, no alterations in basal or stimulated lipolysis were detected in TW (Fig. 3c, e, g) regardless of whether lipolysis was expressed as per gram lipid, per cell unit for basal, noradrenaline-stimulated, isoprenaline-stimulated, or as a ratio of noradrenaline/basal lipolysis (p = 0.06–0.99) (Table 3).Fig. 3 Fat cell volume and lipolysis. Values for a, b Fat cell volume c, d Basal lipolysis e, f Noradrenaline stimulated lipolysis g, h ratio of Noradrenaline/basal lipolysis for TW (n = 14) and TM (n = 16) are shown. Measurements were performed before initiated treatment (T00), 1 month after initiated gonadal down-regulation (T0), 3 months (T3) and 11 months (T11) after gender-affirming hormonal treatment was started

TM showed no changes in basal lipolysis (Fig. 3d), but an increase in noradrenaline-stimulated lipolysis following gonadal downregulation that returned to baseline levels at the examination 11 months after baseline (Fig. 3f). The ratio NA/basal lipolysis ratio decreased at 11 months compared to baseline in TM both when expressed as per gram triglycerides or per cell (Fig. 3h, p = 0.04). No other alterations in basal or stimulated lipolysis were detected in TM when lipolysis was expressed as per gram lipid, per cell unit for basal or isoprenaline-stimulated lipolysis, (Table 2, p = 0.10–0.95).

Discussion

To our knowledge, this is the first study to examine the effects of gonadal downregulation and gender-affirming sex hormone treatment on adipose tissue cytokine levels, and the second to study the effects on lipolysis in both TW and TM. We report transiently elevated levels of C3a and RBP-4 following gonadal downregulation in TW, which was normalized by estrogen treatment. In TM, two cytokines were altered by the treatment (RBP4 and IL1RA), and a transient increase in NA-stimulated lipolysis was seen. No significant changes in fat cell size was observed in either TW or TM.

The effect of estrogen on lipolysis is controversial. Previous in vitro studies have shown that both estrogen and testosterone treatment resulted in inhibition of stimulated lipolysis [6, 19]. On the other hand, another study showed that estradiol treatment of subcutaneous adipocytes resulted in increased lipolysis [28]. Furthermore, some of the biological effects traditionally attributed to testosterone acting via the androgen receptor may in part be dependent on estradiol produced in the testis or aromatized from testosterone in several tissues, as discussed previously [29]. One might speculate that the transient increase of stimulated lipolysis in TM may be a result of decreased inhibition of stimulated lipolysis by estrogen. However, the fact that this was not observed in TW and data showing that estrogen does not regulate lipolysis directly does not support this hypothesis [30]. These discrepancies between the studies could be due to sampling methods, hormone administration and/or gender-specific adrenergic receptor distribution [31].

A study similar to ours reported an increase in abdominal and gluteal subcutaneous fat cell size in TW, a decrease in abdominal subcutaneous fat cells in TM, a decrease in basal lipolysis in TW and increase in basal lipolysis in TM [22]. It should be noted that both our study and Elbers et al. [22] examined relatively small cohorts where interindividual variation was quite high and treatment protocols were different.

Previous findings regarding insulin sensitivity following gender-affirming hormone treatment are also conflicting; both unchanged [32, 33] (and our study), increased [12] (in TM) and decreased [34] insulin sensitivity have been reported [12]. Furthermore, in a recent large retrospective study, transgender individuals did not display an increased incidence of type 2 diabetes compared with the general population [35]. The changes in lipid profile in our study, i.e., a tendency towards decreased HDL cholesterol and increased triglycerides in TM and transiently increased HDL cholesterol levels in TW, are in line with earlier reports [36, 37]. In addition, the ENIGI (European Network for the Investigation of Gender Incongruence) study, including over 2600 individuals, reported unfavorable lipid changes and increased risk of cardiovascular disease in TM, but decrease of total cholesterol, HDL-c, LDL-c. and triglycerides in TW [38].

The observed increase in RBP4 and C3a secretion caused by a GnRH antagonist in TW could be explained not only by suppression of testosterone but also lower levels of estradiol (in connection to decreased production in the testis and aromatization of testosterone to estradiol in other tissues). One might speculate that estrogen has a negative effect on RBP4 and C3a secretion, which is reversed by GnRH antagonist treatment. However, the decrease of both RBP4 and the anti-inflammatory IL-1RA (and a trend for C3a) by testosterone treatment in TM suggests that testosterone may directly regulate these proteins and provides a novel insight into the potential direct effect of androgens.

While there are previous studies that appear to link these three secreted factors to sex hormones, most of them are cohort-based and do not distinguish between direct and confounding effects. The RBP4 has been found to be higher in non-obese and non-diabetic men compared to women [39] and correlated with higher testosterone levels in women with polycystic ovary syndrome [40], suggesting a positive association between testosterone and RBP4, whereas here we show a negative effect of testosterone treatment. The observed positive correlations reported by previous studies may not be due to hormone levels but rather to a confounding factor related to either sex or the presence of polycystic ovary syndrome. Furthermore, the changes in RBP4 levels reported therein could be adipose tissue-specific and would not necessarily be consistent with serum measurements used in previous studies. Hence, our finding may have implications for future disentangling of the direct effects of sex hormones on RBP4 as well as for distinguishing between local versus systemic effects.

Comparatively little research has been published on the relationship between sex hormones and IL-1RA. A recent study suggested that IL-1RA might protect androgen-dependent cancer cells from inflammatory damage and therefore contribute to prostate cancer progression [41]. The fact that IL-1RA is a potent anti-inflammatory factor, possibly directly regulated by sex hormones, adds a new and unique insight to our findings.

Interestingly, even C3a regulation by sex hormones can be debated. A 2016 systematic review of 13 studies reported an increase in C3 complement levels in menopausal women receiving hormone replacement therapy [42]. As the authors of the review note, subjects in the studies were not well matched for demographic or health characteristics, making the association less reliable. Another recent study demonstrated that estradiol treatment of rats subjected to brain death procedures lowers C3 levels [43]. Although the latter model is vastly different and include confounding factors such as brain death, it is in line with our findings that estradiol lowers levels of C3 in adipose tissue.

A strength of the current study is the longitudinal design with four different time points that help to distinguish between possible effects of endogenous sex hormone downregulation versus gender-affirming hormone treatment. The translational approach of the present study with both clinical data and investigations of cellular effects in human adipose tissue is another strength.

A limitation of the present study, as with the few previous translational human studies of gender dysphoria, is the relatively small number of subjects and a rather short follow-up period, which makes it difficult to find small effects due to inter-individual variation, and impossible to evaluate long-term effects on possible changes in adipose tissue expansion and metabolism. Therefore, we cannot exclude that there are small effects of gonadal downregulation or gender-affirming hormone treatment on cytokine secretion or lipolysis. Both cytokine secretion profiles and lipolysis in adipose tissue are well known to be dependent on BMI. In this study, the majority of individuals were lean and we did not have enough power to analyze lean and overweight/obese individuals separately. Another limitation is that we only investigated subcutaneous adipose tissue. It is known that adipose tissue function differs when comparing fat from the abdominal subcutaneous, gluteal subcutaneous, or visceral depot [13, 44].

In conclusion, the current study indicate that gender-affirming hormonal treatment does not result in major alterations in the metabolic profile in adipose tissue regarding lipolysis or cytokine production. This could be an indication for clinicians that, at least in the short-term, gender-affirming hormone treatment is safe in terms of metabolic risk in adipose tissue metabolism.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 91 KB)

Acknowledgements

We would like to express our gratitude to Kerstin Wåhlén and Ana Maria Suzuki at Department of Medicine, Karolinska Institutet, Sweden for their skillful assistance in the preparation of the adipose tissue biopsies.

Authors contributions

TG, SA, DPA, JL, NS and AW designed the study. SA, MH, CD included the subjects. AW, MM, TRL, and JF participated in clinical examinations. NS, JL and AD performed the adipokine studies and analyzed the data. DPA, NS and ER performed the statistical analyses. DPA, NS and JL wrote the first version of the manuscript. All authors revised the content of the manuscript and read and approved the final version. DPA is the guarantor of this work and had the primary responsibility for the final content.

Funding

Open access funding provided by Karolinska Institute. This work was supported by the Stockholm County Council grant numbers 963296, 962029, 954970, the Center for Innovative Medicine at Karolinska Institutet, the Swedish Research Council (2016-00694), Novo Nordisk Foundation (NNF210C0069989) and European Foundation for Studies of Diabetes. 

Availability of data and materials

Data are available from the corresponding author for any interested researcher who meets the criteria for access to confidential data and upon reasonable request.

Declarations

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

The study was performed according to the declaration of Helsinki and all subjects gave oral and written informed consent to participate. The study was approved by the regional ethical review board in Stockholm, Sweden (Dnr: 2014/1495-32; 2015/1186-32).

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

J. Laurencikiene and D. P. Andersson have equal senior contribution.
==== Refs
References

1. Zucker KJ Epidemiology of gender dysphoria and transgender identity Sex Health 2017 14 5 404 411 10.1071/SH17067 28838353
Zucker KJ (2017) Epidemiology of gender dysphoria and transgender identity. Sex Health 14(5):404–411. 10.1071/SH1706728838353 10.1071/SH17067
2. Coleman E Radix AE Bouman WP Brown GR de Vries ALC Deutsch MB Ettner R Fraser L Goodman M Green J Standards of care for the health of transgender and gender diverse people, version 8 Int J Transgend Health 2022 23 Suppl 1 S1 S259 10.1080/26895269.2022.2100644 36238954
Coleman E, Radix AE, Bouman WP, Brown GR, de Vries ALC, Deutsch MB, Ettner R, Fraser L, Goodman M, Green J et al (2022) Standards of care for the health of transgender and gender diverse people, version 8. Int J Transgend Health 23(Suppl 1):S1–S259. 10.1080/26895269.2022.210064436238954 10.1080/26895269.2022.2100644
3. Furman D Hejblum BP Simon N Jojic V Dekker CL Thiebaut R Tibshirani RJ Davis MM Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination Proc Nat Acad Sci USA 2014 111 2 869 874 10.1073/pnas.1321060111 24367114
Furman D, Hejblum BP, Simon N, Jojic V, Dekker CL, Thiebaut R, Tibshirani RJ, Davis MM (2014) Systems analysis of sex differences reveals an immunosuppressive role for testosterone in the response to influenza vaccination. Proc Nat Acad Sci USA 111(2):869–874. 10.1073/pnas.132106011124367114 10.1073/pnas.1321060111
4. Miyagi M Aoyama H Morishita M Iwamoto Y Effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes J Periodontol 1992 63 1 28 32 10.1902/jop.1992.63.1.28 1552459
Miyagi M, Aoyama H, Morishita M, Iwamoto Y (1992) Effects of sex hormones on chemotaxis of human peripheral polymorphonuclear leukocytes and monocytes. J Periodontol 63(1):28–32. 10.1902/jop.1992.63.1.281552459 10.1902/jop.1992.63.1.28
5. Cassidy RA Influence of steroids on oxidant generation in activated human granulocytes and mononuclear leukocytes Shock 2003 20 1 85 90 10.1097/01.shk.0000070740.34700.cd 12813374
Cassidy RA (2003) Influence of steroids on oxidant generation in activated human granulocytes and mononuclear leukocytes. Shock 20(1):85–90. 10.1097/01.shk.0000070740.34700.cd12813374 10.1097/01.shk.0000070740.34700.cd
6. Dicker A Ryden M Naslund E Muehlen IE Wiren M Lafontan M Arner P Effect of testosterone on lipolysis in human pre-adipocytes from different fat depots Diabetologia 2004 47 3 420 428 10.1007/s00125-003-1324-0 14752604
Dicker A, Ryden M, Naslund E, Muehlen IE, Wiren M, Lafontan M, Arner P (2004) Effect of testosterone on lipolysis in human pre-adipocytes from different fat depots. Diabetologia 47(3):420–428. 10.1007/s00125-003-1324-014752604 10.1007/s00125-003-1324-0
7. Karalexi MA Frisell T Cnattingius S Holmberg D Holmberg M Kollia N Skalkidou A Papadopoulos FC Cardiovascular outcomes in transgender individuals in Sweden after initiation of gender-affirming hormone therapy Eur J Prev Cardiol 2022 10.1093/eurjpc/zwac133 35778824
Karalexi MA, Frisell T, Cnattingius S, Holmberg D, Holmberg M, Kollia N, Skalkidou A, Papadopoulos FC (2022) Cardiovascular outcomes in transgender individuals in Sweden after initiation of gender-affirming hormone therapy. Eur J Prev Cardiol. 10.1093/eurjpc/zwac13335778824 10.1093/eurjpc/zwac133
8. Klaver M van Velzen D de Blok C Nota N Wiepjes C Defreyne J Schreiner T Fisher A Twisk J Seidell J Change in visceral fat and total body fat and the effect on cardiometabolic risk factors during transgender hormone therapy J Clin Endocrinol Metab 2022 107 1 e153 e164 10.1210/clinem/dgab616 34415999
Klaver M, van Velzen D, de Blok C, Nota N, Wiepjes C, Defreyne J, Schreiner T, Fisher A, Twisk J, Seidell J et al (2022) Change in visceral fat and total body fat and the effect on cardiometabolic risk factors during transgender hormone therapy. J Clin Endocrinol Metab 107(1):e153–e164. 10.1210/clinem/dgab61634415999 10.1210/clinem/dgab616
9. Glintborg D Rubin KH Petersen TG Lidegaard O T’Sjoen G Hilden M Andersen MS Cardiovascular risk in Danish transgender persons: a matched historical cohort study Eur J Endocrinol 2022 187 3 463 477 10.1530/EJE-22-0306 35900321
Glintborg D, Rubin KH, Petersen TG, Lidegaard O, T’Sjoen G, Hilden M, Andersen MS (2022) Cardiovascular risk in Danish transgender persons: a matched historical cohort study. Eur J Endocrinol 187(3):463–477. 10.1530/EJE-22-030635900321 10.1530/EJE-22-0306
10. de Blok CJ Wiepjes CM van Velzen DM Staphorsius AS Nota NM Gooren LJ Kreukels BP den Heijer M Mortality trends over five decades in adult transgender people receiving hormone treatment: a report from the Amsterdam cohort of gender dysphoria Lancet Diabetes Endocrinol 2021 9 10 663 670 10.1016/S2213-8587(21)00185-6 34481559
de Blok CJ, Wiepjes CM, van Velzen DM, Staphorsius AS, Nota NM, Gooren LJ, Kreukels BP, den Heijer M (2021) Mortality trends over five decades in adult transgender people receiving hormone treatment: a report from the Amsterdam cohort of gender dysphoria. Lancet Diabetes Endocrinol 9(10):663–670. 10.1016/S2213-8587(21)00185-634481559 10.1016/S2213-8587(21)00185-6
11. Elbers JM Asscheman H Seidell JC Gooren LJ Effects of sex steroid hormones on regional fat depots as assessed by magnetic resonance imaging in transsexuals Am J Physiol 1999 276 2 E317 E325 10.1152/ajpendo.1999.276.2.E317 9950792
Elbers JM, Asscheman H, Seidell JC, Gooren LJ (1999) Effects of sex steroid hormones on regional fat depots as assessed by magnetic resonance imaging in transsexuals. Am J Physiol 276(2):E317–E325. 10.1152/ajpendo.1999.276.2.E3179950792 10.1152/ajpendo.1999.276.2.E317
12. Shadid S Abosi-Appeadu K De Maertelaere AS Defreyne J Veldeman L Holst JJ Lapauw B Vilsboll T T’Sjoen G Effects of gender-affirming hormone therapy on insulin sensitivity and incretin responses in transgender people Diabetes Care 2020 43 2 411 417 10.2337/dc19-1061 31740479
Shadid S, Abosi-Appeadu K, De Maertelaere AS, Defreyne J, Veldeman L, Holst JJ, Lapauw B, Vilsboll T, T’Sjoen G (2020) Effects of gender-affirming hormone therapy on insulin sensitivity and incretin responses in transgender people. Diabetes Care 43(2):411–417. 10.2337/dc19-106131740479 10.2337/dc19-1061
13. Hoffstedt J Arner E Wahrenberg H Andersson DP Qvisth V Lofgren P Ryden M Thorne A Wiren M Palmer M Regional impact of adipose tissue morphology on the metabolic profile in morbid obesity Diabetologia 2010 53 12 2496 2503 10.1007/s00125-010-1889-3 20830466
Hoffstedt J, Arner E, Wahrenberg H, Andersson DP, Qvisth V, Lofgren P, Ryden M, Thorne A, Wiren M, Palmer M et al (2010) Regional impact of adipose tissue morphology on the metabolic profile in morbid obesity. Diabetologia 53(12):2496–2503. 10.1007/s00125-010-1889-320830466 10.1007/s00125-010-1889-3
14. Andersson DP Eriksson Hogling D Thorell A Toft E Qvisth V Naslund E Thorne A Wiren M Lofgren P Hoffstedt J Changes in subcutaneous fat cell volume and insulin sensitivity after weight loss Diabetes Care 2014 37 7 1831 1836 10.2337/dc13-2395 24760260
Andersson DP, Eriksson Hogling D, Thorell A, Toft E, Qvisth V, Naslund E, Thorne A, Wiren M, Lofgren P, Hoffstedt J et al (2014) Changes in subcutaneous fat cell volume and insulin sensitivity after weight loss. Diabetes Care 37(7):1831–1836. 10.2337/dc13-239524760260 10.2337/dc13-2395
15. Andersson DP Arner E Hogling DE Ryden M Arner P Abdominal subcutaneous adipose tissue cellularity in men and women Int J Obes 2017 41 10 1564 1569 10.1038/ijo.2017.148
Andersson DP, Arner E, Hogling DE, Ryden M, Arner P (2017) Abdominal subcutaneous adipose tissue cellularity in men and women. Int J Obes 41(10):1564–1569. 10.1038/ijo.2017.14810.1038/ijo.2017.148
16. Newell-Fugate AE The role of sex steroids in white adipose tissue adipocyte function Reproduction 2017 153 4 R133 R149 10.1530/REP-16-0417 28115579
Newell-Fugate AE (2017) The role of sex steroids in white adipose tissue adipocyte function. Reproduction 153(4):R133–R149. 10.1530/REP-16-041728115579 10.1530/REP-16-0417
17. Arner P Andersson DP Backdahl J Dahlman I Ryden M Weight gain and impaired glucose metabolism in women are predicted by inefficient subcutaneous fat cell lipolysis Cell Metab 2018 28 1 45 54.e3 10.1016/j.cmet.2018.05.004 29861390
Arner P, Andersson DP, Backdahl J, Dahlman I, Ryden M (2018) Weight gain and impaired glucose metabolism in women are predicted by inefficient subcutaneous fat cell lipolysis. Cell Metab 28(1):45–54.e3. 10.1016/j.cmet.2018.05.00429861390 10.1016/j.cmet.2018.05.004
18. Eriksson Hogling D Petrus P Gao H Backdahl J Dahlman I Laurencikiene J Acosta J Ehrlund A Naslund E Kulyte A Adipose and circulating CCL18 levels associate with metabolic risk factors in women J Clin Endocrinol Metab 2016 101 11 4021 4029 10.1210/jc.2016-2390 27459538
Eriksson Hogling D, Petrus P, Gao H, Backdahl J, Dahlman I, Laurencikiene J, Acosta J, Ehrlund A, Naslund E, Kulyte A et al (2016) Adipose and circulating CCL18 levels associate with metabolic risk factors in women. J Clin Endocrinol Metab 101(11):4021–4029. 10.1210/jc.2016-239027459538 10.1210/jc.2016-2390
19. Pedersen SB Kristensen K Hermann PA Katzenellenbogen JA Richelsen B Estrogen controls lipolysis by up-regulating alpha2A-adrenergic receptors directly in human adipose tissue through the estrogen receptor alpha. Implications for the female fat distribution J Clin Endocrinol Metab 2004 89 4 1869 1878 10.1210/jc.2003-031327 15070958
Pedersen SB, Kristensen K, Hermann PA, Katzenellenbogen JA, Richelsen B (2004) Estrogen controls lipolysis by up-regulating alpha2A-adrenergic receptors directly in human adipose tissue through the estrogen receptor alpha. Implications for the female fat distribution. J Clin Endocrinol Metab 89(4):1869–1878. 10.1210/jc.2003-03132715070958 10.1210/jc.2003-031327
20. Arner P Andersson DP Thorne A Wiren M Hoffstedt J Naslund E Thorell A Ryden M Variations in the size of the major omentum are primarily determined by fat cell number J Clin Endocrinol Metab 2013 98 5 E897 E901 10.1210/jc.2012-4106 23543656
Arner P, Andersson DP, Thorne A, Wiren M, Hoffstedt J, Naslund E, Thorell A, Ryden M (2013) Variations in the size of the major omentum are primarily determined by fat cell number. J Clin Endocrinol Metab 98(5):E897–E901. 10.1210/jc.2012-410623543656 10.1210/jc.2012-4106
21. Subramanian N, Tavira B, Hofwimmer K, Gutsmann B, Massier L, Abildgaard J, Juul A, Ryden M, Arner P, Laurencikiene J (2022) Sex-specific regulation of IL-10 production in human adipose tissue in obesity. Front Endocrinol 13. 10.3389/fendo.2022.996954
22. Elbers JM de Jong S Teerlink T Asscheman H Seidell JC Gooren LJ Changes in fat cell size and in vitro lipolytic activity of abdominal and gluteal adipocytes after a one-year cross-sex hormone administration in transsexuals Metabolism 1999 48 11 1371 1377 10.1016/s0026-0495(99)90146-4 10582544
Elbers JM, de Jong S, Teerlink T, Asscheman H, Seidell JC, Gooren LJ (1999) Changes in fat cell size and in vitro lipolytic activity of abdominal and gluteal adipocytes after a one-year cross-sex hormone administration in transsexuals. Metabolism 48(11):1371–1377. 10.1016/s0026-0495(99)90146-410582544 10.1016/s0026-0495(99)90146-4
23. Wiik A Andersson DP Brismar TB Chanpen S Dhejne C Ekstrom TJ Flanagan JN Holmberg M Kere J Lilja M Metabolic and functional changes in transgender individuals following cross-sex hormone treatment: design and methods of the GEnder Dysphoria Treatment in Sweden (GETS) study Contemp Clin Trials Commun 2018 10 148 153 10.1016/j.conctc.2018.04.005 30023449
Wiik A, Andersson DP, Brismar TB, Chanpen S, Dhejne C, Ekstrom TJ, Flanagan JN, Holmberg M, Kere J, Lilja M et al (2018) Metabolic and functional changes in transgender individuals following cross-sex hormone treatment: design and methods of the GEnder Dysphoria Treatment in Sweden (GETS) study. Contemp Clin Trials Commun 10:148–153. 10.1016/j.conctc.2018.04.00530023449 10.1016/j.conctc.2018.04.005
24. Wiik A Lundberg TR Rullman E Andersson DP Holmberg M Mandic M Brismar TB Dahlqvist Leinhard O Chanpen S Flanagan JN Muscle strength, size, and composition following 12 months of gender-affirming treatment in transgender individuals J Clin Endocrinol Metab 2020 105 3 e805 e813 10.1210/clinem/dgz247
Wiik A, Lundberg TR, Rullman E, Andersson DP, Holmberg M, Mandic M, Brismar TB, Dahlqvist Leinhard O, Chanpen S, Flanagan JN et al (2020) Muscle strength, size, and composition following 12 months of gender-affirming treatment in transgender individuals. J Clin Endocrinol Metab 105(3):e805–e813. 10.1210/clinem/dgz24710.1210/clinem/dgz247
25. Rodbell M Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis J Biol Chem 1964 239 375 380 10.1016/S0021-9258(18)51687-2 14169133
Rodbell M (1964) Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–38014169133 10.1016/S0021-9258(18)51687-2
26. Lofgren P Hoffstedt J Naslund E Wiren M Arner P Prospective and controlled studies of the actions of insulin and catecholamine in fat cells of obese women following weight reduction Diabetologia 2005 48 11 2334 2342 10.1007/s00125-005-1961-6 16222518
Lofgren P, Hoffstedt J, Naslund E, Wiren M, Arner P (2005) Prospective and controlled studies of the actions of insulin and catecholamine in fat cells of obese women following weight reduction. Diabetologia 48(11):2334–2342. 10.1007/s00125-005-1961-616222518 10.1007/s00125-005-1961-6
27. Acosta JR Douagi I Andersson DP Backdahl J Ryden M Arner P Laurencikiene J Increased fat cell size: a major phenotype of subcutaneous white adipose tissue in non-obese individuals with type 2 diabetes Diabetologia 2016 59 3 560 570 10.1007/s00125-015-3810-6 26607638
Acosta JR, Douagi I, Andersson DP, Backdahl J, Ryden M, Arner P, Laurencikiene J (2016) Increased fat cell size: a major phenotype of subcutaneous white adipose tissue in non-obese individuals with type 2 diabetes. Diabetologia 59(3):560–570. 10.1007/s00125-015-3810-626607638 10.1007/s00125-015-3810-6
28. Palin SL McTernan PG Anderson LA Sturdee DW Barnett AH Kumar S 17Beta-estradiol and anti-estrogen ICI: compound 182,780 regulate expression of lipoprotein lipase and hormone-sensitive lipase in isolated subcutaneous abdominal adipocytes Metabolism 2003 52 4 383 388 10.1053/meta.2003.50088 12701046
Palin SL, McTernan PG, Anderson LA, Sturdee DW, Barnett AH, Kumar S (2003) 17Beta-estradiol and anti-estrogen ICI: compound 182,780 regulate expression of lipoprotein lipase and hormone-sensitive lipase in isolated subcutaneous abdominal adipocytes. Metabolism 52(4):383–388. 10.1053/meta.2003.5008812701046 10.1053/meta.2003.50088
29. Russell N Grossmann M Mechanisms in endocrinology: estradiol as a male hormone Eur J Endocrinol 2019 181 1 R23 R43 10.1530/EJE-18-1000 31096185
Russell N, Grossmann M (2019) Mechanisms in endocrinology: estradiol as a male hormone. Eur J Endocrinol 181(1):R23–R43. 10.1530/EJE-18-100031096185 10.1530/EJE-18-1000
30. MacDonald TL MacPherson R Castellani L Cervone D Anderson E Wright DC Dyck DJ Estradiol does not directly regulate adipose lipolysis Adipocyte 2017 6 2 76 86 10.1080/21623945.2017.1287638 28425842
MacDonald TL, MacPherson R, Castellani L, Cervone D, Anderson E, Wright DC, Dyck DJ (2017) Estradiol does not directly regulate adipose lipolysis. Adipocyte 6(2):76–86. 10.1080/21623945.2017.128763828425842 10.1080/21623945.2017.1287638
31. Ramis JM Salinas R Garcia-Sanz JM Moreiro J Proenza AM Llado I Depot- and gender-related differences in the lipolytic pathway of adipose tissue from severely obese patients Cell Physiol Biochem 2006 17 3–4 173 180 10.1159/000092079 16543734
Ramis JM, Salinas R, Garcia-Sanz JM, Moreiro J, Proenza AM, Llado I (2006) Depot- and gender-related differences in the lipolytic pathway of adipose tissue from severely obese patients. Cell Physiol Biochem 17(3–4):173–180. 10.1159/00009207916543734 10.1159/000092079
32. Olson-Kennedy J Okonta V Clark LF Belzer M Physiologic response to gender-affirming hormones among transgender youth J Adolesc Health 2018 62 4 397 401 10.1016/j.jadohealth.2017.08.005 29056436
Olson-Kennedy J, Okonta V, Clark LF, Belzer M (2018) Physiologic response to gender-affirming hormones among transgender youth. J Adolesc Health 62(4):397–401. 10.1016/j.jadohealth.2017.08.00529056436 10.1016/j.jadohealth.2017.08.005
33. Wierckx K Van Caenegem E Schreiner T Haraldsen I Fisher AD Toye K Kaufman JM T’Sjoen G Cross-sex hormone therapy in trans persons is safe and effective at short-time follow-up: results from the European network for the investigation of gender incongruence J Sex Med 2014 11 8 1999 2011 10.1111/jsm.12571 24828032
Wierckx K, Van Caenegem E, Schreiner T, Haraldsen I, Fisher AD, Toye K, Kaufman JM, T’Sjoen G (2014) Cross-sex hormone therapy in trans persons is safe and effective at short-time follow-up: results from the European network for the investigation of gender incongruence. J Sex Med 11(8):1999–2011. 10.1111/jsm.1257124828032 10.1111/jsm.12571
34. Elbers JM Giltay EJ Teerlink T Scheffer PG Asscheman H Seidell JC Gooren LJ Effects of sex steroids on components of the insulin resistance syndrome in transsexual subjects Clin Endocrinol (Oxf) 2003 58 5 562 571 10.1046/j.1365-2265.2003.01753.x 12699437
Elbers JM, Giltay EJ, Teerlink T, Scheffer PG, Asscheman H, Seidell JC, Gooren LJ (2003) Effects of sex steroids on components of the insulin resistance syndrome in transsexual subjects. Clin Endocrinol (Oxf) 58(5):562–571. 10.1046/j.1365-2265.2003.01753.x12699437 10.1046/j.1365-2265.2003.01753.x
35. van Velzen D Wiepjes C Nota N van Raalte D de Mutsert R Simsek S den Heijer M Incident diabetes risk is not increased in transgender individuals using hormone therapy J Clin Endocrinol Metab 2022 107 5 e2000 e2007 10.1210/clinem/dgab934 34971391
van Velzen D, Wiepjes C, Nota N, van Raalte D, de Mutsert R, Simsek S, den Heijer M (2022) Incident diabetes risk is not increased in transgender individuals using hormone therapy. J Clin Endocrinol Metab 107(5):e2000–e2007. 10.1210/clinem/dgab93434971391 10.1210/clinem/dgab934
36. Leemaqz SY Kyinn M Banks K Sarkodie E Goldstein D Irwig MS Lipid profiles and hypertriglyceridemia among transgender and gender diverse adults on gender-affirming hormone therapy J Clin Lipidol 2022 10.1016/j.jacl.2022.11.010 36473821
Leemaqz SY, Kyinn M, Banks K, Sarkodie E, Goldstein D, Irwig MS (2022) Lipid profiles and hypertriglyceridemia among transgender and gender diverse adults on gender-affirming hormone therapy. J Clin Lipidol. 10.1016/j.jacl.2022.11.01036473821 10.1016/j.jacl.2022.11.010
37. Valentine A Nokoff N Bonny A Chelvakumar G Indyk J Leibowitz S Nahata L Cardiometabolic parameters among transgender adolescent males on testosterone therapy and body mass index-matched cisgender females Transgend Health 2021 6 6 369 373 10.1089/trgh.2020.0052 34993308
Valentine A, Nokoff N, Bonny A, Chelvakumar G, Indyk J, Leibowitz S, Nahata L (2021) Cardiometabolic parameters among transgender adolescent males on testosterone therapy and body mass index-matched cisgender females. Transgend Health 6(6):369–373. 10.1089/trgh.2020.005234993308 10.1089/trgh.2020.0052
38. Cocchetti C Romani A Collet S Greenman Y Schreiner T Wiepjes C den Heijer M T’Sjoen G Fisher AD The ENIGI (European Network for the Investigation of Gender Incongruence) Study: overview of acquired endocrine knowledge and future perspectives J Clin Med 2022 11 7 1784 10.3390/jcm11071784 35407392
Cocchetti C, Romani A, Collet S, Greenman Y, Schreiner T, Wiepjes C, den Heijer M, T’Sjoen G, Fisher AD (2022) The ENIGI (European Network for the Investigation of Gender Incongruence) Study: overview of acquired endocrine knowledge and future perspectives. J Clin Med 11(7):1784. 10.3390/jcm1107178435407392 10.3390/jcm11071784
39. Lee ES Yoo JS Lim JS Yadav D Cho EJ Choi YS Kim HM Chung CH Differences in adipokine and hepatokine levels among non-diabetic population classified by age and sex J Lifestyle Med 2013 3 1 62 67 26064839
Lee ES, Yoo JS, Lim JS, Yadav D, Cho EJ, Choi YS, Kim HM, Chung CH (2013) Differences in adipokine and hepatokine levels among non-diabetic population classified by age and sex. J Lifestyle Med 3(1):62–6726064839
40. Mellati AA Sharifi F Sajadinejad M Sohrabi D Mazloomzadeh S The relationship between retinol-binding protein 4 levels, insulin resistance, androgen hormones and polycystic ovary syndrome Scand J Clin Lab Invest 2012 72 1 39 44 10.3109/00365513.2011.626071 22023042
Mellati AA, Sharifi F, Sajadinejad M, Sohrabi D, Mazloomzadeh S (2012) The relationship between retinol-binding protein 4 levels, insulin resistance, androgen hormones and polycystic ovary syndrome. Scand J Clin Lab Invest 72(1):39–44. 10.3109/00365513.2011.62607122023042 10.3109/00365513.2011.626071
41. Fan YC Lee KD Tsai YC Roles of interleukin-1 receptor antagonist in prostate cancer progression Biomedicines 2020 8 12 602 10.3390/biomedicines8120602 33322099
Fan YC, Lee KD, Tsai YC (2020) Roles of interleukin-1 receptor antagonist in prostate cancer progression. Biomedicines 8(12):602. 10.3390/biomedicines812060233322099 10.3390/biomedicines8120602
42. Abdi F Mobedi H Mosaffa N Dolatian M Ramezani Tehrani F Effects of hormone replacement therapy on immunological factors in the postmenopausal period Climacteric 2016 19 3 234 239 10.3109/13697137.2016.1164136 27086591
Abdi F, Mobedi H, Mosaffa N, Dolatian M, Ramezani Tehrani F (2016) Effects of hormone replacement therapy on immunological factors in the postmenopausal period. Climacteric 19(3):234–239. 10.3109/13697137.2016.116413627086591 10.3109/13697137.2016.1164136
43. Ricardo-da-Silva FY Armstrong-Jr R Vidal-Dos-Santos M Correia CJ Coutinho ESRDS Anunciacao LFD Moreira LFP Leuvenink HGD Breithaupt-Faloppa AC Long-term lung inflammation is reduced by estradiol treatment in brain dead female rats Clinics 2021 76 e3042 10.6061/clinics/2021/e3042 34406272
Ricardo-da-Silva FY, Armstrong-Jr R, Vidal-Dos-Santos M, Correia CJ, Coutinho ESRDS, Anunciacao LFD, Moreira LFP, Leuvenink HGD, Breithaupt-Faloppa AC (2021) Long-term lung inflammation is reduced by estradiol treatment in brain dead female rats. Clinics 76:e3042. 10.6061/clinics/2021/e304234406272 10.6061/clinics/2021/e3042
44. Tchoukalova YD Votruba SB Tchkonia T Giorgadze N Kirkland JL Jensen MD Regional differences in cellular mechanisms of adipose tissue gain with overfeeding Proc Natl Acad Sci USA 2010 107 42 18226 18231 10.1073/pnas.1005259107 20921416
Tchoukalova YD, Votruba SB, Tchkonia T, Giorgadze N, Kirkland JL, Jensen MD (2010) Regional differences in cellular mechanisms of adipose tissue gain with overfeeding. Proc Natl Acad Sci USA 107(42):18226–18231. 10.1073/pnas.100525910720921416 10.1073/pnas.1005259107
