Testosterone treatment is associated with reduced adipose tissue dysfunction and nonalcoholic fatty liver disease in obese hypogonadal men

Obesity, in particular abdominal obesity, is currently one of the greatest public health issues worldwide, owing to its high prevalence and substantial morbidity and mortality. The functional abnormalities of adipocytes, more than the excess of adipose mass itself, represent the major pathogenic link between obesity and non-transmittable chronic diseases, including type 2 diabetes, metabolic syndrome (MetS) and non-alcoholic fatty liver disease (NAFLD), which are all associated, to different degrees, with an increased risk of cardiovascular disease (CVD).

In obesity, excessive calorie intake finally often leads to an impaired insulin signaling, adipocyte hypertrophy, and lipid handling resistance [1‐4]. In particular, the increased lipolysis from adipose tissue promotes hepatic insulin resistance by over-activation of hepatic gluconeogenesis and de novo lipid synthesis with a parallel deterioration of fatty acid oxidation. One of the primary mechanisms responsible for insulin resistance is the dysfunction of the main cellular organelles dedicated to metabolic control, i.e. mitochondria [5, 6]. Indeed, in adipocyte, dysfunctional mitochondria causes the spillover of lipids into non-adipose tissues, thus contributing to insulin resistance via lipotoxicity in other districts, including liver and vascular beds [7‐10]. Therefore, these pathogenic mechanisms crucially link obesity to NAFLD and cardio-metabolic disorders [8, 11, 12]. Noteworthy, NAFLD is nowadays considered as a multisystem disease, affecting extra-hepatic organs and regulatory pathways, therefore amplifying the risk of type 2 diabetes mellitus (T2DM), cardiovascular (CVD) and cardiac diseases [13, 14].

Adding a layer of complexity, MetS and visceral obesity are also strongly associated with the onset of hypogonadotropic hypogonadism in males [15‐17], due to functional abnormalities in the hypothalamus. Preclinical animal models demonstrated that MetS-induced hypogonadotropic hypogonadism is associated with a hypothalamic metainflammation, and with a substantial reduction of GnRH neurons [18, 19]. On the other hand, existing evidence consistently shows that testosterone therapy (TTh) induces a beneficial effect on metabolic parameters. In particular, several randomized [20‐24] and observational [25, 26] trials demonstrated that TTh is able to improve body composition [27] and insulin sensitivity [20]. In an experimental rabbit model of high fat diet-induced MetS, our group previously demonstrated that in vivo T dosing prevented visceral adipose tissue (VAT) expansion [18, 28] and counteracted its derangements, normalizing preadipocyte maturation, lipid handling and insulin sensitivity [29]. In the same animal model, in vivo T dosing was also effective in counteracting all NAFLD features induced by MetS, such as lipid accumulation, inflammation and initial fibrosis within the liver [29]. This is in keeping with other preclinical studies in animal models [30]. Finally, corroborating preclinical evidence indicated that testosterone treatment exerts beneficial metabolic effects mainly by improving MetS-related liver and adipose tissue dysfunctions [29, 31]. In humans, the ameliorating effects of androgens on MetS-associated adipose tissue and liver disease is supported by limited data [32, 33]. Therefore, perhaps surprisingly, the mechanism underlying how TTh improves whole-metabolic control was still missing. To further ascertain the effect of TTh on visceral adipose tissue and liver in a human setting, we designed an observational, prospective study enrolling severely obese male subjects on a waiting list to undergo bariatric surgery and stratified them, at baseline, as having T above (eugonadal) or below (hypogonadal) the normal value. A subgroup of hypogonadal subjects that also presented sexual symptoms were treated with TTh, according to guidelines, during the period before the surgical procedure. Therefore, our study design allows us to investigate the effect of in vivo TTh on VAT and liver dysfunction in obese hypogonadal men undergoing surgery for weight loss, as compared to untreated hypogonadal and eugonadal subjects. The assessment of the main morpho-functional features of human VAT preadipocytes, isolated from the three different treatment groups, was then performed, through analysis of glucose uptake, mitochondrial morphology and mRNA expression. We also studied the effect of TTh by evaluating morphological alterations and mRNA expression profiles of major pathogenic pathways related to NAFLD. These results are part of an observational study aimed at evaluating the effect of TTh on different complications of obesity in a population of severely obese men, candidates for bariatric surgery for weight loss.

Experimental procedures were carried out using the facilities of the Molecular Medicine Facility, Department of Biomedical Experimental and Clinical Sciences “Mario Serio”, University of Florence.

The study is the first to demonstrate that in vivo treatment with the long-lasting T preparation T undecanoate in symptomatic hypogonadal obese patients is associated with less severe NASH and visceral adipose tissue dysfunction as compared to untreated-hypogonadal ones. Liver biopsies from T-treated hypogonadal patients showed significantly lower intrahepatic triglyceride content, macrovesicular lipid accumulation and NAS score for steatosis, as compared to untreated-hypogonadal subjects. Accordingly, the clinical and biochemical algorithm which reflects fatty liver was also significantly reduced in TTh-treated hypogonadal patients over time, as compared to untreated-hypogonadal ones. However, the most remarkable results emerge from the experiments in differentiating preadipocytes isolated from human visceral adipose tissue samples of the different treatment groups (eugonadal, TTh-hypogonadal and untreated-hypogonadal subjects). In this large set of experiments, we demonstrated that preadipocytes from TTh patients showed a metabolically healthy phenotype as evidenced by a significantly healthier mitochondrial function, along with a higher insulin sensitivity and oxygen consumption paralleled by a lower superoxide accumulation. Preadipocytes from TTh-hypogonadal subjects also showed a higher expression of all the genes related to mitochondrial function, beige-brown adipogenesis, insulin signaling, and lipid metabolism, as compared to those from untreated-hypogonadal ones. These cross-sectional data derived from liver and fat biopsies were substantiated further by prospective data on biochemical and metabolic parameters. TTh significantly reduced serum cholesterol levels over time (pre-surgery vs. baseline visit), whilst a significant increase with time of triglycerides and transaminases along with a decrease in insulin serum levels (despite higher fasting glucose) was observed in untreated, but not in TTh-treated, hypogonadal subjects. Therefore, our findings further support pre-existing high-quality evidence that TTh induced a range of beneficial metabolic effects in hypogonadal patients [49]. In a recent study by Di Nisio and colleagues [50], a significant deregulation of gene responsiveness to testosterone in obese subcutaneous adipose tissue (SAT) was observed, with testosterone accumulation resulting in lower expression of genes involved in lipolytic and anti-adipogenic pathways, thus suggesting an altered response of dysfunctional fat cells already present in mild obesity. These data corroborate our finding relative to VAT and preadipocytes derived from class III obesity subjects, in which hypogonadism was associated with a decreased expression of genes involved in brown adipogenesis and lipid catabolism. Consistent with these positive metabolic effects of testosterone, a recent meta-analysis of RCTs demonstrated that TTh significantly reduced fasting glycaemia, HOMA index, and fat mass, while increasing lean mass, as compared to placebo [49]. A nonsignificant trend toward reduced total cholesterol and triglyceride level was also induced by TTh [49]. In accordance, a meta-analytic evaluation of observational studies demonstrated that TTh also improves glyco-metabolic control (reduction of HOMA, fasting glycaemia), lipid profile (reduction of triglycerides and total cholesterol level and increased HDL), and body composition (reduction of waist circumference, BMI, body weight and fat mass, and increased lean mass) [27]. A relevant study on a similar population of severely obese hypogonadal men reported an improvement in several cardio-metabolic parameters after 1-year TTh, with only some retained after 24 weeks from withdrawal (i.e. fat, but not lean mass amelioration, was maintained) [51]. Despite these clinically relevant findings, there is limited evidence on the putative biological and molecular mechanisms underlying TTh metabolic effects. In this context, the key advantage of the present study design is that it allowed us to explore, not only the systemic, but also the direct, local (within liver and adipose tissue) impact of TTh in severely obese patients, by analyzing and comparing tissue samples collected during the surgical procedure from the three different study arms (eugonadal, T-untreated hypogonadal and T-treated hypogonadal). Interestingly, from adipose tissue samples, we were also able to isolate and differentiate preadipocytes, whose dysfunction has a central role in driving insulin resistance [52]. One of the most intriguing findings was that preadipocytes isolated from TTh subjects showed significantly improved mitochondrial morpho-functional features, as compared to those form untreated- hypogonadal subjects. Mitochondria dysfunctions are nowadays considered as the major underlying driver of several metabolic diseases [53, 54]. Mitochondria constantly undergo fusion and division processes to form a tubular network, which is crucial for health in most eukaryotic cells [55, 56]. In unhealthy cells, division (fission) becomes predominant and the mitochondrial network fragments, thus reducing energy efficiency, ATP production and fitness to respond to environmental stress buffering reactive oxygen species (ROS) [57‐62]. Accordingly, mitochondria in differentiating preadipocytes from untreated-hypogonadal subjects were small, immobile and highly fragmented with reduced networking, oxygen consumption and increased superoxide formation. In contrast, preadipocytes from T-treated hypogonadal patients showed increased mitochondria dynamics and interconnectivity, coupled with increased oxygen consumption and reduced superoxide content. A significantly higher mRNA expression level of mitochondrial biogenesis (NRF1, TFAM), networking (MFN2, FIS1) and function (NDUFB3, NDFUB5, SDHB, FOXC2) markers was also observed in preadipocytes from TTh-hypogonadal patients as compared to untreated-hypogonadal ones. Interestingly, in a rodent model, synergistic activation of two of these markers, namely TFAM and FOXC2, enhanced adipose tissue expression of core proteins of mitochondrial fusion (MFN1, MFN2, and OPA1) consequently leading to a lean and insulin-sensitive phenotype [63‐66]. Noteworthy, given that TFAM, one of the most important mitochondrial DNA transcription factors [67‐69], contains AR-responsive elements, it is now considered a relevant AR target gene through which testosterone might regulate mitochondrial homeostasis [70]. In line with this evidence, a recent study demonstrated that AR, besides being nuclear, also localizes into mitochondria, where it modulates some of the non-genomic effects of androgens [71]. Therefore, our data further corroborated the positive effects of T on mitochondrial function. A marked increase of genes related to lipid metabolism/handling (including PPARα, PRKACA, PRAKCB, SNAP23, STX5) and reactive oxygen species removal (such as PPARGC1β) were also observed in preadipocytes from T-treated hypogonadal patients. Lipid handling and lipid droplet formation are closely related to mitochondria processes [61]. Essentially, a direct delivery of lipids from lipid droplets into mitochondria represents an efficient way to maintain ROS within physiological levels, thus shielding other cell organelles from lipotoxicity, while ensuring energy supply as well as insulin sensitivity [11, 61, 72, 73]. Consistent with this view, we observed a reduced superoxide formation, which was associated with an increase of oxygen consumption and insulin sensitivity in preadipocytes after in vivo TTh. In particular, these preadipocytes demonstrated a significantly improved insulin-stimulated glucose uptake, as compared to preadipocytes from untreated-hypogonadal obese subjects, reaching a level that was even higher than that of eugonadal ones. Noteworthy, the ability of preadipocytes to respond to increasing concentrations of insulin was tightly dependent upon T level, measured at the pre-surgery visit 1. The improved metabolic functions of preadipocytes from TTh patients were further supported by a significant increase of all the genes related not only to insulin signaling, mitochondrial biogenesis and lipid handling, but also to brown/beige adipogenesis (ADRB3, CIDEA, CITED1, BMP4, BMP7, HOXC9, TBX1) [74, 75]. In particular, a marked induction of UCP1 and UCP2 was observed in PADs derived from adipose tissue of T-treated hypogonadal obese men. The development of brown/beige adipocytes can be induced in white adipose tissue (WAT), because of a sustained stimulation with cold, ß3-adrenergic (ADRB3), peroxisome proliferator-activated receptor γ (PPARγ) agonists, or BMP7. This program is accompanied by induction of uncoupling proteins, especially UCP1, along with an elevated mitochondrial respiration and thermogenesis [76, 77]. In our study, we also found a significant and huge increase of the UCP isoform 2 (UCP2). Interestingly, UCP2 is an ion/anion transporter located in the mitochondrial inner membrane, with a central function in the regulation of oxidative stress, cellular metabolism and whole energy homeostasis [78]. A reduced UCP2 expression and activity has been linked to obesity-associated metabolic disorders, such as T2DM, hypertension and atherosclerosis [79‐82]. In hPADs collected from TTh men, we also found a concomitant increase of crucial factors (e.g. DKK1, DIO2) for adipose tissue differentiation and the maintenance of adipocyte homeostasis, which allows for the balance between lipolysis and adipogenesis [83]. The mRNA expression of PDE5 in adipose tissue, where it is known to be abundantly expressed [84], did not show significant changes. Finally, the observed increase of UCP1 mRNA expression in VAT from both untreated and treated hypogonadal patients, although not significant, could represent an attempt to counterbalance the potentially reduced UCP1 activity in the hypogonadal phenotype. This phenomenon would find its major efficacy in isolated preadipocytes, which display a significant UCP1 mRNA expression increase only in the TTh group, although the evaluation of UCP1 phosphorylation, rather than mRNA expression, would provide more useful information with regard to its activity. These observations would merit to be analyzed in further studies, perhaps including specific researches on enzymatic activity in a similar model setting.

The aforementioned observations depict a metabolically healthier phenotype for hPADs collected from T-treated obese hypogonadal subjects, as compared with untreated-hypogonadal men. Our data also corroborated the pivotal role of T in maintaining the homeostasis of mitochondria, the most energetically active organelles in the eukaryotic cells [85]. Previous studies, both in animal models and in humans, demonstrated that androgens stimulate glucose utilization and ATP production, as well as mitochondrial cytochrome c oxidase activity in muscle [86‐88], and in several other tissues and cell types [89‐92]. However, to our knowledge, this is the first demonstration that in vivo TTh is associated with a healthier phenotype of human preadipocytes. Accordingly, a significantly increased expression of several genes related to mitochondria biogenesis and function, as well as brown/beige adipogenesis, was also observed in the homogenates of visceral adipose tissue samples from TTh-hypogonadal obese subjects, as compared to those of eugonadal and untreated-hypogonadal ones.

Another striking observation of our study is that untreated hypogonadism was associated with a three-fold higher intrahepatic level of triglycerides (assessed in liver biopsies), as compared to eugonadism. This marked increase was not observed in TTh-subjects, whose levels were comparable to eugonadal ones. It is noteworthy that intrahepatic triglyceride content was closely correlated to the histological score of steatosis and with the NAFLD activity score. The fact that we did not observe a parallel decrease in triglycerides levels in TTh-subjects suggests that liver changes could be considered not as the consequence of lower circulating triglycerides, but indeed as the primum movens of the metabolic actions of TTh. As a result, the reduction in hepatic steatosis may be detectable before its consequences appear systemically. Importantly, TTh was also associated with an increased liver expression of all the genes related to insulin signaling, such as GLUT4, IRS1, STAMP2 [93‐95], insulin-independent glucose transport (GLUT2) and genes related to glycogen synthesis (of note, glycogen synthase 2, GYS2), and intrahepatic lipid handling. The data relative to the increased expression of GYS2 are substantiated by previous results showing that testosterone replacement favored glycogen synthesis in the skeletal muscle in animal models [96]. Liver from TTh-hypogonadal subjects showed a significant upregulation of FAS, the rate-limiting enzyme in the fatty acid biosynthesis and HMGCs, a catalytic enzyme of ketogenesis that provides lipid-derived energy. Noteworthy, the liver specific FAS knockout aggravates hepatic steatosis, thus suggesting that FAS has a prominent protective effect in the liver [97, 98].

SCD1, an enzyme catalyzing the synthesis of oleate, was also increased in the TTh group, as compared to the untreated one. SCD1 is involved in carbohydrate-induced activation of FAS and other lipogenic enzymes, and recently proposed as a potential therapeutic target to control obesity and the progression of related metabolic diseases including hepatic steatosis [99]. Therefore, the SCD1 mRNA increase in a context of histologically- and biochemically-proven intrahepatic lipid reduction, as observed in T-treated subjects, should be interpreted cautiously. It is plausible that its mRNA increase, in this context, represents a compensatory mechanism in response to a general reduction of liver lipogenesis, rather than a marker of an increased fatty acid formation occurring upon TTh. In line with this interpretation, mRNA expression level of several genes related to liver lipid handling, metabolism and β-oxidation showed significant positive correlations with calculated free T, as assessed at the pre-surgery visit. The vast majority of these genes were also closely associated with AR mRNA expression. More importantly, histological total NAS score, as well as the subscores related to steatosis and inflammation within the liver, decreased as a function of calculated free T level at the pre-surgery visit. TTh-hypogonadal subjects showed also a significant reduction of fatty liver index (FLI) and total cholesterol level overtime. In particular, fatty liver index (FLI)—calculated by a specific algorithm based on BMI, waist circumference, triglycerides and yGT—is recognized as an accurate predicting parameter of histologically proven fatty liver [43]. Therefore, given that our main findings are based on differences among treatment groups as assessed within surgical samples, the significant change over time of FLI further strengthened the suggested beneficial effect of TTh on the liver. An association between lower levels of total T and the development of NAFLD has already been described in the clinical [100‐102] and preclinical setting [31, 103‐105]. A significant improvement of non-alcoholic liver steatosis upon treatment of hypogonadal elderly men with parenteral T undecanoate has already been reported [32, 33, 106]. However, having a liver biopsy collected from patients receiving TTh before the surgical procedure, which demonstrated a significant reduction of liver abnormalities, is one of the main strengths of our study.

In contrast to earlier findings [27], we did not find any significant differences between TTh and untreated-hypogonadal subjects in terms of BMI, or glyco-metabolic indices. These discrepancies might be attributed to the shorter therapy duration of our study, as compared to the previous observational trials [27]. Indeed Corona and co-workers found that the strength of the beneficial effects of TTh on weight increased as a function of trial duration. To make an example, body weight showed a significant reduction after 24 months of TTh, with an estimated loss of − 3.50 [− 5.21; − 1.80] kg [27]. Our observational study was designed to evaluate the effect of TTh based on the time interval spent on the waiting list before the patients received bariatric surgery (roughly 12 months). Another important limitation of the present study lies in the fact that it cannot establish causality due to its observational design. Therefore, it could be hypothesized that men in the eugonadal and hypogonadal (asymptomatic) untreated groups are healthier than the hypogonadal treated (symptomatic) patients. Hence, it could be hypothesized that men in the hypogonadal untreated group are intrinsically less exposed to the detrimental effects of T deficiency than the investigated treated group, made up of hypogonadal symptomatic patients. This establishes a possible selection bias. However, baseline value of body weight, waist circumference, glycol-lipid parameters and blood pressure of the three groups were not statistically different, therefore minimizing the risk of a baseline better cardio-metabolic profile in the TTh arm. Similarly, other possible confounders, including the prevalence of comorbidities (type 2 Diabetes Mellitus, hypertension, cardiovascular diseases), the use of glucose- and lipid-lowering drugs and of PDE5 inhibitors did not show a significant difference between the three groups. It should also be considered that liver and visceral fat biopsies were performed only at the time of bariatric surgery; therefore, data on hepatic inflammation, steatosis and triglyceride content do not allow for a comparison between post-treatment and baseline. This limitation, due to the invasiveness of tissue sample collection, is, at least partially, overcome by the use of the eugonadal group as the comparator for the analyses on these tissues and cells. Finally, a Magnetic Resonance Imaging (MRI) study of liver at baseline and after TTh or a FibroScan evaluation of hepatic tissue would have add interesting data.

In conclusion, we provide evidence that TTh in hypogonadal symptomatic severely obese patients is associated with a reduced preadipocytes dysfunction through an increase of insulin sensitivity, an improvement in mitochondrial function and lipid handling, eventually leading to their differentiation towards a metabolically healthier phenotype. Similarly, testosterone was associated with reduced hepatic steatosis and intrahepatic content of triglycerides. Randomized clinical trials are warranted to confirm these observations.