This study showed that ectopic fat distribution is associated with the presence of CAN in participants with AD but not in T2D. More specifically, we found that a visceral body fat accumulation phenotype in participants with CAN compared to those without CAN among people with AD, with greater total fat mass, trunk fat mass, VAT mass and TLR in the group of participants affected by CAN. Overall, our study corroborates the existing literature suggesting a relationship between central obesity and autonomic dysfunction [12, 13, 17,18,19,20,21, 24]. However, to the best of our knowledge, this is the first study providing an in-depth evaluation of body fat distribution in relation to CAN in people with AD, also providing a comparison with people with T2D. Before us, Voulgari and colleagues found that waist circumference was higher among people with CAN in both in AD and T2D [14]. Also in this case, the relationship was stronger in AD than in T2D [14]. Our results expand this knowledge by providing a more detailed evaluation of body fat distribution through DXA method, rather than limiting only to waist circumference, also including estimates of VAT, trunk fat and adipokines. Since overweight and obesity are becoming much more common among people with AD [27,28,29,30,31], there is a need for studies like this, elucidating the relationship between adiposity and the clinical course of AD. Some evidence suggest that obesity is associated with poorer glycemic control, higher total insulin daily dose and increased risk for atherosclerotic CVD in patients with AD [27, 29], all factor potentially associated with CAN. However, in our study the relationship between adiposity and CAN was independent from confounders such as age, BMI, sex and HbA1c%, diabetes duration.

In people with T2D, Jang et al. observed that higher CT-scan derived VAT level was associated with poorer heart rate response to Valsalva maneuver [21]. Also, an association between CT-scan derived VAT levels and signs of CAN, evaluated with 123 I-MIBG scintigraphy, was found in an even smaller population of 24 Japanese people with T2D [15]. Differently from previous studies, we used three cardiovascular autonomic reflex tests among those recommended for the clinical diagnosis of CAN, providing a more robust evaluation of clinically relevant CAN, which was not associated with ectopic fat distribution in people with T2D, but only in AD. This crucial difference in the diagnostic evaluation of CAN, as well as the different populations enrolled, might explain the contrasting results with regards to the relationship between body fat and CAN in T2D.

The mechanisms underlying the stronger association between ectopic fat distribution and CAN observed in AD compared to T2D remain to be addressed. On the one hand, CAN might be a consequence of fat mass excess and abnormal distribution [12, 13, 17,18,19,20,21, 24]. However, in this case, worse autonomic dysfunction should have been found in people with greater amount of fat and VAT mass, such as those with T2D. On the other hand, preclinical and clinical models showed that sympathetic and parasympathetic systems regulate body fat storage and distribution [24] and modulate processes of lipogenesis, thermogenesis and inflammation [25, 26], suggesting that the dysautonomia observed in patients diagnosed with CAN might itself exert a detrimental effects on AD distribution and function. Based on our results, we could speculate that such detrimental effects could be observed in a population with a lower amount of fat and without the metabolic derangements classically observed in T2D, while they could have been blunted in people with an already higher amount of ectopic fat due to other causes.

Our study should be read in the light of some limitations. First, CARTs strictly depends on participants’ compliance and might miss very early stages of autonomic impairment; however, they have been broadly recommended as a screening tool for CAN in patients at risk [8].In addition, CAN was assessed only by three reflex tests (heart rate response to deep breathing, heart rate response to lying-to-standing test, orthostatic hypotension test). Since cardiovascular autonomic reflex tests are time-consuming, a clinical rather than research-focused CAN assessment without resting heart rate variability measures was performed.

In this regard, a simplified battery encompassing a reduced number of tests is suggested, and having one positive heart rate test out of two or three is an internationally recognized sufficient criterion for early CAN diagnosis [8]. Furthermore, according to a recent study by Pafili et al., heart rate response to deep breathing has the highest sensitivity and sensibility for CAN diagnosis, emerging as the most useful diagnostic test alone for accurate screening [36]. In addition, although we are aware that the presence of abnormalities in more than one test on several occasions is preferable for CAN diagnosis [5], we performed CARTs only once due to clinical practice limitations. To note, current guidelines suggest that a single abnormal result among the two or three heart rate tests represent a sufficient criterion for CAN diagnosis [5]. Finally, since this is a cross-sectional study, we cannot draw conclusions about any cause-effect relationship between CAN and abnormal body fat distribution.

On the other hand, we recognize that our work has the significant strength of being the first study evaluating the relationship between adiposity distribution and CAN in a population composed of both AD and T2D. Moreover, we focused not only on fat mass excess, but also on body fat distribution parameters, including estimates of VAT and trunk fat and adipokines. In fact, it is widely recognized that ectopic fat deposition, more than parameters of absolute adiposity, is associated with unhealthy metabolic profile and cardiometabolic complications [37].

In conclusion, our findings suggest that ectopic fat distribution is more strongly associated with CAN in people with AD than in those with T2D. While the causative relationship between CAN and ectopic fat deposition cannot be established, our study highlights the significance of abnormal adipose tissue expansion in the cardiometabolic risk profile of people with AD. These findings emphasize the need for further research to clarify the impact of overweight and obesity on cardiometabolic health in AD.