Bone density and genomic analysis unfold cold adaptation mechanisms of ancient inhabitants of Tierra del Fuego Mikiko Watanabe1 na1, Renata Risi1 na1, Mary Anne Tafuri2, Valentina Silvestri3, Daniel D’Andrea4, Domenico Raimondo3, Sandra Rea5, Fabio Di Vincenzo6,7, Antonio Profico2, Dario Tuccinardi8, Rosa Sciuto5, Sabrina Basciani1, Stefania Mariani1, Carla Lubrano1, Saverio Cinti9, Laura Ottini3, Giorgio Manzi2 na1 & Lucio Gnessi1 na1 Scientific Reports 11, Article number: 23290 (2021) Cite this article EndocrinologyEnvironmental sciencesEvolution The Fuegians, ancient inhabitants of Tierra del Fuego, are an exemplary case of a cold-adapted population, since they were capable of living in extreme climatic conditions without any adequate clothing. However, the mechanisms of their extraordinary resistance to cold remain enigmatic. Brown adipose tissue (BAT) plays a crucial role in this kind of adaptation, besides having a protective role on the detrimental effect of low temperatures on bone structure. Skeletal remains of 12 adult Fuegians, collected in the second half of XIX century, were analyzed for bone mineral density and structure. We show that, despite the unfavorable climate, bone mineral density of Fuegians was close to that seen in modern humans living in temperate zones. Furthermore, we report significant differences between Fuegians and other cold-adapted populations in the frequency of the Homeobox protein Hox-C4 (HOXC4) rs190771160 variant, a gene involved in BAT differentiation, whose identified variant is predicted to upregulate HOXC4 expression. Greater BAT accumulation might therefore explain the Fuegians extreme cold-resistance and the protection against major cold-related damage. These results increase our understanding of how ecological challenges have been important drivers of human–environment interactions during Humankind history. Subsequently to the dispersal of Homo sapiens out of Africa during the Paleolithic1, human populations required adaptations to diverse climatic conditions2 achieved through morphological and cultural adjustments and metabolic mutations3, such as brown adipose tissue (BAT) upregulation in cold climates4. BAT is a highly heterogeneous energy-expending tissue that generates heat, characterized by extraordinary plasticity5. BAT helps maintaining core temperature in a cold environment without shivering. In humans, BAT is found primarily in infants and young children. Defined regions of functionally active BAT are present in some but not all adults (3–10%)6,7 in thermoneutral conditions (approximately 18–24 °C) and may be quantified noninvasively with the use of (18)F-fluorodeoxyglucose ((18)F-FDG) positron-emission tomographic and computed tomographic (PET-CT) scans. BAT negative individuals do not have measurable metabolically active BAT, provided that the temperature conditions the BAT measurement was carried out are given. For humans in typical living conditions (with clothing and control over environmental temperature) the contribution of BAT to total energy expenditure is believed to be very small. BAT can be activated with various levels of intensity depending on sex, age, and ethnicity by cold exposure6,8.The interest in understanding the mechanisms of cold adaptation comes both from obvious anthropological and evolutionary implications, and from theories that attribute to populations with higher BAT activity an increased resistance to obesity and diabetes9. When Europeans reached Tierra del Fuego in 1520, it was inhabited by four ethnic groups: the Yamana/Yaghan, Haush and Kaweskar/Alakaluf, all hunter-fisher-gatherers spending a significant portion of their time in the canoes and swimming, and the Selknam, terrestrial hunters, altogether generally grouped under the term Fuegians10. These ethnic groups were identified as different communities in the XX century11. The descendants of Fuegians show a population continuity as supported by the genetic affinity of modern Yamana and Alakaluf with ancient individuals from their respective regions and a mixture of European ancestry reflecting post-colonial admixture12,13,14. What is known of the ancient inhabitants of Tierra del Fuego mostly comes from navigators and ethnographers’ reports15,16. All were nomadic hunter-gatherers, and they had an exceptional cold resistance: despite the extremely harsh climate, they did not use any closed clothing, and open fires and animal fat smeared on bare skin appeared inadequate means of protection to those describing them10.The climate in this region is inhospitable. It is a subpolar oceanic climate (Köppen climate classification Cfc) with short, cool summers and long, wet, moderate winters: the northeast is characterized by strong winds and little precipitation, in the south and west it is very windy, foggy, and wet for most of the year and there are very few days without rain, slush, hail or snow. Temperatures are steady throughout the year and across several centuries17: in Ushuaia they hardly surpass 9 °C (50 °F) in summers and average 0 °C (30 °F) in winters. Snowfall can occur in summer. The cold and wet summers help preserve the ancient glaciers.Cultural and physiological mechanisms implicated in cold adaptation include clothing and shelter, and bodily changes such as peripheral vasoconstriction, muscle and subcutaneous fat-mediated insulation, lower surface area-to-mass ratio18, thyroid hormone levels variation19,20, high-fat and -protein diet19,21, BAT over-activity, and white adipose tissue browning18,22,23. In humans, BAT increases upon cold exposure6,24, and it generates heat via upregulation of uncoupling protein-1 (UCP1), increasing basal metabolic rate (BMR)25. Interestingly, in 1960 Hammel studied nine Fuegians belonging to the Alakaluf group, showing that their BMR was about 160% of that reported for the cold exposed “average white man” (p. 24)26. This report raised the suspicion that the Fuegians might have had increased BAT activity at the root of their amazing cold adaptation.Beyond the interest in the role of BAT in thermoregulation and as a target for the prevention and treatment of obesity and type 2 diabetes, cold-activated BAT is also positively related to bone mineral density (BMD)27 and femoral cross-sectional area (CSA)28. BAT volume parallels BMD in some29, although not all reports30, and predicts femoral CSA and cortical bone area (CBA)28,31. Moreover, BAT-impaired cold-exposed mice show more bone loss than WT via β2 adrenergic receptor activation, this loss being reduced through β2 adrenergic receptor pharmacologic blockade32; and UCP1 knock-out (KO) mice under permanent cold stress have lower bone formation and mineralization compared to WT33. These altered bone phenotypes are not seen in thermoneutral UCP-1 KO and WT mice, indicating that BAT may prevent, although not eliminate, an otherwise massive cold-induced bone loss33. Finally, it is well established that populations living in cold geographic areas have reduced bone density despite milder temperatures during summers34,35,36, and recent evidence shows that they also have increased BAT activity37.To test the hypothesis that BAT could have contributed to the exceptional cold adaptation of the ancient Fuegians, and in the absence of more direct means to investigate BAT in this population, we leveraged the reported correlation between bone and BAT activation by evaluating bone morphology and density of the skeletal remains of the collection of Fuegians preserved in the Museum of Anthropology ‘G. Sergi’ of the Sapienza University of Rome (Italy). To deepen the possible link, we then analyzed genomes of this population available in public repositories for variants in genes involved in BAT modulation.To confirm BAT and bone physiology interconnection, widely reported in animal models but less established in human beings, we first explored the correlation between BAT and femoral cortical thickness in a living population composed of 34 BAT expressing individuals from a previously described large cohort6. In line with previous reports, BAT volume and activity were directly related to femoral CBA (Table S1). Moreover, BAT volume independently predicted CBA after height and muscle area (Table S2). After confirming this association in the living population, we analyzed Fuegians’ bones for comparison.The Fuegians skeleton series preserved in Rome is composed of 14 complete skeletons; it represents one of the largest collections outside Argentina and is in an exceptionally good state of preservation. Twelve adults were included, and their bones were analyzed (Table S3). Fuegians femoral CBA and CSA were significantly lower than those of BAT positive and negative living humans, whereas the endocortical bone area (EndA) was comparable (Fig. 1A). Gender stratification suggests similar differences across groups, although the small sample size does not allow for analysis (Fig. 1B–D). A dual-energy X-ray-absorptiometry (DXA) of the Fuegians femur and lumbar spine remains showed no different BMD compared with a subpopulation of 12 matched BAT expressing and 12 non-expressing subjects living in temperate zones (mean yearly temperature of 16 °C) and selected among the cohort described above6 (Fig. 1E).Figure. 1Bone geometry and density comparison of a modern BAT+ and BAT− population living in a temperate area and cold adapted Fuegian skeletal remains. (A) Living subjects expressing Brown Adipose Tissue (BAT+) are not significantly different in terms of femoral geometry [height (h) standardized Cross sectional (CSA), Cortical Bone (CBA) and Endocortical Area (EndA)] compared to living subjects not expressing BAT (BAT−). Conversely, Fuegians CSA and CBA are lower, with EndA not being significantly different. (B–D) Height standardized CSA, CBA, and ENDa stratified by gender. (E) Living subjects expressing Brown Adipose Tissue (BAT+) are not significantly different in terms of bone mineral density (BMD) compared to living subjects not expressing BAT (BAT−) and the Fuegians at both lumbar and femoral level. LS, Lumbar spine; FN, Femoral Neck; TH, Total Hip; ***P < 0.001; ns, not significant.The observation that, despite their exposure to environmental conditions unusually detrimental for bone34,35,36, the Fuegians showed similar BMD compared to modern subjects living in temperate zones supports the hypothesis that these peoples had some sort of mechanism capable of protecting their bones from even more negative consequences. Similar to what observed in mouse models, where BAT is capable of partially limiting bone loss consequent to chronic cold exposure38, the hypothesis that a high expression of BAT could have contributed to protect the bones of the Fuegians in terms of BMD, though not enough to avoid thinner cortical bone, was still standing, and led us to test whether the Fuegians might have had an abundance of BAT through a genomic approach.We therefore analyzed available genomes of this population, and we obtained reliable results for five of the 11 ancient Fuegian genomes retrieved, of which three were Yamanas, one was Selknam and one Alakaluf39. First, we selected a panel of genes involved in the differentiation, metabolism, and accumulation of BAT, as these pathways are reasonably the ones responsible for differential thermoregulation and thermogenesis in human populations22. The panel was identified by Sazzini et al. based on literature revision and after exploration of protein–protein interactions in the String database (http://string-db.org/). Some genes with unknown biological functions were included if they had been associated with obesity in genome-wide association studies22. Coding and non-coding variants in these genes involved in BAT functional pathways were identified in the Fuegian genomes (Dataset S1). All variants found in the Fuegians were searched in a control population of 14 individuals including 10 Siberians, 2 Athabascan and 2 Greenlanders39. These individuals, all living in cold areas of the globe (modern Alaska, Greenland and Siberia), were selected for the less striking cold adaptation leading to different lifestyle and habits, such as the use of warm clothing, compared to Fuegians, whose main protections from the harsh climate were open fires, animal fat smeared on bare skin and at most a small piece of fur over the shoulders.Interestingly, a coding variant previously associated with cold adaptation22, the cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) rs75385144, was identified in four out of five Fuegians, with an allelic frequency of 0.4 (Table 1). This candidate variant is reported with a frequency of 0.33 in the gnomAD database, however it has a very low frequency in the African population (0.07) compared with European, American and Asian populations (0.3–0.4)22. Notably, the variant was found in a frequency comparable to Fuegians in our control population including Siberians, Athabascan and Greenlanders (11 out of 14 individuals, allelic frequency of 0.39, p-value for comparison = 0.95). These results suggest that this variant might be implicated in cold adaptation, as the low frequency in African population suggested, but not in different ways across populations.Table 1 Summary of relevant genomic variants.Moreover, a statistically significant difference in frequency between Fuegians and the control population emerged for two non-coding variants, PR domain containing 16 (PRDM16) rs2493270 and Homeobox protein Hox-C4 (HOXC4) rs190771160, both genes being involved in BAT differentiation22,40 (Table 1, Fig. 2).Figure 2Non-coding variants of a panel of 28 genes involved in BAT functional pathways identified in at least 2 Fuegian genomes. All variants found in the Fuegians (890, 894, 895, MA577, Nr74) were searched in control population of 14 individuals including 10 Siberians (Alt1, Alt2, Esk17, Esk20, Ket1, Ket 2, Yak1, Yak2), 2 Athabascan (Athabascan_1, Athabascan_2) and 2 Greenlanders (Greenlander_1, Greenlander_2). Red arrows: variants of interest described within the manuscript.PRDM16 is present in brown adipocytes and is able to upregulate UCP-1 expression, thus increasing the activity of BAT. Its overexpression is also associated with browning in mouse models41. Its variant rs2493270 was found in three out of five Fuegians and was not identified in controls (p = 0.002) (Table 1, Fig. 2). In gnomAD, the variant is reported with a very low frequency in the Finni
https://www.nature.com/articles/s41598-021-02783-1
Bone density and genomic analysis unfold cold adaptation mechanisms of ancient inhabitants of Tierra del Fuego
