Romain Walcker
email: romain.walcker@univ-tlse3.fr

Dov Corenblit

Frédéric Julien

Jean-Michel Martinez

Johannes Steiger

Contribution of meandering rivers to natural carbon fluxes: evidence from the Ucayali River, Peruvian Amazonia

Keywords: Amazon, carbon, floodplain, forest age, meandering river, remote sensing

Contribution of meandering rivers to natural carbon fluxes: evidence from the Ucayali

River, Peruvian Amazonia

Abstract

Better understanding the fate of the atmospheric carbon (C) captured by plant photosynthesis is essential to improve natural C flux modelling. Soils are considered as the major terrestrial bioreactor and repository of plant C, whereas channel networks of floodplain rivers collect and transport, throughout the aquatic continuum, a significant part of plant primary production until its export through outgassing or sequestration in marine sediments. Here, we show that river meandering in forested floodplains is a crucial and widely overlooked Earth surface process promoting C fluxes from the atmosphere to the aquatic continuum, via the floodplain vegetation. Over a recent period of 35 years , we quantified those C fluxes in one of the most active meandering rivers on Earth, the Ucayali River, Peru, South America. We used map time series combined with above-ground forest C data to derive the amount of C that is annually captured by the growing floodplain vegetation within the active meander belt, as well as exported to the aquatic continuum by lateral channel erosion. We found that the annual building and erosion of forested floodplain areas was nearly balanced over time with 19.0±7.7×10 3 ha -1 yr -1 and 19.8±6.7×10 3 ha -1 yr -1 , respectively. While growing forests within the active meander belt annually captured 0.01±0.05×10 6 Mg C yr -1 , lateral channel erosion provided the nearly 100-fold amount of C to the river channel and its streamflow, i.e. 0.9±0.4×10 6 Mg C yr -1 . Our findings revealed that the migration of the Ucayali River channel provided nearly 10-times more lignified C per unit area to the aquatic continuum (44.7±21.4

Mg C ha -1 yr -1 ) than non-meandering central Amazonian floodplains do. Together, these findings point to the importance of quantifying the overall contribution of meandering rivers to natural C fluxes worldwide.

Introduction

Floodplains are areas of high primary production which provide fresh autochthonous carbon (C) to river waters through litterfall, plant and micro-organism respiration [START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF].

Levels of plant production in floodplains and the extent of the floodplain area can therefore considerably modulate the overall C quantity found in river waters [START_REF] Ward | Where Carbon Goes When Water Flows: Carbon Cycling across the Aquatic Continuum[END_REF]. Hence, floodplain vegetation, algae and floating macrophytes in highly productive and preserved tropical areas, directly and rapidly provide organic and inorganic C to rivers [START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF][START_REF] Sawakuchi | Carbon dioxide emissions along the lower Amazon River[END_REF]. [START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF] further suggested that central Amazonian wetlands export half of their gross primary production to rivers, being the primary sources for CO2 outgassing by the Amazon.

Over the last decade, several studies successively updated regional and global estimates of river C fluxes from land to oceans [START_REF] Drake | Terrestrial carbon inputs to inland waters: A current synthesis of estimates and uncertainty[END_REF]. These studies underscored large data gaps and uncertainties, suggesting that C fluxes might be much larger than previously thought. In particular, uncertainties in the contribution of wetlands and macrophytes as a source of C for rivers has been highlighted [START_REF] Spencer | Chromophoric dissolved organic matter export from US rivers[END_REF] and the need for new investigations identified. Furthermore, river meandering, among other processes contributing to C input into rivers, river meandering remains however a largely overlooked Earth surface process [START_REF] Peixoto | Spatial and temporal dynamics of river channel migration and vegetation in centralAmazonian white-water floodplains by remote-sensing techniques[END_REF]).

Yet, this process is recognized in sculpting the Earth surface since more than 5 centuries, and studies have recently pointed out its potential role in biogeochemical fluxes (Iepli & Lapôtre, 2019). Abiotic factors, like sedimentary dynamics in meandering rivers, have been suggested as essential than biotic factors, like productivity and respiration, in the terrestrial organic C cycle [START_REF] Torres | Model predictions of long-lived storage of organiccarbon in river deposits[END_REF][START_REF] Ielpi | A tenfold slowdown in river meander migration driven by plant life[END_REF].

Typically, meandering rivers are characterized by lateral bank erosion occurring at the outer meander bends, whereas lateral accretion processes form point bars within inner meander bends [START_REF] Leopold | River meanders[END_REF][START_REF] Allen | A review of the origin and characteristics of recent alluvial sediments[END_REF]. Within the Amazon basin, lateral channel migration generates meander scrolls forming a sequence of ridges and swales, on which a sequential primary forest succession of high productivity rapidly takes place [START_REF] Salo | River dynamics and the diversity of Amazon lowland forest[END_REF][START_REF] Junk | The flood pulse concept in riverfloodplain systems[END_REF][START_REF] Puhakka | River Types, Site Evolution and Successional Vegetation Patterns in Peruvian Amazonia[END_REF]. Forest succession, leading eventually to the mature stage, continues when the channel further migrates and point bars are integrated into the floodplain. Conversely, channel migration also causes bank erosion which in turn promotes direct massive inputs of coarse woody material to river channels, thus providing substantial quantities of autochthonous C, potentially exceeding those imported through litterfall and respiration. Active river meandering closely linked to point bar formation, floodplain aging and lateral channel erosion, thus presents a relevant Earth surface process to consider for accurately quantifying and understanding floodplain C sequestration and its export to the aquatic continuum [START_REF] Wohl | Organic carbon export in the form of wood during an extreme tropical storm, Upper Rio Chagres, Panama[END_REF][START_REF] Sutfin | Banking carbon: A review of organic carbon storage and physical factors influencing retention in floodplains and riparian ecosystems[END_REF].

Here, we aimed to provide evidence that river meandering in Amazonian floodplains significantly contributes to river C budgets. Our objective was to quantify the amount of C that annually accumulates in the forest biomass, as well as the part that is annually exported to the river channel, within the active meander belt of the river, i.e. the area where contemporary channel migration occurred. We investigated one of the major active tributaries of the Amazon, the Ucayali River, Peru, over a period of 35 years . We used the yearly water classification history dataset provided by the Joint Research Centre to produce annual maps of point bar formation, floodplain ages and river channel erosion in the 1984-2019 active meander belt. Annual maps were combined with a model predicting the living above-ground forest C stock from the floodplain age to derive amounts of C annually fixed by net primary production and exported to river waters after bank erosion. The quantity, quality and fate of the C provided by Amazonian meandering rivers to the aquatic continuum is discussed in respect to the existing literature.

Method

Study area

The Ucayali River is located in the western part of the Amazon basin (Fig. 1a). It is a major tributary of the Amazon River extending approximately 900 km from its upstream confluence with the Rio Tambo and Rio Urubamba to its downstream confluence with the Marañón River.

The mean annual water discharge is about 12 100 m 3 s -1 and water depth between low and high flood flows can range from 8 to 15 m [START_REF] Santini | An index concentration method for suspended load monitoring in large rivers of the Amazonian foreland[END_REF]. The maximum and minimum monthly flows, recorded over the 1996-2018 period at the Requena gauging station, is of 22 900 and 2 500 m 3 s -1 respectively (HYBAM, 2020). Close to the town of Pucallpa, the floodplain width is about 25 km and the active meander belt is approximately 10 km in width (Fig. 1b,c).

The Ucayali is considered as one of the last pristine free meandering white-water rivers worldwide, characterized by high suspended sediment loads, nutrient-rich waters and high lateral channel migration rates within an unconstrained, forested floodplain [START_REF] Constantine | Sediment supply as a driver of river meandering and floodplain evolution in the Amazon Basin[END_REF]. Within meandering floodplain rivers, forest primary succession is distinctly age-zonated [START_REF] Salo | River dynamics and the diversity of Amazon lowland forest[END_REF][START_REF] Junk | The flood pulse concept in riverfloodplain systems[END_REF][START_REF] Puhakka | River Types, Site Evolution and Successional Vegetation Patterns in Peruvian Amazonia[END_REF]. The vegetation succession progresses from early successional stages dominated by short-lived species to latter successional stages dominated by long-lived species along a cross-river and age gradient [START_REF] Salo | River dynamics and the diversity of Amazon lowland forest[END_REF][START_REF] Lamotte | Fluvial dynamics and succession in the Lower Ucayali River basin, Peruvian Amazonia[END_REF]Fig. 2). The early stage of primary succession begins on newly formed alluvial bars, mainly point bars, rapidly colonized by Tessaria integrifolia L. and Gynerium sagittatum, forming dense canopies of about 5-8 m in height. This early pioneer stage is replaced by a second stage dominated by Cecropia tessmannii, forming a closed canopy of about 14-18 m in height. The Cecropia forest is located on older meander scrolls in backwater depressions.

During these two first stages, vegetation contributes to sediment and organic matter trapping, which leads to floodplain construction and soil C sequestration (Fig. 2; [START_REF] Salo | River dynamics and the diversity of Amazon lowland forest[END_REF]. The following stage is a mixed forest dominated by Ficus insipida and Cedrela odorata, forming a closed canopy of about 20-25 m in height. At this stage, floodplain surface elevation is about 5 m above the low water level. Its topography is more homogeneous compared to the pioneer zone in the previous stages of succession. This is due to the deposition of large amounts of silt in backwater depressions during seasonal flooding. The later successional mixed forest is more diversified and composed of large trees with a canopy of about 40 m in height. Soil topography of the floodplain at this level (> 5-10 m above the low water level) is generally flat with only minor remaining depressions. This forest succession trajectory is basically the same at each meander of the Ucayali River [START_REF] Kalliola | New Site Formation and Colonizing Vegetation in Primary Succession on the Western Amazon Floodplains[END_REF]. Lateral channel erosion leads to a continuous reset and regeneration of the primary vegetation succession over large areas [START_REF] Salo | River dynamics and the diversity of Amazon lowland forest[END_REF][START_REF] Kalliola | New Site Formation and Colonizing Vegetation in Primary Succession on the Western Amazon Floodplains[END_REF]. Since point bars of the Ucayali River are either rapidly eroded or integrated into the forested floodplain during lateral channel migration, we will, hereafter, exclusively distinguish the river channel from its floodplain, including point bars.

Mapping floodplain formation, aging and erosion

We used the yearly water classification history dataset (v1.2) provided by the Joint Research Centre (JRC) and freely available on the Google Earth Engine (GEE) platform [START_REF] Gorelick | Google Earth Engine: Planetary-scale geospatial analysis for everyone[END_REF]. This dataset was firstly created by [START_REF] Pekel | High-resolution mapping of global surface water and its long-term changes[END_REF] and now consists in 36 annual raster maps spanning the 1984-2019 period. Pixels size approximately 900 m² at ground level and are classified in 'no data', 'non-water', 'seasonal water' or 'permanent water' (Fig. S1 in Appendix A). In order to create an envelope for data extraction, we firstly downloaded geometries (i.e. polylines shapefiles) of the Ucayali River from the Open Street Map database available at https://download.geofabrik.de. Then, we created a 10-km buffer across the river using the ArcGIS software package (ESRI, Redland, USA). The yearly water classification history dataset overlaying the river buffer was finally downloaded from the GEE platform.

Using the MatLab software package (Mathworks, Natick, USA), the 36 annual raster maps were proceeded in order to map annual areas of floodplain formation, ages and erosion. The algorithm was applied pixel-by-pixel and year-by-year as illustrated in Figure 3. Pixels that were classified as 'seasonal water' or 'permanent water' at a given year (e.g. 1984; Fig. 3a), and classified 'non-water' the following year (e.g. 1985), were mapped as 1-year-old pixels.

Each year from 1985 to 2019, every pixel age-determined at a given year (e.g. 1-year-old in 1985) and remaining 'non-water' the following year (e.g. 1986) was incremented with one additional year (e.g. 2-year-old in 1986). Remaining 'non-water' and 'no data' pixels with no age between successive years (e.g. between 1984 and 1985) were set to age 'non-determined' (ND). Typically, ND pixels corresponded to floodplain and upland areas created before 1984.

Each year, from 1985 to 2019, every pixel age-determined at a given year (e.g. 2-year-old in 1986) and classified 'seasonal water' or 'permanent water' the following year (e.g. 1987) was considered as an eroded area (Fig. 3b) and reset to age 0 (e.g. 0-year-old in 1987).

Deriving C-stocks from floodplain ages

In line with Pahukka et al. (1992), we assumed that on the Ucayali River, pioneer vegetation colonizes bare alluvial bars from the year of their creation (Fig. S2 in Appendix A). We thus considered that the vegetation age is equal to the floodplain age. We used published data to predict the forest above-ground carbon stock (C-stock in Mg C ha -1 ) from the floodplain age (age in years), and constructed a model predicting C-stock as a function of age (Fig. 3c). The data were published by [START_REF] Schöngart | Biomass and Net Primary Production of Central Amazonian Floodplain Forests[END_REF] and based upon thousands of in situ vegetation structure inventories and tree ring measurements within floodplains of central Amazonia. Forest above-ground carbon stocks were estimated in several, almost undisturbed successional stages, with stands varying between 7 and 240 years of age. Forest structure data and several allometric models were used to construct a data series of C-stocks per forest age.

First, we extracted data from the figure 18.10 presented by [START_REF] Schöngart | Biomass and Net Primary Production of Central Amazonian Floodplain Forests[END_REF] using Web Plot Digitizer 4.3 software (Ankit Rohatgi, Pacifica, California, USA). Second, we fitted a 3 rd order polynomial model to the 35 first years of the data series. These 35 years corresponded to the forest establishment that we analysed on the Ucayali River between 1984 and 2019. For floodplain forests established before 1984 (ND ages; Fig. 3a,b), we assumed a constant C-stock, i.e. old-growth forests with ages > 70-year-old in the data series. This assumption was based on the floodplain age structure of eroded outer meander bends presented in the results section.

Results

Annual vegetated floodplain area formation and erosion

Over the 1984-2019 period, the water surface area of the Ucayali River accounted for 220±25 ×10 3 ha (annual average and its standard deviation; Appendix B). Maximum water surface area extensions were observed in 1988, 1994, 2001, 2011, 2017and minimums in 1987, 1992, 1997[START_REF] Mayorga | Young organic matter as a source of carbon dioxide outgassing from Amazonian rivers[END_REF][START_REF] Schöngart | Biomass and Net Primary Production of Central Amazonian Floodplain Forests[END_REF], 2013and 2018 (Fig. 4a (Fig. 4a). Negative anomalies appeared mainly before 2000 and positives anomalies mainly after 2000. Yearly formation of floodplain areas occurred mainly within inner meander bends and accounted in average for 19.0±7.7×10 3 ha -1 yr -1 (Fig. 4b; Fig. 5; Appendix B and C). Conversely, floodplain erosion was located mainly in outer meander bends and closely balanced floodplain formation with an average of 19.8±6.7×10 3 ha -1 yr -1 (Fig. 4c; Appendix B and C).

Age structure of vegetated floodplain areas

We found that meandering promotes rejuvenation and recurrent formation of large areas of bare alluvial bars that are quickly colonized by pioneer vegetation. Over the 1984-2019 period, ages of floodplain areas were unevenly distributed (Fig. 6a). One-year-old floodplain areas largely dominated. However, about 1/3 of these newly formed floodplain areas were eroded the following year. Indeed, we found that older age classes generally decreased in areas following a negative logarithmic trend (Fig. 6a). This trend resulted from floodplain erosion (Fig. 6b).

Newly established floodplain areas at inner meander bends were primarily affected by erosion at a rate of 6.4±3.3×10 3 ha -1 yr -1 , i.e. 1/3 of the yearly formed floodplain surface area (19.0±7.7×10 3 ha -1 yr -1 ). The rate of erosion decreased from younger to older floodplain areas (Fig. 6b). Floodplain erosion occurred equally in outer meander bends and in areas established before 1984, i.e. non-determined in age (ND) in the present study. Erosion in areas of ND ages accounted annually for about 7.0±3.8×10 3 ha -1 yr -1 (Fig. 6b; ND).

Annual changes in floodplain forest C-stocks

Our model used to derive C-stocks from floodplain ages showed a very good adjustment to the data (R² = 0.99; Fig. 7). One-year-old vegetation accounted for an average of 0.7 Mg C ha -1 and increased to 95.3 Mg C ha -1 at 35-year-old. Old-growth floodplain forests, i.e. those that established on areas older than 70-year-old, levelled off at nearly 115 Mg C ha -1 (Fig. 7). We combined annual maps of floodplain ages with our C-stock model and produced annual maps of above-ground C-stocks (Fig. 8). The mean annual C accumulation rate of floodplain forests established after 1984 in the Ucayali 1984-2019 active meander belt was estimated at 0.01×10 6 Mg C yr -1 with a RMSE of 0.04×10 6 Mg C yr -1 (Fig. 9a). Given the relation between available floodplain area and C-stock, the generation with the most effective C-sequestration capacity was the 23-year-old forest (Fig. 9b). Annual floodplain erosion caused by lateral channel migration accounted approximately for 0.9±0.4×10 6 Mg C yr -1 (Fig. 10a). Floodplain areas which established before 1984 largely dominated eroded C-stocks with 0.8±0.4×10 6 Mg C yr - 1 (ND; Fig. 10b).

Discussion

Annual floodplain formation balances annual floodplain erosion in area but not in C budget

We found that rates of annual floodplain formation and rates of annual floodplain erosion appeared to be in balance over time with 19.0±7.7×10 3 ha -1 yr -1 and 19.8±6.7×10 3 ha -1 yr -1 , respectively. Our results confirm those reported by [START_REF] Puhakka | River Types, Site Evolution and Successional Vegetation Patterns in Peruvian Amazonia[END_REF] who showed that outer banks and point bars have, respectively, roughly equivalent rates of erosion and sediment deposition, maintaining a relatively constant channel width. Similarly, [START_REF] Peixoto | Spatial and temporal dynamics of river channel migration and vegetation in centralAmazonian white-water floodplains by remote-sensing techniques[END_REF] and [START_REF] Schwenk | High spatiotemporal resolution of river planform dynamics from landsat: The rivMAP toolbox and results from the Ucayali river[END_REF] reported an annual balance of erosion and deposition over large study areas and multidecadal periods. [START_REF] Peixoto | Spatial and temporal dynamics of river channel migration and vegetation in centralAmazonian white-water floodplains by remote-sensing techniques[END_REF] suggested that annual release of C caused by river dynamics would be much higher than the C annually sequestrated within the floodplain.

Our results have quantitatively confirmed their hypothesis. Indeed, within the 1984-2019 active meander belt of the Ucayali River, the floodplain forest annually accumulates 0.01±0.04×10 6 Mg C yr -1 and exports 0.9±0.4×10 6 Mg C yr -1 . Although it appeared that there is a balance in areas between floodplain formation and erosion, we show that there is however a strong imbalance in rates of floodplain forest C fixation and export to river waters caused by lateral channel migration. This imbalance is mainly due to massive erosion of old-growth floodplain forests with high C-stock densities (about 115 Mg C ha -1 ).

Lateral channel migration as a significant C provider to the aquatic continuum

There are multiple C sources for the aquatic continuum, i.e. between aquatic ecosystems from the atmosphere, headwater streams and inland waters, to coastal and marine systems. First, the C conveyed to streams and rivers can be provided during precipitation where water drops can be enriched in dissolved and particulate organic C (DOC, POC; [START_REF] Ward | Where Carbon Goes When Water Flows: Carbon Cycling across the Aquatic Continuum[END_REF]. Then, when entering forest canopies, drops capture DOC and POC at the surface of leaves, branches and trunks. By instance, in the Rio Negro region, throughfall fluxes of 27.5 kg C ha -1 yr -1 have been reported [START_REF] Filoso | Composition and deposition of throughfall in a flooded forest archipelago[END_REF]. Throughfall fluxes that range between 68.4 and 195.1 kg C ha -1 yr -1 and stem flow fluxes of 1.5 kg ha -1 yr -1 have been also reported in Amazonia [START_REF] Neu | Dissolved organic and inorganic carbon flow paths in an amazonian transitional forest[END_REF]. In addition to the C provided by rainfall, root and canopy litterfall and respiration in floodplain forests together with algal and floating macrophyte communities are considered as the main C pathways to the aquatic continuum [START_REF] Ward | Where Carbon Goes When Water Flows: Carbon Cycling across the Aquatic Continuum[END_REF].

Even though they represent only about 14 % of the Amazon basin, floodplains are highly productive compared to terrestrial uplands and are hydrologically connected to river channels during yearly overbank flooding [START_REF] Richey | Organic carbon: oxidation and transport in the Amazon River[END_REF][START_REF] Junk | The Amazon floodplain-a sink or source for organic carbon[END_REF][START_REF] Hoffmann | A carbon storage perspective on alluvial sediment storage in the Rhine catchment[END_REF]. It has been reported that in Central Amazonia, floodplains can provide an amount of 59.6±38.7

Mg C ha -1 yr -1 to the channel network and streamflow [START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF]. Here, we point out that lateral channel erosion is a crucial and overlooked physical driver delivering large amounts of C to rivers. We show that channel migration in the upper Amazonian floodplain provides between 0.7 and 115 Mg C ha -1 yr -1 to streamflow depending on the age of the forested floodplain areas eroded. Over the studied period and area, we found that in average 44.7±21.4

Mg C ha -1 yr -1 was delivered by lateral channel erosion to the aquatic continuum (0.9±0.4×10 6 Mg C yr -1 delivered over 19.8±6.7×10 3 ha -1 yr -1 ). Our result is roughly equivalent to the amount provided by the entire wetland system of the central Amazonian floodplain (i.e. flooded forest and macrophytes litterfall and respiration; 59.6±38.7 Mg C ha -1 yr -1 ), and roughly 10 times higher than the amount of the woody part considered by [START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF] i.e. 5.3±2 Mg C ha -1 yr -1 .

The amount of C provided by meandering rivers is dependant on annual rates of floodplain erosion. In the Amazon basin, different rates of channel migration of white-water rivers have been observed to exponentially increase from east to west [START_REF] Kalliola | Upper Amazon channel migration: implications for vegetation disturbance and succession using bitemporal Landsat MSS images[END_REF][START_REF] Mertes | Spatial patterns of hydrology, geomorphology, and vegetation on the floodplain of the Amazon river in Brazil from a remote sensing perspective[END_REF][START_REF] Peixoto | Spatial and temporal dynamics of river channel migration and vegetation in centralAmazonian white-water floodplains by remote-sensing techniques[END_REF][START_REF] Salo | Hierarchy of landscape patterns in western Amazon[END_REF]. Therefore, inputs of woody materials as a consequence of lateral channel erosion may certainly increase along a downstream-upstream gradient. Further studies are thus required to quantify the spatial variability and overall contribution of Amazonian meandering rivers. Here, we have calculated the contribution of above-ground forest biomass to streamflow, but further amounts of C input may originate from (i) eroded floodplain soils (i.e. belowground total organic matter; Fig. 2), and (ii) the Andean mountains situated upstream the study area, where torrential streams potentially accumulate and transport large amounts of coarse woody debris provided by landslides occurring in particular during tropical storm events [START_REF] Mcclain | Andean influences on the biogeochemistry and ecology of the Amazon River[END_REF][START_REF] Clark | New views on "old" carbon in the Amazon River: Insight from the source of organic carbon eroded from the Peruvian Andes[END_REF][START_REF] Wohl | Organic carbon export in the form of wood during an extreme tropical storm, Upper Rio Chagres, Panama[END_REF].

Nature and fate of the C provided by meandering rivers

The nature of the C that is annually exported into the aquatic continuum is a fundamental aspect when considering the fate of this C and its role in the global cycle. It is usually considered that river C is advected downstream and returned to the atmosphere via degassing by both microbial and photochemical activity, ultimately resulting in a net zero exchange, especially in the tropics where warm temperatures stimulate organic matter decomposition [START_REF] Raymond | Global carbon dioxide emissions from inland waters[END_REF][START_REF] Ward | Degradation of terrestrially derived macromolecules in the Amazon River[END_REF]. For instance, [START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF] proposed that central Amazonian waters receive at least as much C from wetlands as they emit to the atmosphere, making a nearly neutral balance in atmosphere-biosphere CO2 exchanges. However, the C provided by meandering rivers of the upper Amazon basin most probably differs from the C provided by wetlands of the central Amazon. On the one hand, it has been shown for wetlands of the central Amazon that the microbial communities decompose young organic C (> 5-year-old; [START_REF] Mayorga | Young organic matter as a source of carbon dioxide outgassing from Amazonian rivers[END_REF][START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF]. On the other hand, the organic C found in the water column mainly originates from litterfall with low lignin content [START_REF] Mayorga | Young organic matter as a source of carbon dioxide outgassing from Amazonian rivers[END_REF][START_REF] Abril | Amazon River carbon dioxide outgassing fuelled by wetlands[END_REF]. Our results suggest that in free meandering Amazonian rivers, such as the Ucayali River, older lignin is present in substantial quantities. Higher concentrations of lignin are found in tree trunks, branches and roots of ligneous terrestrial plants, and we estimated that mature and old growth floodplain forests accounted for nearly half of the eroded floodplain area within the Ucayali 1984-2019 meander belt (Fig. 6b). However, the fate of these large C quantities of lignin remains still uncertain. One hypothesis could be that this C is mineralized by microbial and photochemical activity and/or exported downstream the aquatic continuum and/or accumulated in the river sediment. Following [START_REF] Ward | Degradation of terrestrially derived macromolecules in the Amazon River[END_REF], half of these quantities could be degraded and mineralized in the Amazon River. Anyhow, according to [START_REF] Galy | Global carbon export from the terrestrial biosphere controlled by erosion[END_REF], the erosion and transportation capacities of rivers are to be considered as the main control of the biospheric C export to oceans. Many large water storage reservoirs have been recently proposed for the Amazon Basin [START_REF] Finer | Proliferation of Hydroelectric Dams in the Andean Amazon and Implications for Andes-Amazon Connectivity[END_REF]. Since meandering rivers are known to be highly sensitive to the disruption of sediment supply caused by such engineering projects [START_REF] Constantine | Sediment supply as a driver of river meandering and floodplain evolution in the Amazon Basin[END_REF], it is highly suspected that such human infrastructures will greatly alter the natural C fluxes we have here quantified.

Conclusion 11. Tables and figures

Figure 1 The freely meandering Ucayali River in the upper Amazonian region, Peru.

3. 4 .

 4 Annual accumulation of floodplain forest C-stocks and its erosion Floodplain areas were converted into above-ground forest C-stocks based on our model (Fig. 3c). The annual total C-stock (in Mg C ha -1 ) was obtained by summing C-stocks of all vegetated floodplain ages since 1984. An annual C accumulation rate was obtained by (1) subtracting the total C-stock of year n by the one of year n-1, and (2) by applying a linear regression and the calculation of the root mean square error (RMSE). C-accumulation in floodplain and upland forest areas established before 1984 were not considered in the present study because they were outside of the 1984-2019 active-meander belt. Annual C-stock lost through floodplain erosion was obtained by summing eroded C-stocks of determined and non-determined (ND) floodplain ages.

  Figure 1 The freely meandering Ucayali River in the upper Amazonian region, Peru. (a) Location map: the yellow rectangle indicates the extent of the study area and the red rectangle indicates the extensions of panels (b) and (c), located approximately 120 km upstream of Pucallpa, Peru. (b) False colour composite satellite image showing the Ucayali meander belt extension in light green. This cloud-free satellite Landsat 8 image was obtained through the selection of the greenest pixels over the year 2018. The first short wave infra-red satellite band was affected to the red colour on screen, the near infra-red band to the green and the red band to the blue. (c) Floodplain ages range from 1-year (green colour) to 35-year (brown colour) within the 1984-2019 meander belt. Blue and grey colours correspond respectively to surface water and upland areas formed before 1984 (non-determined ages), respectively.

Figure 2

 2 Figure2Simplified model of the floodplain vegetation succession in the study area which comprises four main interrelated carbon reservoirs (modified from[START_REF] Salo | River dynamics and the diversity of Amazon lowland forest[END_REF] Kayranli et al., 2010; Stufin et al., 2016): (i) aboveground living terrestrial and seasonally flooded forest biomass (trunk, branches and foliage); (ii) aboveground necromass (dead wood and litter); (iii) belowground living biomass (roots and micro-organisms); and (iv) total organic matter in the soil (decomposed dead organic flora and fauna). The present study focused on the first carbon reservoir of the floodplain, i.e. aboveground living terrestrial and seasonally flooded forest biomass.

Figure 3

 3 Figure 3 Conceptual approach used to derive forest above ground carbon stock (C-stock) from floodplain area age. a) Pixel-by-pixel and year-by-year algorithm used to obtain yearly maps of area ages based on the analysis of the yearly water classification history dataset (Pekel et al., 2016). b) Pixel-by-pixel and year-by-year algorithm used to obtain yearly maps of eroded floodplain area ages. c) Data series used to model and derive C-stocks from floodplain area ages (Schöngart and Wittmann, 2010).

Figure 4

 4 Figure 4 Annual area fluctuations of a) water surfaces, b) floodplain formation and c) floodplain erosion between 1984 and 2019. Values are standardized, i.e. the 1984-2019 average was subtracted and the result was divided by the 1984-2019 standard deviation.

Figure 5

 5 Figure 5 River meandering and floodplain forest dynamics according to a 5-yr time interval between 1984 and 2019 in the vicinity of Sampaya (Peru). Annual maps were created using the algorithm described in Fig. 3a. Non-eroded, remaining floodplain and upland forests which established before 1984 are indicated as age non-determined (ND; white colour).

Figure 6

 6 Figure 6 Age structure of vegetated floodplain areas observed in the 1984-2019 active meander belt. a) Floodplain areas according to floodplain ages established since 1984. b) Eroded floodplain areas since 1984 according to floodplain ages. Non-determined ages (ND) corresponding to floodplain or upland areas established before 1984, were considered for erosion. Together, mean annual erosion accounted for 19.8±6.7×10 3 ha -1 yr -1 . Points and bars indicate annual mean values for the period 1984-2019 and their standard deviations, respectively.

Figure 7

 7 Figure 7 Model used to derive above-ground forest C-stocks from floodplain forest age. The model between 1 and 35 years (3rd polynomial; black line) is based on data (dots) published by Schöngart and Wittman (2010). The data error range is ±2% (not indicated in the figure).

Figure 9

 9 Figure 9 Floodplain forest carbon stocks (C-stocks) during the 1984-2019 period in the Ucayali active meander belt. a) Annual C accumulation in the floodplain forest established since 1984 and quantified annually until 2019. The black line indicates the annual C accumulation rate as a linear trend of 10256 Mg C yr -1 (R² = 0.87, p value < 0.01 and RMSE = 44939 Mg C yr -1 ). b) Annual C-stock accumulated in the floodplain forest according to the floodplain age established since 1984.

Figure 10

 10 Figure 10 Erosion of floodplain forest carbon stocks (C-stocks) caused by lateral channel migration. a) Annual C-stocks eroded from the floodplain forest during lateral channel migration between 1984 and 2019. b) Annual C-stock eroded from the floodplain forest during lateral channel migration according to forest age. ND indicates floodplain and upland forests established before 1984.

Although the area of the Ucayali's 1984-2019 meander belt represents only a small fraction of the Amazonian basin area, it significantly contributes to the amount of C that is transferred from the floodplain forest to the aquatic continuum. The amount of woody C per unit area provided by floodplain erosion caused by lateral channel erosion is nearly ten-fold higher than that provided by central Amazon wetlands. Extrapolation of our results suggests that free meandering white-water rivers may act as major agent in the global C cycle. These findings point to the importance to quantify the overall contribution of free meandering forested floodplain rivers worldwide to the global C cycle. In addition to variations in channel migration rates, different floodplain tree species may show contrasting C-stock potentials and related forest carbon export rates according to climate zones and biogeographical regions.

Competing interest statement

The authors have no competing interests to declare.

Acknowledgements


Camille Desgrouas
email: camille.desgrouas@univ-amu.fr

Alice-Anaïs Varlet
email: alice-anais.varlet@univ-amu.fr

Anne Dutour
email: anne.dutour@ap-hm.fr

Damien Galant
email: damien.galant@etu.univ-amu.fr

Françoise Merono
email: francoise.merono@univ-amu.fr

Nathalie Bonello-Palot
email: nathalie.bonello@ap-hm.fr

Patrice Bourgeois
email: patrice.bourgeois@ap-hm.fr

Adele Lasbleiz
email: adele.lasbleiz@ap-hm.fr

Cathy Petitjean
email: petitjean.cathy@wanadoo.fr

Patricia Ancel
email: patricia.ancel@univ-amu.fr

Adèle Lasbleiz

Nicolas Levy
email: nicolas.levy@univ-amu.fr

Catherine Badens
email: catherine.badens@univ-amu.fr

Bénédicte Gaborit
email: benedicte.gaborit@ap-hm.fr

Unraveling LMNA Mutations in Metabolic Syndrome: Cellular Phenotype and Clinical Pitfalls

Keywords: dyslipidemia, metabolic syndrome, insulin resistance, lamin A/C, nuclear anomalies and premature senescence

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Familial partial lipodystrophy (FPLD) is a genetic condition characterized by partial lipoatrophy and metabolic dysfunctions, such as insulin resistance and hypertriglyceridemia [START_REF] Vatier | Monogenic forms of lipodystrophic syndromes: Diagnosis, detection, and practical management considerations from clinical cases[END_REF]. This condition predisposes to diabetes type 2, cardiovascular diseases and hepatic or pancreatic comorbidities. FPLD type 2 (FPLD2), commonly named Dunnigan syndrome, is the most frequent FPLD and results from LMNA mutations transmitted in a dominant pattern [START_REF] Hussain | Lipodystrophies, dyslipidaemias and atherosclerotic cardiovascular disease[END_REF][START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF][START_REF] Vatier | What the genetics of lipodystrophy can teach us about insulin resistance and diabetes[END_REF]. LMNA encodes for lamin A and C proteins as the result of an alternative splicing. These proteins are type V intermediate filaments located both in the nucleoplasm and at the nuclear lamina, a meshwork underlying the inner nuclear envelope. Lamins participate in the nuclear structure and in the chromatin-nuclear lamina connection, and consequently, to the set up and maintenance of gene expression pattern [START_REF] Mounkes | The laminopathies: Nuclear structure meets disease[END_REF]. LMNA-related diseases, gathered in a group called laminopathies, display a large spectrum of clinical expression, from FPLD to extremely severe phenotypes, such as progeria syndrome and restrictive dermopathy. At the cellular level, and in vitro, all pathogenic LMNA mutations are associated with a decrease of cell proliferation rate, premature senescence, misshapen nuclei, and chromatin remodeling [START_REF] Vigouroux | Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin A/C gene[END_REF][START_REF] Muchir | Nuclear envelope alterations in fibroblasts from patients with muscular dystrophy, cardiomyopathy, and partial lipodystrophy carrying lamin A/C gene mutations[END_REF][START_REF] Gruenbaum | Nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation[END_REF]. The level of these alterations is roughly correlated with the severity of clinical expression, cells from patients with progeria, and restrictive dermopathy presenting with the most severe cellular senescence.

Regarding FPLD2, mutations located on the residue 482 are responsible for 80% of the cases and are associated with the classical clinical form [START_REF] Guillín-Amarelle | Lipodystrophic laminopathies: Diagnostic clues[END_REF]. Interestingly, women are more often diagnosed than men due to the android appearance characterized by protruding and well-defined musculature [START_REF] Decaudain | New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome[END_REF][START_REF] Gonzaga-Jauregui | Clinical and Molecular Prevalence of Lipodystrophy in an Unascertained Large Clinical Care Cohort[END_REF]. They also suffer from more severe and more precocious metabolic perturbations and from gynecological disorders (polycystic ovarian syndrome, miscarriage, stillbirth) [START_REF] Vantyghem | Fertility and obstetrical complications in women with LMNA-related familial partial lipodystrophy[END_REF]. Among men, phenotypes are more attenuated and induce less severe metabolic complications [START_REF] Vatier | Monogenic forms of lipodystrophic syndromes: Diagnosis, detection, and practical management considerations from clinical cases[END_REF].

Besides mutations on residue 482, other mutations located all along LMNA can also be responsible for FPLD2, but are often associated with atypical forms of the disease. Particularly, in some very rare cases, mutations have been associated with an attenuated phenotype of lipodystrophy without lipoatrophy and corresponding to severe metabolic syndrome (MS), a condition that shares common features with FPLD2, such as dyslipidemia, insulin resistance and central adiposity [START_REF] Decaudain | New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome[END_REF][START_REF] Galant | A Heterozygous ZMPSTE24 Mutation Associated with Severe Metabolic Syndrome, Ectopic Fat Accumulation, and Dilated Cardiomyopathy[END_REF][START_REF] Dutour | High prevalence of laminopathies among patients with metabolic syndrome[END_REF]. In these cases, the pathogenicity of the mutations must be firmly demonstrated to confirm that they are not simply silent polymorphisms, as could often be the case for missense mutations.

In this study, we report the detailed phenotypes of two patients with mutations in LMNA associated with severe metabolic syndrome and no lipoatrophy, together with the results of functional studies, to document the effect of these two rare heterozygous missense mutations on cell proliferation capacity and on nuclear phenotype.

Material and Methods

Patients

The two patients reported here were initially referred to the Endocrinology, Metabolic Diseases and Nutrition Department at the Assistance Publique des Hôpitaux de Marseille for insulin resistance diabetes care. One of them was part of a cohort described previously [START_REF] Dutour | High prevalence of laminopathies among patients with metabolic syndrome[END_REF]. They gave their written informed consent for this study. The patients underwent a subcutaneous fat biopsy (needle aspiration) for RNA sequencing and adipocytes diameter quantification. DNA samples and primary fibroblast were available for both patients and stored at the certified Biological Resource Center (CRB) tissue, DNA and cells components (NF S96-900 & ISO 9001 v2015 Certification).

Plasmids and siRNAs

The two LMNA missenses mutations were introduced using the QuickChange II XL Site-Directed Mutagenesis Kit (Agilent technologies, Santa Clara, CA, USA) in the cDNA of prelamin A cloned in the pEGFP-C1 vector (Clontech Laboratories Inc., Mountain View, Santa Clara, CA, USA). Mutagenic primers were designed using the QuikChange Primer Design Program available online at https: //www.genomics.agilent.com/primerDesignProgram.jsp (sequences available on request). The presence of the two mutations was checked by direct sequencing, according to standard procedures on ABI3500XL (Life Technologies, Carlsbad, CA, USA). Mutations were numbered according to the GenBank reference sequence NM_170707.3 and the Human Genome Variation Society recommendations (http://varnomen.hgvs.org/). SiRNA treatment was directed against lamin A 3 UTR (3 -UUAUUGAAGAGAAUCUUU-5 , Eurogentec) and a scrambled siRNA (Eurogentec ref SR-CL005-005) was used as a negative control. In addition, 40,000 and 150,000 fibroblasts were seeded in coverslips (Lab-Tek, SPL Life Science, Gyeonggi-do, Korea) and 6-well plates (VWR Plates, Fontenay-sous-Bois, France) for immunofluorescence and western blot analysis.

Cell Culture and Cell Transfection

Fibroblasts were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) (Biowest, Nuaillé, France), supplemented with 15% fetal bovine serum (Eurobio, Courtaboeuf, France), 1% L-glutamine 200 mM (Life Technologies, Paisley, UK), and 1% Penicillin-Streptomycin-Amphotericin (PAA Laboratory, Pasching, Austria), in a culture flask of 25 cm 2 (SPL Life Sciences, Gyeonggi-do, Korea), under controlled atmospheric conditions (10% O 2 , 5% CO 2 , and 85% N 2 ) at 37 • C with 95% humidity. Control fibroblasts were provided by the Coriell Institute (Control 1: AG07095 and Control 2: AG09309) (Camden, New Jersey, USA). For cell transfection, fibroblasts were seeded on coverslips (Lab-tek, SPL Life Sciences, Gyeonggi-do, Korea) at a density of 2.5 × 10 4 cells/well. Control 1 was transfected with 250 ng of plasmids containing control (pEGFP-C1) or mutated cDNA using jetPRIME ® transfection reagent (Polyplus Transfection, Illkirch, France), following the manufacturer's instructions. The same reagent was used to transfect 30 nM of small interference RNA silencing specifically lamin A through interference in the 3 UTR of prelamin A transcript [START_REF] Vantyghem | Fertility and obstetrical complications in women with LMNA-related familial partial lipodystrophy[END_REF]. The efficiency of siRNA was analyzed 48 h post-transfection by immunofluorescence, using a primary antibody anti-lamin A/C (SC 6215 or SC 376248, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) associated with a proper secondary antibody (donkey anti-goat IgG H & L, Alexa Fluor ® 594, ab150132, Abcam ® , or goat anti-mouse Alexa Fluor ® 488, A-11001, Thermofisher, Rockford, IL 61105 USA) [START_REF] Galant | A Heterozygous ZMPSTE24 Mutation Associated with Severe Metabolic Syndrome, Ectopic Fat Accumulation, and Dilated Cardiomyopathy[END_REF][START_REF] Dutour | High prevalence of laminopathies among patients with metabolic syndrome[END_REF]. Western blotting was performed using a rabbit polyclonal anti-lamin A/C (sc-20681, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and monoclonal anti-GAPDH (MAB374, Merck Millipore, Darmstadt, Germany) as loading control. Odyssey Infrared Imaging System (LI-COR Biosciences, GmbH, Bad Homburg, Germany) was used to reveal the membranes. Quantification of expression levels was performed using Image J software (National Institutes of Health, Bethesda, Maryland, MD, USA).

Senescence Evaluation

To evaluate the senescence rate, cells were seeded on coverslips (Lab-tek, SPL Life Sciences, Gyeonggi-do, Korea) and beta-galactosidase activity was measured using the Senescence beta-Galactosidase Staining Kit (Cell Signaling Technology ® , WZ Leiden, The Netherlands) following the manufacturer's instructions. To assess the replication capacity of patient and control cells, we measured 5-bromo-2 -deoxyuridine (BrdU) incorporation. To do so, cells were seeded on a 96-well plate at a density of 1.10 4 cells/well, and BrdU was provided by the Cell proliferation ELISA; BrdU (colorimetric) Kit (Roche Applied Science, Mannheim, Germany) was added to the cell medium for a 24-h period, after which the ELISA assay was performed following the manufacturer's instructions [START_REF] Galant | A Heterozygous ZMPSTE24 Mutation Associated with Severe Metabolic Syndrome, Ectopic Fat Accumulation, and Dilated Cardiomyopathy[END_REF].

To quantify nuclear shape anomalies, lamin A/C immunofluorescence was performed on transfected and fixed cells as described elsewhere [START_REF] Bonello-Palot | Prelamin A accumulation in endothelial cells induces premature senescence and functional impairment[END_REF]. Phenotypes were monitored routinely by at least two independent investigators by visual inspection of fixed specimens, using an ApoTome system equipped with a 100× objective, and a charged coupled device (CCD) camera Axiocam MRm controlled by the Zen software (Carl Zeiss Microscopy, LLC, White Plains, NY, USA). Confocal images of fixed samples were performed with a LSM 800 Airyscan Axio Observer.Z1 7 confocal microscope, equipped with a 63×/1.20 W Korr UV VIS IR C-Apochromat objective, and driven by Zen 2.3 system software. Around 100 nuclei were examined for each condition; nuclear anomaly criteria consisted of aberrant nuclear lamin A staining pattern, aberrant lamin A cytoplasmic localization, and aberrant nuclei shape. The Image J 1.48v software (National Institute of Health, Bethesda, Maryland, MD, USA) was used for processing entire images before cropping to emphasize the main point of the image. Processing was limited to background subtraction and brightness/contrast adjustment.

RNA Sequencing and RT-qPCR

RNAseq was performed in duplicate on adipose tissue from the patients and 3 non-diabetic controls using the kit Trio RNAseq from Tecan-NuGen (Redwood City, California, CA, USA) based on human depletion on total RNA degraded. Libraries were constructed by fragmentation of the ds-cDNA, end repair, and adapter ligation. Ribodepletion was performed following the manufacturer's instructions. The 8 indexed libraries were pooled and sequenced on an Illumina NextSeq 500 platform, using paired-end mode (2 × 75 bp reads), in order to reach 25 million reads (clusters) for each library, as an average. Total information of 70.7 Gb was obtained with a cluster passing quality control filters of 92.3%. The Gene Ontology (GO) analysis was performed with the ConsensusPathDB website (http://cpdb.molgen.mpg.de).

Quantitative RT-PCR was performed with 200 ng of RNA, 20 U of reverse transcriptase M-MLV (Life Technologies), 50 ng of random primers, 10 mM of nucleotides, and 40 U of the ribonuclease inhibitor RNaseOUT (Life Technologies Courtaboeuf, France). Primers for quantitative PCR (RT 2 qPCR Primer assay) were designed by Qiagen. Quantitative PCR was performed using primers labelled with fluorescent EvaGreen (Eurogentec, Angers, France) and a real-time thermocycler, Light Cycler 480 (Roche, Meylan, France). The 18s ribosomal and GAPDH RNAs were used as reference genes to normalize cycle thresholds (Ct) and to get relative quantification (∆∆Ct).

Statistical Analysis

For (a) cell proliferation ELISA experiments, and (b) abnormally shaped nuclei, the mean values from at least 3 independent experiments were analyzed using the Mann-Whitney test, which is a non-parametric test that compares two unpaired groups of ordinal or numerical variables. For experiments examining the proportions of cells with SA-beta-galactosidase positive staining, data from individual experiments were analyzed using the Fisher's exact test, which is used for small sample sizes with nominal/categorical variables. Statistical calculations were performed using Prism 6.07 (GraphPad Software, San Diego, CA, USA) software. The p-values < 0.05 considered significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Results

Patients' Clinical Description

Patient 1 is a 55-year-old woman referred for early-onset insulin-resistant diabetes mellitus, diagnosed at 26, initially treated with metformin, glucagon-like peptide 1 analogs, followed by insulin (continuous subcutaneous insulin infusion, daily insulin requirement 2 UI/kg/day with HbA1c = 7.3% (56 mmol/mol)). She had a family history of diabetes mellitus (parents, sister, and one paternal uncle, Figure 1A). Her nephew had a stroke at 15 years old. She presented high blood pressure (treated by tritherapy), combined dyslipidemia, severe hypertriglyceridemia from 4.5 to 12.5 mmol/L, and LDL-cholesterol above 2.58 mmol/L, despite high dose statins (rosuvastatin 10 mg/day), and fibrates (fenofibrate 200 mg/day), sleep apnea syndrome, and asthma. At age 42, she suffered from acute coronary syndrome, and received three stents on her left anterior descending artery. She fulfilled all the diagnosis criteria for metabolic syndrome (MS) [START_REF] Grundy | Diagnosis and management of the metabolic syndrome: An American Heart Association/National Heart, Lung, and Blood Institute scientific statement: Executive Summary[END_REF].

A physical examination revealed visceral adiposity, with an accumulation of truncal fat, cushingoid morphotype with facial hirsutism, and android obesity, but no strict lipoatrophy of lower limbs (Figure 1B). Her anthropometrical features were body mass index (BMI) = 37.1 kg/m 2 , waist circumference (WC) =118 cm, waist to hip ratio (WHR) = 1.08. A dual energy X-ray absorptiometry (DEXA) revealed 44.3% of fat mass (Figure 1C andD) and an abdominal computed tomography (CT) confirmed the presence of subcutaneous adipose tissue: superficial (196 cm 2 ) and deep (201 cm 2 ), with excessive visceral fat accumulation (204 cm 2 ) (Figure 1D). The mean adipocyte diameter obtained by the needle subcutaneous fat biopsy was 125.7 ± 32.5 µm (Figure 1E).

Cardiac investigations revealed severe coronary artery disease and concentric left ventricular hypertrophy with preserved ejection fraction (> 60%), and neither cardiac rhythm nor conduction disturbances. Hormonal investigations ruled out the diagnosis of polycystic ovarian syndrome and Cushing's syndrome. Leptin was high at 39.3 ng/mL (reference 3.3-8.7) and adiponectin at 1.34 µg/mL (reference 3.8-11.8). Liver enzymes were in the normal range, but an abdominal echocardiography revealed diffuse hepatic steatosis.

The patient underwent bariatric surgery because of severe obesity with multiple comorbidities and a failure of lifestyle interventions. She had a sleeve gastrectomy in 2016. One year on, weight loss was 14 kilos (42 percent excess weight lost (%EWL) and the insulin doses were cut by three. Nevertheless, she put on weight again (at 24 months +6 kilos, %EWL 24%), and her insulin needs increased again. Hypertension and hypertriglyceridemia persisted after surgery and required the continuation of lipid lowering (Atorvastatine 80 mg) and antihypertensive drugs (bitherapy). Hepatic steatosis remained after the intervention, but sleep apnea syndrome improved (apnea hypopnea index decreased from 27 to 16 per hour).

Patient 2 is a 45-year-old man treated with high insulin doses (100 UI/day) for diabetes mellitus diagnosed at 34, complicated with non-proliferative retinopathy and microalbuminuria. He also suffered from hypertriglyceridemia (from 5.46 mmol/L to 13.45 mmol/L) despite high doses of statins (Rosuvastatin 10 mg/day). Leptin was measured at 8.49 ng/mL (reference 3.3-8.7). He had a family history of diabetes mellitus (Figure 2A). His mother was diabetic and died of a myocardial infarction at 44 and his father died of a brain hemorrhage at 72. On his mother's side, diabetes mellitus was described for his grandparents and three uncles.

A physical examination revealed a BMI = 26.5 kg/m 2 , WC = 107 cm with no lipodystrophy (Figure 2B). The patient complained of occasional muscular cramps. The percentage of fat mass was 31.1% with DEXA (Figure 2C). The mean subcutaneous adipocyte diameter was 109.9 ± 24.3 µm (Figure 2D). Abdominal CT scan revealed the presence of superficial (103 cm 2 ) and deep (224 cm 2 ) subcutaneous adipose tissue with excessive visceral fat accumulation (167 cm 2 ) (Figure 2E). The anti -glutamic acid decarboxylase (GAD) and anti-tyrosine phosphatase-like insulinoma antigen 2 IA-2 antibodies and Maturity Onset Diabetes of the Young (MODY) testing were negatives. An abdominal echocardiography revealed liver steatosis and hepatomegaly (right hepatic arrow at 20 cm). A myocardial evaluation showed no cardiac or coronary artery disease. The exercise stress testing was normal.

Molecular Findings

A molecular analysis of LMNA showed mutations c.1892G > A p.(Gly631Asp) (rs267607648) and c.1745G > T p.(Arg582Leu), respectively, for patients 1 and 2 as the only potentially pathogenic variations in a panel of 82 genes involved in premature aging, lipodystrophy syndromes, and diabetes (list available on request). Both mutations are very rare (allelic frequency in GnomAD: 8.037 × 10 -6 and 1.095 × 10 -5 https://gnomad.broadinstitute.org/) with no clinical data available, and are predicted to be pathogenic (ACMG class 5), or likely pathogenic (ACMG class 4), by several bioinformatics tools (http://umd-predictor.eu/, https://varsome.com/) [START_REF] Salgado | UMD-Predictor: A High-Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution[END_REF][START_REF] Kopanos | The human genomic variant search engine[END_REF].

Patient Fibroblasts Functional Analysis

After immunostaining with lamin A/C antibodies, we observed for both patient cells, an altered lamin A/C staining with reduced signal, and heterogeneous staining with aggregates. Nuclear shape anomalies were quantified in primary fibroblasts at passage 16, and showed a 2-fold increase of the percentage of nuclear anomalies, such as misshapen nuclei or nuclear membrane blebs in patient cells when compared to two control cells (Figure 3A). To confirm that mutant lamin A expression is responsible for increased abnormal nuclear shape, we first evaluated the effect of lamin A down-expression on the nuclear morphology of patient fibroblasts. We transfected the patient cells with a siRNA specifically targeting the 3 UTR of LMNA transcripts, which induces a significant decrease of lamin A level expression [START_REF] Galant | A Heterozygous ZMPSTE24 Mutation Associated with Severe Metabolic Syndrome, Ectopic Fat Accumulation, and Dilated Cardiomyopathy[END_REF][START_REF] Pongsakul | A/C in renal tubular cells is important for tissue repair, cell proliferation, and calcium oxalate crystal adhesion, and is associated with potential crystal receptors[END_REF]. While the LMNA-specific siRNA did not induce a significant modification of the proportion of abnormal nuclei in the control cells, it did lead to a significant improvement of nuclear phenotypes in the patient cells (Figure 3B). Indeed, under siRNA treatment, the lamin A expression level was reduced by a third, leading to a 10-15% nuclear anomalies reduction in patient fibroblasts. This reduction was moderate but significant as it was observed in five independent experiments. Secondly, in another experiment, we evaluated the effect of LMNA mutant's expression on nuclear morphology, by transient transfection of mutation-expressing plasmids in wild type cells. To do so, we transfected plasmids expressing wild type or LMNA mutant cDNA carrying either patient 1 or 2 mutations, and tagged with GFP at the N-terminal end. A significant higher level of nuclear abnormalities was observed in cells expressing LMNA mutants compared to control cells transfected with wild type lamin A (Figure 3C). Thus, while the depletion of lamin A in patient cells significantly improved nuclear abnormalities, the expression of lamin A mutants mimics these phenotypes, suggesting that mutants are at least partially responsible for nuclear deformations observed in patient fibroblasts.

Next, we assessed senescence by two different tests: first, by evaluating the replication profile of each patient's cells through the estimation of BrdU incorporation. As shown in Figure 3D, BrdU incorporation was significantly lower in patient 1 cells compared to both controls, and in patient 2 cells only when compared to control 1. In a second set of experiments, the production of beta-galactosidase was evaluated as a common marker of cellular senescence. The results showed a significantly higher rate of beta-galactosidase staining, indicating increased senescence for both patient cells (Figure 3E).

RNA Sequencing Analysis in Adipose Tissue

We further compared the transcriptome of the whole subcutaneous adipose tissue of the two patients and three controls paired for age and sex using RNA sequencing. The genes presenting the most important fold change in expression are presented in Figure 4A. Data analysis showed upregulation of genes implicated in immunity, such as TREM2, DCSTAMP, FCRL2, and down-regulation of genes involved in the development and cell differentiation, such as SLPI, CDK2AP1, BMP7, FOS. Interestingly, two genes (one down-(B3GNT4) and one upregulated (SLC22A12)) were implicated in uric acid homeostasis, and two down-regulated genes were found to be implicated in glucose metabolism (KSR2 and GPR1). Remarkably, GO analysis identified that genes deregulated in patients were implicated mainly in immunity and development pathways, such as T cell selection, leucocyte mediated cytotoxicity, lymphocyte co-stimulation, migration and activation, inflammatory response, and cell proliferation (Figure 4B). Separate analysis, one-to-one patient compared to controls, showed in the top-20 deregulated genes, two genes similarly deregulated in the two patients. One is SPLI, which has been validated by q-PCR, the other is GATD3A, a potential mitochondrial protein, which function remains to be fully determined (data not shown). Differentially expressed genes (SLPI, TREM2, FOS) were validated by RT-qPCR to confirm the RNAseq results (Figure 4C). By contrast, these genes were not differentially expressed in fibroblasts of patients and controls, and as expected, TREM2 was not expressed in fibroblasts (Figure 4D).

Discussion

We have shown two rare missense mutations in LMNA; both were associated with severe metabolic phenotypes, resistance to bariatric surgery-induced weight loss in patient 1, alterations in the shape of nuclei, increased beta-galactosidase staining and modifications of gene expression patterns in subcutaneous adipose tissue and fibroblasts. As suggested by Caron et al. [START_REF] Caron | Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence[END_REF] these cellular and phenotypic alterations could be suggestive of metabolic laminopathy.

Both mutations are located on exon 11 corresponding to the C-tail specific domain of lamin A, consequently, lamin C is not affected, and observed alterations can be attributed to the presence of mutated lamin A only. Here, the phenotype induced by the mutation p.(Gly631Asp) (patient 1) seem clinically more severe than the one associated with mutation p.(Arg582Leu) (patient 2). This could be explained either because of the mutation nature in itself or the patient's sex, as women are generally more affected in genetic lipodystrophy than men [START_REF] Decaudain | New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome[END_REF]. Remarkably, patient 1 did not benefit from the expected weight loss after bariatric surgery (<50% EWL) and the metabolic abnormalities (hypertension, dyslipidemia, and insulin-requiring type 2 diabetes) remained, despite an important caloric restriction induced by the sleeve gastrectomy, showing that her metabolic phenotype was not only due to an environmental Western lifestyle. The effect of bariatric surgery on metabolic laminopathy had never been reported to date. This should encourage researchers to try to understand the role of molecules derived from fat accumulation to the onset and progression of the disease.

Only a few cases of MS linked to LMNA mutations have been reported up to now, suggesting that this condition, given the high proportion of patients with MS or metabolic abnormalities with early onset, is under-diagnosed [START_REF] Decaudain | New metabolic phenotypes in laminopathies: LMNA mutations in patients with severe metabolic syndrome[END_REF][START_REF] Galant | A Heterozygous ZMPSTE24 Mutation Associated with Severe Metabolic Syndrome, Ectopic Fat Accumulation, and Dilated Cardiomyopathy[END_REF][START_REF] Dutour | High prevalence of laminopathies among patients with metabolic syndrome[END_REF][START_REF] Ambonville | A complex case of diabetes due to LMNA mutation[END_REF]. Indeed, a recent study based on the analysis of variant frequencies in several public databases gathering data on 60, 706 unrelated individuals, revealed an unexpected high number of LMNA variants (n = 132), including novel variants predicted to perturb lamin A assembly or function. Among them, the variant p.Gly602Ser was identified as a potential risk factor for type 2 diabetes mellitus in African Americans [START_REF] Florwick | LMNA Sequences of 60,706 Unrelated Individuals Reveal 132 Novel Missense Variants in A-Type Lamins and Suggest a Link between Variant p.G602S and Type 2 Diabetes[END_REF].

It is particularly important to pay attention to metabolic derangements induced by LMNA mutations because specific clinical follow-up and therapeutic approaches can be set up for these patients, as mentioned in the multi-society practice guidelines published in 2016 [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF]. This is the first work reporting the poor effect of a restrictive bariatric surgery on a patient with LMNA mutation.

Furthermore, an anticipation of metabolic complications over three generations of patients carrying mutated LMNA has been described, such as hypertriglyceridemia, which appeared 15 years earlier in the second generation, highlighting the importance of pre-symptomatic genetic diagnosis [START_REF] Jeru | LMNA-associated partial lipodystrophy: Anticipation of metabolic complications[END_REF]. Finally, one important finding of this work was the persistence of subcutaneous adipose tissue in the described patients, which was consistent with the measurable serum leptin level that could make the diagnosis of laminopathy harder, but also extend the pattern of LMNA associated phenotypes to cardio-metabolic syndrome without subcutaneous lipoatrophy.

We observed a slight but constant decrease in the nuclear abnormalities after siRNA treatments, over several independent experiments. Moderate reduction of mutated (and WT) lamin A/C improved to some extent the fibroblast cellular phenotype, possibly due to the lowering of prelamin A levels. Using experiments commonly performed to study senescence in vitro, we showed that patient cells exhibit premature senescence, but to a lesser extent than the senescence induced by the progeria mutation, or the canonical FPLD2 mutation R482W. Indeed, when compared to control cells issued from a person with comparable age, we observed no significant differences in population doubling level (data not shown), and the modifications of the beta-galactosidase and BrdU tests results, although significant, were moderate. These results are in accordance with the clinical phenotypes described here, which are milder than progeria, or even typical FPLD2.

In previous studies, lamin A/C has been shown to mediate the activation of adipose tissue macrophage inflammation by regulating the proinflammatory factors, such as NF-κB, or TNFα, contributing to the development of insulin resistance [START_REF] Kim | Macrophage Lamin A/C Regulates Inflammation and the Development of Obesity-Induced Insulin Resistance[END_REF]. In addition, the depletion of lamin A/C in macrophages or myeloid cells reduced the lipopolysaccharide LPS-induced expression of proinflammatory genes and adipose tissue inflammation [START_REF] Kim | Macrophage Lamin A/C Regulates Inflammation and the Development of Obesity-Induced Insulin Resistance[END_REF]. Interestingly, in our study, RNA sequencing of the whole subcutaneous adipose tissue of patients revealed an upregulation of genes involved in immunity (Figure 4B). More specifically, we showed that TREM2 was upregulated in the adipose tissue of both patients carrying lamin A mutations with a fold change of 7 (p < 0.001). TREM2 gene encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) receptor superfamily and was shown to be a major macrophage sensor of extracellular lipids. Furthermore, we observed a drastic decrease of SLPI in the adipose tissue of the two patients. SLPI gene encodes a secretory leukocyte protease inhibitor, which is an important anti-inflammatory adipokine that counteracts adipose tissue inflammation and enhances browning of adipose tissue [START_REF] Liu | Fat-Specific Knockout of Mecp2 Upregulates Slpi to Reduce Obesity by Enhancing Browning[END_REF].

Recent advances on pathological mechanisms involved in progeroid or tissue specific laminopathies, report the activation of the AKT/mTOR pathway, ultimately impairing the autophagic activity [START_REF] Chiarini | The Cutting Edge: The Role of mTOR Signaling in Laminopathies[END_REF]. In our experiments using RNAseq, we did not notice any alteration in the expression level of genes involved in this pathway.

Conclusions

Our work has shown that two heterozygous rare missense mutations in LMNA are associated with severe metabolic alterations, such as hypertriglyceridemia and insulin resistance, and are associated with premature senescence at the cellular level. Altogether, our results confirm that the pattern of LMNA associated phenotypes should be extended to cardio-metabolic phenotypes without subcutaneous lipoatrophy.

Future studies are needed to define which patients should be genetically screened, as it is likely that this condition is under-diagnosed, given the high prevalence of metabolic diseases worldwide. Further advances may provide the insights necessary to understand the many mechanisms and pathways by which A-type lamins influence metabolism.

Figure 1 .

 1 Figure 1. Patient 1 clinical description. (A) Pedigree of patient 1 family, (B) Photographs of patient 1 showing central/android shape obesity with fat accumulation in the facial and supraclavicular regions, and cushingoid morphotype. (C) Dual energy X-ray absorptiometry (DEXA) showing body fat distribution. (D) Percentage of fat mass compared to the reference curve and abdominal computed tomography (CT) scan confirming the presence of a small amount of subcutaneous adipose tissue (SAT) (superficial and deep), and an excessive accumulation of visceral adipose tissue (VAT). (E) Needle fat biopsy showing the same adipocytes diameter as a type 2 diabetic population matched for age, sex, and body mass index (BMI) and higher adipocytes diameter than obese non diabetic (ND) population matched for age, sex and BMI.

Figure 2 .

 2 Figure 2. Patient 2 clinical description (A) Pedigree of patient 2 family. (B) Photographs of patient 2 showing no lower limb lipoatrophy but a high waist circumference. (C) DEXA showing body fat distribution and the moderate excess of fat mass (31.1%) compared to the reference curve. (D) Needle fat biopsy showing smaller adipocytes than a matched for age sex and BMI obese non-diabetic (ND) and a type 2 diabetic population. (E) Abdominal CT scan confirming the presence of an excess of deep subcutaneous fat.

Figure 3 .

 3 Figure 3. Nuclear shape anomalies and senescence studies. (A) Percent of nuclear shape anomalies counted by immunofluorescence in control (in black), and patient (in grey) cells (left panel), and representative pictures of control and patient cells after staining with DAPI and anti-lamin A fluorescent antibody (right panel). (B) Percent of nuclear shape anomalies after transfection in control and patient fibroblasts with a siRNA targeting 3 UTR of LMNA (upper panel); representative pictures of patient cells showing improved nuclear shape after siRNA transfection (lower panel); * represents low staining in nuclei with normal shape. (C) Percent of nuclear shape anomalies after transfection of control fibroblasts with plasmids expressing either wild type prelamin A (in black) or mutants prelamin A (in grey) tagged with GFP at the N-terminal end (left panel) and representative pictures of each conditions (right panel). (D) BrdU incorporation of control and patient cells. (E) Senescence associated to beta-galactosidase production. In these experiments, controls correspond to fibroblasts from healthy donors. White arrows on the pictures show cytoplasmic staining and scale bars 5 µm. The number of independent experiments was between three and seven. Asterisks correspond to significant p-values: * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4 .

 4 Figure 4. RNAseq on whole adipose tissue biopsies. (A) Heat-map and top-20 of deregulated genes in the two patients with LMNA mutations compared to three controls paired for age and sex. (B) GO analysis showing the main pathways deregulated in patients with lamin A mutation. (C) Validation by RT-qPCR of three regulated genes in controls and patients (TREM2, SLPI, FOS) in adipose tissue, * p < 0.05 vs. controls; ** p < 0.01 vs. controls. (D) RT-qPCR of SLPI and FOS in fibroblasts. TREM2 was not expressed in fibroblasts.

Funding: This research received no external funding.

Acknowledgments:

The authors warmly thank the patients who participated in this study and the members of the Genomics and Bioinformatics Platform (GBiM) from the unit 1251/Marseille Medical Genetics facility for performing and analyzing the RNA-Seq experiments.

Conflicts of Interest:

The authors declare no conflict of interest.