Keywords: Yttrium, synchrotron X-ray fluorescence, fossil imaging, apatite

The development of X-ray tomography in the last decade has led to a revolution in palaeontology in providing a means of imaging 3D fossils. In turn, imaging of flat fossils has strongly benefitted from critical improvement of synchrotron X-ray fluorescence (XRF). The latter, which allows mapping the 2D distributions of major-to-trace elements over decimetrescale objects, usually targets metals with atomic number (Z) up to the strontium (K-shell emission lines); in the same energy domain, L-shell emission lines of heavier elements (particularly lead) can also be analysed. Fluorescence signal from strontium can escape from a depth of few 100 µm in fossils, thereby revealing, due to its substitution for calcium in calcium phosphates (apatite group minerals and bone), hidden fossil bone or phosphatized remains. Nonetheless, strontium similarly substitutes for calcium in calcium carbonates, resulting in the absence of contrast when fossils are preserved in limestone. Here, we show that this issue can easily be overcome by using X-rays slightly higher in energy (17.2 keV and above), to also excite and detect yttrium fluorescence. Together with lanthanides (collectively known as the rare earth elements), yttrium preferentially substitutes for calcium in calcium phosphates, offering anatomical contrasts for a wider range of fossils. This is demonstrated here for three fossils, both vertebrates and invertebrates, from different ages and depositional environments. We further discuss the chemistry behind the different behaviour of strontium and yttrium in calcium phosphates and carbonates. Although yttrium is also found in other (rarer) minerals, its mapping using synchrotron XRF could be used as a proxy to pinpoint calcium phosphates in fossils and other geological materials.

ADVANCED imaging techniques are now used on a regular basis in palaeontology as they can provide more accurate views of, or reveal new, anatomical characters preserved in fossils that are difficult or impossible to describe using optical photography and microscopy.

Actually, since the early microscopic observations of petrified wood by Robert Hooke in 1665, palaeontologists have often been pioneers in using, and sometimes even developing, new methodologies to extract as much information as possible from their fossils.

The last ten years has witnessed the development of a series of X-ray-based imaging techniques that have proved to be fantastic sources of new anatomical information with no, or only limited, sample preparation (see e.g. [START_REF] Sutton | Techniques for virtual palaeontology[END_REF]Gueriau et al. 2016 for recent reviews). X-ray tomography, which provides 3D reconstructions of fossils based on local contrasts due to the absorption of X-rays, allows access to the internal morphology of threedimensionally preserved fossils with an unprecedented level of detail (e.g. [START_REF] Tafforeau | Applications of X-ray synchrotron microtomography for nondestructive 3D studies of paleontological specimens[END_REF]). Yet, it suffers from a physical limitation when it comes to the study of flattened fossils, due to the extremely high difference in X-ray absorbance in different directions (along the flat and long dimension of the sample respectively).

Imaging of flat fossils has strongly benefitted from critical improvements in the field of synchrotron rapid scanning X-ray fluorescence (XRF), which produces 2D distributions of major-to-trace elements over decimetre-scale objects, including entire fossil birds (e.g. [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF]Bergmann et al. , 2012;;[START_REF] Wogelius | Trace metals as biomarkers for eumelanin pigment in the fossil record[END_REF][START_REF] Manning | Synchrotron-baser chemical imaging reveals plumage patterns in a 150 million year old early bird[END_REF][START_REF] Egerton | The mapping and differentiation of biological and environmental elemental signatures in the fossil remains of a 50 million year old bird[END_REF], with a micrometric lateral resolution or even better. It appears particularly promising as an approach to reveal anatomical details preserved as 'chemical ghosts', such as the feather shaft (rachis) in the Thermopolis Archaeopteryx [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF], the pigmentation pattern in the early bird Confusiusornis [START_REF] Wogelius | Trace metals as biomarkers for eumelanin pigment in the fossil record[END_REF] or the dentition in a fossil lizard [START_REF] Edwards | Mapping prehistoric ghosts in the synchrotron[END_REF], as well as restored areas [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF]. Beyond anatomical information, the chemical information provided by synchrotron-based XRF also offers unexpectedly detailed palaeobiological, palaeoenvironmental and taphonomic information through the characterisation of the organic, elemental and mineralogical compositions of fossils [START_REF] Wogelius | Trace metals as biomarkers for eumelanin pigment in the fossil record[END_REF]Bergmann et al. 2012;[START_REF] Anné | Synchrotron imaging reveals bone healing and remodeling strategies in extinct and extant vertebrates[END_REF]Anné et al. , 2016;;Edwards et al. 2014;[START_REF] Barden | Geochemical evidence of the seasonality, affinity and pigmentation of Solenopora jurassica[END_REF][START_REF] Egerton | The mapping and differentiation of biological and environmental elemental signatures in the fossil remains of a 50 million year old bird[END_REF]Gueriau et al. 2016).

The principle of synchrotron-based XRF is very similar to elemental mapping under a scanning electron microscope (SEM-EDX): X-ray photons of the incident beam interact with the inner electrons in some of the sample atoms, creating a vacancy and leaving the atom in an ionised state; the atom returns to its ground state when the vacancy is filled by an outer shell electron, emitting a characteristic XRF photon in the process (Fig. 1D-E). However, instead of electrons, X-ray emission in synchrotron-based XRF is produced by bombarding atoms with X-rays generated by an X-ray source, here a synchrotron. Synchrotrons are largescale light source facilities that exist in many countries around the world (www.lightsources.org). A synchrotron possesses several tens of independent experimental end stations, so-called beamlines, which use different energies and / or different setups. A schematic view of a synchrotron, a beamline and a typical XRF setup are shown in Figure 1, together with the XRF principle. The main advantages are that the synchrotron setups can offer an efficient focussing of the X-rays down to sub 100-nm sizes (see e.g. Stangl et al. 2015), can accommodate much larger fossils (Bergmann et al. 2012), and, due to their very large intensity (brightness), are far more sensitive to trace elements. Indeed, the detection limits are in the vicinity of the part per million level [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF][START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF]Gueriau et al. , 2015)), compared to the ca 0.1-5 wt% level achievable using EDX [START_REF] Friel | X-ray mapping in electron-beam instruments[END_REF]. Another great advantage of the method is its non-destructiveness and non-invasiveness in most situations.

The important experimental parameter when performing XRF mapping is the excitation energy/wavelength (often expressed as acceleration voltage under a SEM, since the bombarding particles are electrons). The energy limits the range of elements that will be excited, and consequently that can be detected. The very broad and continuous spectral range of the synchrotron radiation up to the hard X-rays offers the ability to finely change the energy of the beam (tunability), allowing the element(s) of interest to be preferentially excited. Synchrotron-based XRF experiments are usually performed using excitation energies in the 3.15-17 keV (kilo electron volts) range, optimised for mapping low-Z elements (Ca and lighter) and high-Z elements (Ca and heavier) depending on the targeted elements. As strontium is known to substitute for calcium in bone apatite, vertebrate palaeontologists use X-rays up to 17 keV X-rays, optimised to excite fluorescence originating from elements up to strontium. Sr maps notably allowed tracking fossil bone physiology [START_REF] Anné | Synchrotron imaging reveals bone healing and remodeling strategies in extinct and extant vertebrates[END_REF](Anné et al. , 2016(Anné et al. , 2017 and references therein), as well as to virtually reveal fossil bones hidden under a fine layer of sediment, without the need for any prior delicate preparation [START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF].

However, the main limitation of the latter approach is that Sr indistinctly substitutes for Ca in calcium phosphates (apatite group minerals; bone) and calcium carbonates, resulting in the absence of Sr contrast in the case of fossils preserved in limestone. Here, we show that this issue can easily be overcome by using X-rays slightly higher in energy (i.e. 17.2 keV and above), to also excite yttrium, which preferentially substitutes for calcium in calcium phosphates.

[Fig. 1 about here]

RESOLVING THE ANATOMY OF FLAT FOSSILS

The reason why synchrotron XRF offers a good alternative to X-ray tomography for resolving the anatomy of flat fossils is that XRF information is not restricted to the very surface of the sample, but can be assimilated from a depth up to several hundred µm. Indeed, X-rays can penetrate the substrate deeply, depending on their energy (the higher the energy, the deeper the penetration) and on the density of the materials (the higher the density, the weaker the penetration). For most fossils (excluding unusual preservation in high-density minerals), Xrays at 16-17 keV penetrate up to a couple of millimetres. Nevertheless, while XRF photons are produced across the whole interaction volume, they have lower energies than the exciting X-rays and can therefore surface and reach the detector only if originating from a shallower depth (photons produced deeper are totally absorbed by the material itself before getting out of the sample; Fig. 2). Still, the detectable information depth depends on two main physical parameters. First, the heavier the element, the more energetic the XRF photons and the deeper they can be detected (note that, consequently, air between the sample and the detector also absorbs XRF photons, so that good low-Z element detection requires vacuum or helium gas environments). Second, several fluorescence emissions can be generated for each element, depending on the electronic orbitals (electron shells) involved in the process; the most energetic arise from the L→K (Kα) and M→K (Kβ) transitions, whereas M→L (Lα) transitions are less intense and ca 7 times less energetic. Consequently, information depth is higher for K-lines than L-lines, and using different fluorescent lines and/or different elements allows tuning of the probed depth and distinguishing surface from volume. Information depth from Sr K-lines in apatite is 200-250 µm. Thus, fossils or anatomic features hidden under a fine layer of sediment can be revealed [START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF]).

[Fig. 2 about here]

Another important parameter to consider for anatomical reconstructions is the spatial resolution (i.e. the pixel size), which defines the precision and level of details achievable.

Synchrotron XRF major-to-trace elemental distributions are collected using mapping (or raster-scanning), i.e. the sequential acquisition of spectra from adjacent regions of the fossil by moving each region (by a lateral quantity of the size of the probe, i.e. the size of the X-ray beam) into the photon beam, line by line. Mapping can be performed following two modalities. The first mode, primarily implemented at synchrotrons, is known as the step-bystep mode: a XRF spectrum is recorded for each position of the sample, after the sample has moved by the defined scanning step. This implies important dead timesperiods of time when no data are recordedin the acquisition sequence, as the scanning stage has to reach the exact next position to trigger the measurement. Dead times easily reach a few tenth of seconds between each pixel, resulting in scanning times of 1-2 h/cm 2 at 100 μm resolution (depending on the amount of dead time and the chosen counting time per pixel). The second mode, socalled rapid scanning or flyscan (see [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF]Leclercq et al. 2015, respectively), is at the origin of the increasing use of synchrotron-based XRF mapping in palaeontology, particularly for anatomical reconstruction. It consists of scanning each line by continuously moving the sample: each spectrum (i.e. each pixel) does not arise from a single spot (corresponding to the beam size) but from the sample area scanned during a defined time interval (dwell time). As dead times are suppressed (or at least greatly reduced), this mode allows scanning times of a few min/cm 2 at 100 μm resolution (up to ~30 s/cm 2 using a dwell time of ~3 ms and by only recording integrated intensities in 16 spectral regions of interest; [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF][START_REF] Edwards | Mapping prehistoric ghosts in the synchrotron[END_REF]. Such a reduction of the counting time per pixel results in an important loss of signal-to-noise ratio at each pixel, yet the corresponding increase in map speed and map size (or spatial resolution) produces much more XRF spectra that can be summed over a homogeneous area to partly compensate the loss of signal-tonoise. Acquisition times, which depend on the fossil size and the desired spatial (pixel size) and spectral (dwell time) resolutions, are typically of a few to many hours (see Table 1 for indicative examples). Consequently, when high lateral resolution is mandatory to recover fine anatomical details, one has to consider that only a few samples can be mapped over a few days of beam time (typical duration of a synchrotron experiment), and the technique remains restricted to specimens particularly important, rare, exceptional and/or difficult to prepare.

[Table 1 about here]

YTTRIUM MAPPING

Using 17 keV X-rays (the maximal energy usually used for fossil mapping), it is possible to excite K-lines up to strontium (electron binding energy, 16.1 keV), and L-lines up to palladium (electron binding energy, 16.73 keV). Tuning the energy of the impinging X-rays to 17.2 keV (and above) also allows exciting of yttrium K-lines (electron binding energy, energy than Sr photons, the information depth for Y photons is also higher, ca 250-300 µm in apatite, offering even better views of flat fossils (see [START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF]. In addition to the difference in information depth, Y maps have the potential to resolve fossil anatomies in a wider range of fossils, particularly where fossils are preserved in limestone. Indeed, while Sr substitutes for calcium equally well in bone and calcium carbonates, Y and the lanthanides (collectively known as the rare earth elements; REE), preferentially substitute for calcium in calcium phosphates (see also discussion below).

We present here three cases where anatomical, palaeobiological or taphonomical information could only be inferred from Y mapping (Figs 345). Data were collected at the DiffAbs beamline of the SOLEIL synchrotron (Gif-sur-Yvette, France) using an excitation energy of 17.2-18 keV. The incoming X-ray beam was collimated by two bendable mirrors, monochromatised using a Si(111) double-crystal monochromator (energy resolution ΔE/E ≈ 10 -4 ) and focused using Kirkpatrick-Baez (KB) mirrors [START_REF] Kirkpatrick | Formation of optical images by X-rays[END_REF] down to a spot size of 9×7 μm 2 (H×V, full width at half maximum). The fossils were mounted on a xyz scanner stage with μm accuracy, and orientated at 45° to the incident beam and at 45° to the XRF detector, a 4-element silicon drift XRF detector (Vortex ME4, Hitachi High-Technologies Science America, Inc., total active area: 170 mm 2 ) placed in the horizontal plane. The fossil in Fig. 3 was mapped in step-by-step mode, and fossils in Figs 4 & 5 in flyscan mode [START_REF] Leclercq | Flyscan: a fast and multitechnique data acquisition platform for the SOLEIL beamlines[END_REF]. Details about other scan parameters and data processing are available in the figure captions.

Looking for 'Chemical ghosts' in Palaeospondylus

Palaeospondylus gunni Traquair, 1890 is a mysterious, fish-like fossil vertebrate of highly debated affinities, which has been assigned to various jawless and jawed vertebrate groups, including both larval and adult stages (Moy-Thomas 1940; [START_REF] Thomson | A larval Devonian lungfish[END_REF][START_REF] Joss | Is Palaeospondylus gunni a fossil larval lungfish? Insights from Neoceratodus forsteri development[END_REF][START_REF] Newman | New information on the enigmatic Devonian vertebrate Palaeospondylus gunni[END_REF]Johanson et al. 2012, 2017[START_REF] Gardiner | The fossil record of tadpoles[END_REF][START_REF] Hirasawa | Palaeospondylus as a primitive hagfish[END_REF]. It is found in the Middle Devonian (~390 Myr) Achanarras fish bed, Achanarras Quarry (Scotland). The fish bed consists of lacustrine laminites of organic, carbonate (dolomite and calcite) and clastic types [START_REF] Trewin | Palaeoecology and sedimentology of the Achanarras fish bed of the Middle Old Red Sandstone, Scotland[END_REF], so that Ca and Sr maps offer no contrast between the fossil (mineralised cartilage, see [START_REF] Johanson | No bones about it: an enigmatic Devonian fossil reveals a new skeletal framework-a potential role of loss of gene regulation[END_REF]) and the sedimentary matrix (Fig. 3). However, the Y map clearly resolves the fossil morphology.

Although the latter does not reveal any new anatomical details, it was here critical to precisely localise an unexpected distribution of copper, surrounding the vertebrae and very specifically located posterior to the paired rostral sensory structures (see Johanson et al. 2017). Such organised distribution very likely represents palaeobiological (in vivo incorporation) or taphonomic (post mortem incorporation) tissue-specific chemistry, possibly representing the 'chemical ghosts' of soft-tissues that could provide new arguments for P. gunni taxonomic assignment.

[Fig. 3 about here]

Visualising the anatomy of a new, rare fossil fish from the Cretaceous of Brazil

The Late Cretaceous Bauru continental basin in Brazil has delivered remains of various dinosaurs, crocodiles, turtles and fishes. Among newly discovered fossils, a teleost fish, largely hidden under a fine layer of sediment, appears particularly interesting as it represents one of the very few articulated specimens ever found at this locality. The new fossil preserves the anterior part of the animal (skull and first vertebrae) in a fine-to medium-grained arkosic limestone (Fig. 4). Due to the arkosic content, the Ca and Sr maps here provide a good view of the fossil, yet the ventral part of the skull and the first vertebrae remain partly hidden under what is probably more concentrated carbonate grains. Only the Y map clearly reveals the anatomy of this new taxon, which appears be a 'primitive' teleost. A formal description is in preparation.

[Fig. 4 about here]

Highlighting fossil anatomy for point analyses

Besides vertebrates, yttrium mapping can also be applied to many fossil invertebrates, particularly those from exceptional preservation deposits that preserve soft-tissues as apatite group minerals (phosphatisation), and consequently have incorporated yttrium and the other rare earth elements. For instance, it provided a clearer view of the cephalic and thoracic anatomy of a decapod shrimp from the Cretaceous of Morocco [START_REF] Gueriau | Trace elemental imaging of rare earth elements discriminates tissues at microscale in flat fossils[END_REF].

Anatomical reconstructions are rarely the primary aim of a synchrotron-based XRF experiment. It often serves as a support for further point analyses, aiming at recovering palaeobiological, palaeoenvironmental and taphonomic information from the fossils, e.g., through the collection of a high signal-to-noise ratio XRF spectrum for elemental quantifications [START_REF] Bergmann | Archaeopteryx feathers and bone chemistry fully revealed via synchrotron imaging[END_REF][START_REF] Anné | Synchrotron imaging reveals bone healing and remodeling strategies in extinct and extant vertebrates[END_REF]Anné et al. , 2016;;Gueriau et al. 2015), or through X-ray absorption spectroscopy, an associated technique that provides detailed information about the oxidation state and coordination environment of a target atom (e.g. [START_REF] Wogelius | Trace metals as biomarkers for eumelanin pigment in the fossil record[END_REF]Edwards et al. 2014). Points of interest can be selected by using a microscope, set at the beamline to indicate the exact position where the beam hit the sample.

However, the sample roughness can easily make this useful tool quite imprecise to finely define such points of interest. The best way to circumvent this problem is to first perform XRF mapping, and then move the sample to a pixel of interest, read on the XRF maps.

Furthermore, collecting first a XRF map remains necessary when interested in the chemistry of fossils, and/or 'chemical ghosts', i.e. anatomical features which are not visible under optical light (such as, e.g., the unexpected Cu distribution in Palaeospondylus gunni, Fig. 3).

We investigated on the preservation modes of a gladius-bearing coleoid (Dorateuthis syriaca Woodward, 1883, specimen MNHN.F.A68134) from the Upper Cretaceous (~85 Myr) marine Lagerstätten of Sahel Alma, Lebanon (Fig. 5). As it is embedded in lithographic limestone, the Ca map and the Sr map only show a topographical contrast (due to sample roughness), but do not resolve the fossil morphology, which is only seen using the Y map as the fossil is partially phosphatised. The apparent pattern of phosphatisation raises questions regarding differential soft-tissue preservation in such localities.

[Fig. 5 1) is nine, and that of Ca(2) is seven. The coordination number of Ca 2+ in carbonates (XCO3) is mostly six (e.g. calcite and dolomite), but is nine in aragonite. Ionic radii for Ca 2+ , Sr 2+ and Y 3+ in these coordinations are given in Table 2.

However, and as exemplified by the contrasting distributions observed in our elemental maps (though information depth is comparable), the incorporation behaviours of Sr and Y differ between calcium phosphates and carbonates. Incorporation of Sr, Y and the other REEs in fossil bone apatites and geological apatites has been extensively investigated.

Substitution and adsorption mechanisms (difficult to distinguish from each other) constitute the most important mechanisms for incorporation of these elements into bone during diagenesis (Trueman and Tuross 2002). Relative Sr, Y and REEs amounts in fossils (hundred to thousand ppm concentration or greater for Sr, Y and the light REEs, less for the heavy REEs) reflect simultaneously the connectivity of the environmental water network, local redox, the specific surface area of the bioapatite nanocrystals and physico-chemical conditions and properties of substituted apatite [START_REF] Kocsis | Protracted diagenetic alteration of REE contents in fossil bioapatites: direct evidence from Lu-Hf isotope systematics[END_REF]Trueman et al. 2011;[START_REF] Trueman | Chemical taphonomy of biomineralized tissues[END_REF][START_REF] Herwartz | Rare earth element systematics of fossil bone revealed by LA-ICPMS analysis[END_REF]. In contrast, very few data exist in the literature for REEs incorporation mechanisms in calcium carbonates. [START_REF] Rankama | Geochemistry[END_REF] reported that, although Y can replace calcium in calcite, the substitution is extremely slight. [START_REF] Dawson | Trace-element content and partitioning in calcite, dolomite and apatite in carbonatite, Phalaborwa, South Africa[END_REF] further showed that while Sr is partitioned to only a slightly greater extent into the calcite than apatite, apatite incorporates higher concentrations of REEs than calcite and dolomite. However, Sr and Y concentrations in naturally occurring apatites and calcites point in that same direction: impoverishment of Y in carbonates compared to Sr, whereas their concentrations in apatite minerals is similar [START_REF] Rakovan | Differential incorporation of trace-elements and dissymmetrization in apatitethe role of surface-structure during growth[END_REF][START_REF] Borsato | Trace element distribution in annual stalagmite laminae mapped by micrometer-resolution Xray fluorescence: Implications for incorporation of environmentally significant species[END_REF][START_REF] Morteani | Geochemistry of phlogopite, diopside, calcite, anhydrite and apatite pegmatites and syenites of southern Madagascar: Evidence for crustal silicocarbonatitic (CSC) melt formation in a Panafrican collisional tectonic setting[END_REF].

[Table 2 about here]

SUMMARY AND PERSPECTIVES

We introduce synchrotron X-ray fluorescence mapping of yttrium as a novel means of resolving the anatomy and chemistry of flattened vertebrate and other phosphatised fossils embedded within limestone and carbonate-rich sediments. In the three examples given herein, from different ages and depositional environments, yttrium is the only element that thoroughly reveals the fossil anatomy. Yttrium distributions can then be used to describe anatomical characters previously hidden under a fine layer of sediments due to its information depth of a few hundred microns in fossils (e.g. in the new teleost fish from the Late Cretaceous of Brazil, Fig. 4), to allow precise localisation of unexpected elemental distribution (e.g. in the mysterious Devonian Palaeospondylus gunni, Fig. 3), as well as to serve as a support for further point analyses (e.g. in the gladius-bearing coleoid from the Cretaceous of Lebanon, Fig. 5).

Although we focus here on yttrium mapping, one has to remember that XRF mapping at 17.2 keV and above does not excite only yttrium but actually excites most elements from the periodic table (K-lines up to yttrium, and L-lines up to uranium), so that it also allows assessing the major-to-trace elemental composition of the fossil under investigation. Since the full XRF spectrum is recorded in each pixel of the map, the presence of other trace elements can be detected, and a full spectral decomposition of the spectra (often performed using the PyMCA data-analysis freeware; [START_REF] Solé | A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra[END_REF] allows their distribution to be displayed and their concentration to be estimated. For instance, this could be particularly interesting in the case of the REEs in offering the ability to non-destructively extract REE patterns, ratios and anomalies in fossils, which potentially can provide palaeoredox and palaeoenvironmental information (see Gueriau et al. 2015 and references therein).

Finally, we discuss the chemistry controlling the different behaviours of strontium and yttrium in calcium phosphates and carbonates, for which little is actually known.

Nevertheless, their dichotomous behaviour is clearly shown from our datasets, and at least two highly promising perspectives arise from our results: (1) synchrotron X-ray fluorescence mapping of yttrium could be used as a proxy (in association with complementary laboratory analyses such as petrography and electron microscopy) to pinpoint calcium phosphates in fossils, as well as other geological materials; (2) the high sensitivity (up to the ppm range) offered by the synchrotron beam could enhance the search for traces of early calcium phosphate (vertebrate) skeletons in the fossil record, particularly in the putative basal chordates from the Cambrian.

TABLES

  about here] DIFFERENTIAL INCORPORTION OF Sr AND Y Sr and Y are known to substitute in trace quantities (up to several wt%) for Ca in the crystal structure of Ca-based minerals, their ionic radii being close to that of Ca in these minerals. In minerals from the apatite family [Ca(1)4][Ca(2)6][(PO4)6][OH,F,Cl]2, calcium is present as Ca 2+ and occupies two distinct sites -Ca(1) and Ca(2) -in the apatite crystal structure. The coordination number (CN) of Ca(

FIGURESFIG. 1 .

 1 FIGURES

TABLE . 1

 . . Indicative acquisition times for synchrotron rapid scanning XRF mapping, depending on the size of the area of interest to be mapped, pixel size and dwell time.

	map size (cm 2 )	pixel size (µm 2 )	dwell time (ms)	acquisition time
	2×2	25×25	30	5h20'
	2×2	15×15	15	7h24'
	2×2	15×15	10	4h56'
	5×5	100×100	30	2h05'
	5×5	50×50	30	8h20'
	5×5	50×50	15	4h10'
	10×10	100×100	30	8h20'
	10×10	75×75	20	9h53'
	10×10	50×50	10	11h07'
	50×50	200×200	20	34h43'
	50×50	200×200	10	17h22'
	50×50	100×100	3	20h50'

TABLE . 2

 . . Ionic radii of Ca 2+ , Sr 2+ and Y 3+ depending on the coordination number [CN] of

	the Ca sites in carbonates [VI; IX in aragonite] and apatite group minerals [VII and IX] (from
	Shannon 1976).
	Ion	Ionic radius (Å) [CN]
	Ca 2+	1.00 [VI]
		1.06 [VII]
		1.18 [IX]
	Sr 2+	1.18 (+18%) [VI]
		1.21 (+14.1%) [VII]
		1.31 (+11%) [IX]
	Y 3+	0.90 (-10%) [VI]
		0.96 (-9.4%) [VII]
		1.075 (-8.9%) [IX]

Acknowledgements. We acknowledge SOLEIL Synchrotron for provision of beamtime, S. Réguer, D. Thiaudière and P. Joly for assistance at the DiffAbs beamline, as well as N.

Leclercq, J. Berthaud and teams at the "control data acquisition" and "electronics" groups of SOLEIL for the development of the flyscan platform. G. Clément (MNHN, Paris) provided access to the Palaeospondylus specimen, J.-M. Pacaud and S. Charbonnier (MNHN, Paris) to the gladius-bearing coleoid, identified by R. Jattiot (Université de Bourgogne, France). P. M. Brito (UERJ, Brazil) collected and provided the new teleost fish from Bauru, and information about the fossil locality. D. Germain (MNHN, Paris), C. Cupello (UERJ, Brazil) and P. Loubry (MNHN, Paris) took the photographs, respectively of the Palaeospondylus specimen, the new teleost fish from Bauru and the gladius-bearing coleoid. The authors also warmly thank S. Thomas, N. Edwards and an anonymous reviewer, whose comments, suggestions and corrections helped to improve the manuscript. This work was developed as part of the IPANEMA / MNHN agreement on collaborative research. IPANEMA is supported by ANR within the PATRIMEX EquipEx (ANR-l1-EQPX-0034) and by Région Île-de-France in the context of the DIM "Matériaux anciens et patrimoniaux".


Yang Yang

Jingjing Wang

Yongmin Zhang

Jing Li

Wenji Sun

Black Sesame Seeds Ethanol Extract Ameliorates Hepatic Lipid Accumulation, Oxidative Stress and Insulin Resistance in Fructose-induced Nonalcoholic Fatty Liver Disease (NAFLD)

Keywords: black sesame seeds, nonalcoholic fatty liver disease, fructose, insulin tolerance, hepatic oxidative stress

The aim of the present study was to investigate the effect of black sesame seeds ethanol extract (BSSEE) against nonalcoholic fatty liver disease (NAFLD) in fructose-fed mice. Mice were fed diet without or with 30% fructose for 8 consecutive weeks, while mice in three BSSEE tested groups received different dose of BSSEE (0.5, 1 and 2 mL/kg) once a day from the 5th week to the 8th weeks. Administration of BSSEE dose-dependently exerted antiobesity and protective effect against metabolism disorder in fructose-fed mice. Liver histological results indicated that administration of BSSEE significantly reduced hepatic lipid accumulation. Insulin tolerance tests (ITT) and glucose tolerance tests (GTT) along with decrease of serum insulin and glucose levels by BSSEE treatment suggested the improvement of body insulin resistance, and administration of 1 and 2 mL/kg BSSEE mitigated liver insulin resistance as the evidence of down-regulated expressions of phospho-JNK1, phospho-NF-κB p65, phospho-IRS1 (Ser 307) and phospho-IKK, and up-regulated XBP1 expression, as well as reductions of hepatic TNF-α and IL-6 levels. In addition, BSSEE treatment reduced hepatic oxidative stress through increasing GSH, vitamin C and Nrf2 levels, enhancements of SOD, CAT and GSH-Px activities, and decreasing MDA and NO levels. These results demonstrated that black sesame seeds showed good effects against NAFLD-related metabolic diseases in fructose-fed mice, and may be a potent dietary supplements in the prevention of the diseases.

■ INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) comprises a wide pathologic spectrum of liver pathologies ranging from steatosis to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma, which is characterized by the common central feature of the fatty degeneration in liver cells [START_REF] Floros | Non-alcoholic fatty liver disease (NAFLD): summary of NICE guidance[END_REF] . Nowadays, the disease is considered as one of the most prevalent chronic liver disease all over the world with a reported prevalence ranging 20-30% according to the studied populations [START_REF] Do | Epidemiology of nonalcoholic fatty liver disease: a primer[END_REF] . The important reasons for this high prevalence is closely associated with the increasing prevalence of obesity, diabetes and metabolic syndrome [START_REF] Al-Dayyat | Non-alcoholic fatty liver disease and associated dietary and lifestyle risk factors[END_REF] . Although the molecular mechanisms involved in the pathopoiesis of NAFLD are not clearly understood, it has been found that impaired insulin function is a very risk factor in the development of NAFLD. In the progression of NAFLD, pro-infiammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) that drive insulin resistance is closely linked with the aggravation of NAFLD through activation of specific intracellular signalling pathways, involving in not only the c-Jun NH2-terminal kinase 1 (JNK1), iκB kinase (IKK), nuclear factor kappa B (NF-κB), x-box binding protein 1 (XBP1), but also reactive oxygen species (ROS) and free fatty acids (FFA), where all these pathways could interact with insulin signalling via serine/threonine inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) [START_REF] Hotamisligil | Endoplasmic reticulum stress and the inflammatory basis of metabolic disease[END_REF] . Notably, some food supplement is rich in antioxidants and have good anti-inflammatory effect, which may be more beneficial in prevention of NAFLD.

Fructose is one of main sugars consumption in diet. In the United States, Europe and China, high-fructose corn syrup (HFSC), containing more than 42% fructose, has became a replacement of refined sugar in many foods and most sweetened beverages, such as soft drinks, juice beverages, canned fruits, jellies, jams, breakfast cereals and baked goods [START_REF] Spruss | Dietary fructose and intestinal barrier: potential risk factor in the pathogenesis of nonalcoholic fatty liver disease[END_REF] . Fructose intake can cause more productions of glucose, glycogen, lactate and pyruvate, which contribute to the formation of triglycerides in liver cells [START_REF] Spruss | Dietary fructose and intestinal barrier: potential risk factor in the pathogenesis of nonalcoholic fatty liver disease[END_REF][START_REF] Castello | Regulation of GLUT5 gene expression in rat intestinal mucosa: regional distribution, circadian rhythm, perinatal development and effect of diabetes[END_REF] . Further studies indicated that high-fructose diets activates lipogenesis at least partly linked to insulin resistance [START_REF] Spruss | Dietary fructose and intestinal barrier: potential risk factor in the pathogenesis of nonalcoholic fatty liver disease[END_REF][START_REF] Nomura | The role of fructose-enriched diets in mechanisms of nonalcoholic fatty liver disease[END_REF] . In addition, dietary high fructose facilitates oxidative damage in various tissues. More production of ROS has been claimed as a persistent contributor for liver cell damage and impaired glucose tolerance in fructose-induced NAFLD [START_REF] Suganthi | Effect of food seasoning spices mixture on biomarkers of oxidative stress in tissues of fructose-fed insulin-resistant rats[END_REF][START_REF] Armutcu | Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats[END_REF][START_REF] Bagul | Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats[END_REF][START_REF] Gaetano | Free radical biology for medicine: learning from nonalcoholic fatty liver disease[END_REF] . Long term intake of high fructose has been repeatedly proved to be a risk factor for the pathopoiesis of NAFLD both in animals and human [START_REF] Spruss | Dietary fructose and intestinal barrier: potential risk factor in the pathogenesis of nonalcoholic fatty liver disease[END_REF][START_REF] Suganthi | Effect of food seasoning spices mixture on biomarkers of oxidative stress in tissues of fructose-fed insulin-resistant rats[END_REF][START_REF] Bagul | Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats[END_REF] .

Sesame seed (sesamum indicum L.) is one of the world's most important and oldest oil seed crops known to humanity [START_REF] Raghav | Sesame: New Approaches for Crop Improvement[END_REF] . Sesame seeds and its oil are popularly used in cookery to flavour foods and drinks [START_REF] Bbc | Black sesame seeds recipes[END_REF] . Notably, sesame seed have many species (most being white sesame seeds and brown (golden) sesame seeds), one of which is black sesame seeds. The black sesame are rich in fatty oil, containing mainly oleic acid and linoleic acid, and other chemical constituents including sesamin, sesamolin, sesamol, vitamin E, phytosterols, lecithin, pedaliin, protein and minerals [START_REF] Herbs | Black Sesame Seeds (Semen Sesami Nigrum[END_REF] .

The black sesame seeds are more flavorful and have a stronger aroma than white or brown sesame seeds. Toasting black sesame seeds releases beneficial chemicals as well as enhancing the flavor. In addition, black sesame seeds are believed to have medicinal properties. In China, black sesame seeds is superior to other species as food for health and often acts as a traditional Chinese medicine for promoting shiny and dark hair, invigorating liver and kindy, benefiting life essence, nourishing blood and moistening the intestines [START_REF] Herbs | Black Sesame Seeds (Semen Sesami Nigrum[END_REF] . In addition, a comparative study found that black sesame seeds showed potent antioxidant activity than white sesame seeds [START_REF] Zhou | Phytochemical Contents and Antioxidant and Antiproliferative Activities of Selected Black and White Sesame Seeds[END_REF][START_REF] Hu | Antioxidant activity of extracts of black sesame seed (Sesamum indicum L.) by supercritical carbon dioxide extraction[END_REF][START_REF] Shahidi | Antioxidant activity of white and black sesame seeds and their hull fractions[END_REF] . It is worth mentioning that sesame oil mainly from white or brown sesames [START_REF] Periasamy | Sesame oil mitigates nutritional steatohepatitis via attenuation of oxidative stress and inflammation: a tale of two-hit hypothesis[END_REF][START_REF] Periasamy | A single dose of sesame oil alleviates liver injury and steatosis in steatohepatitis via modulation of oxidative stress, cytokines and PPAR[END_REF] , as well as its phytochemical compounds such as sesamin [START_REF] Zhang | Sesamin ameliorates hepatic steatosis and inflammation in rats on a high-fat diet via LXRα and PPARα[END_REF][START_REF] Liu | Sesamin Protects Mouse Liver against Nickel-Induced Oxidative DNA Damage and Apoptosis by the PI3K-Akt Pathway[END_REF][START_REF] Sirato-Yasumoto | Effect of sesame seeds rich in sesamin and sesamolin on fatty acid oxidation in rat liver[END_REF] , sesamolin [START_REF] Kang | Sesamolin inhibits lipid peroxidation in rat liver and kidney[END_REF][START_REF] Liu | Sesamol ameliorates diet-induced obesity in C57BL/6J mice and suppresses adipogenesis in 3T3-L1 cells via regulating mitochondria-lipid metabolism[END_REF] and sesamol [START_REF] Singh | Hepatoprotective effects of sesamol loaded solid lipid nanoparticles in carbon tetrachloride induced sub-chronic hepatotoxicity in rats[END_REF][START_REF] Ryu | LIQUID CHROMATOGRAPHIC DETERMINATION OF ANTIOXIDANT LIGNAN GLYCOSIDES IN SOME VARIETIES OF SESAME[END_REF] , have been reported that they show hepatoprotective effect and can alleviate steatosis in steatohepatitis by regulating lipid metabolism and enhancing anti-oxidative stress effect in animals 18-21, 24, 25 . However, limited studies have been performed on the investigation of the protective effect of black sesame seeds against NAFLD and its molecular biological mechanism in fructose-induced NAFLD. Since the high prevalence of NAFLD and lack of satisfactory treatments, finding the best diet to replace therapies may be a good choice for preventing and treating the disease.

Therefore, the aim of the present study was to investigate the effect of black sesame seeds ethanol extract (BSSEE) against NAFLD-related metabolic diseases.

■ MATERIALS AND METHODS

BSSEE Extraction and Main Ingredient Analysis.

Black sesame seeds (sesamum indicum L.) were obtained from Yurun Global Sourcing Center for Agricultural Products (Xi'an, Shaanxi, China), and pulverized using a Chinese herbal remedier (FW117, Nanchang Jiedao Scientific Instruments Co., Ltd., Jiangxi, China). 1 kg of this powder was extracted with 10000 mL of 95% ethanol (Analytical grade, Xi'an Chemical Reagent Factory, Shaanxi, China) at 25 °C and repeated three times. The ethanol extract was concentrated on a rotavapor (RE52CS-1, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) at 50 °C. Then 20.2 mL of the crude extract was obtained and stored at -20 °C for the analysis, and 1 mL of this extract weights 0.83 g. The yield of the obtained the black sesame seeds ethanol crude extract (BSSEE) from fresh black sesame seeds was 1.68% (w/w), calculated by the weight of the seeds used in the extraction procedure. BSSEE were analyzed using a LiChrosorb C18 column (4.6 mm i.d. × 250 mm, 5 μm, Merck, Darmstadt, Germany) in the analytical high performance liquid chromatography (HPLC) instrument (Waters 2695 HPLC system equipped with a PDA detector was used in addition to an Empower chromatographic workstation, Waters Corporation, Milford, Massachusetts, USA). Chromatographic separation was performed using an gradient elution at a flow rate of 1.0 mL/min with mobile phase for (A) methanol (HPLC grade, Merck, Darmstadt, Germany)/(B) distilled water: 30-100 % A ( 0-60 min). The column temperature was controlled at 30 °C, and the effluents were monitored at 280 nm. 0.1 mL of the black sesame seeds extract was dissolved in 1 mL methanol (Analytical grade, Xi'an Chemical Reagent Factory, Shaanxi, China) and filtered with 0.20 μm Nylon filter (Beijing Kaiyuan Guochuang technology Co., Ltd., Beijing, China), and then 20 μl of the dilute solution was injected for analysis. Calibration curves were constructed with reference standards and used to determine the concentrations of sesaminol, sesamin and sesamolin in BSSEE.

Sesaminol, sesamin and sesamolin (Purity ≥ 97%) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). As shown in the Figure 1, the three major components of BSSEE, sesaminol (1.92%), sesamin (16.33%) and sesamolin (13.06%) accounted for 31.31% of the total extract. This result is similar to previous studies [START_REF] Ryu | LIQUID CHROMATOGRAPHIC DETERMINATION OF ANTIOXIDANT LIGNAN GLYCOSIDES IN SOME VARIETIES OF SESAME[END_REF] .

Animals and Experimental Design.

Fifty healthy adult male kunming mice (20 ± 3 g) were purchased from the Experimental Animal Center, Xi'an Jiaotong University (SCXK2012-003, Xi'an, Shaanxi, China). Animals were housed at standard conditions: normal light/dark (12h/12h) cyclic conditions and 25 ± 3 ℃, 30-70% humidity. Use of the mice was reviewed and approved by both Northwest University and the local animal Ethics Committee.

Mice in all groups were fed a standard diet (Jiangsu Cooperative Medical Biological Engineering Co., Ltd., Nanjing, Jiangsu, China). The composition of diet was as follows: 63% carbohydrate, 18% crude protein, 5% crude fat, 3% crude fiber, 6% ash and 5% predefined minerals plus amino acids and vitamins. Representative ingredients of diet are ground wheat, ground corn, dehulled soybean meal, corn gluten meal, fish meal, chicken meal, soybean oil, methionine, lysine, cystine, calcium carbonate, brewers dried yeast, dicalcium phosphate and iodized salt. The energy content of the diet was 3.64 kcal/g. BSSEE was prepared into soybean oil (Zhejiang Tian Yushan Medicinal Oils Co., Ltd., Quzhou, Zhejiang, China) before administration to the mice. Animals were acclimated to housing conditions for one week. Subsequently mice were randomly divided into 5 groups with 10 each, including the control group, fructose group and three fructose + BSSEE (0.5, 1 and 2 mL/kg) tested groups. Except for the mice in the control group freely drank water without fructose, mice in all groups freely drank water containing 30% fructose (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China) for 8 consecutive weeks (From the 1st week to the 8th week). However, animals in those fructose + BSSEE (0.5, 1 and 2 mL/kg) tested groups received different dose of BSSEE (0.5, 1 and 2 mL/kg) by intragastric administration once a day at the end of the 4th week for 4 consecutive weeks (From the 5th week to the 8th week) respectively, while mice in the control group and the fructose group received the same amount of soybean oil.

During the whole experimental period, the body weight of each mouse was noted at three days intervals, and food intake and water consumption in each group was recorded every day.

On the last day, at the end of the drug administration, mice in all groups were fasted for 8 hours. Then all animals were anesthetized and blood of each mouse was collected through retro-orbital plexus. Subsequently all animals were euthanized, and the liver, epididymal adipose, subcutaneous adipose and perirenal adipose were collected and weighed. The liver of each mouse were collected and cut into two parts, with one part of liver lobe immersed in 4% paraformaldehyde solution for histological examinations and immunohistochemistry (IHC), while the other part of liver lobe stored at -80 ℃ for the biochemical analysis.

Serum Biochemical Analysis.

Serum was separated by centrifugation at 1000 g (4 ℃) for 10 min. The levels of biochemical parameters, including aspartate transaminase (AST), alanine transaminase (ALT), glucose, total cholesterol, and triglycerides, were measured colorimetrically and assayed by an automatic biochemistry analyzer (AU5800, Beckman Coulter, Inc., California, USA). Free fatty acids (FFA) were measured using a commercial kit (BC0590, Solarbio, Beijing, China), and insulin level was measured by a mouse insulin ELISA Kit (TWp001983, Shanghai Tongwei Industrial Co., Ltd., Shanghai, China), in which the results were obtained by a DNM-9602 Reader (Beijing Prang New Technology Co., Ltd., Beijing, China).

Histological Staining.

Liver tissues were fixed in 4% paraformaldehyde solution (137 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 2.7 mM KCl, 1000 mL deionized water, 4% paraformaldehyde, pH 7.4) for 24 hours, embedded in paraffin and cut into 5 μm sections using YD-1508R Tissue Slicer (Beijing Jiayuan Industrial Technology Co., Ltd., Beijing, China), and then selected good tissue sections were mounted on adhesion microscope slides (Citotest Labware Manufacturing Co., Ltd., Haimen, Jiangsu, China) and stained with hematoxylin & eosin (H&E) for the examination of the morphology of liver. In order to evaluate lipid droplet accumulation, liver tissues were treated with 4% paraformaldehyde solution, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek, Torrance, CA, USA), and frozen sections (10 μm) of liver tissues were washed with sterile water, infiltrated with 60% isopropanol for 2 min, and then stained with oil red O (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). The severity of liver injury were evaluated by visual estimate under a LIRI-2006 microscope according to the method reported previously [START_REF] Liang | Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology[END_REF] : Ballooning degeneration of hepatocytes: grade=0 (< 5%), grade=1 (5% -30%), grade=2 (30% -50%), grade=3 (50% ≤); Steatosis: grade=0 (< 5%), grade=1 (5%-30%), grade=2 (30% -50), grade=3 (50% ≤). Three sections and 3 fields per tissue were observed (n= 3 in each group). Photographs taken from tissue sections were digitized using CMOS camera equipped with a GMS image analysis system (Shanghai optical instrument factory, Shanghai, China).

Insulin Tolerance Tests (ITT) and Glucose Tolerance Tests (GTT)

GTT (A) and ITT (B) were performed on weeks 7, respectively. In the ITT, mice from all five group were fasted for 8 hours and then injected intraperitoneally with 1 U human insulin/kg body weight, and blood glucose values were measured at (0 min) and at 5, 10, 30, 60, and 120 min. For GTT, mice fasted 8 hours were injected intraperitoneally with d-glucose (2 g/kg body weight), and blood glucose was measured at (0 min) and at 5, 15, 30, 90, 60, and 120 min. Glucose levels both in GTT and ITT were measured using a glucometer (Sinocare Inc., Changsha, Hunan, China).

The area under the curve (AUC) for GTT and ITT tests were calculated according to the trapezium rule [START_REF] Matthews | Analysis of serial measurements in medical research[END_REF] .

Analyses of TNF-α and IL-6 Levels in Liver and Serum.

TNF-α and IL-6 levels in serum and liver homogenates were determined by commercial ELISA kits (KET7015 and KET7009, Abbkine Scientific Co., Ltd., California, USA), respectively.

Measurement of Hepatic Triglycerides Level

Liver tissue was homogenized in ice-cold PBS buffer (137 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 2.7 mM KCl, 1000 mL deionized water, pH 7.4).

Hepatic triglycerides was extracted with the method described by Fossati [START_REF] Fossati | Serum glycerides determined colourimetrically with an enzyme that produces hydrogen peroxide[END_REF] , and determined on a DNM-9602 Reader (Beijing Perlong New Technology Co., Ltd., Beijing, China) by a assay kit (MAK266-1KT, Sigma-Aldrich Corp., St. Louis, MO, USA).

Hepatic Oxidative Stress Analysis.

Liver tissues (100 mg) were homogenized with 1 mL of cold PBS buffer (137 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 2.7 mM KCl, 1000 mL deionized water, pH 7.4) and then centrifuged at 10000g for 30 min at 4 °C. The homogenates were subjected to assays for nitric oxide (NO), vitamin C, glutathione (GSH), glutathione peroxidase (GSH-px), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) using commercially available enzymatic assay kits (Wuhan ColorfulGene Biological Technology Co., Ltd., Wuhan, Hubei, China). All data acquisition was performed on an A6 semiautomatic biochemistry analyzer (Beijing Shining Sun Technology Co., Ltd., Beijing, China).

Western Blot Analysis.

Hepatic protein expressions of phospho-JNK1, phospho-IRS1 (Ser 307), Phospho-IKK alpha/beta (Ser180/181) and XBP1 were evaluated by western blot analysis. Primary antibody against phospho-JNK1 (AF3318), phospho-IRS1 (AF3272), Phospho-IKK alpha/beta (AF3013) and XBP1 (AF5110) were obtained from Affinity Biosciences (Affinity Biosciences, Cincinnati, USA) and used for the western bolt analysis, respectively. Liver tissues were homogenized in cold radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.4, 1% w/v Triton X-100, 0.1% w/v sodium dodecyl sulfate, 150 mM NaCl, 1% sodium deoxycholate, 1 mM NaF, 5 mM EDTA, protease inhibitors, and 1 mM sodium orthovanadate). The lysates were cleared by centrifugation at 15000g at 4 °C for 30 min. The protein concentrations were quantified with a BCA assay kit (Huaying Biotechnology Co., Ltd., Hubei, China). Aliquots of lysates containing 25-40 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes by electrophoretic transfer (Bio-Rad Laboratories, Inc., California, UAS).

After blocking with 5% BSA (0332, Amresco, USA) in TBST buffer (20 mM Tris-base, 150 mM NaCl, 0.05% Tween 20, pH 8.0) for 2 h and rinsed three times with TBST buffer for 5 min each, the membranes were probed overnight at 4 °C with primary antibodies (Dilution 1:1000), respectively. After removal of the unbound primary antibody with TBST buffer, the membranes were washed and incubated with the secondary horseradish peroxidase-conjugated antibodies (dilution 1:50000, S0001, Affinity Biosciences, Cincinnati, USA) at room temperature for 2 h. Chemiluminescence was visualized using an ECL western blot detection kit (KF001, Affinity Biosciences, Cincinnati, USA). The signal was captured with a UVP BioSpectrum AC imaging system (UVP, Upland, CA, USA). Quantitation of protein expression was evaluated by densitometry using ImageJ software version k1.47 (NIH, Bethesda, MD, USA). β-actin expression with an anti-β-actin mouse antibody (YM3028, ImmunoWay Biotechnology Company, Plano, USA) was used as an internal control for equal loading of protein.

Immunohistochemical Analysis

NF-E2-related factor 2 (Nfr2) and phospho-NF-κB p65 expressions were examined by immunohistochemical analysis, respectively. The paraffin sections (5 μm) were dewaxed and hydrated followed by antigen retrieval through heat mediation in Citrate Plus solution (ab94674, Abcam Trading (Shanghai) Company Ltd., Shanghai, China.) to recover protein structure. Then sections were rinsed with 3% H2O2 and TBS buffer (50 mM Tris, 100 mM NaCl, pH 7.6) to block endogenous peroxidase activity and excessive protein, and incubated with primary Nfr2 antibody or primary phospho-NF-κB antibody (AF0639 or AF2006, dilution 1:100, Affinity Biosciences, Cincinnati, USA) at 37℃ for 30 minutes. After rinsing two times (5 minutes each time) with TBS buffer, sections were incubated with an horseradish peroxidase conjugated goat anti-rabbit antibody (dilution 1:100, S0001, Affinity Biosciences, Cincinnati, USA) for 30 minutes at 37℃. Then, sections were washed using TBS buffer for 3 times with 5 minutes each, and incubated in diaminobenzidine (DAB) solution (ab127055, Abcam Trading (Shanghai) Company Ltd., Shanghai, China.), followed by counter-staining with 0.2% modified hematoxylin solution (Sigma-Aldrich Corp., St. Louis, MO, USA). The stained sections were examined under a LIRI-2006 microscope (Shanghai Optical Instrument Factory, Shanghai, China). The IHC analysis for Nrf2 and phospho-NF-κB p65 was evaluated according to the staining intensity of positive reaction and percentage of positive cells in a blind manner with a reported method [START_REF] Zhu | miR-199b-5p Regulates Immune-Mediated Allograft Rejection after Lung Transplantation Through the GSK3β and NF-κB Pathways[END_REF] . The staining intensity was graded from 0 to 3 (score= 0: colorless, score= 1: pale-yellow, score= 2: brown-yellow, and score= 3: saddle-brown). The score for the percentage of positive cells was graded from 0 to 4 (score= 0: negative, score= 1: fewer than 10% positive cells, score= 2: 10-50% positive cells, score= 3: 51-75% positive cells, and score= 4: over 75% positive cells). Representative five animals (n = 5) were analyze.

Statistical Analysis.

The results were presented as the means ± S.E.M (standard error of mean). The data were statistically analyzed using one-way ANOVA for multiple group comparison followed by Newman-Keuls test using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA). P ≤ 0.05 was considered statistically significant.

■ RESULTS

Changes in Body Weight, Food Intake, Liver Weight and Fat Weight.

In the whole experimental period, the changes in the body weight of mouse in each group were expressed as the average daily body weight of mouse in each week.

The body weight of mice both in the control group and the fructose group continued to gain from beginning to end (Figure 2A and Figure 2B). Compared with mice in the fructose group, the body weight were inhibited at dose of 0.5 and 1 mL/kg BSSEE and trends to decrease at dose of 2 mL/kg of BSSEE after the BSSEE administration (Figure 2B). Before the BSSEE administration, the body weigh in mice received diet without fructose increased to 35.17 g from an initial weight of 16.7 g, whereas the body weight in fructose-fed mice increased to 39.60 (Figure 2A) and showed significant difference compared to the control group. After oral administration of the BSSEE for consecutive 4 weeks, the body in fructose-fed mice slightly increased to 41.83 g from 39.67 g at the dose of 0.5 mL/kg BSSEE and 40.17 g at the dose of 1 mL/kg BSSEE from 39.17 g (Figure 2B), while oral administration of BSSEE at the dose of 2 mL/kg slightly decreased body weight of mice to 38.00 g from the body weight of 40.09 g at the end of the 4th week (Figure 2B). However, mice received fructose without the BSSEE administration markedly increased to 45.83 g at the end of experiment (Figure 2B). The average body weight of mice treated with BSSEE (0.5, 1 and 2 mL/kg) significantly decreased compared with mice in the fructose group at the end of experiment, and significant difference was observed in mice administrated with BSSEE at 1 and 2 mL/kg compared with the control group.

For the changes in the food intake, fructose-fed mice showed the decrease of food intake throughout the experiment, compared with the mice in the control group.

We measured the average amount of food intake per day after the BSSEE treatment, in which the average amount of food intake in those fructose-fed mice administrated with BSSEE (1 and 2 mL/kg) showed significant reduction compared with mice in the fructose group (Table 2). Although energy intake in mice administrated with BSSEE at 1 and 2 mL/kg showed less than mice in the fructose group, no significant difference were observed (Table 2).

At the end the of experiment, the liver weight, perinephrit fat weight, subcutaneous fat weight and epididymal fat weight in fructose group displayed significant increase compared to the control group, and the relative liver and total fat weights also significantly increased in fructose group (Table 1). Compared with mice in the fructose group, BSSEE treated mice showed the decrease in the liver weight, perinephrit fat weight, subcutaneous fat weight, epididymal fat weight, and the relative liver and total fat tissue weight in a dose dependent manner (Table 1).

Effect of BSSEE on Serum Biochemical Parameters.

At the end of the experiment, fructose-fed mice showed significant high levels of serum cholesterol, triglycerides, glucose, insulin and FFA levels (Table 2), which may imply that fructose feeding cause metabolic disorders. However, mice administrated with BSSEE (0.5, 1 or 2 mL/kg) significantly decreased serum cholesterol, triglycerides, glucose, insulin and FFA levels in a dose-dependent manner (Table 2). Decreased these serum parameters by BSSEE indicated that BSSEE administration effectively improved metabolic disorders. In addition, AST and ALT levels in the fructose group are significant higher than that in the control group, whereas administration of BSSEE (0.5, 1 and 2 mL/kg) dose-dependently reversed AST and ALT levels (Table 2). Furthermore, we determined the serum TNF-α and IL-6 levels in mice from all group because the increase of serum TNF-α and IL-6 levels is closely associated with the progression of liver fat deposition [START_REF] Herbert | Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis[END_REF] . Notably, fructose fed-mice in the fructose group showed higher levels of TNF-α and IL-6 than that in the control group. However, treatment with BSSEE (1 and 2 mL/kg) significantly reduced TNF-α and IL-6 in serum.

Effect of BSSEE on Liver Inflammation Response and Histopathological

Changes.

Triglycerides are the main lipids deposited in the liver of patients with NAFLD.

We measured the hepatic triglycerides levels. Fructose administration significantly increased triglycerides level compared with mice in the control group, whereas mice administrated with BSSEE (1 and 2 mL/kg) significantly decreased hepatic triglycerides level (Figure .3E). Furthermore, the morphological changes of the hepatocytes of lobule were examined with H&E staining and oil red O staining, and the severity of histological changes was evaluated by the histopathological scoring system. Liver tissue stained with H&E showed some marked changes in mice from the fructose group, where the hepatocytes of lobule in some sections showed significantly increased compared with mice in the control group, and there are some inflammatory cells infiltration in liver tissue sections stained with H&E. Compared to mice in the fructose group, significant decrease in IL-6 and TNF-α levels with a dose-dependent manner was observed in mice administrated with BSSEE (0.5, 1 and 2 mL/kg), and no inflammatory cells infiltration were observed in liver tissue sections stained with H&E in BSSEE (0.5, 1 and 2 mL/kg) treated mice.

BSSEE improved glucose homeostasis in fructose-fed mice

To investigate the effect of BSSEE on body insulin sensitivity, mice were fasted and were intraperitoneally injected with either glucose (GTT) or insulin (ITT). As shown in Figure .4A, blood glucose level in fructose-fed mice without the treatment were constantly higher (statistically significant at 30 and 60 minutes) than mice in the control group mice, which was accompanied by higher levels of blood glucose level in ITT test at 60 and 90 min ( In order to explore whether BSSEE can improve hepatic oxidative stress, immunohistochemical staining of Nrf2 expression was carried out. Nrf2 is a transcription factor in the protection against any oxidative stress, which is translocated from sequestration in thecytoplasm to the nucleus during oxidative stress, promoting several antioxidant enzymes to enhance antioxidant defense system. Nrf2 are low expressed in liver cell of nuclear in mice form the fructose group, whereas mice treatment with BSSEE at doses of 1 and 2 mL/kg elevated Nrf2 expression in cells of nuclear, suggesting that the anti-oxidant effect of BSSEE linked with increase of Nrf2 expression in cells of nuclear (Figure .5H and Figure .5H).

Effect of BSSEE on Liver Protein Expression.

Insulin resistance is a risk factor for the aggravation of NAFLD, in which

■ DISCUSSION

Long term intake of high fructose has been proven to be a risk factor for the development of obesity, adiposity, liver inflammation and insulin resistance, as well as NAFLD, insulin resistance and metabolic syndrome [START_REF] Spruss | Dietary fructose and intestinal barrier: potential risk factor in the pathogenesis of nonalcoholic fatty liver disease[END_REF][START_REF] Castello | Regulation of GLUT5 gene expression in rat intestinal mucosa: regional distribution, circadian rhythm, perinatal development and effect of diabetes[END_REF][START_REF] Nomura | The role of fructose-enriched diets in mechanisms of nonalcoholic fatty liver disease[END_REF][START_REF] Suganthi | Effect of food seasoning spices mixture on biomarkers of oxidative stress in tissues of fructose-fed insulin-resistant rats[END_REF][START_REF] Armutcu | Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats[END_REF][START_REF] Bagul | Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats[END_REF] . Since HFCS consumption has been found that it is closely associated with the increasing prevalence of NAFLD, obesity, insulin resistance and metabolic syndrome, we investigated the effect of BSSEE on NAFLD in fructose-fed mice. In this study, 30% of fructose intake over a period of 8 weeks caused fast body weight gain together with increase of fat tissue and liver weights in mice, which is similar to previous report [START_REF] Bagul | Attenuation of insulin resistance, metabolic syndrome and hepatic oxidative stress by resveratrol in fructose-fed rats[END_REF] . Overweight that presents an increase of lipids in adipose tissue is considered to be one of the key events occurring in the first steps of hepatic steatosis, where adipose tissue-derived signals (e.g. classical cytokines TNF-α, IL-6) due to excess storage of fat has been demonstrated to play an essential role in the pathopoiesia of NAFLD [START_REF] Al-Dayyat | Non-alcoholic fatty liver disease and associated dietary and lifestyle risk factors[END_REF][START_REF] Herbert | Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis[END_REF] . However, BSSEE intake decreased body weight, adipose tissue weight and liver weight, implying that BSSEE may contribute to the improvement of NAFLD in fructose-fed mice. Importantly, in addition to the improvement of serum biochemical parameters levels after BSSEE intake in fructose-fed mice, further histopathological examinations showed that BSSEE supplementation reduced ballooning degeneration and fat deposition in liver cells compared with the fructose-fed mice without the treatment. Certainly, our experimental results indicate that BSSEE intake could alleviate hepatic steatosis in fructose-fed mice.

In the present study, the major active components of BSSEE were analyzed with HPLC-PDA. On the basis of the BSSEE chromatogram (Figure 1), the results indicated that the major constituent of BSSEE was lignans (19.8%), which mainly contained sesaminol (1.92%), sesamin (16.33%) and sesamolin (31.31%). Previously, vitamins (such as vitamins D, vitamins C and vitamins E) [START_REF] Eliades | Meta-analysis: vitamin D and non-alcoholic fatty liver disease[END_REF][START_REF] Oliveira | Vitamin C and Vitamin E in Prevention of Nonalcoholic Fatty Liver Disease (NAFLD) in Choline Deficient Diet Fed Rats[END_REF] and linoleic acids [START_REF] Monteiro | Oils rich in α-linolenic acid independently protect against characteristics of fatty liver disease in the Δ6-desaturase null mouse[END_REF] were found to be beneficial for the improvement of NAFLD. In the present study, sugar, protein, vitamins and fatty acids in BSSEE were did not detected under that chromatographic conditions. Since soybean oil mainly contain linoleic acid (55%~67%), oleic acid (17%~26%), palmitic acid (9%~13%), stearic acid (3%~6%) as well as vitamins, mice in the fructose group received soybean oil showed no significant improvement in fructose-induced NAFLD. Therefore, lignans including sesaminol (1.92%), sesamin (16.33%) and sesamolin (13.06%) might possess beneficial effect against NAFLD in this animal model.

In the present study, fructose consumption increased serum levels of FFA, triglyceride and total cholesterol. Disorganized these serum parameters altered change in energy metabolism in liver, and are risk factors in the development of diseases such as NAFLD, insulin resistance and metabolic syndrome. Increased free fatty acids, triglyceride and cholesterol, especially accumulation in mitochondria, are considered the "aggressive" lipids leading to TNF-α and IL-6-mediated liver damage and ROS formation [START_REF] Herbert | Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis[END_REF][START_REF] Feldstein | Free fatty acids promote hepatic lipotoxicity by stimulating TNF-alpha expression via a lysosomal pathway[END_REF] . These lipids could also shows in a nonsteatotic liver and act as early "inflammatory" hits, resulting in the whole spectrum of NAFLD pathologies [START_REF] Malhi | Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease[END_REF][START_REF] Alkhouri | Lipotoxicity in nonalcoholic fatty liver disease: not all lipids are created equal[END_REF] . However, we observed BSSEE supplementation decreased free fatty acid, triglyceride and total cholesterol, which is beneficial to the prevention in the development of NAFLD.

In addition, fructose consumption by mice led to insulin resistance as the results presented the increase of serum insulin and glucose levels, and ITT and GTT tests.

Insulin resistance is the condition that a cell, tissue, or organism fails to respond appropriately to a given dose of insulin, in which it is an important feature of NAFLD and type 2 diabetes, and are often found in metabolic syndrome [START_REF] Asrih | Metabolic syndrome and nonalcoholic fatty liver disease: Is insulin resistance the link?[END_REF] . The improvement of NAFLD may occur independent of interference with insulin resistance [START_REF] Amaro | Dissociation between intrahepatic triglyceride content and insulin resistance in familial hypobetalipoproteinemia[END_REF][START_REF] Sanyal | or placebo for nonalcoholic steatohepatitis[END_REF] .

However, presence of insulin resistance may aggravate NAFLD because patients with insulin resistance are often worse in terms of liver histopathological examinations such as hepatic ballooning, steatosis and degree of fibrosis [START_REF] Eguchi | Visceral fat accumulation and insulin resistance are important factors in nonalcoholic fattyliver disease[END_REF][START_REF] Ota | Insulin resistance accelerates a dietary rat model of nonalcoholic steatohepatitis[END_REF] . In contrast, oral administration of BSSEE (0.5, 1 and 2 mg/kg) dose-dependently decreased serum insulin and glucose levels and improved body insulin resistance, which is important for the prevention of NAFLD complicated with type 2 diabetes and metabolic syndrome.

In addition to plasma insulin resistance, we found that fructose intake over a long period of time may impair hepatic insulin function as evidenced by the increase expression of phospho-IRS1 (Ser 307) in liver. Increased serine phosphorylation of IRS1 (Ser 307) may prevent insulin signal transduction, thereby resulting in impairment of insulin functions in liver [START_REF] Hotamisligil | Endoplasmic reticulum stress and the inflammatory basis of metabolic disease[END_REF] . Impaired hepatic insulin function is linked with the exacerbation of NAFLD [START_REF] Sabio | A stress signaling pathway in adipose tissue regulates hepatic insulin resistance[END_REF] . Mice treated with BSSEE improved insulin function, showing decreased serine phosphorylation of IRS1 (Ser 307), which was contribute to the metabolism of glucose and lipid in liver. In order to further understand whether BSSEE could improve insulin signal transduction, we examined hepatic oxidative stress and the expressions of NF-κB, JNK1, IKK and XBP1 in liver.

One of the key aspects that disrupt insulin signaling links to major inflammatory signaling networks, including the activation of the JNK, IKK/NF-κB signaling pathways and production of ROS and NO [START_REF] Hotamisligil | Endoplasmic Reticulum Stress and the Inflammatory Basis of Metabolic Disease[END_REF] . The activation of JNK, IKK-NF-κB pathways can lead to the inhibition of insulin action in part through serine phosphorylation of IRS1 [START_REF] Cai | Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB[END_REF][START_REF] Arkan | IKK-beta links inflammation to obesity-induced insulin resistance[END_REF] . However, mice by BSSEE treatment reduced phosphorylation of NF-κB p65, JNK1 and IKK, and consequently a decrease of serine phosphorylated IRS1 (Ser 307), suggesting that BSSEE could improve insulin signal transduction via its effect on JNK and IKK-NF-κB pathways. In addition, XBP1 has evolved as a critical molecule, which interacts with inflammatory cascades at various stages, including IRE1-mediated activation of JNK, activation of IKK/NF-κB signaling pathways and production of ROS [START_REF] Hotamisligil | Endoplasmic Reticulum Stress and the Inflammatory Basis of Metabolic Disease[END_REF] . XBP1 haploinsufficiency in mice exhibit markedly increased succumb to increase of body weight, hyperinsulinemia, hyperglycemia, and impaired glucose and insulin tolerance, at least in part, through activation of JNK and serine phosphorylation of IRS1 [START_REF] Park | The regulatory subunits of PI3K, p85alpha and p85beta, interact with XBP-1 and increase its nuclear translocation[END_REF][START_REF] Winnay | A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response[END_REF] . XBP1 has proved as a critical molecule in the regulation all aspects of NAFLD [START_REF] Ozcan | Endoplasmic reticulum stress plays a central role in development of leptin resistance[END_REF] . Notably, BSSEE increased expression of XBP1 in liver compared with mice in fructose group, which may be, in part, responsible for the regulation of the lipid synthesis/accumulation, inflammation and insulin signaling/resistance in liver in fructose-fed mice.

As above mentioned, production of ROS and NO has been considered as strong contenders for the cause of exacerbating NAFLD and insulin resistance [START_REF] Gaetano | Free radical biology for medicine: learning from nonalcoholic fatty liver disease[END_REF] . The liver is rich in antioxidant defense system, which include chemicals such as GSH, vitamins C, and enzymes such as SOD, CAT, GSH-px [START_REF] Leclercq | Antioxidant defence mechanisms: new players in the pathogenesis of nonalcoholic steatohepatitis?[END_REF] . Even though ROS and nitrogen species are normally produced by the metabolism of normal cells, in NAFLD the occurrence that an overproduction of free radicals which overcomes the antioxidant defenses aggravate liver injury [START_REF] Muriel | Role of free radicals in liver diseases[END_REF] . The pathogenesis of NAFLD suffers multiple parallel hits, including oxidative stress [START_REF] Herbert | Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis[END_REF][START_REF] Day | Steatohepatitis: a tale of two "hits[END_REF] . Hepatic oxidative stress is also partly responsible for insulin resistance through inflammatory response [START_REF] Houstis | Reactive oxygen species have a causal role in multiple forms of insulin resistance[END_REF] . In the present study, fructose-fed mice showed decrease of GSH and vitamins C levels, reductions of SOD, CAT and GSH-px activities, and high levels of MDA and NO in liver, suggesting decrease of antioxidant defenses function. However, BSSEE enhances antioxidant defenses function, as increase of GSH and vitamins C levels, enhancements of SOD, CAT and GSH-px activities and consequently reduction of MDA and NO level. To understand thoroughly the molecular mechanism behind the antioxidative effect of BSSEE, we further examined hepatic Nrf2 protein. Nrf2 is a transcription factor binds antioxidant response elements (AREs) in the regulatory regions of target genes, which act as a main player in the inducible expression of our cellular defense enzymes [START_REF] Itoh | An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements[END_REF][START_REF] Itoh | Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain[END_REF] . Nrf2 activators have already been developed for the treatment of liver diseases involving oxidative stress and inflammatory response [START_REF] Bellezza | Nrf2-Keap1 signaling in oxidative and reductive stress[END_REF][START_REF] Yu | Oltipraz upregulates the nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant system and prevents insulin resistance and obesity induced by a high-fat diet in C57BL/6J mice[END_REF] . Similar to previous studies, the present study found that BSSEE could increased Nrf2 expression in liver cell, which might have a significant protective effect against fructose-induced oxidative stress in liver as can be seen in this study, possibly through enhancing hepatic antioxidant defense enzymes.

In conclusion, the present study demonstrates for the first time that BSSEE showed good beneficial effects against NAFLD complicated with insulin resistance and metabolic syndrome in fructose-fed mice. These effects are most likely related to the regulation of insulin signaling molecules expressions, antioxidant defenses system and inflammatory response. Thus, black sesame seeds could be considered as a potent dietary supplements in the prevention of NAFLD-related metabolic diseases.

■ AUTHOR INFORMATION

  ballooning degeneration (Figure.3F), and the ballooning degeneration score significantly increased compared with mice in the control group (Figure.3C). Meanwhile, liver tissue stained with oil red O showed fatty deposition of hepatocyte and high fatty deposition score in mice from the fructose group (Figure.3D). In contrast, H&E and oil red O staining displayed that the liver sections from fructose-fed mice treated with BSSEE (1 and 2 mL/kg) alleviated these histological alterations (Figure.3F) and significantly decreased the ballooning degeneration and fatty deposition scores (Figure.3C and Figure.3D) in a dose-dependent manner. Moreover, TNF-α (Figure.3A) and IL-6 (Figure.3B) are two important inflammatory cytokines, in which they drive the development of hepatic steatosis and inflammation in NAFLD. IL-6 and TNF-α levels in fructose-fed mice without the treatment

  Figure. 4C), suggesting that insulin sensitivity in fructose-fed mice without the treatment was relatively decreased. This notion was further strengthened by the analysis of AUC summarized in Figure.4B and Figure.4D, in which the area under the curve for the GTT and ITT in fructose group significantly increased to 29.19 and 9.24 compared with mice in the control group, respectively. However, administration with BSSEE (0.5, 1 and 2 mL/kg) lead to a significant decrease at 30 min for GTT (Figure. 4A) and at 60 and 90 min for ITT (Figure. 4C), and the total prevention of the increase in GTT area under the curve or ITT area under the curve was significantly decreased in a dose dependent manner (Figure. 4B and

Figure

  Figure. 4D), indicating that BSSEE treatment improved decreased insulin sensitivity

  inflammatory mediators and lipids activate a signaling cascade that triggers inflammatory kinases such as JNK, IKK and NF-κB, as well as XBP1. The activation of JNK, IKK and NF-κB and down-regulation of XBP1 can result in the inhibition of insulin action in part through serine phosphorylation of insulin receptor substrates IRS1. In the present study, fructose-fed mice showed the high expressions of phospho-JNK1 (Figure.6C) and phospho-IKK (Figure.6B), along with low expressions of XBP1 (Figure.6A) and phospho-IRS1 (Figure.6D), compared with mice in the control group. In addition, immunohistochemical staining of phospho-NF-κB p65 expression obviously increased in the fructose-fed mice without the treatment (Figure.6E). However, mice treated with BSSEE decreased expressions of phospho-NF-κB p65, phospho-JNK1, phospho-IRS1 and phospho-IKK, and increased expression of XBP1 (Figure.6A-E), indicating that BSSEE administration markedly improved insulin resistance in liver of mice by its effect on these signalling molecules.

  Author *(Yongmin Zhang) Postal address: Sorbonne Université, Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, 4 place Jussieu, 75005 Paris, France. Tel: +86 29 88304569. Fax: +86 29 88304368. E-mail: 15102930802@163.com.

Figure 1 .

 1 Figure 1. HPLC-PDA chromatogram of black sesame seeds ethanol extract (BSSEE) at 280 nm. Retention times

Figure. 2 .

 2 Figure.2. Changes in body weight and amount of food intake. (A) Curve showing changes in average body weight

Figure. 3 .

 3 Figure.3. Effects of BSSEE (0.5, 1 and 2 mL/kg) on hepatic (A) TNF-α, (B) IL-6 and liver histopathological

Fig. 4 .

 4 Fig. 4. Effects of BSSEE (0.5, 1 and 2 mL/kg) on GTT and ITT responses in fructose-fed mice. GTT (A) and ITT

Figure. 5 .

 5 Figure.5. Effects of BSSEE (0.5, 1 and 2 mL/kg) on (A)MDA, (B) NO, (C) GSH, (D) vitamin C, (E) SOD, (F)

Figure. 6 .

 6 Figure.6. Effects of BSSEE (0.5, 1 and 2 mL/kg) on the expressions of hepatic (A) XBP1, (B) phospho-IKK, (C)

Table 1 .

 1 Final tissue weights. The relative tissue weight is expressed as percentage of body weight (tissue weight/final body weight × 100). Values are shown as means ± SEM (n=10 per group). Results were statistically analyzed using one-way ANOVA coupled with Newman-Keuls's multiple-comparison test (p < 0.05).

				Absolute weight (g)			Relative weight (/body, %)
	Groups
		Liver	Perinephrit fat	Epididymal fat	Subcutaneous fat	Total fat	Liver	Total fat
	Control	1.40 ± 0.07c	0.16 ± 0.03c	0.72 ± 0.05b	0.82 ± 0.05c	1.71 ± 0.06c	3.28 ± 0.25c	4.03 ± 0.15d
	Fructose	2.20 ± 0.05a	0.50 ± 0.13a	1.21 ± 0.04a	1.41 ± 0.05a	3.12 ± 0.09a	5.17 ± 0.2a	6.75 ± 0.16a
	Fructose + BSSEE (0.5 mL/kg)	2.13 ± 0.08ab	0.37 ± 0.06b	0.92 ± 0.15ab	1.12 ± 0.18ab	2.42 ± 0.13ab	4.98 ± 0.15ab	5.96 ± 0.26ab
	Fructose + BSSEE (1 mL/kg)	1.80 ± 0.04bc	0.33 ± 0.04b	0.80 ± 0.06b	0.92 ± 0.08bc	1.98 ± 0.08bc	4.65 ± 0.13bc	5.30 ± 0.22bc
	Fructose + BSSEE (2 mL/kg)	1.50 ± 0.07c	0.25 ± 0.08bc	0.70 ± 0.05b	0.85 ± 0.07c	1.87 ± 0.09c	3.82 ± 0.19c	4.36 ± 0.24cd

Table 2 .

 2 Metabolic parameters in fructose-fed mice with or without treatment with BSSEE (0.5, 1 and 2 mL/kg).Liquid intake, food intake and total energy intake show during BSSEE treatment. Cholesterol, triglycerides, glucose, insulin, FFA, AST, ALT, TNF-α and IL-6 were measured in serum. Values are shown as means ± SEM (n=10). Results were statistically analyzed using one-way ANOVA coupled with Newman-Keuls's multiple-comparison test (p < 0.05).

	Group	Control	Fructose	Fructose + BSSEE (0.5 mL/kg)	Fructose + BSSEE (1 mL/kg)	Fructose + BSSEE (2 mL/kg)

Funding

This work was financially supported by the Natural Science Foundation of Shaanxi Province of China, One Hundred Person Expert Project of Shaanxi Province of China (No. 2014JM4125), Special research program of Shaanxi Provincial Department of Education (No. 2013JK0809).

Notes

The authors declare no competing financial interest.

■ ABBREVIATIONS USED

Black sesame seeds ethanol extract (BSSEE), nonalcoholic fatty liver disease (NAFLD), free fatty acids (FFA), aspartate transaminase (AST), alanine transaminase (ALT), insulin tolerance tests (ITT), glucose tolerance tests (GTT), nitric oxide (NO), malondialdehyde (MDA), NF-E2-related factor 2 (Nrf2), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), phospho-c-Jun NH2-terminal kinase 1 (phospho-JNK1), phospho-nuclear factor kappa B (NF-κB), phospho-insulin receptor substrate 1 (phospho-IRS1), phospho-IκB kinase (phospho-IKK), X-box binding protein 1 (XBP1), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), reactive oxygen species (ROS), high performance liquid chromatography (HPLC), high fructose corn syrup (HFCS).