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Am J Physiol Gastrointest Liver Physiol 293: G585-G590, 2007. First published July 19, 2007; doi:10.1152/ajpgi.00175.2007
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LIVER AND BILIARY TRACT

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4{alpha} and PGC1{alpha}

Christoph G. Dietrich,* Ina V. Martin,* Anne C. Porn, Sebastian Voigt, Carsten Gartung, Christian Trautwein, and Andreas Geier

Department of Internal Medicine III (Division of Gastroenterology and Hepatology), Aachen University Hospital (UKA), Aachen, Germany

Submitted 19 April 2007 ; accepted in final form 16 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4{alpha}/PGC-1{alpha}-pathway. Because HNF4{alpha} has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4{alpha} is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1{alpha} in electrophoretic mobility shift assays remained unchanged, HNF4{alpha} binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4{alpha} and PGC-1{alpha}. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4{alpha}, PGC-1{alpha}, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4{alpha}/PGC-1{alpha} pathway.

bile salt transport; HNF4; PGC-1; Ntcp; Oatp

FASTING AS A (TEMPORARY) PHYSIOLOGICAL state leads to massive adaptive changes in metabolic and endocrine pathways in the body, especially in the liver (42). Hepatic gluconeogenesis starts 4–6 h after the onset of fasting and, along with glycogenolysis, supports the liver in providing a stable blood glucose level. Fatty acids, liberated from the adipose tissue during fasting, undergo oxidation in the liver, which forms the basis for ATP production during food deprivation. Additionally, ketogenesis is implemented during rapid beta-oxidation. Ketones provide an alternative food source for high-energy tissues such as the brain, when glucose is low. These key processes of adaptive metabolic reorganization during fasting are induced via specific transcriptional activators.

The nuclear receptor peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) is one of these activators and controls hepatic beta-oxidation via binding as a heterodimer with RXR to its target genes (21, 35). Other pathways controlled by this receptor comprise formation (25), uptake (19) of bile acids, and phospholipid secretion at the hepatocellular canalicular membrane (18). Amino acid metabolism is also influenced (16). PPAR{gamma} is another nuclear receptor active in development and differentiation of fat tissue and fat tissue-specific gene expression (1, 32). The hypothesis that differentiation of fat cells to white or brown adipocytes needs a specific coactivator led to the discovery and cloning of PPAR{gamma} coactivator-1{alpha} (PGC-1{alpha}) (28), a factor preferentially expressed in brown fat and responsible for the activation of thermogenic genes (27).

Obviously, PGC-1{alpha} also serves as a cofactor for PPAR{alpha} by inducing beta-oxidation in adipocytes and the heart (22, 41). Additionally, PGC-1{alpha} is involved in glucose homeostasis by coactivating several important pathways. PGC-1{alpha} induces GLUT4, an insulin-sensitive glucose uptake transporter in muscle cells (23). PGC-1{alpha} stimulates the mRNA of all three key genes for hepatic gluconeogenesis (27), PEPCK, fructose 1,6-bisphosphatase, and glucose-6-phosphatase and is induced in the liver in several states requiring or promoting gluconeogenesis, including fasting (44).

Recently it was established that PGC-1{alpha}-induced gluconeogenesis during fasting requires the presence of hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}) and that the observed effect of PGC-1{alpha} in gluconeogenesis relies on coactivation of the respective enzymes via combined binding of HNF-4{alpha} to specific control elements in the promoter (30, 44). Mutations in HNF-4{alpha} contribute to a specific type of maturity onset diabetes of the young (MODY 1), indicating the importance of this nuclear receptor in glucose homeostasis (34). Additionally HNF-4{alpha} is a key induction activator for basolateral uptake transport systems in the hepatocyte (13, 14, 26; Geier et al., unpublished observations). These uptake systems comprise a high-affinity Na+-dependent bile salt transporter Ntcp/NTCP (Slc10a1/SLC10A1) and a familiy of multispecific organic anion transporters (Oatps/OATPs; Slc21a/SLC21A) that mediate Na+-independent uptake of mostly amphipathic organic compounds, including conjugated and unconjugated bile acids, bromosulfophthalein, and bilirubin. The transport characteristics of Oatp1 (Slc21a1, new nomenclature Oatp1a1), Oatp2 (Slc21a5, new nomenclature Oatp1a4), and Oatp4 (Slc21a10, new nomenclature Oatp1b2) can account for the majority of Na+-independent bile acid uptake in rodent liver. In this respect, HNF-4{alpha} and PGC-1{alpha} might contribute to regulation of these transporters at the basolateral hepatocellular membrane as another adaptive change in hepatic gene expression during fasting. Dumaswala and coworkers (4) showed that fasting leads to decreased bile flow and intestinal reabsorption of bile acids but increases hepatocellular uptake of bile acids from the sinusoidal blood, a key step in enterohepatic cycling of bile constituents and adaptive changes in this context.

We therefore hypothesized that HNF-4{alpha} and PGC-1{alpha} mediate increased hepatocellular bile acid uptake via upregulation of basolateral transporters.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animal model. Male Sprague-Dawley rats (200–250 g, Janvier, Le Genest-St. Isle, France) were used for organ removal and isolation of hepatic microsomes, RNA, and nuclei. All animals were kept on a 12:12-h light-dark cycle and had free access to water. Rats (n = 5 each group) either were starved for up to 72 h or had free access to standard rat diet. The livers were harvested after 48 or 72 h under general anesthesia, immediately snap frozen, and stored in liquid nitrogen until microsomal and RNA extraction or freshly used for isolation of liver nuclei and isolation of nuclear proteins. All study protocols were approved by the local Government's Animal Care Committee. The feeding protocols were approved by the local Animal Care and Use Committee, according to criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences, as published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Northern blot analysis. RNA was isolated from liver by standard phenol chloroform extraction procedure (8). Total RNA (10–20 µg) was analyzed by Northern blotting with specific and constitutively expressed probes of Ntcp, Oatp1, Oatp2, Mrp2, and Gapdh as previously described (7, 10). mRNA levels were detected by exposure of the membrane to a Phosphoimager screen and quantified by using a Phosphoimager and the Quantity One software (Bio-Rad).

EMSA. Preparation of nuclear extracts and electrophoretic mobility shift assays were performed as previously described (10). Protein (5–10 µg) was incubated with [32P]end-labeled oligonucleotide probes.The following double-stranded oligonucleotide probes were used (sense strand): 1) HNF1{alpha}: 5'-GATCTGCTGGTTAATCTTTTATTT-3' [rat Ntcp –11/+9 (15)], 2) HNF4{alpha}: 5'-AGAGGGCCAAAGGTCAGTT-3' [HNF4 consensus (29)], 3) STAT5: 5'-GAAGTTGTCATTCTTGGAAAAATAACAAT-3' [rat Ntcp –922/–892 (6)], 4) GR 5'-TTGTCCACAAACTCTGTCCTG-3' [rat homolog sequence according to hNTCP –32/–22 (5)]. For competition assays, 100-fold molar excess of each specific unlabeled oligonucleotide was added with the labeled oligonucleotide to the binding reaction.

Real-time RT-PCR analysis. Quantitative PCR on a LightCycler (Roche, Basel, Switzerland) was done as previously described (2, 31). Primers were used for rat Hnf4{alpha} (38) sense 5'-TGGCAAACACTACGGAGCCT-3', antisense 5'-CTGAAGAATCCCTTGCAGCC-3' and Pgc-1{alpha} sense 5'-ATGAATGCAGCGGTCTTAGC-3', antisense 5'-TGGTCAGATACTTGAGAAGC-3'. GAPDH primers were used as internal control, sense 5'-GAACCACGAGAAATATGAC-3', antisense 5'-GCAGCACCAGTGGATGCAG-3'.

Western blot analysis. Preparation of liver microsomes and Western blot analysis were performed as previously described (7, 10). Microsomal protein blots were incubated with Ntcp, Oatp1, and Oatp2 peptide antisera as well as monoclonal anti-Na+/K+-ATPase {alpha}-1 antibodies (11). Immune complexes were detected by using horseradish peroxidase-conjugated polyclonal swine anti-rabbit immunoglobulins according to the ECL Western blotting kit (GE Healthcare Lifesciences). Immunoreactive bands were quantified by laser densitometry and the Quantity One software (Bio-Rad).

Cell culture, transfections, RNA isolation, and real-time RT-PCR. HepG2 cells were routinely maintained as described (9). Cells were plated on six-well plates to a confluency of 40–60% and transfected with the Lipofectamine LTX (Invitrogen) reagent according to the manufacturer's instructions. Each well was transfected with 2.5 µg of pMT2-hHNF4{alpha} (33), pcDNA3.1-PGC1{alpha}-HA (kindly provided by Dr. A. Kralli, The Research Institute of Scripps Clinic, La Jolla, CA), or empty pMT2 as a control. All transfections were performed in triplicate. At 24 h posttransfection, total RNA was isolated by using Ultraspec (Biotexx) reagent according to the manufacturer's instructions. We used a Transcriptor First Strand cDNA synthesis kit (Roche) to reverse transcribe 1 µg RNA to yield cDNA, which was diluted to 60 µl. cDNA (2.5–5 µl) was used for real-time PCR with SYBRgreen reagent (Invitrogen) and specific primer pairs on a 7300 ABI PRISM Real-Time PCR system and with ABI PRISM 7300 SDS software (Applied Biosystems). The sequences for the primers for human genes are as follows: hHNF4{alpha}, sense 5'-gcc tac ctc aaa gcc atc at-3', antisense 5'-gac cct ccc agc agc atc tc-3'; hNTCP, sense 5'-ggg aca tga acc tca gca tt-3', antisense 5'-cgt ttg gat ttg agg acg at-3' (20). A BLAST search for the primer sequences against the human genome revealed no other matching sequences outside hNTCP. The data were normalized to 18S RNA, sense 5'-cgc cgc tag agg tga aat tc-3', antisense 5'-ttg gca aat gct ttc gct c-3', and analyzed by the comparative threshold cycle (CT) method.

Statistical analysis. Statistical analysis was performed by using Student's t-test. Statistical significance was considered at P values of <0.05. Data represent means ± SD of five animals per group.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
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DISCUSSION
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mRNA expression of basolateral uptake transporters. We first determined mRNA concentration of Ntcp, Oatp1, and Oatp2 in rats which were left without food but with water for 2 days. Food deprivation of 48 h increased mRNA concentration of all three investigated transporters, with Oatp2 being the most induced (316 ± 6% of fed controls, P < 0.001), while the induction of Oatp1 (181 ± 4%, P < 0.01) and Ntcp (154 ± 8%, P < 0.05) was less, but still significant (n = 5) (Fig. 1). Changes in transporter mRNA expression appear to be specific since Mrp2 (data not shown) and Gapdh mRNA expression (loading control) are unchanged by fasting.


Figure 1
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  		Fig. 1. Increased mRNA expression of basolateral uptake transporters during fasting. mRNA concentrations of the basolateral uptake transporters Ntcp, Oatp1, and Oatp 2 in liver tissue as determined by Northern blotting 48 h after onset of fasting in Sprague-Dawley rats and control animals. Co, control. GAPDH is included as loading control. A: representative Northern blots. B: densitometric results in fasted rats compared with controls (controls set as 100%). All results represent mean ± SD of 5 animals; *P < 0.05.

 
Protein mass of basolateral uptake transporters during fasting. Since mRNA of all investigated transporters was induced after 48 h of fasting, we studied protein expression. Oatp1 protein mass was induced to 128 ± 6% after a fasting period of 48 h. Because protein levels of Ntcp and Oatp2 were not significantly different after 48 h of fasting despite increased mRNA concentrations and changes in protein expression are often found to be delayed in other conditions of transporter gene regulation, we additionally determined protein mass of Ntcp, Oatp1, and Oatp2 72 h after the onset of fasting. In parallel to the mRNA data at 48 h of fasting, protein expression of all three investigated transporters was induced. Induction of protein expression was most prominent for Oatp2, which reached 176 ± 10% after 72 h of fasting (n = 5; P < 0.05). Ntcp was induced to 160 ± 20% (P < 0.05), whereas Oatp1 was only moderately induced to 119 ± 6% after 72 h of fasting (Fig. 2).


Figure 2
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  		Fig. 2. Protein mass of basolateral uptake transporters is upregulated during fasting. Hepatic protein mass of all investigated transporters as determined by Western blotting 48 and 72 h after onset of fasting in Sprague-Dawley rats and control animals. A: representative Western blots after 72 h of fasting. d, Days. B: densitometric results in controls (open bars = 100%) and after 48 (solid bars) and 72 h (shaded bars) of fasting. All results represent mean ± SD of 5 animals; *P < 0.05.

 
Binding activity of transactivators. To identify transcription factors involved in this regulation of transporter genes, we determined the binding activity of relevant gene transactivators of basolateral transporters under fasting conditions by EMSA using oligonucleotides with binding sites for Hnf4{alpha}, Hnf1{alpha}, glucocorticoid receptor (GR), and Stat5. Binding activity of Hnf4{alpha} was elevated to 193 ± 18% of fed controls after 48 h fasting (n = 5; P < 0.05) (Fig. 3). In contrast, binding activity of neither Hnf1{alpha} nor GR was significantly altered, whereas Stat5-binding activity was not significantly reduced compared with fed controls (Fig. 3). This indicated that Hnf4{alpha} but not Hnf1{alpha} paralleled basolateral transporter induction during fasting.


Figure 3
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  		Fig. 3. Binding activity of transporter gene transactivators in fasted rats. Binding activities of different transactivators from liver tissue at the Ntcp promoter (see MATERIALS AND METHODS) in EMSA 48 h after onset of fasting in Sprague-Dawley rats and control animals. A: representative EMSA gels with specific (SC) and nonspecific competition (NSC). GR, glucocorticoid receptor. B: densitometric results in controls (solid bars set as 100%) and animals fasted for 48 h (open bars). All results represent means ± SD of 5 animals; *P < 0.05.

 
mRNA concentrations of Pgc-1{alpha} and Hnf4{alpha} after 48 h of fasting. To determine whether increased binding activity of Hnf4{alpha} is based on a transcriptional activation of the gene and whether Pgc-1{alpha} is also increased as a known coactivator of Hnf4{alpha}, mRNA levels of these two important transcription factors were quantified by real-time RT-PCR (Fig. 4). In accordance with the above results, mRNA concentration of Hnf4{alpha} was increased to 232 ± 10% of fed controls. Likewise, the transcript level of Pgc-1{alpha} was upregulated to 360 ± 210% of fed controls (n = 5; P < 0.05 each).


Figure 4
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  		Fig. 4. mRNA concentrations of Pgc-1{alpha} and Hnf4{alpha} after 48 h of fasting. mRNA concentrations of Hnf4{alpha} and Pgc-1{alpha} in liver tissue as determined by RT-PCR (see MATERIALS AND METHODS for details) 48 h after onset of fasting. Controls are shown with closed bars (set as 100%), fasted rats with open bars. All results represent mean ± SD of 5 animals; *P < 0.05.

 
hNTCP mRNA expression after transfection of hHNF4{alpha} and/or hPGC1{alpha} in HepG2 cells. To undermine the functional mechanism that HNF4{alpha} and PGC1{alpha} activate NTCP expression, HepG2 cells were transfected with HNF4{alpha} and/or PGC1{alpha} expression plasmids (Fig. 5). At 24 h posttransfection, RNA was prepared, reverse transcribed, and used in a real-time PCR reaction with primers specific for HNF4{alpha} and NTCP, respectively. HNF4{alpha} mRNA expression was induced 12-fold upon transfection of the expression plasmid as expected whereas transfection of the PGC-1{alpha} plasmid alone did not affect HNF4{alpha} mRNA expression. Basal NTCP mRNA expression in HepG2 cells is of low abundance (CT values of 32–34 compared with 13.5 for 18S RNA) but tended to be induced around threefold by transfection of either Hnf4{alpha} alone, PGC-1{alpha} alone, or 50% of each plasmid (1:1 ratio) to maintain a constant amount of transfected DNA.


Figure 5
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  		Fig. 5. mRNA concentrations of Hnf4{alpha} and NTCP after transfection of Hnf4{alpha} and/or PGC-1{alpha} expression plasmids into HepG2 cells. HepG2 cells, grown to 40–60% confluency in 6-well plates, were transfected with Hnf4{alpha} and/or PGC-1{alpha} expression plasmids (pHnf4{alpha} alone, pPGC-1{alpha} alone, or 50% of each plasmid in a 1:1 ratio to maintain a constant amount of transfected DNA) as described in MATERIALS AND METHODS. At 24 h later RNA was prepared and reverse transcribed. The cDNA was used in a real-time PCR reaction with HNF4{alpha}- and NTCP-specific primers (A, *P < 0.0001; B, *P < 0.05, #P = 0.13).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 REFERENCES
 
The results of our study indicate that upregulation of hepatocellular basolateral transporters is part of the global fasting response via induction of the Hnf4{alpha}/Pgc-1{alpha} pathway. This fasting response includes the induction of key enzymes involved in hepatic gluconeogenesis such as PEPCK, fructose 1,6-bisphosphatase, and glucose-6-phosphatase (44).

mRNA data at 48 h and protein data at 72 h show that the observed upregulation of the transporters under investigation is mediated transcriptionally (Figs. 1 and 2). Binding activity of Hnf4{alpha}, the central regulator during the fasting response, was doubled during fasting, whereas no other known regulator of Ntcp, especially Hnf1{alpha}, reached this induction level (Fig. 3). Since Ntcp is the quantitatively most important bile acid uptake transporter, a clear link between the systemic fasting response as mediated directly by Hnf4{alpha} (without Hnf1{alpha}) and higher basolateral bile acid uptake has been established by the described experiments.

Hnf1{alpha} and Hnf4{alpha} are connected by an autoregulatory loop, meaning that both activators activate their counterpart via binding its promoter (12, 24). Data from Hnf1{alpha} knockout mice (36) and Hnf4{alpha} conditional knockout mice (13) indicate that expression of both activators is necessary to maintain Ntcp expression in the basolateral membrane of hepatocytes. However, this does not mean that upregulation of one activator alone is not enough to induce Ntcp protein expression. Binding of Hnf1{alpha} to several regulatory elements in the rat and mouse Ntcp promoter leads to activation of transcription (9, 15, 40). Unpublished data from our group demonstrate that Hnf4{alpha} alone directly induces Ntcp expression whereas Hnf1{alpha} upregulation is not required (unpublished observations). The data presented in this study confirm these results and extend them into the in vivo situation as investigated in the physiologically relevant fasting response in rats. Further functional data from transfection studies in HepG2 cells show that expression of either Hnf4{alpha} alone, PGC-1{alpha} alone, or a combination of both increases NTCP mRNA expression approximately threefold (Fig. 5). The fact that transfection of PGC-1{alpha} alone in the presence of just endogenous HNF4{alpha} reveals a similar induction of NTCP as vice versa suggests a role of PGC-1{alpha} as a coactivator of Ntcp/NTCP expression as previously shown for several other hepatic genes. Therefore it is plausible that coactivation of Hnf4{alpha} by Pgc-1{alpha} may contribute to the upregulation of other basolateral transporter genes during fasting in rats. This cooperative effect of Hnf4{alpha} and Pgc-1{alpha} has also been described as an indirect mechanism of mouse Oatp2 gene induction by fasting following an increased Hnf4a/Pgc1a-binding activity to a Hnf4-response element in the mouse CAR promoter (3). Besides this Hnf4{alpha}/Pgc-1{alpha} pathway induction of PPAR{alpha} has also been shown to contribute to Ntcp induction during fasting in mice at 24 h (18) and PPAR{alpha} mRNA is increased up to threefold in our fasting rats at 48 and 72 h as well (data not shown). In addition to HNF4{alpha}, PPAR{alpha} may also activate the Ntcp gene at the DR-1 element as previously shown for the human {Delta}6-desaturase gene (39). Other activators of basolateral transport systems such as Hnf1{alpha}, Stat5, or GR seem to play no significant role in the regulation during fasting. The underlying mechanism of Oatp1 induction remains unidentified in the absence of detail information about its 5'-regulatory region.

The discrepancy between ileal and basolateral hepatic bile acid transport needs further clarification. Several studies showed reduced ileal uptake of bile acids, either due to reduced transporter density (caloric deprivation affects protein synthesis of transporters, too) (17) or due to reduced transporter affinity to taurocholate (4). It is unclear why such limitations of bile acid transport are absent at the basolateral hepatocyte membrane, but even the opposite, upregulation of specific transport proteins, is the consequence of fasting in the liver. It seems possible that different regulatory patterns are responsible for transport proteins in the intestine and the liver, setting priorities during energy expenditure such as in the state of fasting. Although the effect of fasting on carbohydrate and amino acid uptake into hepatocytes is variable (4), bile acids seem important enough to include responsible transporters into the "fasting regulation program." One possible explanation for this upregulation represents the recently discovered function of bile acids as signaling molecules in the liver and peripheral tissues. Bile acids increase energy expenditure in thermogenically important tissues via the TGR5/D2 pathway, which utilizes thyroid hormones to increase mitochondrial activity (43). During fasting, these energy-wasting pathways are not desirable. The physiological role of basolateral bile acid transporter upregulation may therefore not be to accumulate bile acids in the liver, but to remove them from the systemic circulation to avoid signaling functions in peripheral tissues. Nevertheless it is certainly useful in terms of physiological mechanism to maintain micellar functions of the bile by concentrating the remaining bile acids in the liver (37).

Our in vivo data confirm the results of kinetic experiments in membrane vesicles published more than 10 years ago: The higher hepatocellular uptake of bile acids is a result of specific upregulation of the responsible transporters leading to higher transporter density, as indicated by a high Vmax but a constant Km for bile acid transport in vitro (4). This behavior may also lead to lower systemic bile acid concentrations and higher intracellular bile salt retention in the liver despite reduced bile flow during fasting. Given the fact that hepatocellular bile acid uptake is the only step in enterohepatic cycling that is upregulated during fasting, it is straightforward to ascribe an important general regulation principle to this reaction.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Deutsche Forschungsgemeinschaft grant SFB542 TP C1 (to A. Geier), GE 1219/1-1 (to A. Geier and C. G. Dietrich) and DI 729/3-1 (to C. G. Dietrich).


   ACKNOWLEDGMENTS
 
The authors thank Aline Mueller, Petra Schmitz, and Sonja Strauch for excellent technical assistence.

Present address for C. G. Dietrich: Department of Internal Medicine II, Klinikum Aschaffenburg, Aschaffenburg, Germany. Present address for C. Gartung: Department of Internal Medicine I, Klinikum Minden, Minden, Germany.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Geier, Dept. of Internal Medicine III, Univ. Hospital Aachen (UKA), Aachen Univ. (RWTH), Pauwelsstr. 30, 52074 Aachen, Germany (e-mail: ageier{at}ukaachen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* C. G. Dietrich and I. V. Martin equally contributed to this work. Back


   REFERENCES
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 DISCUSSION
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 REFERENCES
 

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