Comparison of the bioavailability of quercetin and catechin in rats

Quercetin and catechin are present in noticeable amounts in human diet and these polyphenolic compounds are supposed to exert beneficial effects on human health. However, their metabolic fates in the organism have never been compared. In the present study, rats were fed a 0.25% quercetin or a 0.25% catechin diet. Quercetin and catechin metabolites were analyzed in plasma and liver samples by high-performance liquid chromatography coupled to an ultraviolet or a multielectrode coulometric detection. All plasma metabolites were present as conjugated forms, but catechin metabolites were mainly constituted by glucuronidated derivatives, whereas quercetin metabolites were sulfo- and glucurono-sulfo conjugates. Quercetin was more intensively methylated than catechin in plasma. The plasma quercetin metabolites are well maintained during the postabsorptive period (approximately 50 microM), whereas the concentration of catechin metabolites dropped dramatically between 12- and 24-h after an experimental meal (from 38.0 to 4.5 microM). In the liver, the concentrations of quercetin and catechin derivatives were lower than in plasma, and no accumulation was observed when the rats were adapted for 14 d to the supplemented diets. The hepatic metabolites were intensively methylated (90-95%), but in contrast to plasma, some free aglycones could be detected. Thus, it clearly appears that studies dealing with the biological impact of these polyphenols should take into account the feature of their bioavailability, particularly the fact that their circulating metabolites are conjugated derivatives.     

Cholesterol feeding alters the metabolism of thoracic-duct lymph lipoprotein cholesterol in rabbits but not in rats

1. Labelled thoracic-duct lymph was collected from rats and rabbits after test meals containing [(14)C]cholesterol and [2-(3)H]glyceryl trioleate. 2. The metabolism of labelled cholesterol and triglyceride was studied in normally fed and cholesterol-fed rats and rabbits injected with radioactive lymph from the same species. 3. In normally fed animals of both species, 10min after intravenous administration, about 80% of lymph cholesteryl ester but only about 10% of triglyceride was recovered in the liver after clearance from the plasma. This distribution is consistent with participation of ;remnant' particles in the metabolism of dietary lymph particles. 4. The metabolism of cleared lymph lipoprotein constituents was unchanged in cholesterol-fed rats, but the recovery of cholesteryl ester in the livers of the cholesterol-fed rabbits was decreased to 30% of the cleared dose. 5. The low recovery in cholesterol-fed rabbits was accounted for mainly by increased hydrolysis of cholesteryl ester. 6. It is proposed that differences between rats and rabbits in metabolism of dietary cholesterol might be partly due to the observed enhancement of hydrolysis of lymph lipoprotein cholesteryl ester in rabbits.     

Cancer cachexia alters intracellular surfactant metabolism but not total alveolar surface area

Dyspnoea is frequently observed in cancer cachectic patients. Little is known whether this is accompanied by structural or functional alterations of the lung. We hypothesized that in analogy to calorie restriction cancer cachexia leads to loss of alveolar surface area and surfactant. Mice were subjected to subcutaneous injection of Lewis lung carcinoma cells (tumour group, TG) or saline (control group, CG). Twenty-one days later blood samples and the lungs were taken. Using design-based stereology, the alveolar surface area and the lamellar body (Lb) content were quantified. Messenger RNA expression of surfactant proteins, ABCA3 and various growth factors was investigated by quantitative RT-PCR. Intraalveolar surfactant subtype composition was analyzed by differential centrifugation. TG mice showed reduced body weight and anaemia but no reduction of lung volume or alveolar surface area. The volume of Lb was significantly reduced and mRNA levels of ABCA3 transporter tended to be lower in TG versus CG. Surfactant protein expression and the ratio between active and inactive intraalveolar surfactant subtypes were not altered in TG. Growth factor mRNA levels were not different between CG and TG lungs but the tumour expressed growth factor mRNA. Vascular endothelial growth factor was significantly enhanced in blood plasma. The present study demonstrates structural alterations of the lung associated with cancer cachexia. These include reduction of Lb content despite normal intraalveolar surfactant and alveolar surface area. The pulmonary phenotype of the cancer cachectic mouse differs from the calorie restricted mouse possibly due to growth factors released from the tumour tissue.     

Mechanisms of antioxidant activities of quercetin and catechin

Abstract

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca²⁺ release from mitochondria. Ebselen stimulates Ca²⁺ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclo-sporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca²⁺ cycling is prevented with ruthenium red, an inhibitor of the Ca²⁺ uniporter, or by chelation of extramitochondrial Ca²⁺ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca²⁺ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca²⁺ release from intact mitochondria via an NAD⁺ hydrolysis-dependent mechanism.     

Electric field exposure alters serum melatonin but not pineal melatonin synthesis in male rats

Sprague-Dawley male rats, maintained in a 14:10 h light:dark cycl were exposed for 30 days (starting at 56 days of age) to a 65 kV/m, 60 Hz electric field or to a sham field for 20 h/day beginning at dark onset. Pineal N-acetyltransferase (NAT), hydroxy-indole-o-methyl transferase (HIOMT), and melatonin as well as serum melatonin were assayed. Preliminary data on unexposed animals indicated that samples obtained 4 h into the dark period would reveal either a phase delay or depression in circadian melatonin synthesis and secretion. Exposure to electric fields for 30 days did not alter the expected nighttime increase in pineal NAT, HIOMT, or melatonin. Serum melatonin levels were also increased at night, but the electric field-exposed animals had lower levels than the sham-exposed animals. Concurrent exposure to red light and the electric field or exposure to the electric field at a different time of the day-night period did not reduce melatonin synthesis. These data do not support the hypothesis that chronic electric field exposure reduces pineal melatonin synthesis in young adult male rats. However, serum melatonin levels were reduced by electric field exposure, suggesting the possibility that degradation or tissue uptake of melatonin is stimulated by exposure to electric fields. © 1994 Wiley-Liss, Inc.     

Chronic amphetamine alters D-2 but not D-1 agonist-induced behavioral responses in rats

In two experiments, using different drug doses and periods of drug administration, rats were given amphetamine (AMPH) either continuously (via slow-release pellets), or intermittently (via injections). In both experiments, only the rats pretreated with intermittent AMPH subsequently showed heightened responsivity to the selective D-2 dopamine agonist LY171555 but not to SKF38393 (a D-1 agonist). This altered response to LY171555 was still present 30 days after the AMPH withdrawal, implying that D-2 dopamine receptors at least partially mediate AMPH inverse tolerance effects. The behavioral response to the D-2 agonist was clearly different in animals receiving high versus low doses of AMPH, suggesting that different drug-state learning may have occurred during pretreatment. In a third experiment, in which rats were given repeated daily injections of either the D-1 or the D-2 agonist, only rats pretreated with the D-2 agonist and subsequently injected with the D-2 agonist clearly showed heightened responsivity. These data imply an important role of D-2 receptors in the AMPH inverse tolerance effect.     

Estrogen alters excitability but not morphology of a sexually dimorphic neuromuscular system in adult rats

In rats, motoneurons of the spinal nucleus of the bulbocavernosus (SNB) innervate the bulbocavernosus (BC) muscle, which surrounds the base of the penis. The SNB/BC is a sexually dimorphic, steroid-sensitive neuromuscular system, which is critically important in male reproductive behavior. Androgens are necessary for the development, morphology, and function of the SNB/BC system. However, estradiol (E) is also necessary for the development of the SNB/BC system, and E is capable of maintaining BC EMG activity in adulthood. In this study, we used electrophysiological and anatomical methods to examine estrogenic effects on BC EMG activity. We used a modified H-reflex testing method to investigate polysynaptic reflex characteristics in intact males, castrates, and castrates treated short term with estradiol benzoate (EB). Measures of EMG activity, response latency, and spike count were altered in castrates, but maintained in EB-treated castrates to the levels of intact males. Furthermore, estrogenic effects were found in EMG activity that could be isolated to the periphery of the SNB/BC system. BC NMJ size and muscle fiber area have been demonstrated to be hormone sensitive, and we examined these for possible correlates of E's effects on BC EMG activity. BC muscles of intact males, castrates, and short-term EB-treated castrates were fixed and stained with zinc iodide and osmium tetroxide. NMJ size and muscle fiber area did not differ between groups. Together, these data suggest that E treatment results in changes in the neuromuscular periphery that maintain BC EMG activity, but this effect cannot be accounted for by changes in NMJ size or muscle fiber area.     

Antioxidative flavonoid quercetin: implication of its intestinal absorption and metabolism

Quercetin is a typical flavonoid ubiquitously present in fruits and vegetables, and its antioxidant effect is implied to be helpful for human health. The bioavailability of quercetin glycosides should be clarified, because dietary quercetin is mostly present as its glycoside form. Although quercetin glycosides are subject to deglycosidation by enterobacteria for the absorption at large intestine, small intestine acts as an effective absorption site for glucose-bound glycosides (quercertin glucosides). This is because small intestinal cells possess a glucoside-hydrolyzing activity and their glucose transport system is capable of participating in the glucoside absorption. A study using a cultured cell model for intestinal absorption explains that the hydrolysis of the glucosides accelerates their absorption in the small intestine. Small intestine is also recognized as the site for metabolic conversion of quercetin and other flavonoids as it possesses enzymatic activity of glucuronidation and sulfation. Modulation of the intestinal absorption and metabolism may be beneficial for regulating the biological effects of dietary quercetin.     

Antagonism of corticotropin-releasing hormone alters serotonergic-induced changes in brain temperature, but not sleep, of rats

Serotonin is involved in many physiological processes, including the regulation of sleep and body temperature. Administration into rats of low doses (25, 50 mg/kg) of the 5-HT precursor l-5-hydroxytryptophan (5-HTP) at the beginning of the dark period of the 12:12-h light-dark cycle initially increases wakefulness. Higher doses (75, 100 mg/kg) increase nonrapid eye movement (NREM) sleep. The initial enhancement of wakefulness after low-dose 5-HTP administration may be a direct action of 5-HT in brain or due to 5-HT-induced activation of other arousal-promoting systems. One candidate arousal-promoting system is corticotropin-releasing hormone (CRH) and the hypothalamic-pituitary-adrenal axis. Serotonergic activation by 5-HTP at the beginning of the dark period also induces hypothermia. Because sleep and body temperature are influenced by circadian factors, one aim of this study was to determine responses to 5-HTP when administered at a different circadian time, the beginning of the light period. Results obtained show that all doses of 5-HTP (25-100 mg/kg) administered at light onset initially increase wakefulness; NREM sleep increases only after a long delay, during the subsequent dark period. Serotonergic activation by 5-HTP at light onset induces hypothermia, the time course of which is biphasic after higher doses (75, 100 mg/kg). Intracerebroventricular pretreatment with the CRH receptor antagonist alpha-helical CRH does not alter the impact of 5-HTP on sleep-wake behavior but potentiates the hypothermic response to 50 mg/kg 5-HTP. These data suggest that serotonergic activation by peripheral administration of 5-HTP may modulate sleep-wake behavior by mechanisms in addition to direct actions in brain and that circadian systems are important determinants of the impact of serotonergic activation on sleep and body temperature.     

Supplementation of L-arginine improves hypertension and lipid metabolism but not insulin resistance in diabetic rats

Nitric oxide (NO) plays an important role in glucose and lipid metabolism. We previously reported that NO synthesis inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME), deteriorate insulin sensitivity and lipid metabolism, while the addition of L-arginine reverses this deterioration. L-arginine is a precursor of NO, and is used as a supplement in the US. In the present study, we evaluated whether the administration of L-arginine alone improves insulin resistance and serum lipid levels in insulin-resistant and hypertriglycemic rat models. Diabetic rats were divided into 3 groups: the control (Cont) group (standard diet), the L-NAME group (diet containing L-NAME), and the Arg group (diet containing L-arginine). After 4 weeks of breeding, urinary NOx, glucose infusion rate (GIR), glucose and lipid tolerance tests were performed. Urinary NOx levels were significantly lower in the L-NAME group than in the Cont group. The GIR in the L-NAME group was significantly lower than that in the Cont group, suggesting increased insulin resistance. However, the administration of L-arginine did not influence insulin resistance in the Arg group. Oral lipid administration significantly increased plasma triglyceride levels in the L-NAME group and plasma triglyceride levels were significantly lower in the Arg group than in the Cont group. The area under the curve of plasma triglyceride levels after oral lipid administration was larger in the L-NAME group than in the Cont group. The administration of L-NAME increased insulin resistance and decreased lipid metabolism. L-arginine significantly increased urinary NO secretion but did not improve insulin resistance, although it did improve lipid metabolism. These findings suggest that supplementation of L-arginine cannot improve insulin resistance in diabetic rats probably due to increased insulin secretion by L-arginine.     

Dietary restriction alters demographic but not behavioral aging in Drosophila

Dietary restriction extends lifespan substantially in numerous species including Drosophila. However, it is unclear whether dietary restriction in flies impacts age-related functional declines in conjunction with its effects on lifespan. Here, we address this issue by assessing the effect of dietary restriction on lifespan and behavioral senescence in two wild-type strains, in our standard white laboratory stock, and in short-lived flies with reduced expression of superoxide dismutase 2. As expected, dietary restriction extended lifespan in all of these strains. The effect of dietary restriction on lifespan varied with genetic background, ranging from 40 to 90% extension of median lifespan in the seven strains tested. Interestingly, despite its robust positive effects on lifespan, dietary restriction had no substantive effects on senescence of behavior in any of the strains in our studies. Our results suggest that dietary restriction does not have a global impact on aging in Drosophila and support the hypothesis that lifespan and behavioral senescence are not driven by identical mechanisms.     

Inhibition of oxidative hemolysis by quercetin,but not other antioxidants

We previously reported that lipid-soluble quercetin, not water-soluble dihydroquercetin, protects human red blood cells against oxidative damage. The objectives of this study were to determine if an antihemolytic effect could be produced by other lipid-soluble antioxidants and if anti-inflammatory activity played a role in antihemolysis by quercetin. This study compared three lipid-soluble polyphenols, muscadine, curcumin and quercetin, and three lipid- (α-tocopherol and α-tocotrienol) or water-soluble (ascorbic acid) vitamins. Among the tested polyphenols, muscadine was the most potent in inhibiting superoxide and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-generated peroxyl radicals, whereas ascorbic acid was the most potent inhibitor of hydrogen peroxide. Activities of the polyphenols after lipid extractions showed that curcumin inhibited superoxide production to a greater extent than quercetin and muscadine. All blood cells were tested 20 min after incubation with the selected compounds. All the polyphenols caused inhibition of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced neutrophil oxidative bursts. Quercetin, but not other polyphenols, significantly reduced AAPH-induced oxidative hemolysis. No significant effect on neutrophil oxidative burst or oxidative hemolysis was found with any of the tested vitamins. These results suggest that quercetin enhances the resistance of membrane to destruction by free radicals. This effect of quercetin is not directly mediated through antioxidative or anti-inflammatory actions. Antioxidant or anti-inflammatory potency may not be used as a simple criterion to select polyphenols for cell protection benefits.     

Photoperiod regulates dietary preferences and energy metabolism in young developing Fischer 344 rats but not in same-age Wistar rats

The effects of photoperiod on dietary preference were examined using young growing Fischer 344 and Wistar rats, which are seasonal and nonseasonal breeders, respectively. Rats were provided a low-fat, high-carbohydrate diet (LFD: 66/10/24% energy as carbohydrate/fat/protein) and high-fat, low-carbohydrate diet (HFD: 21/55/24% energy as carbohydrate/fat/protein) simultaneously under long- (LD: 16 h light/day) and short-day (SD: 8 h light/day) conditions for 3 wk. Fischer 344 rats preferred the LFD to the HFD under the LD condition, whereas preference for both diets was equivalent under the SD condition. Consequently, their body weight and total energy intake exhibited 11-15 and 10-13% increases, respectively, under the LD condition. Calculation of energy intake from macronutrients revealed that rats under the LD condition consumed 20-24 and 9-13% higher energy of carbohydrates and proteins, respectively, than those under the SD condition. In contrast, Wistar rats preferred the LFD to the HFD irrespective of photoperiod and exhibited no photoperiodic changes in any parameters examined. Next, Fischer 344 rats were provided either the LFD or HFD for 3 wk under LD or SD conditions. Calorie intake was 10% higher in the rats fed the LFD than those fed the HFD under SD condition. However, rats under LD condition exhibited 5-10, 14, and 64% increases in body weight, epididymal fat mass, and plasma leptin levels, respectively, compared with those under the SD condition irrespective of dietary composition. In conclusion, photoperiod regulates feeding and energy metabolism in young growing Fischer 344 rats via the interactions with dietary macronutrient composition.     

Lung cancer xenografting alters microRNA profile but not immunophenotype

Lung tumor xenografts grown in immunocompromised mice provide a renewable source of tumor tissue for research and a means to study individualized response to chemotherapy. Critical to this utility is verification that the xenograft cells retain core phenotypic characteristics of the original tumor. We compared eight non-small cell lung carcinomas with their corresponding xenografts grown in mice with severe combined immunodeficiency by way of histology, immunohistochemistry, and microRNA expression profiling. Six of the eight xenografts closely resembled their original tumor by light microscopy. The xenografts also largely retained key immunophenotypic features. With expression profiling of human microRNAs, however, xenografts clustered separately from the original tumors. While this may be partly due to contamination by non-neoplastic human and mouse stroma, the results suggest that miRNA expression may be altered in xenografts and that this possibility should be further evaluated.     

Intestinal metabolism and absorption of cholecystokinin analogs in rats

Intestinal metabolism and poor permeability were known to be major barriers for oral absorption of large peptide drugs. Dimensionless wall permeability values of C-terminal octa- and tetra-peptides cholecystokinin analogs (CCK8 and CCK4) were estimated and found out to be greater than 1, suggesting no permeability-limited absorption for CCK analogs. Thus, a strategy employing enzyme inhibitors and a specific delivery site to improve the absorption was developed and tested with CCK8, followed by identification of metabolites of the analogs and their participating enzymes in rabbit brush-border membrane vesicles. Thiorphan and amastatin, a specific enzyme inhibitor for enkephalinase and aminopeptidase, respectively, in pH 4 buffer solution were coadministered with CCK8 to the ileum in fistulated rats. The absolute bioavailability (F) of CCK8 was 5.4% and increased to 19% in the presence of the enzyme inhibitors, while the F values following oral administration were close to zero. These results indicate that peptide oral delivery is possible.     

A theoretical study of the different radical-scavenging activities of catechin, quercetin, and a rationally designed planar catechin

To improve the radical-scavenging activity of catechin, a planar catechin analogue was designed and synthesized by Fukuhara [J. Am. Chem. Soc. 124 (2002) 5952]. Although the planar catechin is less active than quercetin, it is much more active than catechin in its ability to scavenge galvinoxyl radical, suggesting that the rational design was successful. However, an interesting question remains: what is the basis for the enhanced radical-scavenging activity of the planar catechin? By DFT calculations, we determined that the galvinoxyl radical is scavenged through an electron-transfer mechanism rather than a hydrogen-atom-transfer mechanism. Moreover, the antioxidant anion, derived from proton dissociation, plays a key role in the radical-scavenging process. Hence, the different radical-scavenging activities of the three antioxidants may result from the different ionization potentials of their anions.     

Social technology restriction alters state-anxiety but not autonomic activity in humans

Social technology is extensively used by young adults throughout the world, and it has been suggested that interrupting access to this technology induces anxiety. However, the influence of social technology restriction on anxiety and autonomic activity in young adults has not been formally examined. Therefore, we hypothesized that restriction of social technology would increase state-anxiety and alter neural cardiovascular regulation of arterial blood pressure. Twenty-one college students (age 18-23 yr) were examined during two consecutive weeks in which social technology use was normal or restricted (randomized crossover design). Mean arterial pressure (MAP), heart rate, and muscle sympathetic nerve activity (MSNA) were measured at rest and during several classic autonomic stressors, including isometric handgrip, postexercise muscle ischemia, cold pressor test, and mental stress. Tertile analysis revealed that restriction of social technology was associated with increases (12 ± 2 au; range 5 to 21; n = 7), decreases (-6 ± 2 au; range -2 to -11; n = 6), or no change (0 ± 0 au; range -1 to 3; n = 8) in state-anxiety. Social technology restriction did not alter MAP (74 ± 1 vs. 73 ± 1 mmHg), heart rate (62 ± 2 vs. 61 ± 2 beats/min), or MSNA (9 ± 1 vs. 9 ± 1 bursts/min) at rest, and it did not alter neural or cardiovascular responses to acute stressors. In conclusion, social technology restriction appears to have an interindividual influence on anxiety, but not autonomic activity. It remains unclear how repeated bouts, or chronic restriction of social technology, influence long-term psychological and cardiovascular health.     

Predation threat modifies relationships between metabolism and behavioural traits but not their ecological relevance in Chinese bream

The aims of this study were to test whether the metabolism, behavioural traits, growth and survival under predation of a fish species phenotypically changed under predation threat with the particular emphasis on whether short-time predator exposure would amplify the relationships between metabolic rate and behavioural traits and their fitness consequences (growth and survival). We found that Chinese bream under predation threat for 20 days exhibited a lower specific growth rate (SGR), feeding rate (FR) and feeding efficiency (FE) but a higher standard metabolic rate (SMR) and survival when encountering predators compared to the bream in the no-predator group. Both activity and boldness showed no correlation to SMR in the no-predator group, while it was vice versa in the predator group according to Pearson correlation. It thus demonstrated that short-time predator exposure can mediate the relationships between metabolism and behavioural traits, suggesting that predation may play an important evolutionary role in modifying intraspecific behavioural differences via metabolism. However, no significant effect of predator treatment acted on relationships between behaviour traits and SMR according to ANCOVA, which possibly due to the small sample size of this study. Additionally, the SMR of both groups was positively correlated with survival under predation, whereas the relationships between SMR and fitness cost such as growth and survival are rather complicated and need further investigation.     

Estrogen status alters tissue distribution and metabolism of selenium in female rats

A reported association between estrogen and selenium status may be important in the regulation of selenium metabolism. In this study, the effect of estrogen status on the metabolism of orally administered (75)Se-selenite and tissue selenium status was investigated. Female Sprague-Dawley rats were bilaterally ovariectomized at 7 weeks of age and implanted with either a placebo pellet (OVX) or pellet containing estradiol (OVX+E2), or were sham operated (Sham). At 12 weeks of age, 60 μCi of (75)Se as selenite was orally administered to OVX and OVX+E2 rats. Blood and organs were collected 1, 3, 6 and 24 h after dosing. Estrogen status was associated with time-dependent differences in distribution of (75)Se in plasma, red blood cell (RBC), liver, heart, kidney, spleen, brain and thymus and incorporation of (75)Se into plasma selenoprotein P (Sepp1) and glutathione peroxidase (GPx). Estrogen treatment also significantly increased selenium concentration and GPx activity in plasma, liver and brain, selenium concentration in RBC and hepatic Sepp1 and GPx1 messenger RNA. These results suggest that estrogen status affects tissue distribution of selenium by modulating Sepp1, as this protein plays a central role in selenium transport.     

Climate warming alters subsoil but not topsoil carbon dynamics in alpine grassland

Subsoil contains more than half of soil organic carbon (SOC) globally and is conventionally assumed to be relatively unresponsive to warming compared to the topsoil. Here, we show substantial changes in carbon allocation and dynamics of the subsoil but not topsoil in the Qinghai‐Tibetan alpine grasslands over 5 years of warming. Specifically, warming enhanced the accumulation of newly synthesized (¹⁴C‐enriched) carbon in the subsoil slow‐cycling pool (silt‐clay fraction) but promoted the decomposition of plant‐derived lignin in the fast‐cycling pool (macroaggregates). These changes mirrored an accumulation of lipids and sugars at the expense of lignin in the warmed bulk subsoil, likely associated with shortened soil freezing period and a deepening root system. As warming is accompanied by deepening roots in a wide range of ecosystems, root‐driven accrual of slow‐cycling pool may represent an important and overlooked mechanism for a potential long‐term carbon sink at depth. Moreover, given the contrasting sensitivity of SOC dynamics at varied depths, warming studies focusing only on surface soils may vastly misrepresent shifts in ecosystem carbon storage under climate change.