Background: We previously reported that dietary intake of shiitake mushroom (SM; Lentinus edodes) decreased serum concentrations of polar lipids in male rats.
Objective: This study evaluated the dietary effects of SM on serum cholesterol–related and serum antioxidant indexes in rats of both sexes.
Methods: Sprague-Dawley rats [38 dams and their offspring (20 males and 20 females/diet)] were fed diets containing 0 (control), 1%, 4%, or 10% (wt:wt) SM powder from gestation day 4 through to postnatal day (PND) 126. Biochemical indexes were monitored during the midgrowth phase (PNDs 50–66).
Results: The food consumption by offspring fed the control diet and diets supplemented with SM was not different when measured on PND 65. However, the 4% and 10% SM diets resulted in male rats with 7% lower body weights than those of the other 2 groups on PND 66. SM consumption dose-dependently decreased the concentrations of lipidemia-related factors in sera, irrespective of sex. At PND 50, serum concentrations of total cholesterol, HDL cholesterol, and non-HDL cholesterol in SM-fed male and female rats were generally lower (3–27%) than those in the corresponding control groups. Consumption of the 10% SM diet resulted in significantly decreased (55%) serum triglyceride concentrations relative to the control groups for both sexes. The 10% SM diet elicited a 62% reduction of serum leptin concentrations in females but not in males, and this same diet increased serum insulin (137%) and decreased serum glucose (15%) in males compared with controls. Serum lipophilic antioxidant capacity in males and females fed SM diets was generally lower (31–86%) than that in the control groups.
Conclusion: SM decreased the concentrations of lipidemia-related factors in rat sera irrespective of sex. The SM-elicited reduction of lipophilic antioxidant capacity irrespective of sex may reflect a lower pro-oxidative state and, hence, improved metabolic profile.
Introduction
The beneficial health properties of edible mushrooms have been recorded since 3000 BC. Among mushroom species, shiitake mushroom (SM8; Lentinus edodes) is the second most highly consumed globally (1–6). SM has been reported to possess tumor-inhibiting activities, attributed in part through its promotion of immune surveillance (3, 7–11). However, in a chemically induced rat model of colon cancer we found no effects of dietary SM intake on tumor incidence, multiplicity, or weights (12), although results of this study suggested the potential of SM diet supplementation to enhance energy expenditure. The reported cholesterol-lowering effects of dietary SM have been attributed to the mycochemical eritadenine [2R, 3R-4-(9-adenyl-)-butyric acid] (13–16). Dietary eritadenine regulates lipid metabolism with differing effects on lipid molecular types. For example, eritadenine increased the proportion of the 16:0–18:2 molecular species and decreased that of the 18:0–20:4 species in the plasma lipoprotein phosphatidylcholines of male rats respectively fed cholesterol-free and cholesterol-enriched diets (17). In another study from our laboratory we also found that dietary SM decreased serum concentrations of a number of polar lipids in rats (18). Interestingly, these effects were limited to male rats, suggesting possible sex differences in physiological responses to dietary SM (18). The distinct effects elicited by an eritadenine-supplemented diet from those of an SM-supplemented diet imply that other bioactive components with hypocholesterolemic properties, in addition to eritadenine, are also present in SM (18).
Free radicals and reactive oxygen and reactive nitrogen species play important roles in the pathogenesis of cardiovascular and cerebrovascular diseases and various cancers (19). Antioxidants, which can reduce oxidative stress, are thought to be of central importance in the prevention of such diseases. In this regard, SM extract protected liver cells from paracetamol-induced liver damage by its anti-oxidative effect on hepatocytes and diminished the harmful effects of toxic metabolites of paracetamol (20). Additionally, in another report, SM extract manifested antioxidant activity via the regulation of nitric oxide concentration and thiol redox status (21).
Here we evaluated the effects of SM on a number of lipidemia, hormonal, and oxidative stress parameters in male and female rats exposed to SM-supplemented diets from gestation day 4 through postnatal day (PND) 126. Serum concentrations of insulin and leptin, as well as hydrophilic and lipophilic antioxidant capacities, were measured, given the physiological roles of insulin and leptin in mediating carbohydrate and fat metabolism (22) and the function of antioxidants in providing defense against free-radical damage (23).
Methods
Materials.
SMs (“Snowcap” variety) were grown in oak logs (Shirley Community Development Corporation). Fruiting bodies were harvested and immediately frozen in dry ice. Frozen mushrooms were lyophilized, powdered, bagged under a vacuum, and stored at −20°C until subsequent analyses.
Experimental diets and animals.
Animal use protocols were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Timed-pregnant Sprague Dawley rats of similar mean body weight on gestation day 4 were obtained from Charles River Laboratories and divided into 4 diet groups: control (n = 9 dams), 1% (n = 8 dams), 4% (n = 9 dams), and 10% (n = 12 dams) (wt:wt) SM. Diets were prepared following the AIN-93G formulation as previously described (18). The final protein, carbohydrate, fat, and energy contents in all diets were closely matched (12; Supplemental Table 1). Pregnant rats on gestation day 4 were fed the assigned diets throughout gestation and lactation. Twenty males and 20 females were randomly allocated to each of the 4 diet groups. The offspring were weaned (at PND 21) to the same diet as their dams and fed that diet throughout the remaining experimental period. Offspring were weighed weekly for the duration of the experiment.
At PND 50, randomly chosen subsets of rats from each diet group (5 females and 5 males from each diet group) were anesthetized with pentobarbital and killed, and blood was collected without anticoagulant. Serum was prepared by low-speed centrifugation 2000 × g for 20 min at 4°C. At PND 65, randomly chosen subsets of rats from each diet group (5–7 · sex− 1 · diet− 1) were used to obtain food intake data for individual rats by using the Oxymax Indirect Calorimetry System (Columbus Instruments). Rats were monitored over a 3-d period. Randomly chosen subsets of rats at age PND 66 were used for determination of body composition (5 rats · sex− 1 · diet− 1) by DXA by using a Hologic QDR 4500A instrument. Data from whole body scans were used for analysis of global total mass (sum of fat + lean + bone mineral content), global lean (sum of all muscle and soft organ tissues), global bone mineral content (sum of all skeletal tissues), and global fat (sum of all fat tissues).
Serum biochemical analyses.
Assays were carried out with the use of an Analette Analyzer (Precision Systems, Inc). Serum (300 μL) from PND 50 rats was measured for albumin, HDL cholesterol, total cholesterol, non-HDL cholesterol, TGs, glucose, creatinine, and uric acid. Serum (150 μL) was transferred to a separate vial, and HDL cholesterol reagent was added to precipitate the non-HDL cholesterol including LDL cholesterol, VLDL, and IDL. The resultant mixture was vortexed for 30 s and then centrifuged at 11,292 × g for 5 min. The supernatant was used for HDL cholesterol assay. The non-HDL cholesterol concentration was calculated as follows: non-HDL cholesterol = total cholesterol − HDL cholesterol.
Measurement of leptin and insulin in PND–50 rat serum.
Serum leptin and insulin concentrations were determined by use of rat leptin ELISA and rat/mouse insulin ELISA kits (Linco Research, Inc.), respectively, following the manufacturers’ protocols.
Serum hydrophilic and lipophilic antioxidant capacity.
Extraction of PND-50 rat serum followed previously published procedures (24). In brief, 100 μL of serum, 100 μL water, 200 μL ethanol, and 400 μL hexane were mixed and then centrifuged at 22,000 × g for 5 min. The hexane layer was removed to another vial. An additional 400 μL hexane was added to the original vial for re-extraction, and the hexane extracts were then combined and dried under nitrogen. The dried hexane extract was used in the lipophilic oxygen radical absorbance capacity (ORAC) test. The remaining aqueous phase was depleted of trace amounts of hexane (under nitrogen) and was then used for the hydrophilic ORAC test.
Extraction of SM freeze-dried powder was performed on an ASE 200 Accelerated Solvent Extractor (Dionex Corporation) as previously described (25). SM powder (0.5 g) was extracted by hexane:dichloromethane (1:1) followed by acetone:water:acetic acid (AWA; 70:29.5:0.5). Extracts from the hexane:dichloromethane extraction were dried under nitrogen in a 30°C water bath, and the residues were reconstituted in 10 mL of acetone. This solution was used to measure lipophilic ORAC. Extracts from the AWA extraction were diluted with AWA to 25 mL total volume. The resultant solution was used to measure the hydrophilic ORAC.
Lipophilic and hydrophilic ORAC analyses of sera and freeze-dried SM were carried out on a FLUOstar Galaxy plate reader (BMG Labtechnologies) following methodologies previously established by our group (26). The results were expressed as μM Trolox equivalent (TE)/L for sera and μM TE/g for dried SM.
Statistical analyses.
Linear mixed regression was used to model body weight as a function of age. The model included subject-specific random intercepts and random slopes and used restricted cubic-splines to better accommodate the time relation. Fitted estimates (marginal effects) and corresponding SEs were used to perform Bonferroni-adjusted comparisons at specific time points (27). Continuous outcomes (i.e., food consumption, body composition measurements, serum insulin, serum leptin, serum total antioxidant capacity, and select serum biochemical) are summarized as means ± SEs and were compared across diet groups within sex by using Kruskal-Wallis nonparametric ANOVA. Significant omnibus tests were followed by Bonferroni-adjusted Dunn post hoc tests to identify groups that differed significantly. Nonparametric 2-factor ANOVAs based on ranks were used to test sex-by-diet interactions. Cuzick nonparametric test for trend for ordered groups was used to test for dose response (28). Statistical significance was set at P < 0.05.
Results
The AIN-93G–based diets contained similar amounts of nutrients and calories with the major difference among diets being the amount of added SM powder [0 (control), 1%, 4%, and 10% wt:wt; Supplemental Table 1]. Diets were introduced to pregnant Sprague-Dawley rats on gestation day 4 and were fed throughout gestation and lactation; offspring were weaned to the same diets that their dams were consuming and were then fed that diet through to PND 126 (mature adult stage). This paradigm modeled dietary exposure from gestation through to adulthood. We evaluated diet effects during the midgrowth phase.
Effects of SM on postnatal body weight accretion and body composition
During pregnancy and lactation, the body weights of the dams fed SM-supplemented diets were indistinguishable from those fed the control diet (data not shown). Postnatal growth curves are shown in Supplemental Figure 1. These body weight data were statistically analyzed as a function of age by using a linear mixed model. At PND 126 (early adulthood), body weights were not different with respect to diet within either sex (P > 0.05). However, the 4% and 10% SM diets elicited lower body weights for male rats from PND 37 through to PND 100 (rapid growth phase), when compared with the control and 1% SM diets (P < 0.05). A similar pattern was noted for female rats but only between PND 30 and PND 47 (P < 0.05). Food consumption, measured for a randomly chosen subset of rats at age PND 65, was unaffected by diet composition within each sex (Supplemental Table 2).
A subset of rats at age PND 66 from each group was subjected to DXA. Global total mass for 4% and 10% SM-fed male rats was lower than for the control and 1% SM-fed male rats (Table 1). Global lean tissue content for the 4% SM-fed male rats also was lower than for the 1% and control male groups (Table 1). For the age-matched females, there were no differences in either global total mass or global lean tissue content (Table 1). A nonparametric test for trend indicated a significant (P < 0.05) dose-responsive lowering by SM of global total mass and global lean tissue in the males. Global bone mineral content and global percentage of fat tissue content did not vary as a function of diet within either sex at PND 66 (data not shown).
TABLE 1
Total and lean tissue weights on postnatal day 66 in male and female rats fed 0–10% SM diets1
| Nonparametric ANOVA P | ||||||||
| Control | 1% SM | 4% SM | 10% SM | Kruskal-Wallis P | Diet | Sex | Interaction | |
| Total tissue weight, g | 0.0035 | <0.0001 | 0.31 | |||||
| Males | 347 ± 4.8a | 344 ± 7.8a,b | 322 ± 5.0b | 321 ± 2.1b | 0.005 | |||
| Females | 230 ± 9.6a | 234 ± 7.4a | 221 ± 3.9a | 224 ± 4.0a | 0.58 | |||
| Lean tissue weight, g | 0.0005 | <0.0001 | 0.33 | |||||
| Males | 295 ± 5.1a | 298 ± 5.9a | 266 ± 3.5b | 274 ± 3.4a,b | 0.003 | |||
| Females | 196 ± 7.8a | 198 ± 5.0a | 186 ± 2.6a | 191 ± 3.3a | 0.33 | |||
Effects of dietary SM on serum parameters
We previously reported (18) effects of SM consumption on the concentrations of polar lipid constituents in male rat serum. The present study evaluated the hypolipidemic and hypoglycemic capacities of dietary SM. The insoluble dietary fiber, α-cellulose, can reduce intestinal transit time and possesses some bioactivity (29, 30). In this regard, the final total crude fiber (α-cellulose plus SM fiber) of SM and control diets were equalized (Supplemental Table 1). Serum cholesterol was suppressed by the 4% and 10% SM diets in females and by the 10% SM diet in males at PND 50 (time point representing the young-adult growth phase) (Table 2). A nonparametric test for trend indicated a significant (P < 0.01) dose-responsive suppression of serum total cholesterol in both males and females by added SM. Serum HDL cholesterol closely followed the trends for serum total cholesterol (Table 2); again, a significant (P < 0.05) dose-responsive suppression by SM of serum HDL cholesterol in both males and females was noted. Serum non-HDL cholesterol also exhibited an SM dose-responsive suppression in both sexes (P < 0.05). The serum TG content was suppressed by 55% by the 10% SM diet for both males and females (Table 2); moreover, we noted a significant (P < 0.01) SM dose-responsive suppression of serum TG in both males and females. Serum creatinine and uric acid concentrations were unaffected by diet within each sex (Table 2). Serum albumin concentrations were lowered by the 10% SM diet but only in the males (Table 2). Serum glucose concentrations were lower in male rats fed 10% SM than in the other diet groups (Table 2). We observed a significant (P < 0.01) SM dose-responsive suppression of serum glucose in male rats. In females, diet had either no effect (4% and 10% SM) or an inductive effect (1% SM) on serum glucose concentrations (Table 2).
TABLE 2
Serum metabolites on postnatal day 50 in male and female rats fed 0–10% SM diets1
| Nonparametric ANOVA P | ||||||||
| Control | 1% SM | 4% SM | 10% SM | Kruskal-Wallis P | Diet | Sex | Interaction | |
| Albumin, g/dL | 0.019 | <0.001 | 0.047 | |||||
| Males | 3.25 ± 0.03a | 3.07 ± 0.01a,b | 3.04 ± 0.05a,b | 3.03 ± 0.01b | 0.046 | |||
| Females | 3.48 ± 0.02a | 3.39 ± 0.03a | 3.46 ± 0.05a | 3.47 ± 0.00a | 0.17 | |||
| HDL cholesterol, mg/dL | <0.001 | <0.001 | 0.037 | |||||
| Males | 54.0 ± 6.01a | 38.0 ± 2.00a,b | 36.7 ± 0.67a,b | 26.7 ± 2.91b | 0.024 | |||
| Females | 58.7 ± 2.40a | 50.0 ± 1.16a,b | 43.3 ± 0.67b | 46.0 ± 1.63a,b | 0.023 | |||
| Total cholesterol, mg/dL | <0.001 | <0.001 | 0.013 | |||||
| Males | 95.0 ± 1.53a | 72.3 ± 0.33a,b | 66.7 ± 1.20a,b | 60.7 ± 1.20b | 0.015 | |||
| Females | 104 ± 2.33a | 94.3 ± 1.46a,b | 83.0 ± 1.52b | 82.3 ± 1.20b | 0.025 | |||
| Non-HDL cholesterol, mg/dL | <0.001 | <0.001 | 0.020 | |||||
| Males | 41.0 ± 1.53a | 34.3 ± 0.33a,b | 30.0 ± 1.20b | 34.0 ± 1.20a,b | 0.024 | |||
| Females | 45.7 ± 2.33a | 44.3 ± 1.46a,b | 39.7 ± 1.52a,b | 36.3 ± 1.20b | 0.041 | |||
| Creatinine, mg/dL | 0.034 | 0.57 | 0.019 | |||||
| Males | 0.37 ± 0.01a | 0.30 ± 0.00a | 0.30 ± 0.00a | 0.30 ± 0.01a | 0.052 | |||
| Females | 0.32 ± 0.02a | 0.39 ± 0.03a | 0.31 ± 0.01a | 0.29 ± 0.01a | 0.07 | |||
| Glucose, mg/dL | 0.001 | 0.0013 | 0.001 | |||||
| Males | 146 ± 1.73a | 142 ± 1.46a,b | 132 ± 3.46a,b | 124 ± 3.18b | 0.023 | |||
| Females | 140 ± 1.76a | 148 ± 0.81b | 144 ± 0.67a,b | 140 ± 2.85a,b | 0.033 | |||
| TGs, mg/dL | <0.001 | <0.001 | 0.018 | |||||
| Males | 122 ± 3.18a | 93.0 ± 0.58a,b | 66.0 ± 1.16a,b | 54.3 ± 0.89b | 0.016 | |||
| Females | 97.0 ± 0.89a | 76.3 ± 2.33a,b | 50.7 ± 0.88a,b | 43.3 ± 1.20b | 0.016 | |||
| Uric acid, mg/dL | 0.24 | 0.93 | 0.33 | |||||
| Males | 0.38 ± 0.06a | 0.66 ± 0.29a | 1.75 ± 0.51a | 1.65 ± 0.36a | 0.20 | |||
| Females | 1.48 ± 0.48a | 1.34 ± 0.29a | 1.03 ± 0.26a | 1.66 ± 0.10a | 0.40 | |||
Effects of SM on serum insulin and leptin concentrations
Serum insulin concentrations (Table 3) exhibited an SM dose-responsive suppression in female (P < 0.01) but not male (P = 0.35) rats. Serum leptin concentrations for female rats of the control group were almost twice as high as those for control male rats (Table 3). Serum leptin concentrations in female rats fed SM diets generally were lower than in those fed the control diet, with the 10% SM diet being significantly lower than control (Table 3). Serum leptin concentrations exhibited an SM dose-responsive suppression in female (P < 0.01) but not male (P = 0.24) rats.
TABLE 3
Serum insulin and leptin concentrations on postnatal day 50 in male and female rats fed 0–10% SM diets1
| Nonparametric ANOVA P | ||||||||
| Control | 1% SM | 4% SM | 10% SM | Kruskal-Wallis P | Diet | Sex | Interaction | |
| Insulin, ng/mL | 0.0002 | 0.0002 | 0.0001 | |||||
| Males | 1.78 ± 0.05a,b | 1.75 ± 0.11a,b | 1.34 ± 0.03a | 4.21 ± 0.37b | 0.025 | |||
| Females | 1.76 ± 0.28a | 1.71 ± 0.09a,b | 0.89 ± 0.08a,b | 0.82 ± 0.07b | 0.034 | |||
| Leptin, ng/mL | <0.0001 | 0.0001 | 0.0050 | |||||
| Males | 1.52 ± 0.05a | 1.05 ± 0.07a | 1.08 ± 0.06a | 1.26 ± 0.13a | 0.072 | |||
| Females | 3.05 ± 0.15a | 1.55 ± 0.04a,b | 1.44 ± 0.03a,b | 1.16 ± 0.03b | 0.019 | |||
Hydrophilic and lipophilic antioxidant capacity of serum
The lipophilic and hydrophilic ORAC values of SM powder were 9.3 ± 0.3 μM TE/g and 21.3 ± 0.6 μM TE/g, respectively. Serum hydrophilic ORAC was significantly lowered by 22% by the 10% SM diet (compared with the control diet) in male but not female rats (Table 4). Serum lipophilic ORAC was lowered by the 4% and 10% SM diets in male (65% and 86%, respectively) and female (52% and 51%, respectively) rats (Table 4). Reduction of serum lipophilic ORAC was highly dietary dose-dependent (trend test P < 0.0001 and P = 0.0001 for females and males, respectively).
TABLE 4
Serum total antioxidant capacity on postnatal day 50 in male and female rats fed 0–10% SM diets1
| Non-parametric ANOVA P | ||||||||
| Control | 1% SM | 4% SM | 10% SM | Kruskal-Wallis P | Diet | Sex | Interaction | |
| Hydrophilic ORAC, μM TE | 0.039 | 0.0000 | 0.43 | |||||
| Males | 1620 ± 71.5a | 1480 ± 175a,b | 1350 ± 78.4a,b | 1270 ± 36.1b | 0.08 | |||
| Females | 1740 ± 46.8a | 1770 ± 53.7a | 1740 ± 48.1a | 1610 ± 93.5a | 0.41 | |||
| Lipophilic ORAC, μM TE | 0.0000 | 0.76 | 0.029 | |||||
| Males | 949 ± 49.3a | 423 ± 28.6a,b | 333 ± 72.0b | 133 ± 22.7b | 0.0010 | |||
| Females | 624 ± 67.8a | 430 ± 15.1a,b | 301 ± 42.0b,c | 304 ± 20.0c | 0.0001 | |||
Discussion
We report changes in the serum concentrations of several metabolic biomarkers in male and female rats consuming SM-supplemented diets. Importantly, some effects were found to be sex-specific whereas other effects exhibited dose-dependence. In agreement with earlier work (which focused mainly on male rats or mice), total cholesterol, non-HDL cholesterol, and TGs were generally found at lower concentrations in sera of rats of both sexes fed SM. Our results also demonstrated the dose-dependent effects of SM-supplemented diets in both sexes. Because greater concentrations of total cholesterol and non-HDL cholesterol, especially in association with high TG amounts, are linked to an increased risk of heart disease, results provide further support for the potential human cardiovascular health benefits of regular SM intake. SM has been reported to exhibit beneficial effects on cholesterol metabolism in rats (13, 14), and the mycochemical eritadenine in the mushroom fruiting body has been reported to be hypocholesterolemic (15, 16). It is likely that the effects of SM on the serum cholesterol shown here are due partly to eritadenine. Our findings that SM-supplemented diets also decreased the concentrations of HDL cholesterol, which is considered to be good cholesterol, may be a function of other bioactive components in SMs (18). The dose-dependent reduction in serum TGs with SM consumption is striking and has implications for the utility of SMs in cardiovascular disease prevention. The molecular mechanisms responsible for the noted effects of SMs are undoubtedly complex and likely involve the participation of multiple (e.g., liver, small intestine, biliary tract) tissues.
Leptin is produced and secreted predominantly by white adipose tissue, and serum concentrations of leptin positively correlate with body fat mass (22, 31–33). In the present study consumption of SM-supplemented diets reduced serum leptin concentrations in female rats. The latter predicted decreased adiposity (adipocyte number and/or cell size); however, this was not confirmed by DXA. The lack of dose-dependent changes in leptin concentrations for male rats fed 4% and 10% SM diets despite reduced body weights relative to control groups was unexpected. Interestingly, the reduction in body weights was noted only for lean body mass and not fat mass. The lower amounts of serum insulin with no accompanying differences in serum glucose concentrations for female rats fed 10% SM relative to those of the control females contrasted with the higher concentrations of serum insulin and lower concentrations of serum glucose in the 10% SM-fed males, relative to the control males These collective data suggest that dietary SM may increase tissue sensitivity to leptin and are consistent with the lower TG concentrations in serum of rats fed SM diets and with the role of leptin in preventing TG formation from free FAs and by increasing free FA oxidation (34).
Many studies have shown that free radicals and nonradical reactive oxygen species and reactive nitrogen species play contributory roles in pathogenesis of many chronic diseases. Consumption of diets high in fruits and vegetables can increase blood antioxidant capacity and enhance antioxidant defenses (35, 36). Further, antioxidant capacity has been reported to associate with hypoglycemic and hypolipidemic effects in some tissue contexts, and these may play a synergistic role in physiology and pathophysiology (23, 37). Our reported lipophilic ORAC value (9.3 ± 0.3 μM TE/g) and hydrophilic ORAC value (21.3 ± 0.6 μM TE/g) for SM freeze-dried powder are relatively high and low, respectively, in comparison with most of the other fruits and vegetables measured by our group following the same methodology (26). Nevertheless, the dramatic effects of SM-supplemented diets in reducing serum lipophilic rather than hydrophilic antioxidant capacities, irrespective of sex is consistent with a lower lipophilic pro-oxidative state associated with improved lipid profile.
In summary, SM consumption decreased serum concentrations of lipidemia-related factors in rats, irrespective of sex. The similar reductions in lipophilic antioxidant capacities in sera of both sexes with dietary SM intake may reflect a lower pro-oxidative state and, hence, improved metabolic profile and are consistent with accompanying hypolipidemic states. Our findings highlight endocrine mechanisms (decreased insulin, leptin, and oxidative stress) potentially underlying the cancer-inhibitory actions of SM and that may be dietary dose-dependent (38). Future studies should aim to dissect the dietary components present in SMs and to define their complex endocrine signaling toward improving the clinical management of metabolic disorders and endocrine-related cancers.
Acknowledgments
NF designed the study; SY, XW, and MF conducted the experiments and analyzed the data; MAC performed the statistical analyses; RCMS, FAS, and NF interpreted the data and wrote the paper; and NF had primary responsibility for the final content. All authors read and approved the final manuscript.