Identification of Dihydromyricetin and Metabolites in Serum and Brain Associated with Acute Anti-Ethanol Intoxicating Effects in Mice.

Dihydromyricetin is a natural bioactive flavonoid with unique GABAA receptor activity with a putative mechanism of action to reduce the intoxication effects of ethanol. Although dihydromyricetin's poor oral bioavailability limits clinical utility, the promise of this mechanism for the treatment of alcohol use disorder warrants further investigation into its specificity and druggable potential. These experiments investigated the bioavailability of dihydromyricetin in the brain and serum associated with acute anti-intoxicating effects in C57BL/6J mice. Dihydromyricetin (50 mg/kg IP) administered 0 or 15-min prior to ethanol (PO 5 g/kg) significantly reduced ethanol-induced loss of righting reflex. Total serum exposures (AUC0→24) of dihydromyricetin (PO 50 mg/kg) via oral (PO) administration were determined to be 2.5 µM × h (male) and 0.7 µM × h (female), while intraperitoneal (IP) administration led to 23.8-fold and 7.2- increases in AUC0→24 in male and female mice, respectively. Electrophysiology studies in α5ß3γ2 GABAA receptors expressed in Xenopus oocytes suggest dihydromyricetin (10 µM) potentiates GABAergic activity (+43.2%), and the metabolite 4-O-methyl-dihydromyricetin (10 µM) negatively modulates GABAergic activity (-12.6%). Our results indicate that administration route and sex significantly impact DHM bioavailability in mice, which is limited by poor absorption and rapid clearance. This correlates with the observed short duration of DHM's anti-intoxicating properties and highlights the need for further investigation into mechanism of DHM's potential anti-intoxicating properties.

Metabolic gene signature in white adipose tissue of oral doses raspberry ketone [4-(4-hydroxyphenyl)-2-butanone] that prevent diet-induced weight gain and induce loss of righting reflex.

Raspberry ketone (RK; [4-(4-hydroxyphenyl)-2-butanone]) is a synthetic flavoring agent and dietary supplement for weight control. This study investigated the metabolic signature of oral doses of RK that prevent weight gain or promote loss of righting reflex (LORR) in C57Bl/6J mice. Daily RK 200 mg/kg prevented high-fat diet (HFD; 45% Kcal fat) fed weight gain (∼8% reduction) over 35 days. RNA-seq of inguinal white adipose tissue (WAT) performed in males revealed 12 differentially expressed genes. Apelin (Apln) and potassium voltage-gated channel subfamily C member (Kcnc3) expression were elevated with HFD and normalized with RK dosing, which was confirmed by qPCR. Acute RK 640 mg/kg produced a LORR with a <5 min onset with a >30 min duration. Acute RK 200 mg/kg increased gene expression of Apln, Kcnc3, and nuclear factor erythroid 2-related factor 2 (Nrf2), but reduced acetyl-COA carboxylase (Acc1) and NAD(P)H quinone dehydrogenase 1 (Nqo1) in inguinal WAT. Acute RK 640 mg/kg elevated interleukin 6 (Il 6) and heme oxygenase 1 (Hmox1) expression, but reduced Nrf2 in inguinal and epididymal WAT. Our findings suggest that RK has a dose-dependent metabolic signature in WAT associated with either weight control or LORR.

Grape Polyphenols May Prevent High-Fat Diet-Induced Dampening of the Hypothalamic-Pituitary-Adrenal Axis in Male Mice.

Context : Chronic high-fat diet (HFD) consumption causes obesity associated with retention of bile acids (BAs) that suppress important regulatory axes, such as the hypothalamic-pituitary-adrenal axis (HPAA). HFD impairs nutrient sensing and energy balance due to a dampening of the HPAA and reduced production and peripheral metabolism of corticosterone (CORT). Objective: We assessed whether proanthocyanidin-rich grape polyphenol (GP) extract can prevent HFD-induced energy imbalance and HPAA dysregulation. Methods: Male C57BL6/J mice were fed HFD or HFD supplemented with 0.5% w/w GPs (HFD-GP) for 17 weeks. Results: GP supplementation reduced body weight gain and liver fat while increasing circadian rhythms of energy expenditure and HPAA-regulating hormones, CORT, leptin, and PYY. GP-induced improvements were accompanied by reduced mRNA levels of Il6, Il1b, and Tnfa in ileal or hepatic tissues and lower cecal abundance of Firmicutes, including known BA metabolizers. GP-supplemented mice had lower concentrations of circulating BAs, including hydrophobic and HPAA-inhibiting BAs, but higher cecal levels of taurine-conjugated BAs antagonistic to farnesoid X receptor (FXR). Compared with HFD-fed mice, GP-supplemented mice had increased mRNA levels of hepatic Cyp7a1 and Cyp27a1, suggesting reduced FXR activation and more BA synthesis. GP-supplemented mice also had reduced hepatic Abcc3 and ileal Ibabp and Ostß, indicative of less BA transfer into enterocytes and circulation. Relative to HFD-fed mice, CORT and BA metabolizing enzymes (Akr1d1 and Srd5a1) were increased, and Hsd11b1 was decreased in GP supplemented mice. Conclusion: GPs may attenuate HFD-induced weight gain by improving hormonal control of the HPAA and inducing a BA profile with less cytotoxicity and HPAA inhibition, but greater FXR antagonism.

Weight-gain propensity and morphine withdrawal alters locomotor behavior and regional norepinephrine-related gene expression in male and female mice.

Interactions between obesity and opioid use are poorly understood. The objective of this study was to determine whether phenotypic differences in diet-induced weight gain altered morphine withdrawal responses. Male and female C57BL/6J mice were characterized as obese prone (OP) or obese resistant (OR) based on median split in body weights following exposure to high-fat diet (45% fat). After classification into OP or OR, all mice were fed a low-fat diet (10% fat) for the remainder of the study (≥5 weeks) to remain weight matched. Mice were treated with a 7-day escalating dosing scheme of morphine (20-100 mg/kg; IP) or saline and underwent a spontaneous withdrawal. Morphine-induced weight loss was restored by withdrawal day 7. On withdrawal day 8, male OP demonstrated less total time mobile in the open field test (OFT). In females, OR-morphine traveled less distance than OR-saline, and OR-morphine spent less time mobile compared with all other groups in the OFT. Female OP also increased time spent in the center of the apparatus, regardless of treatment. On withdrawal day 8, relative gene expression was measured by qPCR. For males, expression of dopamine beta-hydroxylase (dbh), alpha-adrenergic receptor 2 a (adra2a), and orexin receptor 1 (orx1) were increased in the locus coeruleus (LC) region of OP mice, regardless of treatment. In comparison, in females, dbh and adra2a were decreased in the LC region of OP mice, regardless of treatment. Also, in the LC region of females, OP-morphine had lower expression of alpha-adrenergic receptor 1 a (adra1a) than OR-morphine and OP-saline. In the hypothalamic paraventricular nucleus (PVN) of females, adra2a was increased in OP-morphine compared with OP-saline and OR-morphine. Our findings suggest morphine withdrawal responses and regional expression of noradrenergic-related genes are differentially influenced by weight gain propensity.

Development and validation of a micro-QuEChERS method with high-throughput enhanced matrix removal followed with UHPLC-QqQ-MS/MS for analysis of raspberry ketone-related phenolic compounds in adipose tissues.

Raspberry ketone (RK) is a major flavor compound in red raspberries, and it has been marketed as a popular weight-loss dietary supplement with high potential in accumulating in fatty tissues. However, challenges in extracting and characterizing RK and its associated phenolic compounds in fatty tissues persist due to the complex matrix effect. In this work, we reported a high-throughput sample preparation method for RK and 25 related phenolic compounds in white adipose tissues using an improved micro-scale QuEChERS (quick, efficient, cheap, easy, rugged and safe) approach with enhanced matrix removal (EMR)-lipid cleanup in 96-well plates, followed by UHPLC-QqQ-MS/MS analysis. The absolute recovery was 73-105% at the extraction step, and achieved 71-96% at the EMR cleanup step. The EMR cleanup removed around 66% of total lipids in the acetonitrile extract as profiled by UHPLC-QTOF-MS/MS. The innovative introduction of a reversed-phase C18 sorbent into the extract significantly improved the analytes' recovery during SpeedVac drying. The final accuracy achieved 80-120% for most analytes. Overall, this newly developed and validated method could serve as a powerful tool for analyzing RK and related phenolic compounds in fatty tissues.

Raspberry Ketone [4-(4-Hydroxyphenyl)-2-Butanone] Differentially Effects Meal Patterns and Cardiovascular Parameters in Mice.

Raspberry ketone (RK; [4-(4-hydroxyphenyl)-2-butanone]) is a popular nutraceutical used for weight management and appetite control. We sought to determine the physiological benefits of RK on the meal patterns and cardiovascular changes associated with an obesogenic diet. In addition, we explored whether the physiological benefits of RK promoted anxiety-related behaviors. Male and female C57BL/6J mice were administered a daily oral gavage of RK 200 mg/kg, RK 400 mg/kg, or vehicle for 14 days. Commencing with dosing, mice were placed on a high-fat diet (45% fat) or low-fat diet (10% fat). Our results indicated that RK 200 mg/kg had a differential influence on meal patterns in males and females. In contrast, RK 400 mg/kg reduced body weight gain, open-field total distance travelled, hemodynamic measures (i.e., reduced systolic blood pressure (BP), diastolic BP and mean BP), and increased nocturnal satiety ratios in males and females. In addition, RK 400 mg/kg increased neural activation in the nucleus of the solitary tract, compared with vehicle. RK actions were not influenced by diet, nor resulted in an anxiety-like phenotype. Our findings suggest that RK has dose-differential feeding and cardiovascular actions, which needs consideration as it is used as a nutraceutical for weight control for obesity.

UHPLC-QqQ-MS/MS method development and validation with statistical analysis: Determination of raspberry ketone metabolites in mice plasma and brain.

Raspberry ketone (RK) (4-(4-hydroxyphenyl)-2-butanone) is the major compound responsible for the characteristic aroma of red raspberries, and has long been used commercially as a flavoring agent and recently as a weight loss supplement. A targeted UHPLC-QqQ-MS/MS method was developed and validated for analysis of RK and 25 associated metabolites in mouse plasma and brain. Dispersion and projection analysis and central composite design were used for method optimization. Random effect analysis of variance was applied for validation inference and variation partition. Within this framework, repeatability, a broader sense of precision, was calculated as fraction of accuracy variance, reflecting instrumental imprecision, compound degradation and carry-over effects. Multivariate correlation analysis and principle component analysis were conducted, revealing underlying association among the manifold of method traits. R programming was engaged in streamlined statistical analysis and data visualization. Two particular phenomena, the analytes' background existence in the enzyme solution used for phase II metabolites deconjugation, and the noted lability of analytes in pure solvent at 4 ℃ vs. elevated stability in biomatrices, were found critical to method development and validation. The approach for the method development and validation provided a foundation for experiments that examine RK metabolism and bioavailability.

Acute feeding suppression and toxicity of raspberry ketone [4-(4-hydroxyphenyl)-2-butanone] in mice.

Raspberry ketone (RK; [4-(4-hydroxyphenyl)-2-butanone]) is used by the food and cosmetic industry as a flavoring agent. RK is also marketed as a dietary supplement for weight maintenance and appetite control. The purpose of the study was to characterize the acute feeding suppression with RK (64-640 mg/kg) by oral gavage in male and female C57BL/6J mice. Cumulative 24 h food intake was reduced at 200 mg/kg (24% feeding suppression) in males and reliably reduced at 640 mg/kg (49-77% feeding suppression). Feeding suppression was not associated with pica behavior over the range of doses or conditioned taste aversion. In a separate experiment, a single oral gavage of RK (640 mg/kg) resulted in approximate 43% mortality rate (6 out 14 male mice) within 2 days. Atrophy of white adipose tissue, splenic abnormalities, and thymus involution were noted after 2-4 days after oral gavage RK. Total white blood cell count, lymphocytes, monocytes, eosinophils were significantly lower, while mean red blood cells, hemoglobin, and hematocrit were significantly higher with RK treatment. Our findings indicated a dose-dependent feeding suppression with acute RK, but doses that reliable suppress food intake are associated with pathological changes.

Influence of Diet-Induced Obesity on the Bioavailability and Metabolism of Raspberry Ketone (4-(4-Hydroxyphenyl)-2-Butanone) in Mice.

OBJECTIVES: Raspberry ketone (RK) is the primary aroma compound in red raspberries and a dietary supplement for weight loss. This work aims to 1) compare RK bioavailability in male versus female, normal-weight versus obese mice; 2) characterize RK metabolic pathways. METHODS: Study 1: C57BL/6J male and female mice fed a low-fat diet (LFD; 10% fat) receive a single oral gavage dose of RK (200 mg kg-1 ). Blood, brain, and white adipose tissue (WAT) are collected over 12 h. Study 2: Male mice are fed a LFD or high-fat diet (45% fat) for 8 weeks before RK dosing. Samples collected are analyzed by UPLC-MS/MS for RK and its metabolites. RESULTS: RK is rapidly absorbed (Tmax  ≈ 15 min), and bioconverted into diverse metabolites in mice. Total bioavailability (AUC0-12 h ) is slightly lower in females than males (566 vs 675 nmol mL-1 min-1 ). Total bioavailability in obese mice is almost doubled that of control mice (1197 vs 679 nmol mL-1 min-1 ), while peaking times and elimination half-lives are delayed. Higher levels of RK and major metabolites are found in WAT of the obese than normal-weight animals. CONCLUSIONS: RK is highly bioavailable, rapidly metabolized, and exhibits significantly different pharmacokinetic behaviors between obese and control mice. Lipid-rich tissues, especially WAT, can be a direct target of RK.

A highly sensitive ultra-high performance liquid chromatography/tandem mass spectrometry method with in-source fragmentation for rapid quantification of raspberry ketone.

Raspberry ketone (RK) is the characteristic aromatic compound in raspberry (Rubus idaeus L.) with wide applications as food additive and anti-obesity agent. However, quantification of RK has presented difficulties in MS detection and reliable LC-MS method for RK analysis in literature is in limit to date. In order to facilitate quality control of raspberry derived products and RK metabolomics study, this study aimed to develop a validated and sensitive UHPLC-MS/MS method. Strong in-source fragmentation was noted and the fragmental ion of 107 m/z produced was selected as the precursor ion for MRM detection, and as such the electrospray ionization performance was optimized by fractional factorial design to accommodate such ion-source dissociation behavior as well as its moderate volatility. A pathway involving the formation of quinone-like structure with strong conjugation was proposed to explain the intense in-source fragmentation. The MRM transition was optimized with product ion of 77 m/z selected as the quantifier ion. The method featured low limit of quantification of ∼2 ng/mL and allowed for rapid detection of RK in fresh raspberries following direct sample preparation. RK contents were found to be higher from locally grown and harvested farm sources compared to commercial products shipped into the state, and higher in those at late-stage compared with early-stage maturity. No correlations in RK content between organic and non-organic labels were noted.

Phenolic-enriched raspberry fruit extract (Rubus idaeus) resulted in lower weight gain, increased ambulatory activity, and elevated hepatic lipoprotein lipase and heme oxygenase-1 expression in male mice fed a high-fat diet.

Red raspberries (Rubus idaeus) contain numerous phenolic compounds with purported health benefits. Raspberry ketone (4-(4-hydroxyphenyl)-2-butanone) is a primary raspberry flavor phenolic found in raspberries and is designated as a synthetic flavoring agent by the Food and Drug Administration. Synthetic raspberry ketone has been demonstrated to result in weight loss in rodents. We tested whether phenolic-enriched raspberry extracts, compared with raspberry ketone, would be more resilient to the metabolic alterations caused by an obesogenic diet. Male C57BL/6J mice (8 weeks old) received a daily oral dose of vehicle (VEH; 50% propylene glycol, 40% water, and 10% dimethyl sulfoxide), raspberry extract low (REL; 0.2 g/kg), raspberry extract high (REH; 2 g/kg), or raspberry ketone (RK; 0.2 g/kg). Coincident with daily dosing, mice were placed on a high-fat diet (45% fat). After 4 weeks, REH and RK reduced body weight gain (approximately 5%-9%) and white adipose mass (approximately 20%) compared with VEH. Hepatic gene expression of heme oxygenase-1 and lipoprotein lipase was upregulated in REH compared with VEH. Indirect calorimetry indicated that respiratory exchange ratio (CO2 production to O2 consumption) was lower, suggesting increased fat oxidation with all treatments. REH treatment increased total ambulatory behavior. Energy expenditure/lean mass was higher in REH compared with REL treatment. There were no treatment differences in cumulative intake, meal patterns, or hypothalamic feed-related gene expression. Our results suggest that raspberry ketone and a phenolic-enriched raspberry extract both have the capacity to prevent weight gain but differ in the preventative mechanisms for excess fat accumulation following high-fat diet exposure.