Pseudocannabinoid H4CBD enhances lipid catabolism to reduce visceral adiposity and large adipocyte size in advanced metabolic syndrome

Pseudocannabinoid H4CBD enhances lipid catabolism to reduce visceral adiposity and large adipocyte size in advanced metabolic syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pseudocannabinoid H4CBD enhances lipid catabolism to reduce visceral adiposity and large adipocyte size in advanced metabolic syndrome Jessica N. Wilson, Dora A. Mendez, Marisol Hernandez Garcia, Nikolay Shevchenko, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7274449/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Metabolic Syndrome (MetS) is a precursor for cardiovascular disease (CVD) and type 2 diabetes (T2D) and has a prevalence of 55% among people ≥ 60 years of age in the US. Cannabidiol (CBD) use has grown more popular in the last two decades, particularly amongst adults > 55 years of age. Synthetic analogues of CBD have generated great interest because they can offer safer, chemically pure products with no abuse potential and no regulatory barriers. However, the effects of chronic cannabinoid use during age-associated cardiometabolic dysfunction have not been examined. Methods To assess the effects of H4CBD, a synthetic analogue of CBD, on advanced MetS, a cohort of 41-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were administered 200 mg H4CBD/kg by oral gavage for 4 weeks. Animals were fed ad libitum and monitored alongside vehicle-treated OLETF and lean, strain-control Long-Evans Tokushima Otsuka (LETO) rats. Results Body mass (BM) was reduced 22% in H4CBD group compared to OLETF and was similar to LETO levels within the first week but did not reverse the diabetic phenotype of the aged OLETF. H4CBD also reduced visceral fat mass (FM; 41%) and nearly ablated large adipocyte (> 100µm) abundance compared to OLETF. Plasma triglycerides were more than doubled in OLETF compared to LETO, and H4CBD normalized the levels. Urinary 3-methylhistidine (3-MH) to creatinine ratio tended to be higher (77%; p = 0.07) in H4CBD suggesting that some lean tissue was lost along with FM, which contributed to the reduction in BM. Conclusions Chronic H4CBD treatment increased lipid catabolism resulting in increased FM loss, although some lean mass loss was also observed. These results suggest that synthetic cannabinoids have potential for advanced-age obesity management during severe metabolic dysfunction, even with consideration of possible cachexic effects. 3-methylhistidine cannabinoids insulin resistance MAFLD metabolism metabolic syndrome obesity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The popularity of cannabis use in older adults (> 55 years of age) in the United States has more than doubled over the past two decades. 1 – 4 Alleviation of chronic pain is a condition for which older adults typically use medicinal cannabis. 3 – 5 A common consequence of aging is impaired substrate metabolism, which could result in the onset of metabolic conditions ranging in severity from increases in the individual risk factors for metabolic syndrome (MetS) 6 such as obesity and hypertension to frank type 2 diabetes mellitus (T2DM). 7 The prevalence of MetS is 55% among people ≥ 60 years of age 7 in the US and the average age at diagnosis of T2DM is approximately 46 years, with equal abundance across racial and ethnic groups. 8 The therapeutic benefits and risks of cannabis and cannabinoids in metabolic dysfunction have yet to be delineated, especially in older individuals. Cannabidiol (CBD) is an abundant, non-intoxicating constituent of Cannabis sativa , which is of particular interest for pharmacological investigation. The legislative ambiguity and increasing ease-of-access to unregulated cannabis constituents have prompted endeavors to synthesize analogues of cannabis component molecules of interest without use of whole-plant extract. This circumvents violating laws surrounding production or use of a DEA scheduled compound while simultaneously providing a chemically pure product free from the growing concerns of unregulated pesticide use on whole plants. Synthetically derived CBD analogues, like 1,2,8,9-tetrahydrocannabidiol (H4CBD), are inexpensive to produce and can offer essentially identical therapeutic effects to herbal CBD. For example, it has been shown that the closely related pseudocannabinoid H2CBD (8,9-dihydrocannabidiol) controls seizures in rats with the same efficacy as CBD. 9 Additionally, previous in vivo and in vitro uses of H4CBD have yielded promising benefits consistent with herbal CBD. 10 – 12 We have also recently shown that H4CBD improved glucose tolerance in a rat model of metabolic syndrome (MetS). 13 H4CBD is a synthetic analogue of CBD that differs by the saturation of the two carbon-carbon double bonds of the terpene moiety. A significant advantage of hydrogenated cannabinoids is that, while CBD itself is easily converted into THC, H4CBD cannot undergo this chemical transformation, thus eliminating any abuse potential. Similar to natural CBD, H4CBD has little affinity for the endocannabinoid receptors responsible for cannabis intoxication. 10 However, the potential benefits of H4CBD on lipid metabolism and adiposity associated with age-related metabolic dysfunction remain to be assessed. Observations of cardiovascular and/or metabolic improvements conferred by CBD or CBD-dominant cannabis products have prompted inquiry into other applications for CBD. 14 – 19 Clinical trials determined that 100 mg CBD, twice daily, is well-tolerated in patients with MetS or T2DM and led to improvements in circulating resistin and gastric inhibitory peptide, but did not reverse the T2DM phenotype. 20 A much higher dose (600 mg) of CBD acutely reduced arterial blood pressure in healthy males; 21 however, this effect was lost after a 7-day dose regimen. 22 These examples provide promising clinical evidence for the potential of cannabinoids to ameliorate metabolic disorders. There is sparse pre-clinical data on the effects of CBD in models of metabolic dysfunction. Natural (plant-extracted) CBD improved glucose tolerance or metabolism in mice with varying degrees of metabolic dysfunction including a high fat-high cholesterol diet, 23 – 25 which provides some reasonable evidence that related synthetic cannabinoids have the potential to likewise ameliorate dysregulated metabolism. Moreover, CBD administered over 4 weeks attenuated cardiac dysfunction and cardiac fibrosis in mice with induced type I diabetes. 26 The goals of this study were to assess the effect of H4CBD on MetS risk factors, primarily dyslipidemia, in the advanced MetS condition, consistent with those observed in older adults. Because of the increasing interest in cannabinoids in this population, a critical intersection between their use and advanced metabolic dysfunction exists, which warrants further investigation. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a monogenic model of diet-induced obesity accelerated by a mutation in the CCK receptor. 27 , 28 These rats have a predictable, timed progression toward non-insulin dependent diabetes mellitus (T2DM) (> 20 weeks), marked by a linear phase progression of hypertension (8–20 weeks), which closely resembles symptoms displayed in human T2DM, including visceral adiposity, dyslipidemia, and insulin resistance. 27 – 29 At > 40 weeks of age, OLETF rats suffer from severe metabolic dysfunction and therefore serve as a model of aged, severe MetS. The hypothesis tested here is that H4CBD can improve lipid metabolism during MetS. Materials and methods All animal procedures were reviewed and approved by the institutional animal care and use committees of the University of California Merced. These data complement our previous study of H4CBD on glucose metabolism using samples from the same animals. 13 Animals Male lean Long Evans Tokushima Otsuka (LETO) rats and obese Otsuka Long Evans Tokushima Fatty (OLETF) rats (Japan SLC Inc., Hamamatsu, Japan) at 14 weeks of age were assigned to the following groups (n = 8/group): (1) vehicle-dosed LETO (LETO), (2) vehicle-dosed OLETF (OLETF), and (3) H4CBD-treated (200mg/kg/day x 4 weeks) OLETF (H4CBD). Daily dosing was relaxed to every other day after the first week. Rats were maintained in a specific pathogen-free, climate-controlled facility at the University of California Merced on a 12-hour light:dark cycle (07:00–19:00 and 19:00–07:00, respectively). All animals had free access to water and were fed rat chow (Teklad Global; fat 9.0%; carbohydrate 44.9%; protein 19.0%) ad libitum . Treatment intervention was initiated at 41 weeks of age. Body Mass and Water & Food Intake Body mass was measured daily to calculate the appropriate drug and vehicle doses. Water intake, urine volume, and food consumption were also measured daily throughout the study. Drug Preparation & Administration 1,2,8,9-Tetrahydrocannabidiol (H4CBD) was synthesized as previously described. 13 Purified H4CBD (> 99%) was dissolved in sesame oil and administered by oral gavage at a dose of 200 mg/kg. This dose has been shown to be similarly effective on seizure frequency and severity in rats compared to natural CBD, 9 which gave reasonable cause for efficacy of H4CBD at the same dose, which is well below the documented toxic threshold of CBD (> 600mg/kg) in rodents. 30 Bioavailability of H4CBD in circulation has been demonstrated previously. 13 Tissue Collection After the 4-week study, animals were fasted overnight and tissues (epididymal and retroperitoneal adipose and liver) were collected the following morning as previously described. 31 – 33 Retroperitoneal fat was used to represent white adipose tissue (WAT) in the analyses. Plasma Analysis Plasma concentrations of non-esterified fatty acid (NEFA; Fujifilm Wako Diagnostics; 999-34691, 995-34791, 991-34891, and 993-35191) and triglycerides (TG; Cayman Chemical; 10010303) were measured in fasted, end of study plasma samples. All samples were analyzed in duplicate and run in a single assay with intra-assay and percent CV of < 10% for all assays. Urinalysis Urine was collected as previously described. 34 Urine was thawed on ice to measure 3-methylhistidine (3MH; Abbexa; 257295) and creatinine (Invitrogen; EIACUN). Excretion was calculated by multiplying the 24 hr urine volume by the measured concentration (U x V = V * [x]), where x = creatinine or 3MH. Lipase Activity Assay Lipase activity was assayed from retroperitoneal adipose, liver, and end of study plasma as previously performed. 35 Adipocyte Morphological Quantification Slices of retroperitoneal WAT were mounted and stained using hematoxylin and eosin (H&E) stain. Each slide was imaged at 10X using a Keyence BZ-X Series microscope at three distinct locations and scaled. 32 The images were coded and analyzed for single-blind adipocyte count and area using ImageJ software. 32 , 34 Liver Damage Assessment An aliquot of liver was homogenized and assayed for TG content (Cayman Chemical; 10010303), 4-hydroxynonenal (4HNE) accumulation (myBioSource; MBS736336), and collagen type IV (myBioSource; MBS732756) deposition by ELISA following the manufacturer's recommendation. Western Blot An aliquot of liver was used to measure proteins involved in fatty acid uptake, metabolism, and storage as previously described. 36 Densitometry values were quantified using ImageJ software (NIH) and normalized by correcting for densitometry values of representative protein bands below 37kDa stained with Ponceau S. Results are reported as expression (%) compared to LETO. Calculations & Statistics To estimate total body water (TBW), equations extrapolated from the literature were used. 37 All values are represented as mean ± standard error mean (SEM) unless otherwise indicated. Means were compared by one-way ANOVA followed by Tukey's honest significant difference or unpaired, one-tailed t-test to assess significant differences among groups. Means and regressions were considered significant at p < 0.05. Means and regressions were considered a trend between p = 0.09 − 0.051. Outliers were detected by ROUT (Q = 1.0%) and removed; however, it should be noted that this was necessary for only 6 occurrences. All statistical procedures were performed using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA). Results H4CBD reduced BM despite increased food consumption BM, relative food consumption, water consumption, and activity were measured daily to determine the effect of H4CBD on phenotypic indicators of metabolic dysfunction. H4CBD reduced BM in OLETF to LETO levels within the first week, which was maintained for the duration of the study (Fig. 1 A & B ). BM of untreated OLETF was 16% and 15% greater than LETO and H4CBD-treated OLETF, respectively, after the first week of the study (day 10–30). The reduction in BM in the H4CBD-treated OLETF was independent of food intake as relative food consumption increased 24% and 74% compared to OLETF control and LETO, respectively, between day 10 and day 30 (Fig. 1 C). Water intake was > 200% greater in OLETF compared to LETO for the duration of the study (Fig. 1 D). By day 13, water intake in H4CBD-treated OLETF was 90% and > 600% greater than OLETF control than LETO, respectively, through the end of the study (Fig. 1 D). H4CBD-treated OLETF (n = 3) tended (p < 0.10) toward increased activity that was nearly double that of OLETF control (Fig. 1 E). H4CBD promoted fat loss associated with lean tissue catabolism Urine was collected over a 24-hour period on the final day of treatment and analyzed for 3-MH, creatinine (Cr), and total protein excretion to determine if H4CBD promoted muscle wasting. Urine output and proteinuria in the H4CBD-treated group were comparable to control groups (Fig. 2 A-B). H4CBD treatment reduced creatinine excretion by 42% compared to OLETF (Fig. 2 C). 3-MH excretion is a product of amino acid breakdown and urine 3-MH to Cr ratio (3-MH/Cr) is used clinically to assess muscle wasting. 38 3-MH excretion of the H4CBD group was comparable to OLETF (Fig. 2 D). The changes in 3-MH/Cr ratios trended toward significance, with levels in OLETF 128% (p = 0.06) and 77% (p = 0.07) greater than LETO and H4CBD OLETF, respectively (Fig. 2 E). End of study BM, FM, and lean organ mass were tabulated to determine the degree of lean tissue loss in treated animals. At 45 weeks of age, OLETF BM was 20% greater than LETO, and H4CBD reduced BM by 22% compared to OLETF (Table 1 ). Percent FM was 147% greater in OLETF compared to LETO, and reduced 49% in H4CBD OLETF compared to OLETF control (Table 1 ). Table 1 Mean (± SEM) end of study measurements and calculations. Strain LETO OLETF H4CBD Body mass (BM) (g) 508 ± 10 611 ± 41 * 476 ± 18 Ϯ Lean body mass (g) 486 ± 8.9 546 ± 34 438 ± 13 Ϯ Estimated total body H 2 O (g) 325 ± 4 369 ± 17 * 312 ± 8 Ϯ Fat mass (FM) (%) 4.3 ± 0.3 11 ± 0.8 * 7.9 ± 1.1 *# Lean tissue (g) 145 ± 5 153 ± 16 102 ± 6 *,Ϯ Liver mass (LM) (g) 12 ± 0.3 18 ± 0.9 * 19 ± 0.5 * Epidydimal fat mass (g) 9 ± 0.6 12 ± 2 6 ± 1 Ϯ Retroperitoneal fat mass (g) 12 ± 0.8 54 ± 6 * 33 ± 6 *,Ϯ Plasma albumin (mg/mL) 55 ± 2.7 56 ± 2.8 54 ± 2.6 ΔBM compared to OLETF (g) 108 ± 12 ΔBM compared to OLETF (%) 20 ± 2 ΔFM compared to OLETF (g) 33 ± 4 ΔFM compared to OLETF (%) 49 ± 6 ΔLM compared to OLETF (g) 33 ± 4 ΔLM compared to OLETF (%) 49 ± 6 # p < 0.01 different from LETO, * p < 0.05 different from LETO, † p < 0.05 different from OLETF by one-way ANOVA with Tukey's HSD or unpaired on-tailed t-test Visceral fat masses (retroperitoneal and epidydimal) were dissected and quantified to determine the effect of H4CBD on abdominal adiposity. Fasted plasma lipase activity, as well as plasma NEFA and TG, were measured to assess the effect of H4CBD on parameters of lipid metabolism. Retroperitoneal fat, but not relative epidydimal fat, was 267% more abundant in OLETF than LETO ( p < 0.0001) (Table 1 ). H4CBD reduced relative retroperitoneal and epidydimal fat by 24% and 35%, respectively, compared to OLETF control (Table 1 ). Relative combined adipose was 149% greater in OLETF compared to LETO, which was reduced by 25% in H4CBD-treated OLETF (Table 1 ). H4CBD reduces large adipocyte abundance Adipocyte morphology was comparable below 100 µm among groups (Fig. 3A-D). However, H4CBD treatment reduced the frequency of large adipocytes (100–150µm) by 95% compared to OLETF (Fig. 3D). H4CBD reduced plasma triglycerides Adipose, liver, and plasma lipase activities were measured to determine the effect of H4CBD on their contributions to lipolysis. The reduction in visceral adiposity and adipocyte size suggests that H4CBD activated and enhanced lipid metabolism. Adipose lipase activity was 31% lower in OLETF compared to LETO and H4CBD tended to normalize it, increasing 22% (p = 0.094) compared to OLETF control (Fig. 4 A). Liver endothelial membrane-bound lipases are known to contribute to TG metabolism in the bloodstream. 39 – 41 However, neither cytosolic nor membrane-bound lipase activities in the liver were different among groups (Fig. 4 B & 4 C). Fasted plasma lipase activity also was not significantly different among groups; however, fasted plasma TG, the substrate of lipolysis, was 147% greater in OLETF compared to LETO, and H4CBD treatment reduced it to LETO levels (Fig. 4 E). Fasted plasma NEFA was not different among groups (Fig. 4 F). H4CBD promoted lipid metabolism via increased hepatic CD36 and FATP2 expression Hepatic lipid metabolism signaling proteins were measured to assess the impact of H4CBD on lipid shuttling. FATP5 expression was increased 109% in OLETF compared to LETO and was not affected by H4CBD treatment (Fig. 5 A). FATP2 expression was comparable between LETO and OLETF but increased by 60% in H4CBD-treated OLETF (Fig. 5 B). CD36 expression was 41% lesser in OLETF compared to LETO, and H4CBD treatment reversed CD36 expression by 48% (Fig. 5 C). Downstream signaling proteins, GPAM, DGAT1, CPT1A, ACOX1, ApoB, and PRDX6 were comparable among groups ( data not shown ). Liver triglyceride content was 24% greater in OLETF compared to LETO, and reduced by 28% in H4CBD-treated OLETF (Fig. 5 D). Collectively, the increase in fatty acid transporter expression suggests that H4CBD treatment enhanced FFA uptake to promote fatty acid oxidation. H4CBD did not contribute to liver damage Indicators of damage were measured in liver to assess the effects of H4CBD on hepatotoxicity. End of study liver 4HNE was not different among groups (Fig. 6 A). Type IV collagen deposition is an indicator of liver fibrosis, which is useful in the diagnosis of MAFLD in elderly individuals 42 and models of MAFLD like the OLETF rat. 43 Liver collagen deposition was 33% greater in OLETF compared to LETO and H4CBD treatment had no effect on collagen levels (Fig. 6 B). Discussion The prevalence of MetS has been estimated to be three times more pervasive than T2DM, which extrapolates to account for a population of over 1 billion people globally. 44 Since MetS is associated with development of CVD and T2DM, intervention strategies that affect multiple cluster conditions of MetS are of critical importance. The effects of CBD on MetS pathophysiology are not well established, although a few studies have shown beneficial 26 , 45 or null effects 46 of CBD on isolated MetS risk factors. However, these effects have not been explored in context as cluster factors. Therefore, the aim of the present study was to preclinically assess the therapeutic effect of a CBD analogue on adiposity and the associated dyslipidemia in the condition of advanced MetS, which is most frequently observed in older populations. We chose for this purpose to evaluate the effects of the non-narcotic cannabinoid, 1,2,8,9-tetrahydrocannabidiol (H4CBD) due to its synthetic accessibility in pure form, regulatory advantages, and absence of abuse liability, and thus, potential for wider global adoption than naturally-derived CBD. H4CBD reduced BM despite an increase in food consumption Effective weight loss therapeutics are a significant and growing healthcare market. 47 Body mass (BM) and food intake were consistently greater in OLETF compared to LETO, as expected of a DIO model. 27 – 29 CBD has not been previously shown to reduce body mass de novo but rather inhibit body mass gain in rats without underlying metabolic dysfunction. 45 We establish here that H4CBD dosed daily reduced body mass in OLETF within the first week to LETO levels, which were maintained for the duration of the study. Although relative food consumption was reduced in the treated group in the first week, which in part explains the initial reduction in BM, by day 10 relative food consumption was greater in the treated OLETF than both OLETF and LETO. High doses of CBD administered clinically have been noted to have anorexic and diarrheal effects indicative of gastric upset, 48 which could account for the food intake reduction noted in the first week. Daily dosing was relaxed to every other day after the first week, which may have offset the anorexic effects of the terpene-rich H4CBD, indicated by the increase in relative food consumption. However, all treated animals were observed to have soft stools at multiple points during the dosing window regardless of dose frequency. Thus, this hyperphagia observed following this initial phase of reduced food intake was possibly a compensatory response to the decrease in BM, but was only sufficient to maintain their BM as mass loss did not continue to decrease after this period. BM loss was not offset by increased food consumption, which indicated H4CBD did not exert anorexic effects when dosed every other day. Therefore, energy balance (in vs. out) is thought to have been disrupted through increased physical activity in the H4CBD group, which likely accounted for the loss in BM in conjunction with non-significant increases in lean tissue catabolism as indicated by the increasing trend in 3MH/Cr excretion. Indeed, activity scores were nearly doubled in H4CBD treated OLETFs, suggesting that increased energy expenditure may partially account for the initial reduction in BM as well as the maintenance of BM during the subsequent increased food intake phase. Lipid catabolism was activated by H4CBD during advanced MetS and associated with a reduction in large adipocytes Adipocytes are specialized cells responsible for storing energy in the form of triglycerides (TGs). The expansion of adipose can be influenced by physical inactivity, genetic predisposition, and excessive caloric intake, which can lead to metabolic dysfunction by altering key signaling pathways involved in lipid and glucose homeostasis. 49 – 51 The hypertrophy of adipocytes increases the risk of cellular rupture, resulting in the uncontrolled release of stored triglycerides into circulation, which can contribute to hypertriglyceridemia and subsequent metabolic complications. 52 Reducing the prevalence of large adipocytes has been associated with improved metabolic outcomes as it limits excessive TG accumulation and mitigates MetS risk factors. 52 Catabolism of adipose depots is mediated by lipase activity in multiple locations, which breaks down TGs in circulation to NEFAs for cellular uptake and metabolism. 53 Although consumption of excess NEFAs is implicated in adipocyte proliferation, 54 , 55 the pervasive insulin resistance of the older OLETF likely drives the utilization of NEFA in lieu of homeostatic glucose uptake and metabolism. 56 This is demonstrated here by the reduction in visceral adipose mass and large adipocytes in treated animals. Moreover, hyperlipidemia is characterized by peripheral insulin resistance, which may be partially mediated by an increase in NEFA uptake by skeletal muscle, thereby decreasing glucose uptake. 56 Indeed, obese, insulin-resistant OLETFs older than 25 weeks of age are hyperglycemic compared to age-matched LETO 29 , and one of the most striking effects H4CBD had on OLETF was the complete attenuation of plasma TG levels compared to OLETF control. Adipose lipase activity was reduced in OLETF compared to LETO and H4CBD-treated OLETF suggesting that impaired lipase activity contributes to the elevated adiposity and plasma TG in OLETF. Although none of the lipase activity measured reached statistical significance between the treated and untreated OLETF, there may be an implication of biological effect which did not attain statistical significance. Lipase activity in adipose, liver, and plasma were consistently greater in H4CBD treated OLETF than untreated OLETF, thus suggesting that the collective, non-significant increases within each tissue promoted systemic lipolysis, resulting in reduced plasma and hepatic TG content. Furthermore, the increasing trend in WAT lipase activity in H4CBD-treated OLETF may partially contribute to the reduction in large adipocytes and lower plasma TG levels measured in this group. Collectively, the summation of these non-statistically significant increases in lipase activity was sufficient to translate into reduced plasma TG levels. Hepatic lipid metabolism indicators measured here suggest that H4CBD promotes NEFA uptake, but not storage, for β-oxidation to compensate for reduced glucose uptake as characterized by Randle Cycle biochemistry. 57 , 58 Interestingly, H4CBD increased the protein abundance of FATP2 but not FATP5. Specific increases in FATP2 and CD36 protein expressions indicate the potential for sustained influx of fatty acids for β-oxidation. The biological response to meet the energetic demands altered by H4CBD is characterized by an increase in adipose catabolism to fuel skeletal muscle needs via increased FFA oxidation in the OLETF, which exhibit severe insulin resistance and β-cell exhaustion at this stage. 32 The reciprocal consequence of this increase in FFA oxidation may be sustained hyperglycemia in the presence of irreversible insulin resistance. 13 Thus, the implications here are that chronic H4CBD corrects an impaired Randle Cycle characteristic of MetS by promoting TG catabolism and FFA utilization. The summarized result is that H4CBD promotes reduced BM by reducing visceral adiposity through increased adipose lipase lipolysis, resulting in reduced large adipocytes and plasma TG levels. This increase in lipid utilization then would partially explain the increase in energy expenditure predicted from the increase in physical activity. Chronic H4CBD is not associated with overt hepatotoxicity Liver injury encompasses a range of pathological conditions resulting from damage to hepatocytes, which can progress to liver fibrosis, a condition characterized by excessive extracellular matrix deposition. 59 Hepatotoxicity is a form of liver injury induced by harmful substances that contribute to fibrosis by stimulating collagen production. 59 Type IV liver collagen deposition was increased in the untreated OLETF compared to LETO, indicative of hepatic fibrosis associated with MetS in the strain. Interestingly, H4CBD induced an intermediate phenotype with respect to liver collagen type IV levels as these levels were not different from either LETO or OLETF, suggesting that a spectrum of fibrotic progression exists and, if H4CBD were consumed for a longer duration, it may have led to a statistical reduction in collagen deposition. At the very least, the lack of a difference in collagen and 4-HNE levels between untreated and treated OLETF indicates that H4CBD did not exacerbate hepatic fibrosis and injury. Additionally, in contrast to previous findings in CBD-treated mice where hepatotoxicity was evident at a dosage of 615 mg/kg, 30 H4CBD at 200 mg/kg in the OLETF did not induce hepatoxicity. These findings demonstrate that H4CBD improved the MetS-associated dyslipidemia and adiposity without hepatotoxic effects at this dosage, making it a potential therapeutic candidate for dyslipidemia and associated metabolic derangements. Limitations While this study provides valuable insights into the metabolic effects of H4CBD, some limitations should be considered. The study duration was restricted to four weeks, and longer-term effects of H4CBD remain unknown. While reductions in fat mass were observed, the apparent loss of estimated lean tissue raises concerns about potential cachexic effects, which require further investigation. Although no overt hepatotoxicity was observed, more comprehensive liver function analyses such as liver enzyme measurements and transcriptomic and proteomic assessments would strengthen conclusions about the biosafety of H4CBD. Future research should focus on optimizing dosing strategies to minimize the potential of unintended muscle degradation. Conclusions The present study demonstrated that chronic, high-dose treatment of H4CBD promotes adipose catabolism during advanced MetS; however, this reduction in adiposity is associated with some loss of lean mass (likely skeletal muscle), and thus, a risk of cachexia may exist. Therefore, further research along these lines, especially in aged-animal models, is necessary before safe therapeutic interventions in elderly populations can be recommended. Nonetheless, the reduction in adiposity was associated with significant reductions in plasma and liver TG content suggestive of an improvement in lipid profile and metabolism. Additionally, the reduction in large adipocytes with H4CBD treatment may be indicative of an improvement in adipose phenotype, which may contribute to an overall improvement in lipid metabolism by increasing lipolysis and reducing plasma TG. While we recognize that the human equivalent concentration of H4CBD used here would be slightly greater than that prescribed for Epidiolex 60 , the lack of liver injury and fibrosis in the treated group is indicative of biological or tissue tolerability, which reflects its biosafety at the concentration used here. Future pre-clinical research should determine not only the ideal timepoint to initiate a therapeutic intervention, but minimum dose to effect, ideal dose duration as well as the efficacy of intermittent dosing. The benefits observed here are encouraging and contribute to establishing a foundation from which to inform future studies on the effects of CBD and its synthetic analogues on metabolic disorders and associated dysfunction in lipid metabolism. Abbreviations 3MH 3-Methylhistidine 4HNE 4-Hydroxynonenal Acox1 Acyl-CoA oxidase 1 ApoB Apolipoprotein B BM Body mass CBD Cannabidiol CD36 Cluster of differentiation 36 Cr Creatinine CPT1A Carnitine palmitoyltransferase 1A, CVD Cardiovascular disease DGAT1 Diacylglycerol O-acyltransferase 1 FATP2 Fatty acid transport protein 2 FATP5 Fatty acid transport protein 5 FM Fat mass GPAM Glycerol-3-phosphate acyltransferase 1 H4CBD 1,2,8,9-Tetrahydrocannabidiol HPLC High-performance Liquid Chromatography LETO Long Evans Tokushima Otsuka Rat LM Liver mass MetS Metabolic syndrome MS Mass spectrometry NAFLD Non-Alcoholic Fatty Liver Disease NEFA Non-esterified fatty acid OLETF Otsuka Long Evans Tokushima Fatty Rat PRDX6 Peroxiredoxin 6 T2DM Type 2 diabetes TG Triglycerides TP Total protein Declarations Acknowledgements We thank Dr. M. Cornejo for lending invaluable experience to the in vivo portion of the study and various technical aspects. We thank P. Arana for his assistance in lipase activity experiments and R. Rodriguez and A. Gonzalez for blinded histology measurements. We also thank M. Smith and J. Emery for their assistance with animal husbandry and end of study procedures. Author’s Contributions JW and RMO conceived and designed research. JW and DM performed experiments and JW analyzed data. NS and MM synthesized drug. JW and MHG prepared figures and drafted manuscript. All authors contributed to the interpretation of results and approved the final version of the manuscript. Funding JW and most of the analyses were supported by CMCR pilot grant A21-0086 awarded to RMO. Availability of data and materials The datasets generated for this study are available upon request to the corresponding author. Ethics Approval and Consent to Participate This animal study was reviewed and approved by the institutional care and use committee of the University of California Merced (USA). Consent for publication Not applicable. 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Angiotensin receptor blockade improves cardiac mitochondrial activity in response to an acute glucose load in obese insulin resistant rats. Redox Biol. 2018;14:371–8. https://doi.org/10.1016/j.redox.2017.10.005 . Rodriguez R, Moreno M, Lee AY, et al. Simultaneous GLP-1 receptor activation and angiotensin receptor blockade increase natriuresis independent of altered arterial pressure in obese OLETF rats. Hypertens Res. 2018;41:798–808. https://doi.org/10.1038/s41440-018-0070-0 . Viscarra JA, Vázquez-Medina JP, Rodriguez R, et al. Decreased expression of adipose CD36 and FATP1 are associated with increased plasma non-esterified fatty acids during prolonged fasting in northern elephant seal pups (Mirounga angustirostris). J Exp Biol. 2012;215:2455–64. https://doi.org/10.1242/jeb.069070 . Godoy-Lugo JA, Thorwald MA, Hui DY et al. Chronic angiotensin receptor activation promotes hepatic triacylglycerol accumulation during an acute glucose challenge in obese-insulin-resistant OLETF rats. 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The regulation of adipocyte growth in white adipose tissue. Front Cell Dev Biology, 10. https://doi.org/10.3389/fcell.2022.1003219 Rodriguez R, Lee AY, Godoy-Lugo JA et al. Chronic at1 blockade improves hyperglycemia by decreasing adipocyte inflammation and decreasing hepatic PCK1 and G6PC1 expression in obese rats. Am J Physiology-Endocrinology Metabolism, 321(5). https://doi.org/10.1152/ajpendo.00584.2020 Van de Woestijne AP, Monajemi AP, Kalkhoven H et al. Adipose tissue dysfunction and hypertriglyceridemia: Mechanisms and management. Obes Rev, 12(10), 829–40. https://doi.org/10.1111/j.1467-789x.2011.00900.x Lass A, Zimmermann R, Oberer M, et al. Lipolysis – A highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores. Prog Lipid Res. 2011;50:14–27. https://doi.org/10.1016/j.plipres.2010.10.004 . Matias I, Belluomo I, Cota D. The Fat Side of the Endocannabinoid System: Role of Endocannabinoids in the Adipocyte. Cannabis Cannabinoid Res. 2016;1:176–85. https://doi.org/10.1089/can.2016.0014 . Tripathi YB, Pandey V. Obesity and endoplasmic reticulum (ER) stresses. Front Immunol. 2012;3. https://doi.org/10.3389/fimmu.2012.00240 . Randle PJ, Garland PB, Hales CN, et al. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet (London England). 1963;1:785–9. https://doi.org/10.1016/s0140-6736(63)91500-9 . Randle PJ, Newsholme EA, Garland PB. Regulation of glucose uptake by muscle. 8. Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphragm muscles. Biochem J. 1964;93(3):652–65. Randle PJ. Regulatory interactions between lipids and carbohydrates: the glucose fatty acid cycle after 35 years. Diabetes / Metabolism Reviews. 1998;14(4):263–83. Berumen J, Baglieri J, Kisseleva T, et al. Liver fibrosis: Pathophysiology and clinical implications. WIREs Mech Disease. 2020;13(1). https://doi.org/10.1002/wsbm.1499 . Nair AB, Jacob S. A simple practice guide for dose conversion between animals and humans. J Basic Clin Pharma. 2016;7:27–31. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7274449","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499229320,"identity":"dafc21aa-302b-4365-81b5-f5121b98b649","order_by":0,"name":"Jessica N. Wilson","email":"","orcid":"","institution":"University of California Merced","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"N.","lastName":"Wilson","suffix":""},{"id":499229321,"identity":"fc2f3c04-9f48-407b-808a-9ba7aaa82215","order_by":1,"name":"Dora A. Mendez","email":"","orcid":"","institution":"University of California Merced","correspondingAuthor":false,"prefix":"","firstName":"Dora","middleName":"A.","lastName":"Mendez","suffix":""},{"id":499229322,"identity":"6926c1e6-5e89-42b7-9ca3-8c6eb10c942d","order_by":2,"name":"Marisol Hernandez Garcia","email":"","orcid":"","institution":"University of California Merced","correspondingAuthor":false,"prefix":"","firstName":"Marisol","middleName":"Hernandez","lastName":"Garcia","suffix":""},{"id":499229323,"identity":"9d5ed9b4-0f33-4467-9939-bd6e82bf1ac3","order_by":3,"name":"Nikolay Shevchenko","email":"","orcid":"","institution":"University of California Davis","correspondingAuthor":false,"prefix":"","firstName":"Nikolay","middleName":"","lastName":"Shevchenko","suffix":""},{"id":499229324,"identity":"b177ec6e-4c0f-415c-b397-090b639227be","order_by":4,"name":"Mark Mascal","email":"","orcid":"","institution":"University of California Davis","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Mascal","suffix":""},{"id":499229325,"identity":"0164ea68-78f5-4aba-97d6-841939de8962","order_by":5,"name":"Nicholas V. DiPatrizio","email":"","orcid":"","institution":"University of California Riverside","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"V.","lastName":"DiPatrizio","suffix":""},{"id":499229326,"identity":"97f4863e-96ea-42b8-a067-f49a3215acf7","order_by":6,"name":"Rudy M. Ortiz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYLCCBAYbGJMZzCUEGBsSGNJI1cLAcJgELebtvc8fPPhzPrF/2uFjH35UWDPws+cY4NUic+a4YUMCz+3EGbfTkmf2nElnkOx5g1+LhEQa0C8StxM3SOcYMzO2HWYwuEHAFogWg3NALfmfmRn/HWawJ05LwgGQLczMjA1AWyQIaeE5xjgj4UCyMdAvxow9x9J5JM48K8Cvhb2N4eOPP3ay/bOTHzP8qLGW429P3oBXCwbgIU35KBgFo2AUjAKsAAAeYkPhNQvOTQAAAABJRU5ErkJggg==","orcid":"","institution":"University of California Merced","correspondingAuthor":true,"prefix":"","firstName":"Rudy","middleName":"M.","lastName":"Ortiz","suffix":""}],"badges":[],"createdAt":"2025-08-01 22:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7274449/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7274449/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89392190,"identity":"10181840-8a50-4b77-8992-a70e34cc92bb","added_by":"auto","created_at":"2025-08-19 13:10:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":221967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH4CBD decreased body mass and increased activity. \u003c/strong\u003eMean (±SEM) (A) cumulative sum gain of body mass, (B) daily body mass, (C) relative food intake and (D) water intake in 41–45-week-old Long-Evans Tokushima Otsuka (LETO; n=8), Otsuka Long-Evans Tokushima Fatty (OLETF; n=8) and OLETF+H4CBD (H4CBD; n=8). (E) Activity score [DSI; HD-S10] of OLETF (OLETF; n=2) and OLETF+H4CBD (H4CBD; n=3). *\u003cem\u003ep\u0026lt;0.05 different from LETO, \u003c/em\u003e\u003csup\u003e\u003cem\u003eϮ\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05 different from OLETF by 2-way ANOVA with Tukey’s HSD.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/a1be9d7a70ca2083a99596c8.png"},{"id":89392634,"identity":"e03d6f85-caa9-477d-a4cf-8a845bf816da","added_by":"auto","created_at":"2025-08-19 13:18:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased 3MH/Cr in H4CBD-treated animals suggests partial lean tissue loss. \u003c/strong\u003eMean (±SEM) end of study (A) urine output (mL) (\u003cem\u003en=8 all groups\u003c/em\u003e), (B) urine total protein, (C) urine creatinine (Cr), (D) urine 3-methylhistidine (3MH) and (E) 3MH/Cr ratio in 45-week-old LETO (n=5), OLETF (n=5) and H4CBD-treated OLETF (H4CBD; n=5). Analysis was done on original sample size with exceptions for outliers or missing samples. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u003cem\u003edifferent from LETO, \u003c/em\u003e\u003csup\u003e\u003cem\u003eϮ\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05 different from OLETF \u003c/em\u003eby 1-way ANOVA with Tukey’s HSD or one-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/cbadf2bd49e98c9969a7887d.png"},{"id":89393887,"identity":"4f9d42f4-6179-4816-8ac2-47cc3dbea030","added_by":"auto","created_at":"2025-08-19 13:26:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":122334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH4CBD reduced adipocyte morphology in advanced MetS. \u003c/strong\u003e(A-C) Representative images of adipocytes from retroperitoneal adipose. (D) Adipocyte percent relative frequency distribution by size (µm) in 45-week-old LETO (n=5-8), OLETF (n=5-8) and H4CBD-treated OLETF (H4CBD; n=6-8). Analysis was done on original sample size with exceptions for outliers or missing samples. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u003cem\u003edifferent from LETO, \u003c/em\u003e\u003csup\u003e\u003cem\u003eϮ\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05 different from OLETF \u003c/em\u003eby 1-way ANOVA with Tukey’s HSD or one-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/920feb76dd1e3bf69f8a2054.png"},{"id":89392638,"identity":"b363a43c-858e-4bca-971c-50a290c1e329","added_by":"auto","created_at":"2025-08-19 13:18:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":129062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH4CBD ablated dyslipidemia. \u003c/strong\u003eMean (±SEM) (A) adipose lipase activity, (B) liver cytosol lipase activity and (C) liver membrane lipase activity, (D) plasma triglycerides, (E) plasma non-esterified fatty acids (NEFA) and (F) plasma lipase activity in 45-week-old LETO (n=8), OLETF (n=8) and H4CBD-treated OLETF (H4CBD; n=8). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 \u003cem\u003edifferent from LETO, \u003c/em\u003e\u003csup\u003e\u003cem\u003eϮ\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05 different from OLETF \u003c/em\u003eby 1-way ANOVA with Tukey’s HSD or one-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/343267fb055f260dc4a7e3c4.png"},{"id":89392636,"identity":"c36fe55e-40d4-40ca-890f-0dd09121c75c","added_by":"auto","created_at":"2025-08-19 13:18:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":422627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH4CBD increased fatty acid transporter expression but reduced triglyceride storage. \u003c/strong\u003e(A) Western blot of LETO, OLETF and H4CBD group with PS, ponceau stain. Mean (±SEM) hepatic (B) FATP5 (C) FATP2, (D) CD36 and (E) triglyceride content in 45-week-old LETO (n=5-8), OLETF (n=5-8) and H4CBD-treated OLETF (H4CBD; n=6-7). \u003csup\u003eϮ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 LETO vs. OLETF, ^\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 OLETF vs H4CBD and \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 LETO vs. H4CBD by one-way ANOVA w/ Tukey’s HSD. \u003csup\u003e§\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 LETO vs. OLETF, *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 LETO vs. H4CBD and \u003csup\u003eǂ\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 OLETF vs H4CBD by one-tailed unpaired t-test.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/335a0360ce010254c922f2b7.png"},{"id":89392212,"identity":"576299bc-c162-4a98-b517-84b01a1efbad","added_by":"auto","created_at":"2025-08-19 13:10:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH4CBD did not contribute to liver injury. \u003c/strong\u003eMean (±SEM) hepatic (A) 4HNE and (B) Collagen Type IV in 45-week-old LETO (n=5), OLETF (n=5) and H4CBD-treated OLETF (H4CBD; n=6). Analysis was done on original sample size with exceptions for outliers or missing samples. \u003csup\u003eϮ\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt;0.05 difference from LETO by one-way ANOVA w/ Tukey’s HSD.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/692563b0e9bb26a56637e223.png"},{"id":103712266,"identity":"ef60edf0-0f11-4ab9-a332-56d0f6d855b6","added_by":"auto","created_at":"2026-03-02 04:10:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2272261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/0644ab28-0539-4dec-bd8f-76fd2c14fad5.pdf"},{"id":89392635,"identity":"92e8f75b-c58b-4d49-92c8-f177ff624a52","added_by":"auto","created_at":"2025-08-19 13:18:40","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":585226,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7274449/v1/358df8de9e17699262f2d867.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pseudocannabinoid H4CBD enhances lipid catabolism to reduce visceral adiposity and large adipocyte size in advanced metabolic syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe popularity of cannabis use in older adults (\u0026gt;\u0026thinsp;55 years of age) in the United States has more than doubled over the past two decades.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Alleviation of chronic pain is a condition for which older adults typically use medicinal cannabis.\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e A common consequence of aging is impaired substrate metabolism, which could result in the onset of metabolic conditions ranging in severity from increases in the individual risk factors for metabolic syndrome (MetS)\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e such as obesity and hypertension to frank type 2 diabetes mellitus (T2DM).\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The prevalence of MetS is 55% among people\u0026thinsp;\u0026ge;\u0026thinsp;60 years of age\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e in the US and the average age at diagnosis of T2DM is approximately 46 years, with equal abundance across racial and ethnic groups.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The therapeutic benefits and risks of cannabis and cannabinoids in metabolic dysfunction have yet to be delineated, especially in older individuals.\u003c/p\u003e\u003cp\u003eCannabidiol (CBD) is an abundant, non-intoxicating constituent of \u003cem\u003eCannabis sativa\u003c/em\u003e, which is of particular interest for pharmacological investigation. The legislative ambiguity and increasing ease-of-access to unregulated cannabis constituents have prompted endeavors to synthesize analogues of cannabis component molecules of interest without use of whole-plant extract. This circumvents violating laws surrounding production or use of a DEA scheduled compound while simultaneously providing a chemically pure product free from the growing concerns of unregulated pesticide use on whole plants. Synthetically derived CBD analogues, like 1,2,8,9-tetrahydrocannabidiol (H4CBD), are inexpensive to produce and can offer essentially identical therapeutic effects to herbal CBD. For example, it has been shown that the closely related pseudocannabinoid H2CBD (8,9-dihydrocannabidiol) controls seizures in rats with the same efficacy as CBD.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Additionally, previous \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e uses of H4CBD have yielded promising benefits consistent with herbal CBD.\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e We have also recently shown that H4CBD improved glucose tolerance in a rat model of metabolic syndrome (MetS).\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eH4CBD is a synthetic analogue of CBD that differs by the saturation of the two carbon-carbon double bonds of the terpene moiety. A significant advantage of hydrogenated cannabinoids is that, while CBD itself is easily converted into THC, H4CBD cannot undergo this chemical transformation, thus eliminating any abuse potential. Similar to natural CBD, H4CBD has little affinity for the endocannabinoid receptors responsible for cannabis intoxication.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, the potential benefits of H4CBD on lipid metabolism and adiposity associated with age-related metabolic dysfunction remain to be assessed.\u003c/p\u003e\u003cp\u003eObservations of cardiovascular and/or metabolic improvements conferred by CBD or CBD-dominant cannabis products have prompted inquiry into other applications for CBD.\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Clinical trials determined that 100 mg CBD, twice daily, is well-tolerated in patients with MetS or T2DM and led to improvements in circulating resistin and gastric inhibitory peptide, but did not reverse the T2DM phenotype.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e A much higher dose (600 mg) of CBD acutely reduced arterial blood pressure in healthy males;\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e however, this effect was lost after a 7-day dose regimen.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e These examples provide promising clinical evidence for the potential of cannabinoids to ameliorate metabolic disorders.\u003c/p\u003e\u003cp\u003eThere is sparse pre-clinical data on the effects of CBD in models of metabolic dysfunction. Natural (plant-extracted) CBD improved glucose tolerance or metabolism in mice with varying degrees of metabolic dysfunction including a high fat-high cholesterol diet,\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e which provides some reasonable evidence that related synthetic cannabinoids have the potential to likewise ameliorate dysregulated metabolism. Moreover, CBD administered over 4 weeks attenuated cardiac dysfunction and cardiac fibrosis in mice with induced type I diabetes.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe goals of this study were to assess the effect of H4CBD on MetS risk factors, primarily dyslipidemia, in the advanced MetS condition, consistent with those observed in older adults. Because of the increasing interest in cannabinoids in this population, a critical intersection between their use and advanced metabolic dysfunction exists, which warrants further investigation. The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is a monogenic model of diet-induced obesity accelerated by a mutation in the CCK receptor.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e These rats have a predictable, timed progression toward non-insulin dependent diabetes mellitus (T2DM) (\u0026gt;\u0026thinsp;20 weeks), marked by a linear phase progression of hypertension (8\u0026ndash;20 weeks), which closely resembles symptoms displayed in human T2DM, including visceral adiposity, dyslipidemia, and insulin resistance.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e At \u0026gt;\u0026thinsp;40 weeks of age, OLETF rats suffer from severe metabolic dysfunction and therefore serve as a model of aged, severe MetS. The hypothesis tested here is that H4CBD can improve lipid metabolism during MetS.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e All animal procedures were reviewed and approved by the institutional animal care and use committees of the University of California Merced. These data complement our previous study of\u003c/p\u003e\u003cp\u003eH4CBD on glucose metabolism using samples from the same animals.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eMale lean Long Evans Tokushima Otsuka (LETO) rats and obese Otsuka Long Evans Tokushima Fatty (OLETF) rats (Japan SLC Inc., Hamamatsu, Japan) at 14 weeks of age were assigned to the following groups (n\u0026thinsp;=\u0026thinsp;8/group): \u003cb\u003e(1)\u003c/b\u003e vehicle-dosed LETO (LETO), \u003cb\u003e(2)\u003c/b\u003e vehicle-dosed OLETF (OLETF), and \u003cb\u003e(3)\u003c/b\u003e H4CBD-treated (200mg/kg/day x 4 weeks) OLETF (H4CBD). Daily dosing was relaxed to every other day after the first week. Rats were maintained in a specific pathogen-free, climate-controlled facility at the University of California Merced on a 12-hour light:dark cycle (07:00\u0026ndash;19:00 and 19:00\u0026ndash;07:00, respectively). All animals had free access to water and were fed rat chow (Teklad Global; fat 9.0%; carbohydrate 44.9%; protein 19.0%) \u003cem\u003ead libitum\u003c/em\u003e. Treatment intervention was initiated at 41 weeks of age.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBody Mass and Water \u0026 Food Intake\u003c/h3\u003e\n\u003cp\u003eBody mass was measured daily to calculate the appropriate drug and vehicle doses. Water intake, urine volume, and food consumption were also measured daily throughout the study.\u003c/p\u003e\n\u003ch3\u003eDrug Preparation \u0026 Administration\u003c/h3\u003e\n\u003cp\u003e1,2,8,9-Tetrahydrocannabidiol (H4CBD) was synthesized as previously described.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Purified H4CBD (\u0026gt;\u0026thinsp;99%) was dissolved in sesame oil and administered by oral gavage at a dose of 200 mg/kg. This dose has been shown to be similarly effective on seizure frequency and severity in rats compared to natural CBD,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e which gave reasonable cause for efficacy of H4CBD at the same dose, which is well below the documented toxic threshold of CBD (\u0026gt;\u0026thinsp;600mg/kg) in rodents.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Bioavailability of H4CBD in circulation has been demonstrated previously.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eTissue Collection\u003c/h3\u003e\n\u003cp\u003eAfter the 4-week study, animals were fasted overnight and tissues (epididymal and retroperitoneal adipose and liver) were collected the following morning as previously described.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Retroperitoneal fat was used to represent white adipose tissue (WAT) in the analyses.\u003c/p\u003e\n\u003ch3\u003ePlasma Analysis\u003c/h3\u003e\n\u003cp\u003ePlasma concentrations of non-esterified fatty acid (NEFA; Fujifilm Wako Diagnostics; 999-34691, 995-34791, 991-34891, and 993-35191) and triglycerides (TG; Cayman Chemical; 10010303) were measured in fasted, end of study plasma samples. All samples were analyzed in duplicate and run in a single assay with intra-assay and percent CV of \u0026lt;\u0026thinsp;10% for all assays.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eUrinalysis\u003c/h2\u003e\u003cp\u003eUrine was collected as previously described.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Urine was thawed on ice to measure 3-methylhistidine (3MH; Abbexa; 257295) and creatinine (Invitrogen; EIACUN). Excretion was calculated by multiplying the 24 hr urine volume by the measured concentration (U\u003csub\u003ex\u003c/sub\u003eV = V * [x]), where x\u0026thinsp;=\u0026thinsp;creatinine or 3MH.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLipase Activity Assay\u003c/h3\u003e\n\u003cp\u003eLipase activity was assayed from retroperitoneal adipose, liver, and end of study plasma as previously performed.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eAdipocyte Morphological Quantification\u003c/h3\u003e\n\u003cp\u003eSlices of retroperitoneal WAT were mounted and stained using hematoxylin and eosin (H\u0026amp;E) stain. Each slide was imaged at 10X using a Keyence BZ-X Series microscope at three distinct locations and scaled.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The images were coded and analyzed for single-blind adipocyte count and area using ImageJ software.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLiver Damage Assessment\u003c/h2\u003e\u003cp\u003eAn aliquot of liver was homogenized and assayed for TG content (Cayman Chemical; 10010303), 4-hydroxynonenal (4HNE) accumulation (myBioSource; MBS736336), and collagen type IV (myBioSource; MBS732756) deposition by ELISA following the manufacturer's recommendation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eWestern Blot\u003c/h2\u003e\u003cp\u003eAn aliquot of liver was used to measure proteins involved in fatty acid uptake, metabolism, and storage as previously described.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Densitometry values were quantified using ImageJ software (NIH) and normalized by correcting for densitometry values of representative protein bands below 37kDa stained with Ponceau S. Results are reported as expression (%) compared to LETO.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCalculations \u0026amp; Statistics\u003c/h2\u003e\u003cp\u003eTo estimate total body water (TBW), equations extrapolated from the literature were used.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e All values are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error mean (SEM) unless otherwise indicated. Means were compared by one-way ANOVA followed by Tukey's honest significant difference or unpaired, one-tailed t-test to assess significant differences among groups. Means and regressions were considered significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Means and regressions were considered a trend between p\u0026thinsp;=\u0026thinsp;0.09\u0026thinsp;\u0026minus;\u0026thinsp;0.051. Outliers were detected by ROUT (Q\u0026thinsp;=\u0026thinsp;1.0%) and removed; however, it should be noted that this was necessary for only 6 occurrences. All statistical procedures were performed using GraphPad Prism 7 (GraphPad Software, Inc., San Diego, CA, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD reduced BM despite increased food consumption\u003c/h2\u003e\u003cp\u003eBM, relative food consumption, water consumption, and activity were measured daily to determine the effect of H4CBD on phenotypic indicators of metabolic dysfunction. H4CBD reduced BM in OLETF to LETO levels within the first week, which was maintained for the duration of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u003cb\u003e\u0026amp; B\u003c/b\u003e). BM of untreated OLETF was 16% and 15% greater than LETO and H4CBD-treated OLETF, respectively, after the first week of the study (day 10\u0026ndash;30). The reduction in BM in the H4CBD-treated OLETF was independent of food intake as relative food consumption increased 24% and 74% compared to OLETF control and LETO, respectively, between day 10 and day 30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Water intake was \u0026gt;\u0026thinsp;200% greater in OLETF compared to LETO for the duration of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). By day 13, water intake in H4CBD-treated OLETF was 90% and \u0026gt;\u0026thinsp;600% greater than OLETF control than LETO, respectively, through the end of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). H4CBD-treated OLETF (n\u0026thinsp;=\u0026thinsp;3) tended (p\u0026thinsp;\u0026lt;\u0026thinsp;0.10) toward increased activity that was nearly double that of OLETF control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD promoted fat loss associated with lean tissue catabolism\u003c/h2\u003e\u003cp\u003eUrine was collected over a 24-hour period on the final day of treatment and analyzed for 3-MH, creatinine (Cr), and total protein excretion to determine if H4CBD promoted muscle wasting. Urine output and proteinuria in the H4CBD-treated group were comparable to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). H4CBD treatment reduced creatinine excretion by 42% compared to OLETF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). 3-MH excretion is a product of amino acid breakdown and urine 3-MH to Cr ratio (3-MH/Cr) is used clinically to assess muscle wasting.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e 3-MH excretion of the H4CBD group was comparable to OLETF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The changes in 3-MH/Cr ratios trended toward significance, with levels in OLETF 128% (p\u0026thinsp;=\u0026thinsp;0.06) and 77% (p\u0026thinsp;=\u0026thinsp;0.07) greater than LETO and H4CBD OLETF, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). End of study BM, FM, and lean organ mass were tabulated to determine the degree of lean tissue loss in treated animals. At 45 weeks of age, OLETF BM was 20% greater than LETO, and H4CBD reduced BM by 22% compared to OLETF (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Percent FM was 147% greater in OLETF compared to LETO, and reduced 49% in H4CBD OLETF compared to OLETF control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMean (\u0026plusmn;\u0026thinsp;SEM) end of study measurements and calculations.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eLETO\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c10\" namest=\"c7\"\u003e\u003cp\u003eOLETF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c14\" namest=\"c11\"\u003e\u003cp\u003eH4CBD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody mass (BM) (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e508\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e611\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e476\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eϮ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLean body mass (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e486\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e546\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e438\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eϮ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEstimated total body H\u003csub\u003e2\u003c/sub\u003eO (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e325\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e369\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e312\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eϮ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFat mass (FM) (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e7.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e*#\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLean tissue (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e145\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e102\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e*,Ϯ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLiver mass (LM) (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEpidydimal fat mass (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003eϮ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRetroperitoneal fat mass (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e*,Ϯ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlasma albumin (mg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e2.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eΔBM compared to OLETF (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eΔBM compared to OLETF (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eΔFM compared to OLETF (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eΔFM compared to OLETF (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eΔLM compared to OLETF (g)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eΔLM compared to OLETF (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"14\" nameend=\"c14\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e#\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 different from LETO, * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 different from LETO, \u003csup\u003e\u0026dagger;\u003c/sup\u003e p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 different from OLETF by one-way ANOVA with Tukey's HSD or unpaired on-tailed t-test\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eVisceral fat masses (retroperitoneal and epidydimal) were dissected and quantified to determine the effect of H4CBD on abdominal adiposity. Fasted plasma lipase activity, as well as plasma NEFA and TG, were measured to assess the effect of H4CBD on parameters of lipid metabolism. Retroperitoneal fat, but not relative epidydimal fat, was 267% more abundant in OLETF than LETO (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). H4CBD reduced relative retroperitoneal and epidydimal fat by 24% and 35%, respectively, compared to OLETF control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Relative combined adipose was 149% greater in OLETF compared to LETO, which was reduced by 25% in H4CBD-treated OLETF (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD reduces large adipocyte abundance\u003c/h2\u003e\u003cp\u003eAdipocyte morphology was comparable below 100 \u0026micro;m among groups (Fig.\u0026nbsp;3A-D). However, H4CBD treatment reduced the frequency of large adipocytes (100\u0026ndash;150\u0026micro;m) by 95% compared to OLETF (Fig.\u0026nbsp;3D).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD reduced plasma triglycerides\u003c/h2\u003e\u003cp\u003eAdipose, liver, and plasma lipase activities were measured to determine the effect of H4CBD on their contributions to lipolysis. The reduction in visceral adiposity and adipocyte size suggests that H4CBD activated and enhanced lipid metabolism. Adipose lipase activity was 31% lower in OLETF compared to LETO and H4CBD tended to normalize it, increasing 22% (p\u0026thinsp;=\u0026thinsp;0.094) compared to OLETF control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Liver endothelial membrane-bound lipases are known to contribute to TG metabolism in the bloodstream.\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e However, neither cytosolic nor membrane-bound lipase activities in the liver were different among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Fasted plasma lipase activity also was not significantly different among groups; however, fasted plasma TG, the substrate of lipolysis, was 147% greater in OLETF compared to LETO, and H4CBD treatment reduced it to LETO levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Fasted plasma NEFA was not different among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD promoted lipid metabolism via increased hepatic CD36 and FATP2 expression\u003c/h2\u003e\u003cp\u003eHepatic lipid metabolism signaling proteins were measured to assess the impact of H4CBD on lipid shuttling. FATP5 expression was increased 109% in OLETF compared to LETO and was not affected by H4CBD treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). FATP2 expression was comparable between LETO and OLETF but increased by 60% in H4CBD-treated OLETF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). CD36 expression was 41% lesser in OLETF compared to LETO, and H4CBD treatment reversed CD36 expression by 48% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Downstream signaling proteins, GPAM, DGAT1, CPT1A, ACOX1, ApoB, and PRDX6 were comparable among groups (\u003cem\u003edata not shown\u003c/em\u003e). Liver triglyceride content was 24% greater in OLETF compared to LETO, and reduced by 28% in H4CBD-treated OLETF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Collectively, the increase in fatty acid transporter expression suggests that H4CBD treatment enhanced FFA uptake to promote fatty acid oxidation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD did not contribute to liver damage\u003c/h2\u003e\u003cp\u003eIndicators of damage were measured in liver to assess the effects of H4CBD on hepatotoxicity. End of study liver 4HNE was not different among groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Type IV collagen deposition is an indicator of liver fibrosis, which is useful in the diagnosis of MAFLD in elderly individuals\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and models of MAFLD like the OLETF rat.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Liver collagen deposition was 33% greater in OLETF compared to LETO and H4CBD treatment had no effect on collagen levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe prevalence of MetS has been estimated to be three times more pervasive than T2DM, which extrapolates to account for a population of over 1\u0026nbsp;billion people globally.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Since MetS is associated with development of CVD and T2DM, intervention strategies that affect multiple cluster conditions of MetS are of critical importance. The effects of CBD on MetS pathophysiology are not well established, although a few studies have shown beneficial\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e or null effects\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e of CBD on isolated MetS risk factors. However, these effects have not been explored in context as cluster factors. Therefore, the aim of the present study was to preclinically assess the therapeutic effect of a CBD analogue on adiposity and the associated dyslipidemia in the condition of advanced MetS, which is most frequently observed in older populations. We chose for this purpose to evaluate the effects of the non-narcotic cannabinoid, 1,2,8,9-tetrahydrocannabidiol (H4CBD) due to its synthetic accessibility in pure form, regulatory advantages, and absence of abuse liability, and thus, potential for wider global adoption than naturally-derived CBD.\u003c/p\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eH4CBD reduced BM despite an increase in food consumption\u003c/h2\u003e\u003cp\u003eEffective weight loss therapeutics are a significant and growing healthcare market.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e Body mass (BM) and food intake were consistently greater in OLETF compared to LETO, as expected of a DIO model.\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e CBD has not been previously shown to reduce body mass \u003cem\u003ede novo\u003c/em\u003e but rather inhibit body mass gain in rats without underlying metabolic dysfunction.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e We establish here that H4CBD dosed daily reduced body mass in OLETF within the first week to LETO levels, which were maintained for the duration of the study. Although relative food consumption was reduced in the treated group in the first week, which in part explains the initial reduction in BM, by day 10 relative food consumption was greater in the treated OLETF than both OLETF and LETO. High doses of CBD administered clinically have been noted to have anorexic and diarrheal effects indicative of gastric upset,\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e which could account for the food intake reduction noted in the first week. Daily dosing was relaxed to every other day after the first week, which may have offset the anorexic effects of the terpene-rich H4CBD, indicated by the increase in relative food consumption. However, all treated animals were observed to have soft stools at multiple points during the dosing window regardless of dose frequency. Thus, this hyperphagia observed following this initial phase of reduced food intake was possibly a compensatory response to the decrease in BM, but was only sufficient to maintain their BM as mass loss did not continue to decrease after this period. BM loss was not offset by increased food consumption, which indicated H4CBD did not exert anorexic effects when dosed every other day. Therefore, energy balance (in vs. out) is thought to have been disrupted through increased physical activity in the H4CBD group, which likely accounted for the loss in BM in conjunction with non-significant increases in lean tissue catabolism as indicated by the increasing trend in 3MH/Cr excretion. Indeed, activity scores were nearly doubled in H4CBD treated OLETFs, suggesting that increased energy expenditure may partially account for the initial reduction in BM as well as the maintenance of BM during the subsequent increased food intake phase.\u003c/p\u003e\u003cp\u003e\u003cem\u003eLipid catabolism was activated by H4CBD during advanced MetS and associated with a reduction in large adipocytes\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAdipocytes are specialized cells responsible for storing energy in the form of triglycerides (TGs). The expansion of adipose can be influenced by physical inactivity, genetic predisposition, and excessive caloric intake, which can lead to metabolic dysfunction by altering key signaling pathways involved in lipid and glucose homeostasis.\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e The hypertrophy of adipocytes increases the risk of cellular rupture, resulting in the uncontrolled release of stored triglycerides into circulation, which can contribute to hypertriglyceridemia and subsequent metabolic complications.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Reducing the prevalence of large adipocytes has been associated with improved metabolic outcomes as it limits excessive TG accumulation and mitigates MetS risk factors.\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e Catabolism of adipose depots is mediated by lipase activity in multiple locations, which breaks down TGs in circulation to NEFAs for cellular uptake and metabolism.\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e Although consumption of excess NEFAs is implicated in adipocyte proliferation,\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e the pervasive insulin resistance of the older OLETF likely drives the utilization of NEFA \u003cem\u003ein lieu\u003c/em\u003e of homeostatic glucose uptake and metabolism.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e This is demonstrated here by the reduction in visceral adipose mass and large adipocytes in treated animals.\u003c/p\u003e\u003cp\u003eMoreover, hyperlipidemia is characterized by peripheral insulin resistance, which may be partially mediated by an increase in NEFA uptake by skeletal muscle, thereby decreasing glucose uptake.\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e Indeed, obese, insulin-resistant OLETFs older than 25 weeks of age are hyperglycemic compared to age-matched LETO\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and one of the most striking effects H4CBD had on OLETF was the complete attenuation of plasma TG levels compared to OLETF control. Adipose lipase activity was reduced in OLETF compared to LETO and H4CBD-treated OLETF suggesting that impaired lipase activity contributes to the elevated adiposity and plasma TG in OLETF. Although none of the lipase activity measured reached statistical significance between the treated and untreated OLETF, there may be an implication of biological effect which did not attain statistical significance. Lipase activity in adipose, liver, and plasma were consistently greater in H4CBD treated OLETF than untreated OLETF, thus suggesting that the collective, non-significant increases within each tissue promoted systemic lipolysis, resulting in reduced plasma and hepatic TG content. Furthermore, the increasing trend in WAT lipase activity in H4CBD-treated OLETF may partially contribute to the reduction in large adipocytes and lower plasma TG levels measured in this group. Collectively, the summation of these non-statistically significant increases in lipase activity was sufficient to translate into reduced plasma TG levels.\u003c/p\u003e\u003cp\u003eHepatic lipid metabolism indicators measured here suggest that H4CBD promotes NEFA uptake, but not storage, for β-oxidation to compensate for reduced glucose uptake as characterized by Randle Cycle biochemistry.\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e Interestingly, H4CBD increased the protein abundance of FATP2 but not FATP5. Specific increases in FATP2 and CD36 protein expressions indicate the potential for sustained influx of fatty acids for β-oxidation. The biological response to meet the energetic demands altered by H4CBD is characterized by an increase in adipose catabolism to fuel skeletal muscle needs via increased FFA oxidation in the OLETF, which exhibit severe insulin resistance and β-cell exhaustion at this stage.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e The reciprocal consequence of this increase in FFA oxidation may be sustained hyperglycemia in the presence of irreversible insulin resistance.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Thus, the implications here are that chronic H4CBD corrects an impaired Randle Cycle characteristic of MetS by promoting TG catabolism and FFA utilization. The summarized result is that H4CBD promotes reduced BM by reducing visceral adiposity through increased adipose lipase lipolysis, resulting in reduced large adipocytes and plasma TG levels. This increase in lipid utilization then would partially explain the increase in energy expenditure predicted from the increase in physical activity.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eChronic H4CBD is not associated with overt hepatotoxicity\u003c/h2\u003e\u003cp\u003eLiver injury encompasses a range of pathological conditions resulting from damage to hepatocytes, which can progress to liver fibrosis, a condition characterized by excessive extracellular matrix deposition.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e Hepatotoxicity is a form of liver injury induced by harmful substances that contribute to fibrosis by stimulating collagen production.\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e Type IV liver collagen deposition was increased in the untreated OLETF compared to LETO, indicative of hepatic fibrosis associated with MetS in the strain. Interestingly, H4CBD induced an intermediate phenotype with respect to liver collagen type IV levels as these levels were not different from either LETO or OLETF, suggesting that a spectrum of fibrotic progression exists and, if H4CBD were consumed for a longer duration, it may have led to a statistical reduction in collagen deposition. At the very least, the lack of a difference in collagen and 4-HNE levels between untreated and treated OLETF indicates that H4CBD did not exacerbate hepatic fibrosis and injury. Additionally, in contrast to previous findings in CBD-treated mice where hepatotoxicity was evident at a dosage of 615 mg/kg,\u003csup\u003e30\u003c/sup\u003e H4CBD at 200 mg/kg in the OLETF did not induce hepatoxicity. These findings demonstrate that H4CBD improved the MetS-associated dyslipidemia and adiposity without hepatotoxic effects at this dosage, making it a potential therapeutic candidate for dyslipidemia and associated metabolic derangements.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eWhile this study provides valuable insights into the metabolic effects of H4CBD, some limitations should be considered. The study duration was restricted to four weeks, and longer-term effects of H4CBD remain unknown. While reductions in fat mass were observed, the apparent loss of estimated lean tissue raises concerns about potential cachexic effects, which require further investigation. Although no overt hepatotoxicity was observed, more comprehensive liver function analyses such as liver enzyme measurements and transcriptomic and proteomic assessments would strengthen conclusions about the biosafety of H4CBD. Future research should focus on optimizing dosing strategies to minimize the potential of unintended muscle degradation.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study demonstrated that chronic, high-dose treatment of H4CBD promotes adipose catabolism during advanced MetS; however, this reduction in adiposity is associated with some loss of lean mass (likely skeletal muscle), and thus, a risk of cachexia may exist. Therefore, further research along these lines, especially in aged-animal models, is necessary before safe therapeutic interventions in elderly populations can be recommended. Nonetheless, the reduction in adiposity was associated with significant reductions in plasma and liver TG content suggestive of an improvement in lipid profile and metabolism. Additionally, the reduction in large adipocytes with H4CBD treatment may be indicative of an improvement in adipose phenotype, which may contribute to an overall improvement in lipid metabolism by increasing lipolysis and reducing plasma TG. While we recognize that the human equivalent concentration of H4CBD used here would be slightly greater than that prescribed for Epidiolex\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, the lack of liver injury and fibrosis in the treated group is indicative of biological or tissue tolerability, which reflects its biosafety at the concentration used here. Future pre-clinical research should determine not only the ideal timepoint to initiate a therapeutic intervention, but minimum dose to effect, ideal dose duration as well as the efficacy of intermittent dosing. The benefits observed here are encouraging and contribute to establishing a foundation from which to inform future studies on the effects of CBD and its synthetic analogues on metabolic disorders and associated dysfunction in lipid metabolism.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e3MH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;3-Methylhistidine\u003c/p\u003e\n\u003cp\u003e4HNE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;4-Hydroxynonenal\u003c/p\u003e\n\u003cp\u003eAcox1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Acyl-CoA oxidase 1\u003c/p\u003e\n\u003cp\u003eApoB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Apolipoprotein B\u003c/p\u003e\n\u003cp\u003eBM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Body mass\u003c/p\u003e\n\u003cp\u003eCBD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cannabidiol\u003c/p\u003e\n\u003cp\u003eCD36 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cluster of differentiation 36\u003c/p\u003e\n\u003cp\u003eCr \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Creatinine\u003c/p\u003e\n\u003cp\u003eCPT1A \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Carnitine palmitoyltransferase 1A,\u003c/p\u003e\n\u003cp\u003eCVD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cardiovascular disease\u003c/p\u003e\n\u003cp\u003eDGAT1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Diacylglycerol O-acyltransferase 1\u003c/p\u003e\n\u003cp\u003eFATP2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fatty acid transport protein 2\u003c/p\u003e\n\u003cp\u003eFATP5 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fatty acid transport protein 5\u003c/p\u003e\n\u003cp\u003eFM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fat mass\u003c/p\u003e\n\u003cp\u003eGPAM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Glycerol-3-phosphate acyltransferase 1\u003c/p\u003e\n\u003cp\u003eH4CBD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;1,2,8,9-Tetrahydrocannabidiol\u003c/p\u003e\n\u003cp\u003eHPLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;High-performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003eLETO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Long Evans Tokushima Otsuka Rat\u003c/p\u003e\n\u003cp\u003eLM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Liver mass\u003c/p\u003e\n\u003cp\u003eMetS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Metabolic syndrome\u003c/p\u003e\n\u003cp\u003eMS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Mass spectrometry\u003c/p\u003e\n\u003cp\u003eNAFLD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Non-Alcoholic Fatty Liver Disease\u003c/p\u003e\n\u003cp\u003eNEFA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Non-esterified fatty acid\u003c/p\u003e\n\u003cp\u003eOLETF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Otsuka Long Evans Tokushima Fatty Rat\u003c/p\u003e\n\u003cp\u003ePRDX6 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Peroxiredoxin 6\u003c/p\u003e\n\u003cp\u003eT2DM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Type 2 diabetes\u003c/p\u003e\n\u003cp\u003eTG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Triglycerides\u003c/p\u003e\n\u003cp\u003eTP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Total protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. M. Cornejo for lending invaluable experience to the \u003cem\u003ein vivo\u003c/em\u003e portion of the study and various technical aspects. We thank P. Arana for his assistance in lipase activity experiments and R. Rodriguez and A. Gonzalez for blinded histology measurements. We also thank M. Smith and J. Emery for their assistance with animal husbandry and end of study procedures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJW and RMO conceived and designed research. JW and DM performed experiments and JW analyzed data. NS and MM synthesized drug. JW and MHG prepared figures and drafted manuscript. All authors contributed to the interpretation of results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJW and most of the analyses were supported by CMCR pilot grant A21-0086 awarded to RMO.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated for this study are available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis animal study was reviewed and approved by the institutional care and use committee of the University of California Merced (USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLloyd SL, Striley CW. 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J Basic Clin Pharma. 2016;7:27\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"3-methylhistidine, cannabinoids, insulin resistance, MAFLD, metabolism, metabolic syndrome, obesity","lastPublishedDoi":"10.21203/rs.3.rs-7274449/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7274449/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMetabolic Syndrome (MetS) is a precursor for cardiovascular disease (CVD) and type 2 diabetes (T2D) and has a prevalence of 55% among people\u0026thinsp;\u0026ge;\u0026thinsp;60 years of age in the US. Cannabidiol (CBD) use has grown more popular in the last two decades, particularly amongst adults\u0026thinsp;\u0026gt;\u0026thinsp;55 years of age. Synthetic analogues of CBD have generated great interest because they can offer safer, chemically pure products with no abuse potential and no regulatory barriers. However, the effects of chronic cannabinoid use during age-associated cardiometabolic dysfunction have not been examined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTo assess the effects of H4CBD, a synthetic analogue of CBD, on advanced MetS, a cohort of 41-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were administered 200 mg H4CBD/kg by oral gavage for 4 weeks. Animals were fed \u003cem\u003ead libitum\u003c/em\u003e and monitored alongside vehicle-treated OLETF and lean, strain-control Long-Evans Tokushima Otsuka (LETO) rats.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eBody mass (BM) was reduced 22% in H4CBD group compared to OLETF and was similar to LETO levels within the first week but did not reverse the diabetic phenotype of the aged OLETF. H4CBD also reduced visceral fat mass (FM; 41%) and nearly ablated large adipocyte (\u0026gt;\u0026thinsp;100\u0026micro;m) abundance compared to OLETF. Plasma triglycerides were more than doubled in OLETF compared to LETO, and H4CBD normalized the levels. Urinary 3-methylhistidine (3-MH) to creatinine ratio tended to be higher (77%; p\u0026thinsp;=\u0026thinsp;0.07) in H4CBD suggesting that some lean tissue was lost along with FM, which contributed to the reduction in BM.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eChronic H4CBD treatment increased lipid catabolism resulting in increased FM loss, although some lean mass loss was also observed. These results suggest that synthetic cannabinoids have potential for advanced-age obesity management during severe metabolic dysfunction, even with consideration of possible cachexic effects.\u003c/p\u003e","manuscriptTitle":"Pseudocannabinoid H4CBD enhances lipid catabolism to reduce visceral adiposity and large adipocyte size in advanced metabolic syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 13:10:34","doi":"10.21203/rs.3.rs-7274449/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a324975f-1ffd-475b-99ec-e9c80971e036","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T04:09:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 13:10:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7274449","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7274449","identity":"rs-7274449","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}