Cardiovascular disease (CVD) is a global health concern. Vascular dysfunction is an aspect of CVD, and novel treatments targeting vascular physiology are necessary. In the endothelium, eNOS regulates vasodilation and mitochondrial function; both are disrupted in CVD. (–)-Epicatechin, a botanical compound known for its vasodilatory, eNOS, and mitochondrial-stimulating properties, is a potential therapy in those with CVD. We hypothesized that (–)-epicatechin would support eNOS activity and mitochondrial respiration, leading to improved vasoreactivity in a thermoneutral-derived rat model of vascular dysfunction. We housed Wistar rats at room temperature or in thermoneutral conditions for a total of 16 week and treated them with 1mg/kg body weight (–)-epicatechin for 15 day. Vasoreactivity, eNOS activity, and mitochondrial respiration were measured, in addition to the protein expression of upstream cellular signaling molecules including AMPK and CaMKII. We observed a significant improvement of vasodilation in those housed in thermoneutrality and treated with (–)-epicatechin (p < 0.05), as well as dampened mitochondrial respiration (p < 0.05). AMPK and CaMKIIα and β expression were lessened with (–)-epicatechin treatment in those housed at thermoneutrality (p < 0.05). The opposite was observed with animals housed at room temperature supplemented with (–)-epicatechin. These data illustrate a context-dependent vascular response to (–)-epicatechin, a candidate for CVD therapeutic development.

Diabetes is a life-threatening and debilitating disease with pathological hallmarks, including glucose intolerance and insulin resistance. Plant compounds are a source of novel and effective therapeutics, and the flavonoid (−)-epicatechin, common to popular foods worldwide, has been shown to improve carbohydrate metabolism in both clinical studies and preclinical models. We hypothesized that (−)-epicatechin would alleviate thermoneutral housing-induced glucose intolerance. Male rats were housed at either thermoneutral (30 °C) or room temperature (24 °C) for 16 weeks and gavaged with either 1 mg/kg body weight or vehicle for the last 15 days before sacrifice. Rats housed at thermoneutrality had a significantly elevated serum glucose area under the curve (p < 0.05) and reduced glucose-mediated insulin secretion. In contrast, rats at thermoneutrality treated with (−)-epicatechin had improved glucose tolerance and increased insulin secretion (p < 0.05). Insulin tolerance tests revealed no differences in insulin sensitivity in any of the four groups. Pancreatic immunohistochemistry staining showed significantly greater islet insulin positive cells in animals housed at thermoneutrality. In conclusion, (−)-epicatechin improved carbohydrate tolerance via increased insulin secretion in response to glucose challenge without a change in insulin sensitivity.

Diabetes increases cardiovascular disease (CVD) risk. Diabetes-mediated vascular pathology proceeds differently in men and women. We reported that housing rats at thermoneutral (30°C, TN) conditions alters perivascular adipose tissue (PVAT) phenotype. We also observed related abnormalities in vasoreactivity and mitochondrial function with sex-differences. We hypothesized that TN-mediated PVAT phenotypic transformation alters brown adipose tissue (BAT) regulator PRDM16, fatty acid composition, fatty acid (FA) transporter FATP1, and mitochondrial lipid oxidation in a sex-dependent manner. Male and female Wistar rats were housed at room temperature (24°C, RT) or TN for 16 weeks. Endpoints included PVAT phenotypic characterization, RNA seq analysis, and PVAT mitochondrial respiration. PVAT phenotype was morphologically different between TN and RT rats, with rats housed at TN having 19.7% less BAT phenotype overall (p&lt;0.05). UCP-1 expression was lower in animals housed at TN (p=0.06). PRDM16 was significantly dampened in all animals at TN (p&lt;0.05), and notably decreased in males at TN (80.6%, p&lt;0.05). Loss of BAT markers was associated with changes in FA regulation. Genomic expression of FATP1 was lower in all animals at TN, more prominently in females. Palmitoleic and arachidonic acids were significantly lower in TN females (48.5% and 15.5%, respectively, p&lt;0.05) and males (63.0% and 40.1%, respectively, p&lt;0.05). PVAT of all animals housed at TN showed mitochondrial respiration significantly diminished in lipid substrate experiments for state 3, 4, and uncoupled (p&lt;0.05 for all), aligning with our previous reports in aorta. These data support a model wherein altered PVAT phenotype and FA composition impacts lipid transport and utilization. These changes are associated with differential impact on vascular impairment between females and males. These results provide insights into sex differences in PVAT contributions to vascular disease progression and vascular crosstalk. Disclosure A.C.Keller: None. M.M.Henckel: None. L.Knaub: None. G.Pott: None. G.James: None. L.A.Walker: None. J.E.B.Reusch: Advisory Panel; Medtronic. Funding National Center for Research Resources (UL1RR025780); U.S. Department of Veterans Affairs (BX002046 to J.E.B.R.), (BX003185 to A.C.K.); National Institutes of Health (R01 DK124344-01A1 to J.E.B.R.); Denver Research Institute; Ludeman Family Center for Women’s Health Research at the University of Colorado Anschutz Medical Campus; Junior Faculty Research Development Grant (to A.C.K.)

Objective: Cardiovascular disease is of paramount importance, yet there are few relevant rat models to investigate its pathology and explore potential therapeutics. Housing at thermoneutral temperature (30 8C) is being employed to humanize metabolic derangements in rodents. We hypothesized that housing rats in thermoneutral conditions would potentiate a highfat diet, resulting in diabetes and dysmetabolism, and deleteriously impact vascular function, in comparison to traditional room temperature housing (22 8C).Methods: Male Wistar rats were housed at either room temperature or thermoneutral temperatures for 16 weeks on either a low or high-fat diet. Glucose and insulin tolerance tests were conducted at the beginning and end of the study. At the study's conclusion, vasoreactivity and mitochondrial respiration of aorta and carotid were conducted. Results:We observed diminished vasodilation in vessels from thermoneutral rats (P < 0.05), whereas high-fat diet had no effect. This effect was also observed in endothelium-denuded aorta in thermoneutral rats (P < 0.05). Vasoconstriction was significantly elevated in aorta of thermoneutral rats (P < 0.05). Diminished nitric oxide synthase activity and nitrotyrosine, and elevated glutathione activity were observed in aorta from rats housed under thermoneutral conditions, indicating a climate of lower nitric oxide and excess reactive oxygen species in aorta. Thermoneutral rat aorta also demonstrated less mitochondrial respiration with lipid substrates compared with the controls (P < 0.05). Conclusion:Our data support that thermoneutrality causes dysfunctional vasoreactivity, decreased lipid mitochondrial metabolism, and modified cellular signaling. These are critical observations as thermoneutrality is becoming prevalent for translational research models. This new model of vascular dysfunction may be useful for dissection of targetable aspects of cardiovascular disease and is a novel and necessary model of disease.

Cardiovascular disease (CVD) is a leading cause of hospitalization and death. CVD is characterized by impaired vasoreactivity and mitochondrial dysfunction. Perivascular adipose tissue (PVAT), considered brown adipose tissue (BAT), surrounds the vasculature and regulates its response. Preliminary data with rats housed at either their thermoneutrality (TN, 30°C) or room temperature (RT, 22°C) showed diminished vasodilation in aorta from TN rats as compared with those from RT rats (10.2% ± 4.0% (0.159 g of vasodilation capacity, starting from maximal force constriction of 1.563 g) versus 64.2% ± 5.3% (0.909 g of 1.417 g, p<0.001). TN-housed rat aorta also showed less mitochondrial respiration with lipid substrates in multiple states (p<0.05). We hypothesize that remodeling of PVAT phenotype from BAT to white adipose tissue (WAT) may alter mitochondrial lipid utilization and cause vasoreactivity dysfunction. To test this, we housed male and female rats at either RT or TN and investigated their own PVAT + aorta or PVAT from the oppositely- housed animals along with each rat’s own aorta for vasoreactivity ex situ. There was diminished vasodilation in all TN animals with PVAT + aorta (29.2% ± 3.8% (0.269 g of 0.923 g) versus 37.6% ± 6.0% (0.255 g of 0.677 g), p<0.02), with only male animals showing a significant effect from PVAT (p<0.001). In aorta of TN-housed animals analyzed with PVAT from RT-housed animals, female vessels showed an increase in vasodilation capacity as compared to controls (56.8% ± 13.6% (0.589 g of 1.037 g) versus 5.2% ± 2.3% (0.028 g of 0.534 g), p<0.001), strongly suggesting that PVAT not only regulates vasoreactivity, but can repair TN-induced diminished dilation in a sex-dependent manner. All animals at TN had significantly less mitochondrial respiration with lipid substrates (p<0.05), with no sex differences. We further observed a significantly greater amount of lipids in PVAT from male TN-housed animals as compared to that in RT-housed animals (p<0.05), consistent with a WAT phenotype. Our data support that TN alters PVAT phenotype in a sex-dependent manner, resulting in dysfunctional vasoreactivity and mitochondrial function. These targets of CVD in both male and female animals are exciting avenues for novel therapeutics.