Thermoneutrality induces vascular dysfunction and impaired metabolic function in male Wistar rats: a new model of vascular disease

INTRODUCTION

Cardiovascular disease (CVD), the leading cause of death in the United States [1], includes all conditions impacting the heart and vasculature, such as hypertension, heart failure, and atherosclerotic cardiovascular disease (ASCVD). CVD risk is significantly elevated in those with diabetes compared with the general population [2], and its progression includes impaired vasoreactivity (dilation and constriction), compromised vascular endothelial function, structural stiffness, increased tone, and vascular mitochondrial dysfunction [3–6]. Vasodilation is regulated by endothelial nitric oxide synthase (eNOS), and this enzyme's activity is compromised in CVD and diabetes [7–11]. We and others have demonstrated that mitochondria are critical to vascular function [12–14] and are regulated by eNOS activity in the vasculature [14–16]. Endothelial-mediated vasoreactivity, calcium signaling associated with vascular relaxation, smooth muscle cell proliferation, and apoptosis are processes regulated, in part, by eNOS. Each of these processes require healthy mitochondrial function [17–19]. Vascular mitochondrial dysfunction has been reported in association with smooth muscle cell apoptosis, vascular inflammation, and vascular stiffness [20–22]. In summary, mitochondria are a dominant factor regulating vascular function in myriad ways. Thus, elucidating the connection between mitochondria, eNOS activity, and vascular dysfunction in CVD is critical to a more complete understanding of the pathogenesis of ASCVD.

Animal models demonstrating dysfunction in vascular reactivity and mitochondrial activity within the context of diabetes, metabolic syndrome, or obesity have limitations. Behavioral rat models of diabetes and obesity, such as consuming a high-fat diet, are difficult to calibrate, demonstrate inconsistent vascular structural and functional changes and align poorly with human physiology (such as development of chronic hypertension or development of ASCVD). Rodents can be resistant to the metabolic and cardiovascular impact of cage dwelling (sedentary behavior) and the development of diet-induced obesity and carbohydrate intolerance. As such, most rodent models incompletely and inconsistently recapitulate metabolic disease. Further, many rodent models of ASCVD require genetic changes, such as manipulation of apolipoprotein E or the low-density lipoprotein (LDL) receptor, only available in mice.

Thermoneutrality refers to an environmental temperature where caloric intake is not used to maintain body temperature homeostasis. This temperature is between 14.8 and 24 °C [23] for humans but is 30 °C for rats [24,25]. Animal research environments have traditionally been kept at human thermoneutral temperatures. However, appreciation that this temperature is not thermoneutral for rodents has led to the reevaluation of optimal housing temperatures for rodent research in the modeling of human physiology [26,27]. Thermoneutral housing is now used commonly in rodent metabolic research. Mice and rats housed at thermoneutral conditions show measurable differences in physiology, such as increased energy expenditure rates that align more closely with those of humans, elevated glucose and insulin concentrations, decreased heart rate and blood pressure, and elevated apoptosis proteins [26,28,29]. Thermoneutral housing is reported to increase weight gain and carbohydrate intolerance in rats [26,28,29]. The impact of thermoneutral housing conditions on metabolism and vascular function is somewhat characterized in mice but is unknown in the rat.

We hypothesized that housing rats in thermoneutral conditions would potentiate high-fat diet-induced obesity, resulting in carbohydrate intolerance and impairment in vasoreactivity and mitochondrial function. To test our hypothesis, we housed male rats at either room temperature (RT) or thermoneutrality for 16 weeks and measured metabolic parameters, vasoreactivity, and vascular mitochondrial function. We report here a significant dampening of both vascular function and mitochondrial respiration in aorta, along with metabolic modulation. These effects are present in rats fed either a high-fat or a low-fat diet with little impact of diet on the vascular endpoints. This novel model of thermoneutrality-induced vascular dysfunction is a simple and significant new tool for CVD research and suggests prominent impacts of thermoneutral housing on vascular function.

METHODS AND MATERIALS Reagents

Western blotting gels were from BioRad (Hercules, California, USA) and PVDF membranes were from Millipore (Burlington, Massachusetts, USA). Collagenase, EDTA, ethylene glycol tetraacetic acid (EGTA), sodium pyrophosphate, sodium orthovanadate, sodium fluoride, okadaic acid, 1% protease inhibitor cocktail, dithiothreitol, magnesium chloride, K-lactobionate, taurine, potassium phosphate, HEPES, digitonin, pyruvate, malic acid, glutamic acid, adenosine diphosphate, succinic acid, oligomycin, carbonyl cyanide 4 (trifluoromethoxy)phenylhydrazone (FCCP), antibody to β-actin (mouse), phenylephrine and acetylcholine, trypsin inhibitor and cytochrome c were procured from Sigma-Aldrich (St Louis, Missouri, USA). Dimethyl sulfoxide (DMSO), sodium chloride, sucrose, and bovine serum albumin were purchased from Fisher Scientific (Pittsburg, Pennsylvania, USA).

Antibodies

Antibodies to total adenosine monophosphate kinase (AMPK, Cell Signaling #2532S, 1 : 500, mouse), phosphorylated AMPK (pAMPK, Cell Signaling #2532S, 1 : 500, rabbit), Sirtuin 1 (SIRT1, Cell Signaling #9475S, 1 : 250, rabbit), Sirtuin 3 (SIRT3, Cell Signaling #2627S, 1 : 500, rabbit), total endothelial nitric oxide synthase (eNOS, Cell Signaling #9572S, 1 : 500, mouse), Ser1177 phosphorylated eNOS (Cell Signaling #9571S, 1 : 500 Rabbit), were obtained from Cell Signaling (Danvers, Massachusetts, USA). Nitrotyrosine was purchased from Cayman Chemical (#10189540, 1 : 500, rabbit, Ann Arbor, Michigan, USA). For the ratio of phosphorylated to total protein, alternate host animal antibodies and alternate secondary antibodies were used with different wavelengths, to eliminate the possibility of signal bleed-through. Antibody cocktail to representative subunits of mitochondrial oxidative phosphorylation (Total OXPHOS Rodent WB Antibody Cocktail Abcam #ab110413,1 : 1500, mouse) complexes I (subunit NDUF88), II (subunit SDHB), III (subunit UQCRC2), IV (MTCO1), and V (subunit ATP5A), PPARγ coactivator 1 alpha (PGC-1α, Abcam #ab54481, 1 : 500, rabbit) and MnSOD antibody (anti-SOD2/MnSOD antibody [2a1], Abcam, #ab16956, 1 : 500) were obtained from Abcam (Cambridge, Massachusetts, USA). Secondary antibodies were IRDye 800RD goat antimouse #926-68070 at 1 : 10 000, IRDye 800RD goat antirabbit #926-68071 at 1 : 10 000 were purchased from LI-COR (Lincoln, Nebraska, USA) and Starbright Blue 700 goat antimouse at 1 : 5000 #12004159 from Bio-Rad Laboratories.

In-vivo experiments

Animals (male Wistar rats, 5 weeks old), kept at two animals per cage, were housed at either room temperature (22 °C) or thermoneutrality (29–30 °C). Body temperature was taken superficially and elevated temperature was achieved in those housed at thermoneutral conditions as compared with those house at room temperature (30.4 ± 0.1 vs. 27.4 ± 0.1 °C, P < 0.001, data not shown). Animals were fed either a customized diet containing 13% kcal fat (LFD) or 42% kcal fat (HFD) (Envigo [Teklad]) for 16 weeks. Blood (approximately 50 μl) was collected in 0.5 mol/l EDTA and spun at 12 000g for 10 min at 4 °C. Plasma was extracted and stored at −80 °C. Fasting blood (6 h) was taken at the beginning and end of the study, fed blood was taken biweekly for glucose and insulin concentrations, and body weight and food consumption were measured weekly. Endpoint parameters were taken at sacrifice, and all animals were euthanized in the morning following ad libitum food consumption.

Vasoreactivity

Sacrifice of animals occurred at 16 weeks, and aortae and carotid vessels were taken from rats at sacrifice, cleaned of fat and tissue, and measured for vasoreactivity using force tension as previously described [30–33]. Denuding was completed mechanically; interior tissue was either rubbed gently with tweezers (aorta) or bubbled with air using a syringe (carotid), and we have previously determined that these techniques result in significantly less response to ACh as compared with intact. We also conducted a Student's t test to ensure denuding caused no mechanical damage; we observed no significant differences in paired aorta or carotid. To quantify vasoreactivity, tissue (2 mm rings) was mounted on a stainless steel hook and a force-displacement transducer (Grass Instruments Co., West Warwick, Rhode Island, USA) while incubated in a bath at 1.5 g basal tension for aortae and 1.0 g for carotids; baths contained Krebs buffer (119 mmol/l NaCl, 4.7 mmol/1 KCl, 2.5 mmol/l CaCl2, 1 mmol/l MgCl2, 25 mmol/l NaHCO3, 1.2 mmol/l KH2PO4, and 11 mmol/l D-glucose) and continuously bubbled with 95% O2 and 5% CO2. Constriction was conducted by exposure to 80 mmol/l KCl. A phenylephrine dose–response curve was also done with doses ranging from 0.002 to 0.7 μmol/l. To investigate vasodilation, a dose–response curve with ACh was performed with a range of 0.05–20 μmol/l secondary to phenylephrine constriction. Data was collected using AcqKnowledge software.

Respiration

Mitochondrial respiration was measured using Oroboros Oxygraph-2k (O2k; Oroboros Instruments Corp., Innsbruck, Austria). The aortae and carotid vessels (n = 8) were placed in a mitochondrial preservation buffer [BIOPS (10 mmol/l Ca-EGTA, 0.1 mmol/l free calcium, 20 mmol/l imidazole, 20 mmol/l taurine, 50 mmol/l K-MES, 0.5 mmol/l DTT, 6.56 mmol/l MgCl2, 5.77 mmol/l ATP, 15 mmol/l phosphocreatine, pH 7.1)], stored on ice, cleaned of fat and connective tissue, and permeabilized by incubation with saponin (40 mg/ml) in BIOPS on ice on a shaker for 30 min. The vessels were then washed for 10 min on ice on a shaker in mitochondrial respiration buffer [MiR06 (0.5 mmol/l EGTA, 3 mmol/l magnesium chloride, 60 mmol/l K-lactobionate, 20 mmol/l taurine, 10 mmol/l potassium phosphate, 20 mmol/l HEPES, 110 mmol/l sucrose, 1 g/l bovine serum albumin, 280 U/ml catalase, pH 7.1)]. The vessels were then transferred to MiR06 that had been prewarmed to 37 °C in the chamber of the O2k. Oxygen concentration in the MiR06 started at approximately 400 nmol/ml and was maintained above 250 nmol/ml. Substrates and inhibitors were added to assess respiration rates at several states, including background consumption with carbohydrate or lipid only (state 2), oxidative phosphorylation (+ADP, state 3), maximum oxidative phosphorylation (succinate, state 3S), state 4 (+oligomycin), and uncoupled state (+FCCP). For the carbohydrate experiment, (pyruvate/malate/glutamate-driven), respiration rates were measured with the final concentrations of 5 mmol/l pyruvate + 2 mmol/l malate + 10 mmol/l glutamate, 2 mmol/l adenosine diphosphate (ADP), 6 mmol/l succinate, 4 mg/ml oligomycin, and 0.5 mmol/l stepwise titration of 1 mmol/l carbonyl cyanide 4-trifluoromethoxy) phenylhydrazone (FCCP) until maximal uncoupling (uncoupled state). Only aorta was subjected to the carbohydrate experiment. In the lipid experiment (palmitoylcarnitine-driven respiration), rates were measured with 5 μmol/l palmitoylcarnitine + 1 mmol/l malate, 2 mmol/l ADP, 2 mmol/l glutamate + succinate, 4 mg/ml oligomycin, and 1 mmol/l stepwise titration of FCCP. Cytochrome c (10 mmol/l) was used to determine mitochondrial membrane integrity. Both vessels were subjected to the lipid experiment. Vessels were dried overnight at 60 °C and weighed for dry weight normalization. Using the Oroboros DatLab software, traces were analyzed by averaging a stable trace segment of 3–5 min for each state.

Insulin and glucose intraperitoneal tolerance tests

Insulin tolerance testing (ITT) was done at 1 and 16 weeks of the study, following a 6 h fast, by injection of 1 U/kg body weight of insulin. Blood glucose concentrations were sampled at 0, 15, 30, 45, 60, and 120 min postinjection. Glucose tolerance testing (GTT) followed the same protocol using 1.5 g/kg body weight of glucose, injected intraperitoneally, and was separated from ITT testing by 4 days. Baseline concentrations were subtracted for area under the curve (AUC) analyses. Fasting glucose and insulin concentrations were taken as the 0-min blood collection during GTT.

Western blotting

Aortae were flash-frozen in nitrogen and later processed in mammalian lysis buffer (MPER with 150 mmol/l sodium chloride, 1 mmol/l of EDTA, 1 mmol/l EGTA, 5 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 20 mmol/l sodium fluoride, 500 nmol/l okadaic acid, 1% protease inhibitor cocktail). Aortae were ground under nitrogen with a mortar and pestle, and homogenized at 4 °C and centrifuged first at 1000g for 2 min, and supernatants subsequently centrifuged 16 400g at 4 °C for 10 min. The Bradford protein assay was used to measure the protein concentration of the lysate. Protein samples (15–40 μg) in Laemmli sample buffer [LSB, boiled with 100 mmol/l dithiothreitol (DTT)] were run on precast SDS-4–15% polyacrylamide gels. Proteins were transferred to PVDF membranes. Quantity One, Bio-Rad, was used to evaluate protein loading. Blots were probed with antibodies described above and left overnight at 4 °C. Fluorescent secondary antibodies were applied following the primary antibody incubation (1 : 10 000 IRDye800CW, 1 : 10 000 IRDye680RD and Starbright Blue 700), 1 h at room temperature, protected from light). Total and targeted proteins were detected by fluorescence. Total protein was measured using the stain-free gels and associated protocols on the ChemiDoc Imaging System (Bio-Rad, Hercules, California, USA) using the Quantity One 1-D Analysis software (Bio-Rad). All protein data has been normalized to loading control and total protein expression. For determining the ratio of phosphorylated signal to total signal following total protein normalization, antibodies were probed on the same blot using different animal primary antibodies between the phosphorylated and total protein (rabbit vs. mouse) allowing for two color detection and analysis when used with secondary florescent antibodies with differing wavelengths (IRDye 680RD and IRDye 800CW). For nondenatured western blotting, we followed detailed protocols from a previous study [34]. Briefly, samples were prepared with LSB containing 2.5% β-mercaptoethanol, without SDS and DTT, and not denatured. Gels were loaded and run at 4 °C at 20 V overnight in running buffer containing SDS to protect from heat-related denaturing, and transfers were also conducted at 4 °C at 75 V for 2.5 h. eNOS antibody settings, imaging, and analysis were completed as described above.

Glutathione and thiobarbituric acid reactive substances

GSH and thiobarbituric acid reactive substances (TBARS) concentrations were assessed in plasma using kits and instructions from Abcam (Cambridge, UK) and Cayman Chemical (Ann Arbor, Michigan, USA), respectively. For GSH measurements, samples were deproteinated using trichloroacetic acid (TCA) precipitation, according to the kit protocol.

Statistical analysis

To analyze data with time/dose along with diet and temperature, we employed a repeated measures ANOVA, along with a mixed-effects model. For data without a time or dose component, we employed a two-way ANOVA for the variable temperature and diet. Tukey's post hoc analyses were conducted within ANOVA tests. A P value of less than 0.05 was used as the cutoff for statistical significance in all tests. A P value of equal or less than 0.08 was considered indicative of data trends approaching significance.

RESULTS Metabolic parameters

Rat metabolic parameters were impacted by diet, temperature, or the interaction of these variables across the study (Table 1). Body weights and insulin significantly increased over time (P < 0.05 for all), with a high-fat diet resulting in higher weight gain (P < 0.05, Table 1); Thermoneutral housing resulted in less weight gain than other groups (P < 0.05, Table 1). Fasting insulin concentrations increased to a greater degree in animals housed at thermoneutrality, as well as those on a high-fat diet (P < 0.05 for all, Table 1). There was a significant interaction effect of all variables on fasting glucose concentrations (P < 0.05, Table 1). AUC of GTT and ITT were higher in those housed at thermoneutrality as compared with diet-matched controls, approaching significance (P < 0.05 for all, Table 1). Animals on a high-fat diet had significantly greater gonadal epididymal fat depots than those on a low-fat diet (P < 0.05, Table 2). Cardiac fat was unaffected (Table 2).

TABLE 1 - Animal weight, fasting glucose and insulin concentrations, and glucose and insulin tolerance test area under the curve at 1 and 16 weeks of treatment 1 week 16 weeks Housing RT TN RT TN diet LFD HFD LFD HFD LFD HFD LFD HFD Weight (g)a,b,c,d 199.8 ± 2.6 218.8 ± 3.5 187.5 ± 4.2 182.4 ± 4.3 565.3 ± 12.2 623.3 ± 33.7 516.0 ± 34.1 578.1 ± 24.9 Glucosee (mg/dl) 79.8 ± 1.8 86.8 ± 2.2 84.9 ± 3.0 89.1 ± 3.5 70.4 ± 2.6 67.0 ± 2.9 67.2 ± 2.3 72.0 ± 2.4 Insulin (μg/ml) 0.5 ± 0.1 0.9 ± 0.2 0.5 ± 0.2 0.7 ± 0.1 1.1 ± 0.2 1.7 ± 0.3 0.9 ± 0.2 1.7 ± 0.4 GTT (AUC)c 4031 ± 464 5574 ± 482 4158 ± 523 4693 ± 388 7860 ± 2,361 9599 ± 1271 10 916 ± 1183 12 651 ± 1577 ITT (AUC) 5366 ± 711 4958 ± 454 5214 ± 469 4155 ± 383 4290 ± 509 3754 ± 467 4483 ± 285 4163 ± 330

HFD, high fat diet; LFD, low fat diet; RT, room temperature; TN, thermoneutrality. All parameters had significant effect of time (P < 0.05 for all).

aP < 0.05 diet.

bTemperature.

cTime × temperature.

eTime × diet × temperature effects.†P < 0.08 temperature.‡Diet.§Time × temperature, three-way ANOVA, mean ± SEM. GTT AUC reflects integrated glucose concentrations (mg/dl) at time 0, 15, 30, 45, 60, and 120 minutes following an IP glucose injection and ITT AUC is integrated glucose concentrations at the same timepoints following an IP insulin injection. Data were analyzed with mixed-effects and/or repeated measures three-way ANOVA. Data is mean ± SEM.


TABLE 2 - Animal cardiac and gonadal–epididymal adipose mass after 16 weeks of treatment 16 weeks Housing RT TN Diet LFD HFD LFD HFD Cardiac fat (g)† 0.19 ± 0.02 0.22 ± 0.03 0.28 ± 0.04 0.26 ± 0.02 GE fat (g)a 14.78 ± 1.47 22.02 ± 2.55 12.46 ± 1.98 19.49 ± 1.40

HFD, high fat diet; LFD, low fat diet; RT, room temperature; TN, thermoneutrality.

aP < 0.05 diet.bTemperaturecTime × temperaturedTime × dieteTime × diet × temperature effects.†P = 0.05 temperature effect, two-way ANOVA, mean ± SEM.


Housing temperature significantly alters vascular reactivity

To gauge differences in vasoreactivity, aorta and carotid tissue rings were hung on a force transducer and exposed to dose–response curves of vasoconstrictor phenylephrine and vasodilator acetylcholine (ACh). When exposed ex situ to vasodilator ACh doses, aorta from rats housed at thermonutrality demonstrated significantly less response as compared with aorta from RT-housed animals (∗P < 0.05 for dose × temperature interaction, Fig. 1  Aa). The compromised vasodilation activity was also seen in carotids from rats housed at thermoneutrality (∗P < 0.05, Fig. 1  Ad). When further analyzed, no effect of diet was noted on aorta or carotid (Fig. 1  Ab, c, e, f). In endothelium denuded vessels (Fig. 1  B), aorta from rats in thermoneutral housing showed less vasodilation than those at RT (P < 0.05, temperature effect, Fig. 1  Ba). Diet significantly impacted vasodilation in denuded aorta, regardless of housing temperature (P < 0.05, diet effect, dose × temperature and diet × temperature interactions, Fig. 1  Bb and c). Endothelium denuded carotid from rats housed at thermoneutral condition were significantly less responsive to ACh as compared with those at RT (P < 0.05, Fig. 1  Bd). Diet did not impact vasodilation in carotids ex situ (Fig. 1  Be and f). In animals housed at thermoneutrality, phenylephrine response was significantly elevated in aorta as compared with animals housed at RT (P < 0.05, Fig. 1  C). In aorta, there was a significant effect of both temperature and diet on vasoconstriction (P < 0.05 for both, Fig. 1  C), not observed in carotids (Fig. 1  C).

F1FIGURE 1:

(A–D) Vasoreactivity of aorta and carotid intact (A) and denuded (B) in response to acetylcholine or phenylephrine (C), and aorta protein expression of adenosine monophosphate protein kinase and endothelial nitric oxide synthase, western blot experiments (D), dimer and monomer eNOS protein expression, and total nitrotyrosine. Cleaned, intact vessels were attached to a force transducer and exposed to an increased dose of either ACh or PE (A–C). ACh dose–response is expressed as a percentage of fully PE-constricted vessels (A–C). PE dose–response is expressed as mN/mg normalized to vessel wet weight (C). Effect of dose was significant for both ACh and PE (P < 0.05). Effects of temperature ∗P less than 0.05, ACh/PE × temperature interaction (∗∗P < 0.05), diet (∗∗∗P < 0.05), diet × temperature interaction (∗∗∗∗P < 0.05) mixed-effects and/or repeated measures three-way ANOVA (A–C). All vessels are combined according to housing temperature (A, B, and Ca and d) or analyzed together for temperature and diet effect (A and Bb, c, e, f). For D, aorta tissue was processed for protein analysis via western blot analysis, including specific activity (Dc, f, i) (n = 8). Blots were probed for pAMPK, AMPK, peNOS, eNOS (Da–i), and nondenatured samples were used for eNOS monomer and dimer detection. Interaction effect (long bar) ∗P less than 0.05, effect of temperature (elongated bracket), ∗∗P less than 0.05, †P = 0.07 diet (symbol above data point), two-way ANOVA, (C and D) two-way analysis of variance (ANOVA). Data are mean ± SEM. ACh, acetylcholine; AMPK, adenosine monophosphate protein kinase; eNOS, endothelial nitric oxide synthase; PE, phenylephrine; SEM, standard error of the mean.

F2FIGURE 1 (Continued):

(A–D) Vasoreactivity of aorta and carotid intact (A) and denuded (B) in response to acetylcholine or phenylephrine (C), and aorta protein expression of adenosine monophosphate protein kinase and endothelial nitric oxide synthase, western blot experiments (D), dimer and monomer eNOS protein expression, and total nitrotyrosine. Cleaned, intact vessels were attached to a force transducer and exposed to an increased dose of either ACh or PE (A–C). ACh dose–response is expressed as a percentage of fully PE-constricted vessels (A–C). PE dose–response is expressed as mN/mg normalized to vessel wet weight (C). Effect of dose was significant for both ACh and PE (P < 0.05). Effects of temperature ∗P less than 0.05, ACh/PE × temperature interaction (∗∗P < 0.05), diet (∗∗∗P < 0.05), diet × temperature interaction (∗∗∗∗P < 0.05) mixed-effects and/or repeated measures three-way ANOVA (A–C). All vessels are combined according to housing temperature (A, B, and Ca and d) or analyzed together for temperature and diet effect (A and Bb, c, e, f). For D, aorta tissue was processed for protein analysis via western blot analysis, including specific activity (Dc, f, i) (n = 8). Blots were probed for pAMPK, AMPK, peNOS, eNOS (Da–i), and nondenatured samples were used for eNOS monomer and dimer detection. Interaction effect (long bar) ∗P less than 0.05, effect of temperature (elongated bracket), ∗∗P less than 0.05, †P = 0.07 diet (symbol above data point), two-way ANOVA, (C and D) two-way analysis of variance (ANOVA). Data are mean ± SEM. ACh, acetylcholine; AMPK, adenosine monophosphate protein kinase; eNOS, endothelial nitric oxide synthase; PE, phenylephrine; SEM, standard error of the mean.

F3FIGURE 1 (Continued):

(A–D) Vasoreactivity of aorta and carotid intact (A) and denuded (B) in response to acetylcholine or phenylephrine (C), and aorta protein expression of adenosine monophosphate protein kinase and endothelial nitric oxide synthase, western blot experiments (D), dimer and monomer eNOS protein expression, and total nitrotyrosine. Cleaned, intact vessels were attached to a force transducer and exposed to an increased dose of either ACh or PE (A–C). ACh dose–response is expressed as a percentage of fully PE-constricted vessels (A–C). PE dose–response is expressed as mN/mg normalized to vessel wet weight (C). Effect of dose was significant for both ACh and PE (P < 0.05). Effects of temperature ∗P less than 0.05, ACh/PE × temperature interaction (∗∗P < 0.05), diet (∗∗∗P < 0.05), diet × temperature interaction (∗∗∗∗P < 0.05) mixed-effects and/or repeated measures three-way ANOVA (A–C). All vessels are combined according to housing temperature (A, B, and Ca and d) or analyzed together for temperature and diet effect (A and Bb, c, e, f). For D, aorta tissue was processed for protein analysis via western blot analysis, including specific activity (Dc, f, i) (n = 8). Blots were probed for pAMPK, AMPK, peNOS, eNOS (Da–i), and nondenatured samples were used for eNOS monomer and dimer detection. Interaction effect (long bar) ∗P less than 0.05, effect of temperature (elongated bracket), ∗∗P less than 0.05, †P = 0.07 diet (symbol above data point), two-way ANOVA, (C and D) two-way analysis of variance (ANOVA). Data are mean ± SEM. ACh, acetylcholine; AMPK, adenosine monophosphate protein kinase; eNOS, endothelial nitric oxide synthase; PE, phenylephrine; SEM, standard error of the mean.

Thermoneutral housing impacts cellular signaling associated with vasoreactivity and nutrient sensing

To address effects of housing temperature on cellular signaling upstream of vasoreactivity and mitochondrial function, we measured protein expression of nutrient sensor and regulator of eNOS, AMPK, as well as vasodilator eNOS itself. There was a significant interaction effect of diet and temperature on phosphorylated AMPK protein expression in aorta (P < 0.05, Fig. 1  Da), but no significance was observed in AMPK specific activity (Fig. 1  Da). Phosphorylated eNOS was significantly lower in those housed at thermoneutrality (P < 0.05, Fig. 1  1Da). There was a significant interaction effect of temperature × diet, resulting in a greater difference in expression between the room temperature low-fat diet and room temperature high-fat diet groups than between the thermoneutral groups (P < 0.05, Fig. 1  D). Total eNOS protein expression was not different between groups (Fig. 1  D). eNOS specific activity was lower in rats housed at thermoneutrality (Fig. 1  D). A nondenatured gel was used to measure dimer and monomer eNOS concentrations in rat aorta (Fig. 1  D) to ascertain the presence of the active dimer form or inactive monomer form of eNOS. Monomer protein expression was significantly reduced in those housed at room temperature and on a low-fat diet, indicative of less NO generation, as well as in both groups housed at thermoneutral (P < 0.05, Fig. 1  D). There were no differences between groups in dimer expression (Fig. 1  D). To ascertain whether nitric oxide contributed to a climate of excess reactive nitrogen species, we measured total nitrotyrosine via protein expression. Significantly less nitrotyrosine was observed in aorta from rats housed at thermoneutral (P < 0.05, Fig. 1  Di).

Lower lipid substrate mitochondrial respiration and altered protein expression of mitochondrial complexes with thermoneutrality

To determine the impact of thermoneutrality on mitochondrial function, we measured mitochondria oxygen consumption using an Oroboros Oxygraph 2k closed chamber. Vessels were exposed to substrates and inhibitors mimicking carbohydrate and lipid metabolism in various states of oxidative phosphorylation. In both aorta and carotid vessels, mitochondrial states 3S (ATP production), 4 (membrane potential maintenance), and uncoupled (maximal uncoupled from ATP production) respiration were significantly diminished in thermoneutrally housed animals (P < 0.05 for all, Fig. 2A and B), regardless of diet. In aorta, thermoneutral resulted in significantly less mitochondrial respiration in state 2 (P < 0.05, Table 3) and carotid state 3 was diminished with diet, approaching significance (P < 0.08, Table 3). No impact on RCR was observed in either vessel (Table 3). To ascertain impacts of thermoneutral housing on mitochondrial complex protein expression, western blotting was used. Thermoneutral housing along with a high-fat diet elevated complex III expression, approaching significance (P = 0.08, Fig. 2Cc) Complex IV was also elevated in aorta from animals housed at thermoneutrality, near significance (P = 0.08, Fig. 2Cd).

F4FIGURE 2:

(A–C) Mitochondrial respiration in aorta (Aa–c) and carotid (Ba–c), lipid metabolism, and aorta protein expression of mitochondrial complexes (Ca–e). Permeabilized vessels were exposed to substrates and inhibitors mimicking lipid metabolism and background oxygen consumption or leak state (state 2), oxidative phosphorylation (+ADP, state 3), maximum oxidative phosphorylation [succinate, state 3S (A and Ba)], state 4 [+oligomycin (A and Bb), and uncoupled respiration [+FCCP (A and Bc)] were determined. Respiration rates were normalized to tissue dry weight (n = 7–8). Effect of temperature ∗P less than 0.05, +P less than 0.08, two-way ANOVA. For (Ca–e), aorta tissue was processed for protein analysis via western blot analysis (n = 8). Blots were probed for mitochondrial complexes I–IV using a single antibody containing subunits of all complexes. +P = 0.08 interaction (long bar) and temperature (elongated bracket) effect, two-way ANOVA; (Ca–e) data are mean ± SEM. ANOVA, analysis of variance.

TABLE 3 - Mitochondrial respiration states and respiratory control ratios of aorta and carotid Respiration state/ratio Aorta RT LFD Aorta RT HFD Aorta TN LFD Aorta TN HFD 2∗ 4.034 ± 0.79 3.924 ± 0.55 2.612 ± 0.54 2.527 ± 0.45 3 9.211 ± 2.35 6.277 ± 1.09 6.042 ± 1.13 7.392 ± 0.75 RCR 1.351 ± 0.05 1.343 ± 0.05 1.332 ± 0.05 1.336 ± 0.04 Respiration state/ratio Carotid RT LFD Carotid RT HFD Carotid TN LFD Carotid TN HFD 2 4.955 ± 0.80 5.292 ± 0.66 6.065 ± 0.58 4.289 ± 0.92 3† 11.470 ± 1.31 8.395 ± 1.03 9.412 ± 1.26 7.528 ± 1.23 RCR 1.951 ± 0.15 1.816 ± 0.11 1.952 ± 0.21 2.079 ± 0.18

Vessels were exposed to lipid substrates and inhibitors to assess respiration rates at several states, including background oxygen consumption or leak state (state 2), oxidative phosphorylation (+ADP, state 3), maximum oxidative phosphorylation (succinate, state 3S), state 4 (+oligomycin), and uncoupled respiration (+ FCCP). RCR was calculated as state 3S normalized to state 4. HFD, high-fat diet; LFD, low-fat diet; RCR, Respiratory control ratio; RT, room temperature; TN, thermoneutrality.

∗P less than 0.05, temperature effect.

†P less than 0.05 diet effect, two-way ANOVA. Data are presented in O2 flux per mass pmol (s/mg).


Thermoneutrality is associated with decreased insulin secretion

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