Diurnal Variation in Vascular and Metabolic Function in Diet-induced Obesity

Divergence of Insulin Resistance and Loss of Clock Rhythm

Madhu J. Prasai; Romana S. Mughal; Stephen B. Wheatcroft; Mark T. Kearney; Peter J. Grant; Eleanor M. Scott


Diabetes. 2013;62(6):1981-1989. 

In This Article

Research Design and Methods


Three-week-old male C57BL6J mice were purchased from Harlan Laboratories UK and randomized either to high-fat diet (Bio-serv, Frenchtown, NJ; nutritional content by weight: protein, 20%; fat, 35.5%; carbohydrate, 36.3%; fat content by calories: 60%) or standard laboratory chow (Special Diet Services, Essex, U.K.) as previously described.[3,28] Animals were housed in a standard animal facility under controlled temperature and humidity with a regular lighting schedule of 12 h light:12 h dark (lights on 7:00 A.M. and lights off 7:00 P.M.). Experiments were performed after 10 weeks of diet in accordance with U.K. Home Office regulations for animal care. Aortic vasomotion studies, in vivo metabolic tests, and Akt immunoblotting were performed at 8:00 A.M. and 8:00 P.M., which in nocturnal mice correspond to the beginning of the inactive and active phases, respectively. BP measurement, quantitative PCR, AMPK immunoblotting, and plasma insulin measurement were performed at 8:00 A.M., 2:00 P.M., 8:00 P.M., and 2:00 A.M. to yield 24-h expression profiles.

Quantitative PCR

Tissues were snap-frozen in liquid nitrogen and prepared by mechanical homogenization (TissueLyser; Qiagen, Dusseldorf, Germany). RNA was extracted using Trizol reagent (Invitrogen, Paisley, U.K.) and reverse transcribed using a kit (Applied Biosystems, Warrington, U.K.). Quantitative PCR was performed in triplicate in a thermal cycler (ABI Prism 7900HT; Applied Biosystems) using custom-synthesized oligonucleotides (Invitrogen, Paisley, U.K.) with the SybrGreen fluorescent dye system (Applied Biosystems), and results were normalized against the reference gene Gapdh. Primer sequences are given in Supplementary Fig. 1.

Figure 1.

The effect of obesity on rhythmic transcription of the core clock genes Bmal1 and Per2 in vascular and metabolic tissues. Aorta (A); liver (B); adipose tissue (C); skeletal muscle (D). Results from obese animals are denoted by circles and lean by triangles. Two-way ANOVA with Bonferroni post hoc correction: *P < 0.05 and #P < 0.001; n = 4 in each group.

Ex Vivo Aortic Vasomotion Studies

Vasomotor activity was measured as previously described.[3,5–7,28,29] The thoracic aorta was debrided of adherent connective tissue and cut into rings of 5-mm length. Rings were mounted in an organ bath apparatus (PanLabs, Barcelona, Spain) immersed in Krebs-Henselheit solution (composition in mmol/L: NaCl 119, KCl 4.7, KH2PO4 1.18, NaHCO3 25, MgSO4 1.19, CaCl2 2.5, glucose 11.0) maintained at 37°C and bubbled with 95% O2/5% CO2. After incubation at a resting tension of 3 g for 45 min, vasoconstriction was assessed by cumulative stimulation with the α-adrenoceptor agonist phenylephrine (PE) (1 nmol/L to 1 μmol/L). After washout and reincubation at resting tension, rings were preconstricted with 300 nmol/L PE, and endothelial-dependent vasodilation was assessed by cumulative stimulation with the vasodilator acetylcholine (ACh; 1 nmol/L to 1 μmol/L). Insulin-mediated vasodilation was examined by incubation for 2 h with insulin (100 mU/mL; Actrapid) followed by cumulative stimulation with PE. Basal NO release was examined by incubation with the nonspecific NOS inhibitor N G-monomethyl-l-arginine (L-NMMA; 0.1 mol/L) followed by cumulative stimulation with PE.

Noninvasive BP and Heart Rate Measurement

Systolic and diastolic BP and heart rate were measured by tail-cuff plethysmography (Kent Scientific, Torrington, U.K.) as previously described.[3,5–7] Conscious mice were placed in a cylindrical restraining device on a warmed holding platform, and mean values for systolic and diastolic BP and heart rate were calculated from 30 recorded cycles. Three training sessions were performed before measurements were obtained.

Metabolic Profiling

In vivo metabolic testing was performed as previously described.[3,5–7,28,29] For glucose tolerance testing, mice were fasted for 12 h, followed by intraperitoneal injection of 1 mg/kg glucose. For insulin tolerance testing, mice were fasted for 4 h, followed by intraperitoneal injection of 0.75 units/kg insulin (Actrapid; NovoNordisk, Bagsvaerd, Denmark). Whole-blood glucose was determined sequentially by tail vein sampling using a portable meter (Accu-chek Aviva; Roche Diagnostics, Burgess Hill, U.K.). For plasma insulin quantification, blood was collected by terminal cardiac puncture and plasma was obtained by centrifugation. Insulin was measured using an ultrasensitive mouse ELISA kit (CrystalChem, Downers Grove, IL) as previously described.[6,7,28,29]

Protein Immunoblotting

Protein was extracted in lysis buffer by mechanical homogenization and quantified according to colorimetric assay (BCA protein assay kit, Calbiochem, San Diego, CA). Protein (30 μg) was loaded into 4–12% Bis-Tris gels (NuPage; Invitrogen Life Sciences, Carlsbad, CA), separated by electrophoresis, and blotted onto polyvinylidene fluoride membrane as previously described.[28,29] Blots were probed with primary antibody as follows: eNOS 1:1,000 and Ser1177-phosphorylated eNOS 1:666 (both BD Biosciences, San Jose, CA); AMPK α-subunit 1:5,000 (a gift of Grahame Hardie, University of Dundee, Dundee City, U.K.); Akt 1:1,000 and Thre308-phosphorylated Akt 1:1,000 (both Cell Signaling Technology, Danvers, MA); and β-actin 1:3,000 (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were incubated with 1:1,000 secondary antibody conjugated to horseradish peroxidase (Dako, Glostrup, Denmark) and visualized with SuperSignal West Pico chemiluminescent substrate (ThermoFisher Scientific, Rockford, IL). Protein band densitometry data were normalized against β-actin.

Statistical Analysis

Data are expressed as mean ± SEM. Analysis was performed by unpaired Student t test or ANOVA with Bonferroni post hoc correction as indicated. Significance was accepted at P < 0.05.