Fig 1.
Study design and insulin clamp protocol for the conscious unrestrained mouse.
A: All mice were randomized to either chow or HF diet at 3 weeks of age. Studies were performed at 12 weeks of age. BC: body composition; EB: energy balance. B: Insulin clamp protocol. C: Insulin clamp setup. Mice are never handled nor retrained after they are hooked up to the swivel (t = -90min). [14C]2DG: 2[14C]deoxyglucose.
Table 1.
Characteristics of the clamped WT and SIRT2 KO mice on a chow or HF diet.
Fig 2.
HF SIRT2 KO mice exhibit increased adiposity and energy expenditure.
(A) Body weight of the chow WT and SIRT2 KO mice. Lean (B) and fat mass (C) in WT and SIRT2 KO mice on a chow diet at 12 weeks of age. Energy Expenditure (EE) over time (D) or ANCOVA-adjusted 12h average of EE (E) in chow-fed WT or SIRT2 KO mice. RQ over time (F) or expressed as 12h averages (G) in chow-fed WT or SIRT2 KO mice. (H) Body weight of the HF-fed WT and SIRT2 KO mice. Lean (I) and fat mass (J) in WT and SIRT2 KO mice on a HF diet at 12 weeks of age. Energy Expenditure (EE) over time (K) or ANCOVA-adjusted 12h average of EE, regressed to body weight (L) in HF-fed WT or SIRT2 KO mice. RQ over time (M) or expressed as 12h averages (N) in HF-fed WT or SIRT2 KO mice. @ p<0.05 WT vs. SIRT2 KO by ANCOVA when regression is run against body weight, absolute fat mass or absolute fat-free mass. There was no interaction between body weight, lean mass, fat mass, and Energy Expenditure. (n = 8/group for all panels). Black bars: WT; open bars: SIRT2 KO.
Fig 3.
Feeding behavior was altered in HF SIRT2 KO mice.
Total food intake in kcal per 12h cycle (A, I), time spent eating per 12h cycle (B, J), average individual meal size (C, K), number of meals per 12h cycle (D, L), average individual meal duration (E, M) and average time interval between meals (F, N) in chow and HF-fed mice, respectively, during either dark or light cycle (average of four days). Water intake (G, O) and locomotor activity (H, P) in chow or HF-fed mice respectively, during either dark or light cycle (average of four days). (n = 8/group for all panels). Black bars: WT; open bars: SIRT2 KO.
Fig 4.
SIRT2 KO mice exhibit muscle insulin resistance during the hyperinsulinemic-euglycemic clamp.
A, E: Blood glucose was monitored throughout the clamp at 10-min intervals by sampling from the arterial catheter. Blood glucose was maintained at euglycemia (∼150 mg/dL) in both chow-fed (A) and HF-fed (E) mice. The GIR in the venous catheter needed to maintain euglycemia in chow-fed (B) or HF-fed (F) mice. Plasma insulin levels at baseline and during the clamp in chow (C) or HF-fed mice (G). Rd, rate of glucose disappearance, in chow-fed (D) or HF-fed (H) mice, determined by administration of [3-3H]glucose. Rg in gastrocnemius, vastus lateralis, white epidydimal adipose tissue, and brain is determined by the administration of nonmetabolizable 2[14C]deoxyglucose in chow-fed (I) or HF-fed (J) WT and SIRT2 KO mice. (n = 7-13/group, see Table 1). Black bars: WT; open bars: SIRT2 KO. For clamp data of weight-matched HF subgroup, see Table 1 and Fig 8.
Fig 5.
Insulin-induced skeletal muscle Akt phosphorylation were reduced in SIRT2 KO mice.
A, D: Immunoblots for P-Akt (Ser473), Akt, and β-actin on gastrocnemius homogenates from chow-fed and HF-fed insulin clamped mice. Integrated intensities for P-Akt were obtained by the Odyssey software and normalized to Akt intensities (n = 7/group). B, E: Immunoblots for acetylated lysine and β-actin on gastrocnemius homogenates from chow-fed and HF-fed insulin clamped mice. Integrated intensities were obtained by the Odyssey software and normalized to β-actin intensities (n = 8/group). C, F, G: Immunoblots for acetylated lysine, GAPDH and VDAC on cytosolic or mitochondrial gastrocnemius homogenates. Integrated intensities (F, G) were obtained by the Odyssey software and normalized to GAPDH or VDAC respectively (n = 6/group). Black bars: WT; open bars: SIRT2 KO. H: Immunoblots for SIRT2 in cytosolic (top) and mitochondrial (bottom) protein fractions extracted from gastrocnemius muscle from 5h-fasted WT and SIRT2 KO mice on either a chow of HF diet. SIRT2 intensities were normalized to GAPDH (cytosolic fraction) or VDAC (mitochondrial fraction) (n = 6/group). Black bars: Chow; open bars: HF.
Fig 6.
HF SIRT2 KO mice exhibit hepatic insulin resistance.
A, B: endoRa, a marker of hepatic glucose production, in chow-fed (A) or HF-fed (B) mice, determined by administration of [3-3H]glucose during the insulin clamp (n = 7-13/group, see Table 1). C, D: Liver triglycerides (C) and liver glycogen (D) in chow and HF WT and SIRT2 KO mice (n = 7/group). E: Immunoblots for P-IRS1 (Ser302), IRS1, P-Akt (Ser473), Akt, P-FOXO1 (Ser256), FOXO1 and GAPDH on liver homogenates from clamped mice (n = 7/group). The uncropped Western blot is presented in S5 Fig. F, G, H: Integrated intensities were obtained by the Odyssey software and phospho-proteins were normalized to their respective total protein intensities (n = 7/group). I, J, K: Hepatic mRNA relative expression for adgre (F4/80) (I), il1b (J), il6 (K) from HF WT and SIRT2 KO mice (n = 8/group). Black bars: WT; open bars: SIRT2 KO.
Fig 7.
SIRT2 KO livers exhibit increased protein acetylation in both cytosolic and mitochondrial fractions.
A, B. Immunoblots for acetylated lysine on whole liver (A) or cytosolic and mitochondrial liver fractions (B). Integrated intensities (D, E, F) were obtained by the Odyssey software and normalized to β-actin, GAPDH or VDAC intensities, respectively (n = 6/group).
Fig 8.
Weight-matched HF WT and SIRT2 KO mice only exhibit increased muscle insulin resistance during the insulin clamp.
Mice whose body weights were within 1SD of the average body weight of all HF mice were included (38.4 ± 4.2 grams). Body weights of weight-matched HF subgroup are presented in Table 1. A: Blood glucose was monitored throughout the clamp at 10-min intervals by sampling from the arterial catheter. Blood glucose was maintained at euglycemia (∼150 mg/dL). B: The GIR in the venous catheter needed to maintain euglycemia in weight-matched HF-fed mice. C: Plasma insulin levels at baseline and during the clamp in weight-matched HF-fed mice. D: endoRa, a marker of hepatic glucose production, in weight-matched HF-fed mice, determined by administration of [3-3H]glucose. E: Rg in gastrocnemius, vastus lateralis, white epidydimal adipose tissue and brain is determined by the administration of nonmetabolizable glucose 2[14C]deoxyglucose in weight-matched HF-fed mice. F: GIR plotted against body weight for all WT and SIRT2 KO clamped mice (chow and HF). The regression lines were generated by the ANCOVA statistical tool. For clamp data of entire HF groups, see Figs 4 and 6 and Table 1 (n = 7-10/group for all panels, see Table 1).