Restriction of advanced glycation end products improves insulin resistance in human type 2 diabetes: potential role of AGER1 and SIRT1

Abstract

Objective: Increased oxidative stress (OS) and impaired anti-OS defenses are important in the development and persistence of insulin resistance (IR). Several anti-inflammatory and cell-protective mechanisms, including advanced glycation end product (AGE) receptor-1 (AGER1) and sirtuin (silent mating-type information regulation 2 homolog) 1 (SIRT1) are suppressed in diabetes. Because basal OS in type 2 diabetic patients is influenced by the consumption of AGEs, we examined whether AGE consumption also affects IR and whether AGER1 and SIRT1 are involved.

Research design and methods: The study randomly assigned 36 subjects, 18 type 2 diabetic patients (age 61±4 years) and 18 healthy subjects (age 67±1.4 years), to a standard diet (>20 AGE equivalents [Eq]/day) or an isocaloric AGE-restricted diet (<10 AGE Eq/day) for 4 months. Circulating metabolic and inflammatory markers were assessed. Expression and activities of AGER1 and SIRT1 were examined in patients' peripheral blood mononuclear cells (PMNC) and in AGE-stimulated, AGER1-transduced (AGER1+), or AGER1-silenced human monocyte-like THP-1 cells.

Results: Insulin and homeostasis model assessment, leptin, tumor necrosis factor-α and nuclear factor-κB p65 acetylation, serum AGEs, and 8-isoprostanes decreased in AGE-restricted type 2 diabetic patients, whereas PMNC AGER1 and SIRT1 mRNA, and protein levels normalized and adiponectin markedly increased. AGEs suppressed AGER1, SIRT-1, and NAD+ levels in THP-1 cells. These effects were inhibited in AGER1+ but were enhanced in AGER1-silenced cells.

Conclusions: Food-derived pro-oxidant AGEs may contribute to IR in clinical type 2 diabetes and suppress protective mechanisms, AGER1 and SIRT1. AGE restriction may preserve native defenses and insulin sensitivity by maintaining lower basal OS.

Figure 1

A: AGE restriction reduces IR and improves inflammation in type 2 diabetic patients. Changes after AGE restriction (×4 months) in circulating factors (by ELISAs), plasma insulin, and HOMA, leptin, adiponectin, serum CML, MG, or in PMNCs, and AGER1, SIRT1, RAGE mRNA (by RT-PCR), are shown as percentage (mean ± SEM) above or below the baseline. *P < 0.050. Inset: Leptin (Lep)/adiponectin (Adipo) ratio before and after treatment is shown relative to normal control subjects (NL, at baseline, open bars). B: AGE restriction enhances SIRT1 and AGER1 protein expression in PMNCs of type 2 diabetic patients. PMNCs obtained at entry (1) and at the end of the study (2) are shown for subjects exposed to AGE restriction (AGE-Restr) vs. regular diet (Reg). SIRT1 and AGER1 protein levels were assessed by Western blotting (upper panels), followed by densitometric analysis (lower panels). C: AGE restriction enhances SIRT1 deacetylation of NF-κB p65. Levels of NF-κB p65 acetylation are shown at entry (1) and at the end of study (2) after immunoprecipitation (IP) and immunoblotting (IB) against acetyl-lysine residues. Data are shown as the percentage (mean ± SEM) change from entry (1). P values are as indicated.

Figure 2

AGEs suppress AGER1, SIRT1 protein, and NAD⁺ levels as well as NF-κB p65 deacetylation in THP-1 cells. A: Western blots (upper panels) and densitometry (lower panels) results are shown for AGER1 (black bars) and SIRT1 (gray bars) protein expression in THP-1 cells (wild-type [WT]) stimulated with CML-BSA (150 μg/mL), MG-BSA (60 μg/mL), and BSA (60 μg/mL) for 72 h. B and C: MG-induced effects on SIRT1 are AGER1-dependent. WT or THP-1 cells transfected with AGER1 (AGER1⁺) or short-hairpin RNA for AGER1 (shAGER1) were stimulated by MG (60 μg/mL) for 24 h before Western blots (upper panels) and densitometry plots (lower panels; AGER1, black bars; SIRT1, gray bars; WT, open bars). Data (mean ± SEM) of three to five experiments, derived from test/β-actin ratio, are shown as fold above control (cells alone, WT). *P < 0.001 vs. BSA or cells alone. #P < 0.002 vs. maximal values. D and E: AGE-induced effects on SIRT1 are NAD⁺-dependent and regulated by OS (D) and AGER1 (E). THP-1 cells were cultured with MG-BSA (60 μg/mL) or media (CL) for up to 72 h prior to Western blotting for SIRT1 (top inset) and NAD⁺/NADH ratio in the presence or absence of antioxidants (NAC or apocynin) in WT or AGER1⁺-transduced cells (E). NAD⁺/NADH ratio is shown as fold (mean ± SE) above control (n = 3, each in triplicate). *P < 0.001 vs. control. §P < 0.002 vs. maximal increase. F: NF-κB p65 hyperacetylation is induced by AGEs but is blocked by AGER1. Acetyl-p65 was determined in THP-1 cells, after MG stimulation (60 μg/mL) for 72 h in the presence or absence of SIRT1 inhibitor, sirtinol (10 μmol/L). Western blots and density plots are shown as mean ± SEM from four independent experiments. *P < 0.002 vs. nonstimulated or vs. nontransduced THP-1 cells.