Oral AGE restriction ameliorates insulin resistance in obese individuals with the metabolic syndrome: a randomised controlled trial

Abstract

Aims/hypothesis: We previously reported that obese individuals with the metabolic syndrome (at risk), compared with obese individuals without the metabolic syndrome (healthy obese), have elevated serum AGEs that strongly correlate with insulin resistance, oxidative stress and inflammation. We hypothesised that a diet low in AGEs (L-AGE) would improve components of the metabolic syndrome in obese individuals, confirming high AGEs as a new risk factor for the metabolic syndrome.

Methods: A randomised 1 year trial was conducted in obese individuals with the metabolic syndrome in two parallel groups: L-AGE diet vs a regular diet, habitually high in AGEs (Reg-AGE). Participants were allocated to each group by randomisation using random permuted blocks. At baseline and at the end of the trial, we obtained anthropometric variables, blood and urine samples, and performed OGTTs and MRI measurements of visceral and subcutaneous abdominal tissue and carotid artery. Only investigators involved in laboratory determinations were blinded to dietary assignment. Effects on insulin resistance (HOMA-IR) were the primary outcome.

Results: Sixty-one individuals were randomised to a Reg-AGE diet and 77 to an L-AGE diet; the data of 49 and 51, respectively, were analysed at the study end in 2014. The L-AGE diet markedly improved insulin resistance; modestly decreased body weight; lowered AGEs, oxidative stress and inflammation; and enhanced the protective factors sirtuin 1, AGE receptor 1 and glyoxalase I. The Reg-AGE diet raised AGEs and markers of insulin resistance, oxidative stress and inflammation. There were no effects on MRI-assessed measurements. No side effects from the intervention were identified. HOMA-IR came down from 3.1 ± 1.8 to 1.9 ± 1.3 (p < 0.001) in the L-AGE group, while it increased from 2.9 ± 1.2 to 3.6 ± 1.7 (p < 0.002) in the Reg-AGE group.

Conclusions/interpretation: L-AGE ameliorates insulin resistance in obese people with the metabolic syndrome, and may reduce the risk of type 2 diabetes, without necessitating a major reduction in adiposity. Elevated serum AGEs may be used to diagnose and treat 'at-risk' obesity.

Trial registration: ClinicalTrials.gov NCT01363141 FUNDING: The study was funded by the National Institute of Diabetes and Digestive and Kidney Diseases (DK091231).

Keywords: AGER1; Cardiovascular disease; Diabetes; Glycotoxins; Inflammation; Innate defence; RAGE; SIRT1.

Fig. 1. CONSORT study flow diagram

Fig. 2

Effects of L-AGE diet (black bars) vs Reg-AGE diet (white bars) on metabolic variables and systemic markers of AGEs, oxidative stress and inflammation (also see Table 2). (a) Markers of insulin resistance: per cent changes (means ± SEM) in levels of HOMA, leptin and fasting insulin between baseline and end of study (Reg-AGE, n=43; L-AGE, n=51; *p≤0.05). (b) Plasma insulin after OGTT: per cent changes (means ± SEM) in plasma insulin levels 0 and 120 min after OGTT between baseline and end of study (Reg-AGE, n=43; L-AGE, n=51; *p≤0.05). (c) AGEs and pro-oxidative stress and inflammation markers: per cent changes (means ± SEM) in markers of serum (s) AGEs (CML, MG), plasma 8-isoprostanes (8-iso) and PMNC inflammatory factors (TNFα protein and RAGE mRNA) between baseline and end of study (Reg-AGE, n=33; L-AGE, n=21; *p≤0.05). (d) Anti-oxidative stress and inflammation markers: per cent changes (means ± SEM) in levels of antioxidants and markers of host defence, SIRT1, AGER1 and GLO1 mRNA, between baseline and end of study (Reg-AGE, n=33; L-AGE, n=21; *p≤0.05)

Fig. 3

Baseline ex vivo cellular data: AGEs suppress host defences and promote inflammatory activation of PMNCs in individuals with the metabolic syndrome (MS) compared with normal individuals (NL). (a) Expression levels of anti-oxidative stress and inflammation factors, SIRT1 (white bars) and AGER1 (black bars), and (b) of the pro-oxidative stress and inflammation cytokine TNFα protein in PMNCs freshly obtained at study commencement (baseline), from obese study MS participants (MS-PMNCs) (black bar) compared with that in PMNCs simultaneously obtained from NL (white bar). Data in (a) are from western blots and densitometry, shown as AU or ratio of target protein to β-actin (means ± SEM) and in (b) as (means ± SEM) pg/mg cell protein by ELISA (n=10/group, each in triplicate; *p≤0.05 vs NL). (c) Baseline MS-PMNCs were exposed ex vivo to MG-BSA 60 μg/ml for 72 h, in the presence or absence of a SIRT1 activator, SRT1720 (1 μmol/l), or a SIRT1 inhibitor, sirtinol (10 μmol/l). Western blots, performed on cell extracts, and density analysis (means ± SEM) (n=3–5) are shown as the ratio of SIRT1 (white bars) or AGER1 (black bars) to β-actin; *p≤0.05 vs non-stimulated MS-PMNCs (control, CL); ^† p≤0.05 vs MG alone. (d) MS-PMNC extracts, prepared as in (c), were also probed for levels of p-JNK (black bars) and acetylated NFκB p65 at lys310 (Acl-p65) (white bars) by western blots, using the respective antibodies and density analyses. Total JNK and p65 served as internal controls (CL). Density data (means ± SEM, n=3–5) indicate the ratios of phosphorylated or acetylated protein to total protein; *p≤0.05 vs non-stimulated CL; ^† p≤0.05 vs MG alone. (e) TNFα secreted into the culture medium at 72 h from non-stimulated MS-PMNCs (CL, white bar) or MG-stimulated MS-PMNCs (black bars) at baseline, in the presence or absence of SRT1720 (1 μmol/l) or sirtinol (10 μmol/l), as above. Assays were performed in triplicate for each sample. Data (means ± SEM) are shown as per cent of control (n=7, *p≤0.05 vs CL; ^†p≤0.05 vs MG alone). All PMNC samples were collected at study baseline

Fig. 4

End-of-study ex vivo cellular data: L-AGE diet mitigates inflammatory activation of PMNCs from individuals with the metabolic syndrome (MS-PMNCs) and improves their impact on insulin responses of adipocytes in vitro. (a) TNFα from MS-PMNCs secreted into the conditioned media, after 1 year on L-AGE (black bar, n=5) or Reg-AGE diet (white bar, n=5). Data are shown as means ± SEM pg/ml medium, each in triplicate; *p≤0.05 L-AGE vs Reg-AGE diet. (b) JNK phosphorylation in differentiated 3T3-L1 adipocytes after overnight incubation with conditioned medium either from baseline (BL) or end-of-study PMNCs from L-AGE (black bar, n=5) and Reg-AGE (white bar, n=5) treatment groups. Cell lysates were subjected to western blot and densitometry. As additional control, 3T3-L1 adipocytes were incubated with normal culture medium, shown as 0. Density data are means ± SEM (n=3–5 independent experiments); *p≤0.05, L-AGE or Reg-AGE vs BL; ^† p≤0.05, L-AGE vs Reg-AGE diet. (c) Serine phosphorylation (at Ser473) of Akt and tyrosine phosphorylation of insulin receptor (IR) in 3T3-L1 adipocytes after exposure to conditioned medium (CM) overnight as in (b). Cells were incubated in the presence (black bars) or absence of insulin (Ins) (white bars) (100 nmol/l, 30 min) and subjected to western blot and densitometry (means ± SEM, n=3 independent experiments); *p≤0.05 L-AGE vs BL. PMNC samples (n=7) were from the same individuals evaluated at baseline (Fig. 2, n=7). 3T3-L1 adipocytes incubated with normal CM are shown as 0