Nick Wlazlo

Iron Metabolism Is Associated With Adipocyte Insulin Resistance and Plasma Adiponectin The Cohort on Diabetes and Atherosclerosis Maastricht (CODAM) study

OBJECTIVE Adipocyte insulin resistance (IR) is a key feature early in the pathogenesis of type 2 diabetes mellitus (T2DM), and although scarce, data in the literature suggest a direct role for iron and iron metabolism–related factors in adipose tissue function and metabolism. Serum ferritin and transferrin were shown to be associated with muscle insulin resistance (IR) and T2DM, but little is known about the role of iron metabolism on adipose tissue. We therefore investigated whether markers of iron metabolism were associated with adipocyte IR and plasma adiponectin. RESEARCH DESIGN AND METHODS Serum ferritin, transferrin, total iron, non–transferrin-bound iron (NTBI), transferrin saturation, and plasma adiponectin were determined in 492 individuals. Adipocyte IR was defined by the product of fasting insulin and nonesterified fatty acids (NEFAs). Using linear regression analyses, we investigated the difference in adipocyte IR or adiponectin (in %) according to differences in iron metabolism markers. RESULTS Serum ferritin (β = 1.00% increase in adipocyte IR per 10 μg/L [95% CI 0.66–1.34]), transferrin (4.18% per 0.1 g/L [2.88–5.50]), total iron (1.36% per μmol/L [0.61–2.12]), and NTBI (5.14% per μmol/L [1.88–8.52]) were associated with adipocyte IR after adjustment for several covariates, including inflammatory markers. All markers of iron metabolism were also associated with NEFAs (all P < 0.01). In addition, ferritin and transferrin were inversely associated with adiponectin (both P < 0.01). CONCLUSIONS The observed associations of several markers of iron metabolism with adipocyte IR and adiponectin suggest that factors related to iron and iron metabolism may contribute to adipocyte IR early in the pathogenesis of T2DM.

Complement Factor 3 Is Associated With Insulin Resistance and With Incident Type 2 Diabetes Over a 7-Year Follow-up Period: The CODAM Study

OBJECTIVE Immune dysregulation can affect insulin resistance (IR) and β-cell function and hence contribute to development of type 2 diabetes mellitus (T2DM). The complement system, as a regulator of immune and inflammatory homeostasis, may be a relevant contributor therein. However, longitudinal studies focusing on complement as a determinant of T2DM and IR are scarce. Therefore, we prospectively investigated the association of plasma complement factor 3 (C3) with (estimates of) IR in muscle, liver, and adipocytes, as well as with glucose tolerance, including incident T2DM. RESEARCH DESIGN AND METHODS Fasting C3, nonesterified fatty acids, glucose, and insulin (the latter two during oral glucose tolerance tests) were measured at baseline (n = 545) and after 7 years of follow-up (n = 394) in a prospective cohort study. RESULTS Over the 7-year period, C3 levels (per 0.1 g/L) were longitudinally associated with higher homeostasis model assessment of IR (HOMA2-IR; β = 15.2% [95% CI 12.9–17.6]), hepatic IR (β = 6.1% [95% CI 4.7–7.4]), adipocyte IR (β = 16.0% [95% CI 13.0–19.1]), fasting glucose (β = 1.8% [95% CI 1.2–2.4]), 2-h glucose (β = 5.2% [95% CI 3.7–6.7]), and area under the curve for glucose (β = 3.6% [95% CI 2.7–4.6]). In addition, greater changes in C3 (per 0.1 g/L) were associated with greater changes in HOMA2-IR (β = 0.08 [95% CI 0.02–0.15]) and greater changes in hepatic IR (β = 0.87 [95% CI 0.12–1.61]) over 7 years, but not glucose tolerance. Moreover, baseline C3 was associated with the 7-year incidence of T2DM (odds ratio 1.5 [95% CI 1.1–2.0]). CONCLUSIONS Changes in C3 were associated with changes in several measures of IR and may reflect progression of metabolic dysregulation, which eventually leads to abnormalities in glucose tolerance and T2DM.

Low-grade inflammation and insulin resistance independently explain substantial parts of the association between body fat and serum C3: The CODAM study

OBJECTIVE To investigate the role of low-grade inflammation and insulin resistance (HOMA2-IR) in adiposity-related increases in serum complement factor 3 (C3). Although C3 has been linked to type 2 diabetes and cardiovascular diseases, and C3 levels are closely related to body fat, the underlying mechanisms explaining this association are still unknown. METHODS Adiposity measures (including BMI, waist circumference (WC), sagittal diameter and several skinfolds), HOMA2-IR and markers of inflammation (hs-CRP, IL-6, SAA, haptoglobin, ceruloplasmin, sICAM-1) were determined in 532 individuals (62% men, mean age 59±6.9 yrs) from the Cohort on Diabetes and Atherosclerosis Maastricht study. Markers of inflammation were standardized and compiled into an averaged inflammation score. Cross-sectional associations between adiposity measures and C3 and the mediating role of low-grade inflammation and/or HOMA2-IR herein were analysed with multiple linear regression models. RESULTS Adiposity measurements were significantly associated with C3 levels, with the strongest (adjusted) associations found for WC (β=0.383; 95%CI 0.302-0.464) and sagittal diameter (β=0.412; 95%CI 0.333-0.490). Further adjustment for inflammation and HOMA2-IR attenuated these associations to β=0.115 (95%CI 0.030-0.200) and β=0.163 (95%CI 0.082-0.244) respectively. Multiple mediation analyses showed that inflammation [β=0.090 (95%CI 0.060-0.126)] and HOMA2-IR [β=0.179 (95%CI 0.128-0.236)] each explained, independently of one another, a significant portion of the association between WC and C3 (23% and 47%, respectively). Similar mediation by inflammation (19-27%) and HOMA2-IR (37-56%) was found for other adiposity measures. CONCLUSION Systemic low-grade inflammation and insulin resistance may represent two independent pathways by which body fat leads to elevated C3 levels.

Activated complement factor 3 is associated with liver fat and liver enzymes: the CODAM study.

The complement system may be involved in the pathogenesis of alcoholic and nonalcoholic liver disease, although studies in humans are scarce. For this reason, we investigated whether circulating levels of activated complement factor 3 (C3a) were associated with hepatic steatosis and hepatocellular damage.

Diabetes mellitus at the time of diagnosis of cirrhosis is associated with higher incidence of spontaneous bacterial peritonitis, but not with increased mortality

DM (diabetes mellitus) is present in 20-40% of patients with liver cirrhosis, but its prognostic impact is unclear. Therefore, in the present study, we investigated whether the presence of DM in patients with cirrhosis was associated with increased mortality, and/or with increased incidence of SBP (spontaneous bacterial peritonitis). We reviewed medical and laboratory data of 230 patients with cirrhosis from the period 2001-2011, for whom data were complete in n=226. Follow-up for the outcomes mortality and SBP was performed until May 2012, with only 13 patients lost to follow-up. DM was present at baseline in 78 patients (35%). Median follow-up was 6.2 (interquartile range, 3.1-9.3) years, during which 118 patients died [47 out of 78 with DM (60%), and 71 out of 148 without DM (48%)]. The presence of DM at baseline was not associated with increased mortality after adjustment for age {HR (hazard ratio), 1.00 [95% CI (confidence interval), 0.67-1.50]}. Further adjustment for sex, aetiology of cirrhosis, platelet count and the Child-Pugh or MELD (model for end-stage liver disease) score did not change this finding. During follow-up, 37 patients developed incident SBP (19 with DM and 18 without DM). DM at baseline was associated with incident SBP, even after adjustment for age, sex, aetiology, platelet count and the Child-Pugh [HR, 2.39 (95% CI, 1.10-5.18)] or MELD score [HR, 2.50 (95% CI, 1.16-5.40)]. In conclusion, the presence of DM at baseline in patients with cirrhosis was associated with an increased risk of SBP, which may represent an increased susceptibility to infections. On the other hand, DM was not clearly associated with increased mortality in these patients.

The diagnosis of non-alcoholic fatty liver disease

SIRS, We read with interest the comprehensive review by Downman et al., in which they describe several strategies for the diagnosis and staging of non-alcoholic fatty liver disease (NAFLD). For the diagnosis of NAFLD, the authors describe, in addition to the exclusion of other causes of liver disease, the role of ultrasound and ⁄ or the fatty liver index (FLI) in determining the presence of hepatic steatosis. Ultrasound has a high sensitivity for diagnosing moderate to severe steatosis, but it does not accurately detect mild steatosis. The FLI was developed to predict steatosis on ultrasound, not on liver biopsy, and therefore shares this insensitivity. Although biopsy is the gold standard, modern techniques, such as proton magnetic resonance spectrometry (1H-MRS), quantify hepatic fat content accurately, non-invasively and without sampling error or inter-observer variability. 3 Based on the 95th percentile of a healthy population, steatosis was defined as liver fat content ‡ 5.56% on 1H-MRS. However, 1H-MRS is not yet feasible in clinical practice. To overcome this problem, a score was recently developed to estimate the presence of NAFLD defined as ‡ 5.56% liver fat on 1H-MRS, although this was not reported in the review. This score includes the presence of the metabolic syndrome, type 2 diabetes, and levels of fasting serum insulin and transaminases, and achieved a discriminative value (area under the curve) of 0.86 when validated internally. We calculated its predictive values for the general population and an obese ⁄ diabetic population, 5 using reported sensitivities and specificities at several cutoffs (Table 1). These data suggest that, in addition to the proposed algorithms by Dowman et al., this NAFLD liver fat score may be useful to detect mild to moder-

Lipid metabolism: a role for iron?

DOI:10.1097/MOL.0b013e328354752d Body iron is known to be linked to the development of diabetes mellitus, particularly in hereditary hemochromatosis, but also in individuals with normal iron stores [1]. More recent data have shown that the effects of iron metabolism may not be confined to glucose metabolism, but may also influence lipid metabolism. Two large epidemiological studies in Korean individuals have shown consistent associations of serum ferritin, the most reliable marker of body iron stores, with (components of) the metabolic syndrome in the general population [2,3]. The prevalence of dyslipidemia (triglycerides >1.70 mmol/l and/or HDL cholesterol <1.03 mmol/l and <1.29 mmol/l for men and women, respectively) increased significantly across quartiles of serum ferritin in 6311 individuals, including 2120 premenopausal women [2]. In another Korean cohort of 12 090 individuals, serum ferritin correlated significantly with triglycerides in both men (r1⁄40.203) and women (r1⁄40.167) [3]. These results are in agreement with previous studies in European and American cohorts [4,5], showing that the observed associations are not ethnicity-specific. The French Data From an Epidemiological Study on the Insulin Resistance Syndrome study additionally showed that both ferritin and transferrin were positively associated with the development of hypertriglyceridemia and low HDL cholesterol during 6 years of follow-up [5]. Elevated ferritin levels ( 300 ng/ml in men and 200 ng/ml in women) were also detected in 20% of patients with familial combined hyperlipidemia and familial hypertriglyceridemia, and serum ferritin showed a stronger correlation with serum triglycerides than adiposity measures, homeostasis model assessment insulin resistance (HOMA-IR), or liver enzymes in this population [6]. Although (low-grade) inflammation is a potential confounder in associations of iron metabolism with lipids, adjustment for C-reactive protein (CRP) or exclusion of patients with elevated CRP did not affect the results of the studies above [3,5,6]. Finally, the association between body iron stores and cardiometabolic risk factors has recently been studied inhealthy teenagers (16–17 yearsold)as well, using the ratio of soluble transferrin receptor/ ferritin (sTfR/ferritin) [7]. Across decreasing quartiles of sTfR/ferritin quartiles (indicating higher iron stores), mean serum triglycerides increased from

PS7 - 5. Complement factor 3 is longitudinally associated with insulin resistance, glucose tolerance, and incident type 2 diabetes mellitus over a 7-year follow-up period: the CODAM study.

Complement factor 3 (C3) is an emerging risk factor in obesity-related metabolic and cardiovascular diseases. Several cross-sectional studies have shown that C3 levels are associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM), but longitudinal studies on this subject are scarce.

The association between body fat and circulating complement C3 is largely explained by low-grade inflammation and insulin resistance: the CODAM study

Background and aims: TCF7L2 is both an activator and an inhibitor of transcription and the most highly associated type 2 diabetes gene known to date. It influences beta cell survival and function, i.e. incretin hormonal effects, insulin processing and secretion. However, its target genes in pancreatic islets are not fully described and the molecular mechanism whereby it propagates its effects on islet function is not known. The aim of this study is to identify the molecular mechanisms through which TCF7L2 influence beta cell survival and function. Materials and methods: Wister rat primary islets and INS-1 (832/13) cells were incubated with siRNA against Tcf7l2, both Tcf7l2 and TP53INP1 or both TCF7L2 and TP53 in 5.5 mM and 14.3 mM glucose. TCF7L2 activity, p53 activity and target gene expression (using qPCR) were measured after siRNA treatment. INS-1 cell apoptosis was measured by DNA degradation levels, caspase-3/7 levels and by using antibodies against Annexin V, and 7-AAD, visualized using confocal microscopy. Rat islet viability was estimated measuring metabolic rate. Rat islet apoptosis was estimated by measuring Caspase-3/7 level. Results: The type 2 diabetes associated genes TP53INP1, FTO, GIPR and ADAMTS9 were identified as TCF7L2 potential target gene using chromatin immunoprecipitation on microarrays. In INS-1 cells, siRNA mediated Tcf7l2 knock down (69.5 %) resulted in decreased TCF7L2 activity (91%) and differential expression of the target genes: Tp53 (14.5% increase), TP53INP1 (65.9% increase) and ADAMTS9 (82.8% decrease). TCF7L2 knockdown also lead to reduced cell viability (65%) and increased apoptosis (113%). The TCF7L2 induced cell death was replicated in rat primary islets. When restoring (decreasing) the Tp53inp1 expression level in TCF7L2 depleted islets, the decrease in cell viability and increase in apoptosis were prevented, suggesting that the Tcf7l2 effect is mediated via Tp53inp1. Furthermore, p53 depletion prohibited TCF7L2 down regulation induced cell death and elevation of Tp53inp1 expression in both INS-1 cells and rat primary islets. Conclusion: The type 2 diabetes associated genes TP53INP1 and ADAMTS9 are target genes of TCF7L2 in pancreatic islets. TCF7L2 induced apoptosis and decreased cell viability are mediated through activation of p53 and increased p53INP1 expression.