nature publishing group Translational Investigation Articles Initial hyperinsulinemia and subsequent β-cell dysfunction is associated with elevated palmitate levels Johan Staaf1,2, Sarojini J.K.A. Ubhayasekera3, Ernest Sargsyan1, Azazul Chowdhury1, Hjalti Kristinsson1, Hannes Manell1,2, Jonas Bergquist3, Anders Forslund2 and Peter Bergsten1 BACKGROUND: The prevalence of obesity-related diabetes in obesity with T2DM (5). High NEFA concentrations are observed childhood is increasing and circulating levels of nonesterified in both obese adults (5,6) and children (7) and have been asso- fatty acids may constitute a link. Here, the association between ciated with insulin secretion in vivo (8,9). Among circulating palmitate and insulin secretion was investigated in vivo and NEFAs, the saturated fatty acid palmitate (C16:0) is one of the in vitro. most abundant (10). Palmitate has previously been associated METHODS: Obese and lean children and adolescents (n = 80) with both postprandial insulin secretion and insulin sensitivity were included. Palmitate was measured at fasting; insulin and in vivo (11) and extensively investigated in vitro in connection glucose during an oral glucose tolerance test (OGTT). Human with decline in islet β-cell function and mass after prolonged islets were cultured for 0 to 7 d in presence of 0.5 mmol/l pal- exposure (12). Detrimental effects of chronic palmitate expo- mitate. Glucose-stimulated insulin secretion (GSIS), insulin con- sure include shifts in sphingolipid metabolism as well as ini- tent and apoptosis were measured. tiation of oxidative stress, downregulation of glucokinase, and RESULTS: Obese subjects had fasting palmitate levels triggering of endoplasmic reticulum-stress response (13–18). between 0.10 and 0.33 mmol/l, with higher average levels In addition, palmitate contributes to inhibition of insulin pro- compared to lean subjects. While obese children with elevated cessing, resulting in an increase in the proinsulin to insulin palmitate (>0.20 mmol/l) had accentuated insulin levels dur- ratio (19). In contrast, when palmitate concentration is acutely ing OGTT, obese adolescents with high palmitate had delayed increased, insulin secretion is stimulated (20,21), where free first-phase insulin response. In human islets exposed to palmi- fatty acid receptor 1 plays an important role (22). tate for 2 d GSIS was twofold enhanced, but after 7 d attenu- Here, we investigated how elevated palmitate levels are ated. Intracellular insulin content decreased time-dependently related to the insulin secretory response to glucose in obese in islets cultured in the presence of palmitate and cleaved cas- children and adolescents as well as in isolated human islets. pase 3 increased. The cross-sectional in vivo study was complemented by an in CONCLUSION: The rapid accentuated and delayed insulin vitro approach, where effects of fixed palmitate concentrations secretory responses observed in obese children and ado- were studied in a controlled, longitudinal setting. We hypoth- lescents, respectively, with high palmitate levels may reflect esized that insulin secretion is accentuated in obese children changes in islet secretory activity and integrity induced by with high palmitate levels and later becomes impaired in obese extended exposure to the fatty acid. adolescents. RESULTS besity in children is associated with type 2 diabetes melli- Palmitate and Insulin Concentrations at Fasting Otus (T2DM) and the prevalence of both childhood obesity Fasting palmitate levels showed an approximate fourfold and T2DM have increased (1). Obesity commonly precedes range in the whole study population and were normally dis- manifest T2DM and affects both insulin-secreting β-cells and tributed with a mean of 0.19 mmol/l (Figure 1a). Palmitate insulin-target tissue (2). Indeed, approximately 85% of chil- levels were higher in obese subjects (Figure 1b). Fasting dren with T2DM are overweight or obese (3). Children with palmitate was neither associated with pubertal stage (age) obesity consequently have increased risk of developing meta- (Table 1) nor with gender (data not shown). Fasting insu- bolic complications already in their youth (4), which makes lin levels in obese subjects were approximately threefold the obesity epidemic a serious threat to global health. higher compared to lean subjects (Table 1). Fasting insulin Elevated concentrations of circulating nonesterified fatty correlated with palmitate levels for the whole study popula- acids (NEFAs) have been implicated as a factor connecting tion (P < 0.005) (Figure 1c). When analyzing the obese and 1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden; 2Department of Women’s and Children’s Health, Uppsala University, Uppsala, Sweden; 3Department of Chemistry-BMC, Analytical Chemistry and Science for Life Laboratory, Uppsala University, Uppsala, Sweden. Correspondence: Johan Staaf ([email protected]) Received 8 December 2015; accepted 3 March 2016; advance online publication 11 May 2016. doi:10.1038/pr.2016.80 Copyright © 2016 International Pediatric Research Foundation, Inc. Volume 80 | Number 2 | August 2016 Pediatric REsEarch 267 Articles Staaf et al. ab c 20 0.3 500 400 15 * 0.2 300 10 200 0.1 5 100 Number of subjects Fasting insulin (pmol/l) 0 0.0 0 Palmitate concentration (mmol/l) Lean Obese 0.00.1 0.2 0.30.4 Palmitate (mmol/l) 0.06–0.080.10–0120.14–0160.18–0.200.22–0.240.26–0.280.30–0.320.34–0.36 Palmitate (mmol/l) Figure 1. Palmitate levels in obese and lean subjects. Fasting palmitate concentrations in 40 obese (black bars and circles) and 40 lean (white bars and circles) children and adolescents showed as distribution (panel a), mean concentration ± SEM (panel b), and correlation to fasting insulin (panel c). In panel c; Rspearman = 0.335, P = 0.0029 for whole study population; Rspearman = 0.275, P = 0.0902 for obese children and Rspearman = −0.0553, P = 0.7416 for lean children; regression line for whole study ­population shown in graph. *P < 0.05 vs. lean subjects in panel b. Table 1. Fasting characteristics of study population Lean subjects Obese subjects Pubertal Prepubertal Pubertal Prepubertal Pubertal Prepubertal Pubertal Prepubertal adolescents children high adolescents Total children adolescents Total children adolescents children low PA low PA PA high PA Number of 40 19 21 40 20 20 10 10 10 10 subjects Age 11.6 ± 0.7 9.5 ± 0.4 14.7 ± 0.6 11.6 ± 0.6 8.7 ± 0.6 14.4 ± 0.4 7.3 ± 0.8 (3–12) 14.2 ± 0.5 10.1 ± 0.5 14.6 ± 0.6 (7–17) (6–10) (10–18) (3–18) (3–12) (10–18) (12–18) (8–12) (10–16) Gender 19 females 8 females 11 females 19 females 10 females 9 females 6 females 6 females 4 females 3 females 21 males 11 males 10 males 21 males 10 males 11 males 4 males 4 males 6 males 7 males Palmitate 0.17 ± 0.01 0.17 ± 0.01 0.18 ± 0.01 0.21 ± 0.01* 0.22 ± 0.01* 0.21 ± 0.01* 0.18 ± 0.01 0.16 ± 0.01 0.25 ± 0.01* 0.25 ± 0.01* (mmol/l) (0.08–0.33) (0.10–0.33) (0.08–0.28) (0.10–0.34) (0.11–0.34) (0.10–0.29) (0.11–0.20) (0.10–0.20) (0.20–0.34) (0.22–0.29) Insulin 55 ± 7 34 ± 6 65 ± 7 (26– 154 ± 14* 136 ± 16* 173 ± 23* 92 ± 16 * 186 ± 37* 180 ± 21* 161 ± 30 * (pmol/l) (22–121) (22–59) 121) (33–410) (33–285) (43–410) (33–181) (85–410) (78–285) (43–368) Glucose 5.4 ± 0.1 5.3 ± 0.1 5.4 ± 0.2 5.0 ± 0.1 5.1 ± 0.1 4.9 ± 0.1 5.1 ± 0.1 4.9 ± 0.1 5.1 ± 0.1 4.9 ± 0.1 (mmol/l) (4.6–5.9) (4.6–5.9) (4.7–5.9) (4.4–5.9) (4.6–5.9) (4.4–5.4) (4.6–5.7) (4.4–5.4) (4.7–5.9) (4.4–5.4) Results are means ± SEM and range in parenthesis. *P < 0.05 vs. lean subjects. PA, palmitate. lean sub-populations separately, lean subjects showed no Insulin Secretion During Oral Glucose Tolerance Test correlation. In the obese subjects, a positive relationship was In obese children with high palmitate, insulin increased sharply observed (P = 0.09). Fasting glucose levels were not different with a peak after 30 min with a concentration of 1,597 ± 277 pmol/l between the groups, however (Table 1). (Figure 2a). Insulin levels subsequently declined but remained To further study the associations between fasting palmitate higher than in lean children throughout the oral glucose tolerance and insulin concentrations in children and adolescents with test (OGTT) (Figure 2b). In obese children with low palmitate, obesity separately, the obese subjects were divided into two the insulin levels also peaked after 30 min but merely at 586 ± 91 groups, “low palmitate” and “high palmitate”, with the median pmol/l, which was similar to lean children. In fact, insulin levels 0.20 mmol/l as devisor. The low and high palmitate groups during OGTT of obese children with low palmitate levels were, were further divided into children and adolescents based on apart from fasting levels, only significantly elevated compared to sex hormone levels. In obese children with high palmitate lean children at 90 and 120 min (Figure 2b). Insulin levels dur- levels, fasting insulin was 180 ± 21 pmol/l, which was twofold ing the initial 30 min of the OGTT were further examined. The higher than levels in obese children with low palmitate lev- most prominent change in secretory rate was observed in obese els and fivefold higher than levels in lean children (Table 1). children with high palmitate levels between 5 and 10 min of the Obese adolescents with high palmitate levels had fasting insu- OGTT, when insulin concentration rose with 113 pmol/l/min lin concentration of 161 ± 30 pmol/l, which was not different on average, with a max-value of 204 pmol/l/min (female, 8 y old) from fasting insulin concentration in obese adolescents with (Figure 2c).