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Pancreatic Lipid Content Is Not Associated with Beta Cell Dysfunction in Youth-Onset Type 2 Diabetes
Can J Diabetes 39 (2015) 398e404
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Canadian Journal of Diabetes journal homepage: www.canadianjournalofdiabetes.com
Original Research
Pancreatic Lipid Content Is Not Associated with Beta Cell Dysfunction in Youth-Onset Type 2 Diabetes Brandy A. Wicklow MD, MSc a, b, *, Angella T. Griffith BSc a, Jacqueline N. Dumontet BSc a, Niranjan Venugopal PhD c, Lawrence N. Ryner PhD c, Jonathan M. McGavock PhD a, b a
Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada University of Manitoba, Department of Pediatrics and Child Health, Faculty of Medicine, Winnipeg, Manitoba, Canada c National Research Council Canada Institute for Biodiagnostics, Winnipeg, Manitoba, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2014 Received in revised form 31 March 2015 Accepted 6 April 2015
Objective: To determine whether pancreatic lipid content is associated with type 2 diabetes and beta cell function in Indigenous and Caucasian adolescents. Methods: This was a cross-sectional study comparing 1H-magnetic resonance spectroscopy-derived pancreatic triglyceride content in adolescents 13 to 18 years of age with type 2 diabetes (n¼20) and body mass index-matched normoglycemic controls (n¼34). Beta cell function was measured by the acute insulin response and disposition index derived from intravenous glucose tolerance tests. Results: Pancreatic lipid content was not significantly different in youth with type 2 diabetes and their normoglycemic body mass index-matched peers (2.41 [95% CI: 0.63, 5.60] vs. 1.22 [0.08, 5.93]; p¼0.27). Pancreatic triglyceride levels were not associated with measures of beta cell function in the cohort. In subgroup analyses, pancreatic lipid content was w4-fold higher in youth with type 2 diabetes who were carriers of the G319S mutation in the HNF-1alpha gene (7.45 [2.85, 26.8] vs. 2.20 [0.350, 3.30] % Fat to Water Ratio F/W; p¼0.032). Conclusions: Pancreatic lipid content is not elevated in Indigenous or Caucasian youth with type 2 diabetes compared to normoglycemic youth, nor is it associated with beta cell function. The presence of the G319S mutation in the HNF-1alpha gene in Indigenous youth with type 2 diabetes is associated with higher pancreatic lipid content. Further research is needed to understand the mechanisms that explain beta cell failure in overweight youth with type 2 diabetes. Ó 2015 Canadian Diabetes Association
Keywords: adolescent lipotoxicity nuclear magnetic resonance (NMR) spectroscopy pancreas steatosis type 2 diabetes
r é s u m é Mots clés : adolescent lipotoxicité spectroscopie par résonance magnétique nucléaire (RMN) pancréas stéatose diabète de type 2
Objectif : Déterminer si la teneur en lipides dans les cellules pancréatiques est associée au diabète de type 2 et au fonctionnement des cellules bêta chez les adolescents autochtones et caucasiens. Méthodes : Il s’agissait d’une étude transversale comparant la teneur en triglycérides dans les cellules pancréatiques obtenue par la spectroscopie par résonance magnétique 1H chez des adolescents de 13 à 18 ans souffrant de diabète de type 2 (n¼20) et des témoins normoglycémiques appariés selon l’indice de masse corporelle (n¼34). Le fonctionnement des cellules bêta était mesuré par la réponse insulinique aiguë et l’indice de disposition obtenu par les épreuves d’hyperglycémie provoquée par voie intraveineuse. Résultats : La teneur en lipides dans les cellules pancréatiques n’était pas significativement différente chez les jeunes souffrant du diabète de type 2 et les pairs normoglycémiques appariés selon l’indice de masse corporelle (2,41 [IC à 95 % : 0,63, 5,60] vs 1,22 [0,08, 5,93]; p¼0,27). Les concentrations en triglycérides dans les cellules pancréatiques n’étaient pas associées aux mesures du fonctionnement des cellules bêta dans la cohorte. Dans les analyses en sous-groupes, la teneur en lipides dans les cellules pancréatiques était w4 fois plus élevée chez les jeunes souffrant du diabète de type 2 qui étaient porteurs de la mutation G319S sur le gène HNF-1alpha (7,45 [2,85, 26,8] vs 2,20 [0,350, 3,30] %F/W; p¼0,032).
* Address for correspondence: Brandy A. Wicklow, MD, MSc, Department of Pediatrics and Child Health, Faculty of Medicine, University of Manitoba, FW306, 3rd Floor, Community Services Building, 685 William Avenue, Winnipeg, Manitoba R3E 0Z2, Canada. E-mail address: [email protected] 1499-2671/$ e see front matter Ó 2015 Canadian Diabetes Association http://dx.doi.org/10.1016/j.jcjd.2015.04.001
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Conclusions : La teneur en lipides dans les cellules pancréatiques n’est pas élevée chez les jeunes autochtones ou caucasiens souffrant du diabète de type 2 comparativement aux jeunes normoglycémiques, et n’est pas associée au fonctionnement des cellules bêta. La présence de la mutation G319S sur le gène HNF-1alpha chez les jeunes autochtones souffrant du diabète de type 2 est associée à une teneur en lipides plus élevée dans les cellules pancréatiques. D’autres recherches sont nécessaires pour comprendre les mécanismes qui expliquent l’insuffisance des cellules bêta chez les jeunes souffrant du diabète de type 2 qui ont un excès de poids. Ó 2015 Canadian Diabetes Association
Introduction
Methods
The hallmark pathophysiologic feature of type 2 diabetes is a relative deficit in insulin secretion by pancreatic beta cells (1). The mechanisms to explain the loss of insulin secretory capacity in type 2 diabetes remain poorly understood (2,3). Conventional wisdom suggests that following years of insulin resistance, compensatory hyperinsulinemia results in beta cell exhaustion and eventual failure (1,2). This theory, however, does not explain the rapid loss of glucose-stimulated insulin secretion seen in children and adolescents who develop type 2 diabetes early in life (4). One theory to explain the loss of insulin secretion in the setting of type 2 diabetes is an excessive accumulation of lipid moieties within beta cells. The additional lipid disrupts the energy status of the beta cells (i.e. the adenosine triphosphate:adenosine diphosphate [ATP:ADP] ratio) and leads to the production of toxic lipid intermediates that accelerate cell death (5,6). To explore this pancreatic lipotoxicity theory in a clinical setting, noninvasive imaging modalities have emerged to quantify lipid content in the human pancreas (7e12). Studies in adults with type 2 diabetes and children with genetic disorders involving triglyceride synthesis have demonstrated that 1) noninvasive imaging tools can quantify triglyceride accurately in the pancreas (13); 2) pancreatic lipid content is elevated in human obesity and in type 2 diabetes (14,15); and 3) the degree of lipid within the pancreas is negatively associated with beta cell function (14). Although these results implicate lipotoxicity in the natural history of beta cell dysfunction, the studies did not control for differences in visceral fat content between the groups, which may contribute to both pancreatic lipid content and defects in glucose-stimulated insulin secretion (16). Our cohort represents a clinical model of early, accelerated beta cell failure in the absence of aging and insulin resistance (4,17). We recently demonstrated that adolescents with type 2 diabetes are 4 to 6 times more likely to display ectopic lipid accumulation, in the form of hepatic steatosis, than normoglycemic adolescents matched for body mass index (BMI) (17). In addition, we found that the presence of hepatic steatosis in overweight normoglycemic adolescents predicts metabolic abnormalities, including metabolic syndrome and increased glucose dispersion, following oral glucose challenges (18). Based on these findings, we hypothesized 1) that youth with type 2 diabetes would display a significantly higher proportion of lipid within the pancreas compared to normoglycemic adolescents, independent of adiposity; and 2) that the degree of lipid accumulation in the pancreas would be negatively associated with glucose-stimulated insulin secretion. Finally, because a number of patients with type 2 diabetes in our clinical program are carriers of a private polymorphism in the HNF-1alpha gene G319S (19), resulting in reduced insulin secretory capacity (20e22), we also performed an exploratory subgroup analysis to determine levels of pancreatic lipid among carriers of the G319S polymorphism in the HNF-1alpha gene.
Study design and study population This was a cross-sectional study comparing pancreatic triglyceride content between adolescents with type 2 diabetes and age-, sex- and BMI-matched normoglycemic adolescents. Participants with type 2 diabetes were recruited through the Diabetes Education Resource for Children and Adolescents (DER-CA) in Winnipeg, Manitoba, Canada. Overweight and obese normoglycemic adolescents were recruited through radio advertisements and posters in local community health centers and in the Manitoba Institute of Child Health Research Center. Type 2 diabetes was diagnosed according to the criteria published by the American and Canadian Diabetes Associations (23,24). Participants were classified as overweight or obese according to the age- and sex-specific guidelines established by the International Obesity Task Force (25). Adolescents were excluded from the study if they 1) had used antidiabetes medications in the 12 months prior to participation in the study or 2) had been treated with medication known to affect glucose handling or lipid metabolism, including statins, atypical antipsychotics or corticosteroids. Three youth recruited as normoglycemic controls demonstrated impaired glucose tolerance during a 75 g oral glucose tolerance test and were excluded from further participation. Two participants with type 2 diabetes failed to obtain magnetic resonance imaging (MRI) due to size restrictions and, therefore, were excluded from final analysis. Three normoglycemic participants failed to receive MRI due to scheduling problems with the MRI acquisition and were excluded from the final analysis. A total of 20 adolescents with type 2 diabetes and 34 healthy overweight/obese controls were included in the final analysis. All participants and parents provided written informed consent to participate in the study, which was approved by the biomedical research ethics board at the University of Manitoba and performed according to the Declaration of Helsinki. Pancreatic triglyceride content All MRIs were performed using a 3.0-T whole-body magnet (Siemens USA, Malvern, PA, US) as previously described (17,18). In vivo magnetic resonance spectroscopy with a single voxel volume of 3 cm (axial) 3 cm (coronal) 3 cm (sagittal) (27 cm3) was used to collect 64 consecutive 1H spectra from the tail of the pancreas (15,26). We chose the tail of the pancreas because preliminary analyses revealed that this location provided the most reliable acquisition of spectra, and it is also the anatomic location of the islets of Langerhans. Axial, coronal and sagittal high-resolution images of the pancreas were obtained by using standard clinical techniques. To reduce contamination from surrounding visceral fat, spatial saturation bands were manually placed around the voxel. The use of spatial saturation bands improves the baseline of collected spectra by dramatically reducing the signals from surrounding contaminating lipids (27). In addition, spectra and imaging were collected using respiratory gating to reduce motion
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artefact and contamination by surrounding fat. Images and spectra were collected at 10% of the subjects’ inspiratory efforts (28). Robust automated spectral analysis was performed using the LCModel software , Japan L.A Systems (29). Using this software, triglyceride and water peaks were quantified by fitting known basis-set spectra to measured spectra. The relative standard deviation between the known spectra and the measured spectra are reported by the Cramer-Rao lower bounds (i.e. % SD). This method of acquiring pancreatic lipid content has been published previously and validated in the scientific literature (11,12,15,26). Beta cell function A modified, frequently sampled intravenous glucose tolerance test was used to determine the 2 primary measures of beta cell function: the acute insulin response to exogenous glucose and the disposition index (30). The disposition index is a measure of glucosestimulated insulin secretion adjusted for the prevailing levels of insulin resistance. It was calculated as the product of insulin sensitivity and the acute insulin response (Si AIR) and was used to quantify glucose-stimulated insulin secretion relative to insulin resistance. Fasting blood samples were collected prior to an intravenous bolus of a 25% glucose solution (0.3 g/kg body weight) at time 0, followed by an intravenous bolus of regular human insulin (0.03 U/ kg body weight) at the 20-minute time point. Blood samples to test for glucose and insulin were collected at 1, 2, 3, 4, 5, 6, 8, 10, 14, 19, 22, 25, 30, 40, 50, 70, 100, 140 and 180 minutes. Glucose and insulin kinetics, including acute insulin response and insulin sensitivity, were then modelled using the Bergman minimal model provided in
Table 1 Anthropometric and metabolic characteristics of obese/overweight healthy controls and adolescents with type 2 diabetes Variable
Overweight/obese controls
Type 2 diabetes
n Age (years) Sex (male/female) Ethnicity (FN/Metis/other) Tanner stage (1-2/3-5) Adiposity BMI z score Waist circumference z score % Body fat Visceral fat (cm2) Cardiometabolic risk factors Systolic blood pressure (mm Hg) Total cholesterol (mmol/L) Fasting glucose (mmol/L) HDL (mmol/L) LDL (mmol/L) AST (mmol/L) ALT (mmol/L) Serum triglycerides (mmol/L) Metabolic profile Insulin sensitivity (mU kg1 min1) Disposition index Acute insulin response* Total pancreatic triglyceride (% F/W)*
34 15.41 (14.73e16.09) 7/27 6/5/23 3/31
20 15.45 (14.65e16.25) 7/13 16/2/2y 1/15
1.99 (1.83e2.15) 5.80 (4.90e6.70)
1.72 (1.41e2.03) 5.66 (4.39e6.94)
39.6 (37.5e41.7) 94.5 (77.7e111.3)
34.0 (30.2e37.7)z 126 (87.0e165.5)
114 (110e119)
123 (117e130)z
4.0 4.7 1.13 2.3 19 17 1.35
4.0 6.7 1.05 2.2 22 27 1.66
(3.7e4.3) (4.5e4.9) (1.04e1.22) (2.0e2.5) (17e21) (14e20) (1.14e1.56)
3.37 (2.50e4.25) 2612 (1970e3255) 758.0 (460.0, 1452) 1.22 (0.08, 5.93)
(3.6e4.5) (5.8e7.7)y (0.92e1.18) (1.8e2.6) (17e27) (16e39) (1.22e2.10)
4.93 (1.07e8.80) 493.1 (169.1e817.2)y 33.37 (0.00, 188.0)y 2.41 (0.63, 5.60)
ALT, Alanine aminotransferase; AST, alternate site testing; F/W, fat/water; FN, First Nations; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Data are means (95% CI). * Medians (25%, 75% quartiles) are used to represent non-normally distributed variables. y p<0.001. z p<0.05 vs. overweight/obese.
customized software (MINMOD; RC, Boston, Massachusetts, USA) to quantify insulin sensitivity and beta cell function. Confounding variables Visceral fat mass was assessed using high-resolution MRI at a level between the 3rd and 5th lumbar vertebrae, as previously described (31,32). Images were viewed, and adiposity was quantified offline using Slicer3 software (v. 3.6.3 1.0; pieper at bwh. harvard.edu, Cambridge, Massachusetts, USA). Dual energy x-ray absorptiometry (Hologic; Bedford, Massachusetts, USA) was used to quantify fat mass (kg), fat free mass (kg) and percent body fat. Waist and hip circumferences were measured in duplicate with a flexible tape measure, and the average of the 2 measures was used. Waist circumference was taken at the level of the iliac crest, as previously described, and z-scores were calculated from a nationally representative sample of Canadian youth (33). Pubertal stage was assessed using a pictorial tool for self-assessment of stage of development, and in girls, attainment of menstruation was collected from histories. Ethnicity was identified by self-report. Assessment of HNF-1alpha genotype Genotyping was performed as previously described (34) in youth with type 2 diabetes. Biochemical measurements Plasma glucose was measured on a Roche Modular P analyzer with an ultraviolet test principle (hexokinase method). Insulin was measured on an Immulite (Siemens Healthcare Global; Erlangen, Germany) solid-phase, 2-site chemiluminescent immunometric assay. Serum lipoproteins, liver transaminases and triglycerides were measured on a Roche Modular P analyzer (Roche Diagnostics; Indianapolis, Indiana, USA). Statistical analyses A sample size calculation based on a projected difference in pancreatic lipid between groups of 30% and a standard deviation of MRI-derived pancreatic lipid content of 15% revealed that a sample of 20 patients per group would provide 80% power to detect a 30% difference between the groups [alpha¼0.05, beta¼0.1]. Sample size analysis was performed using SPSS sample power software (v. 3.0.0; IBM, Chicago, Illinois, USA). All data are presented as means with 95% confidence intervals, unless otherwise stated. Data were assessed for normal distribution by using the Kolmogorov-Smirnov test. For non-normally distributed variables, Mann-Whitney U tests were used to test for differences between groups. Comparisons of normally distributed variables between groups were performed using the Student t test. A univariate regression was performed to test for differences in outcome variables between the groups after adjusting for ethnicity, age, sex, visceral adiposity and genetic variance. The Spearman rank correlation tests were used to assess the associations between pancreatic lipid content and measures of beta cell function. Multiple stepwise linear regression analysis was used to assess the independence of the association between pancreatic triglyceride content and beta cell function after adjusting for potential confounding variables, including age, sex, ethnicity, BMI z-scores, pubertal stages and total and visceral fat masses. A second multiple linear regression analysis was performed to determine whether adiposity, hepatic lipid content and ethnicity were independently associated with pancreatic lipid content. All analyses were performed using SPSS v. 19.0; p<0.05 was considered statistically significant.
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Results
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Table 3 Determinants of pancreatic triglyceride in youth
Participants’ demographics and metabolic variables are presented in Table 1. The type 2 diabetes group included a greater proportion of First Nations/Metis youth than did the control group (90% vs. 32%, X2¼16.8; p<0.001). Systolic blood pressure (12313 vs. 11411 mm Hg, p¼0.019) was significantly higher in youth with type 2 diabetes, while percent of total body fat (34.07.6 vs. 39.66.0%; p¼0.005) was significantly lower in youth with type 2 diabetes, despite similar BMIs. However, the proportion of body fat composed of visceral adiposity appeared to be higher in youth with type 2 diabetes (not statistically significant). As expected, fasting glucose was higher in youth with type 2 diabetes compared to normoglycemic controls (6.70.5 vs. 4.70.1 mmol/L; p<0.001). Although insulin sensitivity was not significantly different in the 2 groups, both acute insulin response (33 [interquartile range (IQR): 0, 188] vs. 758 [IQR: 460, 1452]; p<0.001) and disposition index (DI: 493692 vs. 26121841; p<0.001) were significantly lower in those with type 2 diabetes (Table 1). Pancreatic lipid content was 2-fold higher in youth with type 2 diabetes compared to normoglycemic controls; however, this difference was not statistically significant (Table 1). Total pancreatic lipid content was significantly higher in Indigenous youth compared to non-Indigenous youth (2.60 [IQR: 1.06, 5.70] vs. 0.65 [IQR: 0.03, 5.95] %F/W, p¼0.034) (Table 2). Indigenous adolescents with type 2 diabetes who were carriers of the G319S polymorphism in the HNF-1alpha gene displayed a w4-fold higher total pancreatic triglyceride content than their wild-type or non Oji-Cree counterparts (7.45 [IQR: 2.85, 26.8] vs. 2.20 [IQR: 0.35, 3.30] %F/W, p¼0.03) (Table 4). In bivariate analysis, pancreatic fat content was not associated with either the acute insulin response (r¼e0.21; p¼0.13) or the disposition index (r¼e0.08; p¼0.55) adjusted for visceral adiposity. Hepatic lipid content was the only variable associated with pancreatic lipid content in the bivariate analysis (r¼0.31; p¼0.04), adjusted for age, sex, BMI z-score, level of insulin resistance, pubertal stage, ethnicity and visceral fat mass (r¼0.16; p¼0.016) (Table 3).
Table 2 Subgroup analysis of metabolic risk in youth matched for Indigenous ethnicity status Variable
Overweight/obese controls
Type 2 diabetes
n Age (years) Sex (male/female) Tanner stage (1e2/3e5) Adiposity BMI z score Waist circumference z score % body fat Visceral fat (cm2) Metabolic data Insulin sensitivity (mU kg1 min1) Disposition index (AIR Si) Acute insulin response* (mU/L) Total hepatic triglyceride (% F/W) Total pancreatic triglyceride (% F/W)*
11 15.1 (1.4) 9/2 1/10
20 15.9 (1.7) 8/12 1/19
2.06 (1.82e2.30) 7.26 (5.44e9.07) 40.89 (37.16e44.62) 87.2 (64.4e109.0) 3.03 (1.05e5.00) 2169 (1669e3735) 234 (132,870)
1.69 (1.39e1.98) 5.58 (4.40e6.76) 34.87 (31.16e38.58)z 112.4 (83.8e139.9) 4.97 (1.15e8.82) 330 (281e1009)y 97 (22 780)y
20.2 (4.2e23.3)
14.0 (0e30.5)
3.3 (1.7,6.6)
2.6 (1.2,5.0)
AIR, Acute insulin response; BMI, body mass index; F/W, fat/water; Si, insulin sensitivity. Data are means (95% CI). * Medians (25%, 75% quartiles) represent non-normally distributed variables. y p<0.001. z p<0.05 vs. overweight/obese.
Variable
Standardized beta
Triglyceride
p
Ethnicity BMI z score Insulin sensitivity Tanner stage Hepatic triglyceride
0.20 0.05 0.17 0.18 0.39
1.3 0.30 0.90 1.03 2.59
0.19 0.77 0.37 0.31 0.013
BMI, Body mass index.
Exploratory analyses To determine whether the presence of the G319S polymorphism was associated with differences in ectopic lipid content and beta cell function, we performed a series of exploratory cross-sectional comparisons between Indigenous adolescents who were carriers and noncarriers of the polymorphism. Adolescents with type 2 diabetes who were carriers of the G319S polymorphism in the HNF1alpha gene displayed a w4 fold higher total pancreatic triglyceride content than their wild-type or non Oji-Cree counterparts (7.45 [IQR: 2.85, 26.8] vs. 2.20 [IQR: 0.35, 3.30] %F/W; p¼0.03).
Discussion This study revealed that pancreatic triglyceride content was not associated with a diagnosis of type 2 diabetes in adolescents, nor was it related to pancreatic beta cell function in adolescents. In addition, we found that pancreatic fat content was higher in overweight Indigenous youth, irrespective of glycemic status and unaffected by sex, relative to non-Indigenous peers. Finally, we found that the presence of the G319S polymorphism in the HNF1alpha gene known to attenuate insulin secretory capacity was associated with a w4 fold increase in pancreatic triglyceride content despite similar levels of whole-body and visceral adiposity. It is possible that type 2 diabetes and the HNF-1alpha polymorphism may be associated with ectopic fat distribution in the pancreas, but the latter is not associated with beta cell dysfunction. Interest in the role of excessive pancreatic triglyceride content in the pathogenesis of type 2 diabetes has grown since the seminal work of Roger Unger in the late 1990s (35). Recently, the development of noninvasive imaging tools has made it possible to validate these studies in clinical populations (26,36). Previous studies in adolescents and young adults suggest that although the pancreatic fat fraction derived from computed tomography is associated with components of the metabolic syndrome, it is not associated with beta cell function (36). The data presented here support this observation and extend it by demonstrating that pancreatic triglyceride content, measured directly by proton magnetic resonance spectroscopy (1H-MRS), is not significantly elevated in youth with established type 2 diabetes and is not associated with glucosestimulated insulin secretion. Previous 1H-MRS studies of the pancreas found 2- to 3-fold higher triglyceride content in the pancreases of adults with impaired glucose tolerance or type 2 diabetes (14,26) compared to normoglycemic adults. None of these studies demonstrated an association between triglyceride content in the pancreas and any measure of beta cell function, and it was postulated that type 2 diabetes diagnosed in adults with the prolonged duration of hyperglycemia may be too late in the natural history to identify this early component of pathophysiology. Using a similar 1H-MRS technique, we found that pancreatic triglyceride content is not associated with measures of beta cell function in the early-onset, short-duration model of type 2 diabetes in adolescents. The data presented here suggest that pancreatic triglyceride
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content is not elevated, even in the early stages of beta cell dysfunction in adolescents, after adjusting for measures of adiposity. Similar to previous studies in adolescents (36), we found that lipid content in the pancreas is elevated in ethnic subgroups that are at particularly high risk for type 2 diabetes. Specifically, we found that pancreatic triglyceride content was w4-fold higher in Indigenous youth, who display a 3- to 5-fold greater risk for type 2 diabetes compared to Caucasian peers (37). This observation is consistent with previous studies by our group that showed that overweight Indigenous youth are more susceptible to ectopic triglyceride deposition (17). Ethnic disparity in ectopic lipid accumulation has been described by others (38,39). The ethnic differences in susceptibility may be attributed to the presence of genetic variants associated with increase lipid synthesis or uptake (40,41). It may also be attributed to differences in diet or physical activity patterns between ethnic groups (42e44). Regardless of the mechanisms, the consistent observation that ethnic groups with particularly high susceptibility to type 2 diabetes are characterized by excessive ectopic triglyceride suggests that clinical screening for steatosis in patients from these minority groups may provide insight into their risk for type 2 diabetes. The data presented here suggest that the G319S polymorphism in the HNF-1alpha gene may increase the susceptibility to excessive triglyceride deposition in the pancreas. Mutations in the HNF-1alpha gene are associated with monogenic maturity-onset diabetes in the young (45). The presence of the G319S polymorphism in the HNF-1alpha gene does not result in monogenic diabetes, despite a 50% reduction in insulin protein transcription Table 4 The presence of the G319S polymorphism of the HNF-1alpha gene is associated with elevated pancreatic triglyceride content in youth with type 2 diabetes Variable
G/G
S/G and S/S
n Age (years) Sex (male/ female) (n) Ethnicity (FN/Metis/ other) (n) Adiposity BMI z score Waist circumference z score Visceral fat (cm2) % body fat Cardiometabolic risk factors Systolic blood pressure (mm Hg) Fasting glucose (mmol/L) Total cholesterol (mmol/L) Serum triglycerides (mmol/L) HDL (mmol/L) LDL (mmol/L) AST (mmol/L) ALT (mmol/L) Metabolic profile Insulin sensitivity (mU kg1 min1) Disposition index AIR Total pancreatic triglyceride (% F/W)
15 15.0 (14.0, 17.0) 4/11
5 15.0 (15.0, 16.5) 3/2
11/2/2
5/0/0
1.89 (1.23, 2.27) 5.27 (3.62, 9.42)
1.79 (1.70, 1.98) 4.97 (4.23, 5.89)
114 (79.0, 144) 31.8 (28.6, 42.3)
85.3 (57.7, 148) 35.5 (25.2, 42.2)
121 (112, 128)
126 (111, 150)
6.3 (5.1, 9.1)
5.20 (4.85, 6.65)
4.0 (3.3, 4.9)
3.20 (3.15, 4.65)
1.5 (0.83, 2.6)
1.40 (1.10, 2.05)
1.0 2.1 19 21
1.00 1.70 16 13
(0.90, 1.2) (1.4, 3.2) (14, 29) (9.5, 41)
1.88 (0.919, 2.78) 35.59 (e57.93, 397.1) 12.81 (e98.48, 76.78) 2.20 (0.350, 3.30)
(1.00, 1.15) (1.45, 2.70) (15, 30) (12, 46)
6.89 (3.50, 8.60)* 1060 (162.6, 1694) 116.7 (16.56, 378.7) 7.45 (2.85, 26.8)*
AIR, Acute insulin response; ALT, alanine aminotransferase; AST, alternate site testing; FN, First Nations; F/W, fat/water; G/G, Wild Type; HDL, high-density lipoprotein; LDL, low-density lipoprotein; S/G, Heterozygote for G319S polymorphism; S/S, Homozygote for G319S polymorphism. Data are medians (25%, 75% quartiles). * p<0.05 vs. wild-type.
and insulin secretory capacity (20). Among heterozygotes, the decrement in beta cell reserve is mild and typically requires environmental pressures, such as increasing adiposity or exposure to gestational diabetes, to result in type 2 diabetes (46). However, among homozygotes, beta cell function is profoundly reduced, increasing the risk for type 2 diabetes 10-fold. Youth who are carriers of this polymorphism are characterized by less adiposity and less insulin resistance (19). Further studies are needed to determine whether the predisposition to pancreatic steatosis is evident in carriers of this polymorphism and to discover the potential mechanisms through which the HNF-1alpha gene regulate triglyceride metabolism in human tissues. Ectopic lipid accumulation is reversible with changes in lifestyle behaviours that lead to weight loss. A recent small study in adults with type 2 diabetes (n¼11) investigated the utility of dietary restriction on reversal of pancreatic fat and normalization of beta cell function (47). The authors reported that caloric restriction to 600 kcal per day in overweight individuals over a period of 8 weeks significantly reduced pancreatic (8.0%1.6% to 6.2%1.1%) and hepatic triglyceride content (12.8%2.4% to 2.9%0.2%). The reductions in these ectopic lipid deposits were associated with a near 2-fold improvement in hepatic insulin sensitivity and a 3-fold increase in the first-phase insulin response (0.190.02 to 0.460.07 nmol min1 m2) (47). We did not observe an association between any measures of lifestyle and the degree of pancreatic lipid content in this cohort of youth. It remains unclear whether lifestyle factors play roles in the loss of beta cell function or pancreatic steatosis in overweight youth. Youth with type 2 diabetes had lower percentages of body fat, but the distribution of body fat appeared to favour visceral fat and to be clinically recognized as higher waist circumference. There may be an important ethnic difference in body fat deposition and accumulation of metabolically active adipose. Additional research is needed to confirm that rapid changes in body weight are important in the regulation of ectopic lipid content and the insulin-glucose axis in adolescents with type 2 diabetes. Several limitations of this study require discussion. First, the cross-sectional design of the study precludes determination of a causal nature for the associations described. Differences in ethnicity existed between groups; however, including ethnicity as a covariate in a general linear model did not alter groupwise differences in pancreatic triglyceride content, insulin sensitivity or acutephase insulin secretion. We were unable to match for sex, age and BMI z-score for the subanalysis isolated to the Indigenous population of adolescents. Significant differences existed in the proportion of males within the 2 groups, which may contribute to the among-group differences identified within Indigenous subgroups. The use of an intravenous glucose tolerance test to assess beta cell function does have limitations, and its use in patients with type 2 diabetes has been questioned due to an inhibitory effect of hyperglycemia on glucose-stimulated insulin secretion (i.e. glucotoxicity). To reduce the effects of glucotoxicity on the measures of beta cell function, we recruited patients with good metabolic control (glycated hemoglobin 5.5% to 8.0%). In these studies, 95% of patients with type 2 diabetes presented with a fasting glucose <7.0 mmol/L. Because the inhibitory effects of hyperglycemia on glucose-stimulated insulin secretion become evident only with fasting glucose levels above 7 mmol/L, the use of this method to assess beta cell function was supported in our study cohort of subjects with type 2 diabetes (48). In conclusion, in our study, pancreatic triglyceride content was not associated with type 2 diabetes in adolescents or with pancreatic beta cell function. Our findings are in contrast to the theory of pancreatic lipotoxicity as a mechanism for beta cell failure in the setting of type 2 diabetes in adolescents. Pancreatic steatosis was evident in groups of youth at risk for type 2 diabetes and carriers of a polymorphism in the HNF-1alpha gene that is associated with beta cell dysfunction.
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These data suggest that pancreatic triglyceride accumulation occurs independent of beta cell failure and may be a marker of a more global defect in fatty-acid metabolism in high-risk cohorts of youth. Acknowledgements The authors are grateful for the clinical expertise provided by Dr. Heather Dean and Dr. Elizabeth Sellers (Pediatric Endocrinology, Diabetes Education Resource for Children, Winnipeg Children’s Hospital); Kristy Wittmeier (PhD, clinical investigator, Faculty of Medicine, University of Manitoba); Barry Clabbert (Clinical Chemistry, Faculty of Medicine, University of Manitoba); Lori Berard and Sherri Pockett (Clinical Research Unit, John Buhler Research Centre, Winnipeg, Manitoba); Quan Chung (National Research Council Institute for Biodiagnostics [NRC-IBD], Canada) Neil Pawlyshyn (MRI, St. Boniface Hospital, Winnipeg, Manitoba), Yao Fu (MR Spectroscopy, NRC-IBD, Winnipeg, Canada) and Anita Durksen (research associate, Manitoba Institute of Child Health). We are indebted to the parents and children who volunteered their time and effort for the completion of this study. Author Disclosures This work was supported by a Manitoba Medical Services Foundation grant to BAW; by MHRC, Lawson Foundation, Cosmopolitan Foundation and CIHR New Investigator Award to JMM; and by a Manitoba Health Research Council and Manitoba Institute of Child Health fellowship to BAW; ATRG and JND were supported by Manitoba Institute of Child Health Summer Studentships. Contributions of Authors BAW contributed to study design, researched data and wrote the manuscript; ATRG researched data and wrote the manuscript; JND researched data and contributed to discussion; NV and LNR contributed to the development of spectroscopy methods and analysis and to the discussion; JMM contributed to the concept and study design, researched data and edited the manuscript; BAW is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References 1. Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: Pathogenesis and treatment. Lancet 2008;371:2153e6. 2. Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin Invest 2006; 116:1802e12. 3. Robertson RP, Harmon J, Tran PO, et al. Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes 2004;53(Suppl 1): S119e24. 4. Bacha F, Lee S, Gungor N, et al. From pre-diabetes to type 2 diabetes in obese youth: Pathophysiological characteristics along the spectrum of glucose dysregulation. Diabetes Care 2010;33:2225e31. 5. Lee Y, Hirose H, Ohneda M, et al. Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: Impairment in adipocyte-beta-cell relationships. Proc Nat Acad Sci U S A 1994;91:10878e82. 6. Goh TT, Mason TM, Gupta N, et al. Lipid-induced beta-cell dysfunction in vivo in models of progressive beta-cell failure. Am J Physiol Endo Metab 2007;292: E549e60. 7. Lee Y, Lingvay I, Szczepaniak LS, et al. Pancreatic steatosis: Harbinger of type 2 diabetes in obese rodents. Int J Obes 2010;34:396e400. 8. Perman WH, Balci NC, Akduman I. Review of magnetic resonance spectroscopy in the liver and the pancreas. Top Magn Reson Imaging 2009;20:89e97. 9. Misra D, Gupta V, Sonkar AA, et al. Proton HR-MAS NMR spectroscopic characterization of metabolites in various human organ tissues: Pancreas, brain and liver from trauma cases. Physiol Chem Physics Med NMR 2008;40:67e88. 10. Esclassan J, Chemin-Thomas C, Creach Y, et al. High-resolution 1H NMR study of membrane phospholipids in rat pancreas stimulated by caerulein. Pancreas 1994;9:263e9. 11. Haaga JR. Magnetic resonance imaging of the pancreas. Radiol Clin North Am 1984;22:869e77.
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Canadian Journal of Diabetes journal homepage: www.canadianjournalofdiabetes.com
Original Research
Pancreatic Lipid Content Is Not Associated with Beta Cell Dysfunction in Youth-Onset Type 2 Diabetes Brandy A. Wicklow MD, MSc a, b, *, Angella T. Griffith BSc a, Jacqueline N. Dumontet BSc a, Niranjan Venugopal PhD c, Lawrence N. Ryner PhD c, Jonathan M. McGavock PhD a, b a
Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada University of Manitoba, Department of Pediatrics and Child Health, Faculty of Medicine, Winnipeg, Manitoba, Canada c National Research Council Canada Institute for Biodiagnostics, Winnipeg, Manitoba, Canada b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2014 Received in revised form 31 March 2015 Accepted 6 April 2015
Objective: To determine whether pancreatic lipid content is associated with type 2 diabetes and beta cell function in Indigenous and Caucasian adolescents. Methods: This was a cross-sectional study comparing 1H-magnetic resonance spectroscopy-derived pancreatic triglyceride content in adolescents 13 to 18 years of age with type 2 diabetes (n¼20) and body mass index-matched normoglycemic controls (n¼34). Beta cell function was measured by the acute insulin response and disposition index derived from intravenous glucose tolerance tests. Results: Pancreatic lipid content was not significantly different in youth with type 2 diabetes and their normoglycemic body mass index-matched peers (2.41 [95% CI: 0.63, 5.60] vs. 1.22 [0.08, 5.93]; p¼0.27). Pancreatic triglyceride levels were not associated with measures of beta cell function in the cohort. In subgroup analyses, pancreatic lipid content was w4-fold higher in youth with type 2 diabetes who were carriers of the G319S mutation in the HNF-1alpha gene (7.45 [2.85, 26.8] vs. 2.20 [0.350, 3.30] % Fat to Water Ratio F/W; p¼0.032). Conclusions: Pancreatic lipid content is not elevated in Indigenous or Caucasian youth with type 2 diabetes compared to normoglycemic youth, nor is it associated with beta cell function. The presence of the G319S mutation in the HNF-1alpha gene in Indigenous youth with type 2 diabetes is associated with higher pancreatic lipid content. Further research is needed to understand the mechanisms that explain beta cell failure in overweight youth with type 2 diabetes. Ó 2015 Canadian Diabetes Association
Keywords: adolescent lipotoxicity nuclear magnetic resonance (NMR) spectroscopy pancreas steatosis type 2 diabetes
r é s u m é Mots clés : adolescent lipotoxicité spectroscopie par résonance magnétique nucléaire (RMN) pancréas stéatose diabète de type 2
Objectif : Déterminer si la teneur en lipides dans les cellules pancréatiques est associée au diabète de type 2 et au fonctionnement des cellules bêta chez les adolescents autochtones et caucasiens. Méthodes : Il s’agissait d’une étude transversale comparant la teneur en triglycérides dans les cellules pancréatiques obtenue par la spectroscopie par résonance magnétique 1H chez des adolescents de 13 à 18 ans souffrant de diabète de type 2 (n¼20) et des témoins normoglycémiques appariés selon l’indice de masse corporelle (n¼34). Le fonctionnement des cellules bêta était mesuré par la réponse insulinique aiguë et l’indice de disposition obtenu par les épreuves d’hyperglycémie provoquée par voie intraveineuse. Résultats : La teneur en lipides dans les cellules pancréatiques n’était pas significativement différente chez les jeunes souffrant du diabète de type 2 et les pairs normoglycémiques appariés selon l’indice de masse corporelle (2,41 [IC à 95 % : 0,63, 5,60] vs 1,22 [0,08, 5,93]; p¼0,27). Les concentrations en triglycérides dans les cellules pancréatiques n’étaient pas associées aux mesures du fonctionnement des cellules bêta dans la cohorte. Dans les analyses en sous-groupes, la teneur en lipides dans les cellules pancréatiques était w4 fois plus élevée chez les jeunes souffrant du diabète de type 2 qui étaient porteurs de la mutation G319S sur le gène HNF-1alpha (7,45 [2,85, 26,8] vs 2,20 [0,350, 3,30] %F/W; p¼0,032).
* Address for correspondence: Brandy A. Wicklow, MD, MSc, Department of Pediatrics and Child Health, Faculty of Medicine, University of Manitoba, FW306, 3rd Floor, Community Services Building, 685 William Avenue, Winnipeg, Manitoba R3E 0Z2, Canada. E-mail address: [email protected] 1499-2671/$ e see front matter Ó 2015 Canadian Diabetes Association http://dx.doi.org/10.1016/j.jcjd.2015.04.001
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Conclusions : La teneur en lipides dans les cellules pancréatiques n’est pas élevée chez les jeunes autochtones ou caucasiens souffrant du diabète de type 2 comparativement aux jeunes normoglycémiques, et n’est pas associée au fonctionnement des cellules bêta. La présence de la mutation G319S sur le gène HNF-1alpha chez les jeunes autochtones souffrant du diabète de type 2 est associée à une teneur en lipides plus élevée dans les cellules pancréatiques. D’autres recherches sont nécessaires pour comprendre les mécanismes qui expliquent l’insuffisance des cellules bêta chez les jeunes souffrant du diabète de type 2 qui ont un excès de poids. Ó 2015 Canadian Diabetes Association
Introduction
Methods
The hallmark pathophysiologic feature of type 2 diabetes is a relative deficit in insulin secretion by pancreatic beta cells (1). The mechanisms to explain the loss of insulin secretory capacity in type 2 diabetes remain poorly understood (2,3). Conventional wisdom suggests that following years of insulin resistance, compensatory hyperinsulinemia results in beta cell exhaustion and eventual failure (1,2). This theory, however, does not explain the rapid loss of glucose-stimulated insulin secretion seen in children and adolescents who develop type 2 diabetes early in life (4). One theory to explain the loss of insulin secretion in the setting of type 2 diabetes is an excessive accumulation of lipid moieties within beta cells. The additional lipid disrupts the energy status of the beta cells (i.e. the adenosine triphosphate:adenosine diphosphate [ATP:ADP] ratio) and leads to the production of toxic lipid intermediates that accelerate cell death (5,6). To explore this pancreatic lipotoxicity theory in a clinical setting, noninvasive imaging modalities have emerged to quantify lipid content in the human pancreas (7e12). Studies in adults with type 2 diabetes and children with genetic disorders involving triglyceride synthesis have demonstrated that 1) noninvasive imaging tools can quantify triglyceride accurately in the pancreas (13); 2) pancreatic lipid content is elevated in human obesity and in type 2 diabetes (14,15); and 3) the degree of lipid within the pancreas is negatively associated with beta cell function (14). Although these results implicate lipotoxicity in the natural history of beta cell dysfunction, the studies did not control for differences in visceral fat content between the groups, which may contribute to both pancreatic lipid content and defects in glucose-stimulated insulin secretion (16). Our cohort represents a clinical model of early, accelerated beta cell failure in the absence of aging and insulin resistance (4,17). We recently demonstrated that adolescents with type 2 diabetes are 4 to 6 times more likely to display ectopic lipid accumulation, in the form of hepatic steatosis, than normoglycemic adolescents matched for body mass index (BMI) (17). In addition, we found that the presence of hepatic steatosis in overweight normoglycemic adolescents predicts metabolic abnormalities, including metabolic syndrome and increased glucose dispersion, following oral glucose challenges (18). Based on these findings, we hypothesized 1) that youth with type 2 diabetes would display a significantly higher proportion of lipid within the pancreas compared to normoglycemic adolescents, independent of adiposity; and 2) that the degree of lipid accumulation in the pancreas would be negatively associated with glucose-stimulated insulin secretion. Finally, because a number of patients with type 2 diabetes in our clinical program are carriers of a private polymorphism in the HNF-1alpha gene G319S (19), resulting in reduced insulin secretory capacity (20e22), we also performed an exploratory subgroup analysis to determine levels of pancreatic lipid among carriers of the G319S polymorphism in the HNF-1alpha gene.
Study design and study population This was a cross-sectional study comparing pancreatic triglyceride content between adolescents with type 2 diabetes and age-, sex- and BMI-matched normoglycemic adolescents. Participants with type 2 diabetes were recruited through the Diabetes Education Resource for Children and Adolescents (DER-CA) in Winnipeg, Manitoba, Canada. Overweight and obese normoglycemic adolescents were recruited through radio advertisements and posters in local community health centers and in the Manitoba Institute of Child Health Research Center. Type 2 diabetes was diagnosed according to the criteria published by the American and Canadian Diabetes Associations (23,24). Participants were classified as overweight or obese according to the age- and sex-specific guidelines established by the International Obesity Task Force (25). Adolescents were excluded from the study if they 1) had used antidiabetes medications in the 12 months prior to participation in the study or 2) had been treated with medication known to affect glucose handling or lipid metabolism, including statins, atypical antipsychotics or corticosteroids. Three youth recruited as normoglycemic controls demonstrated impaired glucose tolerance during a 75 g oral glucose tolerance test and were excluded from further participation. Two participants with type 2 diabetes failed to obtain magnetic resonance imaging (MRI) due to size restrictions and, therefore, were excluded from final analysis. Three normoglycemic participants failed to receive MRI due to scheduling problems with the MRI acquisition and were excluded from the final analysis. A total of 20 adolescents with type 2 diabetes and 34 healthy overweight/obese controls were included in the final analysis. All participants and parents provided written informed consent to participate in the study, which was approved by the biomedical research ethics board at the University of Manitoba and performed according to the Declaration of Helsinki. Pancreatic triglyceride content All MRIs were performed using a 3.0-T whole-body magnet (Siemens USA, Malvern, PA, US) as previously described (17,18). In vivo magnetic resonance spectroscopy with a single voxel volume of 3 cm (axial) 3 cm (coronal) 3 cm (sagittal) (27 cm3) was used to collect 64 consecutive 1H spectra from the tail of the pancreas (15,26). We chose the tail of the pancreas because preliminary analyses revealed that this location provided the most reliable acquisition of spectra, and it is also the anatomic location of the islets of Langerhans. Axial, coronal and sagittal high-resolution images of the pancreas were obtained by using standard clinical techniques. To reduce contamination from surrounding visceral fat, spatial saturation bands were manually placed around the voxel. The use of spatial saturation bands improves the baseline of collected spectra by dramatically reducing the signals from surrounding contaminating lipids (27). In addition, spectra and imaging were collected using respiratory gating to reduce motion
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artefact and contamination by surrounding fat. Images and spectra were collected at 10% of the subjects’ inspiratory efforts (28). Robust automated spectral analysis was performed using the LCModel software , Japan L.A Systems (29). Using this software, triglyceride and water peaks were quantified by fitting known basis-set spectra to measured spectra. The relative standard deviation between the known spectra and the measured spectra are reported by the Cramer-Rao lower bounds (i.e. % SD). This method of acquiring pancreatic lipid content has been published previously and validated in the scientific literature (11,12,15,26). Beta cell function A modified, frequently sampled intravenous glucose tolerance test was used to determine the 2 primary measures of beta cell function: the acute insulin response to exogenous glucose and the disposition index (30). The disposition index is a measure of glucosestimulated insulin secretion adjusted for the prevailing levels of insulin resistance. It was calculated as the product of insulin sensitivity and the acute insulin response (Si AIR) and was used to quantify glucose-stimulated insulin secretion relative to insulin resistance. Fasting blood samples were collected prior to an intravenous bolus of a 25% glucose solution (0.3 g/kg body weight) at time 0, followed by an intravenous bolus of regular human insulin (0.03 U/ kg body weight) at the 20-minute time point. Blood samples to test for glucose and insulin were collected at 1, 2, 3, 4, 5, 6, 8, 10, 14, 19, 22, 25, 30, 40, 50, 70, 100, 140 and 180 minutes. Glucose and insulin kinetics, including acute insulin response and insulin sensitivity, were then modelled using the Bergman minimal model provided in
Table 1 Anthropometric and metabolic characteristics of obese/overweight healthy controls and adolescents with type 2 diabetes Variable
Overweight/obese controls
Type 2 diabetes
n Age (years) Sex (male/female) Ethnicity (FN/Metis/other) Tanner stage (1-2/3-5) Adiposity BMI z score Waist circumference z score % Body fat Visceral fat (cm2) Cardiometabolic risk factors Systolic blood pressure (mm Hg) Total cholesterol (mmol/L) Fasting glucose (mmol/L) HDL (mmol/L) LDL (mmol/L) AST (mmol/L) ALT (mmol/L) Serum triglycerides (mmol/L) Metabolic profile Insulin sensitivity (mU kg1 min1) Disposition index Acute insulin response* Total pancreatic triglyceride (% F/W)*
34 15.41 (14.73e16.09) 7/27 6/5/23 3/31
20 15.45 (14.65e16.25) 7/13 16/2/2y 1/15
1.99 (1.83e2.15) 5.80 (4.90e6.70)
1.72 (1.41e2.03) 5.66 (4.39e6.94)
39.6 (37.5e41.7) 94.5 (77.7e111.3)
34.0 (30.2e37.7)z 126 (87.0e165.5)
114 (110e119)
123 (117e130)z
4.0 4.7 1.13 2.3 19 17 1.35
4.0 6.7 1.05 2.2 22 27 1.66
(3.7e4.3) (4.5e4.9) (1.04e1.22) (2.0e2.5) (17e21) (14e20) (1.14e1.56)
3.37 (2.50e4.25) 2612 (1970e3255) 758.0 (460.0, 1452) 1.22 (0.08, 5.93)
(3.6e4.5) (5.8e7.7)y (0.92e1.18) (1.8e2.6) (17e27) (16e39) (1.22e2.10)
4.93 (1.07e8.80) 493.1 (169.1e817.2)y 33.37 (0.00, 188.0)y 2.41 (0.63, 5.60)
ALT, Alanine aminotransferase; AST, alternate site testing; F/W, fat/water; FN, First Nations; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Data are means (95% CI). * Medians (25%, 75% quartiles) are used to represent non-normally distributed variables. y p<0.001. z p<0.05 vs. overweight/obese.
customized software (MINMOD; RC, Boston, Massachusetts, USA) to quantify insulin sensitivity and beta cell function. Confounding variables Visceral fat mass was assessed using high-resolution MRI at a level between the 3rd and 5th lumbar vertebrae, as previously described (31,32). Images were viewed, and adiposity was quantified offline using Slicer3 software (v. 3.6.3 1.0; pieper at bwh. harvard.edu, Cambridge, Massachusetts, USA). Dual energy x-ray absorptiometry (Hologic; Bedford, Massachusetts, USA) was used to quantify fat mass (kg), fat free mass (kg) and percent body fat. Waist and hip circumferences were measured in duplicate with a flexible tape measure, and the average of the 2 measures was used. Waist circumference was taken at the level of the iliac crest, as previously described, and z-scores were calculated from a nationally representative sample of Canadian youth (33). Pubertal stage was assessed using a pictorial tool for self-assessment of stage of development, and in girls, attainment of menstruation was collected from histories. Ethnicity was identified by self-report. Assessment of HNF-1alpha genotype Genotyping was performed as previously described (34) in youth with type 2 diabetes. Biochemical measurements Plasma glucose was measured on a Roche Modular P analyzer with an ultraviolet test principle (hexokinase method). Insulin was measured on an Immulite (Siemens Healthcare Global; Erlangen, Germany) solid-phase, 2-site chemiluminescent immunometric assay. Serum lipoproteins, liver transaminases and triglycerides were measured on a Roche Modular P analyzer (Roche Diagnostics; Indianapolis, Indiana, USA). Statistical analyses A sample size calculation based on a projected difference in pancreatic lipid between groups of 30% and a standard deviation of MRI-derived pancreatic lipid content of 15% revealed that a sample of 20 patients per group would provide 80% power to detect a 30% difference between the groups [alpha¼0.05, beta¼0.1]. Sample size analysis was performed using SPSS sample power software (v. 3.0.0; IBM, Chicago, Illinois, USA). All data are presented as means with 95% confidence intervals, unless otherwise stated. Data were assessed for normal distribution by using the Kolmogorov-Smirnov test. For non-normally distributed variables, Mann-Whitney U tests were used to test for differences between groups. Comparisons of normally distributed variables between groups were performed using the Student t test. A univariate regression was performed to test for differences in outcome variables between the groups after adjusting for ethnicity, age, sex, visceral adiposity and genetic variance. The Spearman rank correlation tests were used to assess the associations between pancreatic lipid content and measures of beta cell function. Multiple stepwise linear regression analysis was used to assess the independence of the association between pancreatic triglyceride content and beta cell function after adjusting for potential confounding variables, including age, sex, ethnicity, BMI z-scores, pubertal stages and total and visceral fat masses. A second multiple linear regression analysis was performed to determine whether adiposity, hepatic lipid content and ethnicity were independently associated with pancreatic lipid content. All analyses were performed using SPSS v. 19.0; p<0.05 was considered statistically significant.
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Results
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Table 3 Determinants of pancreatic triglyceride in youth
Participants’ demographics and metabolic variables are presented in Table 1. The type 2 diabetes group included a greater proportion of First Nations/Metis youth than did the control group (90% vs. 32%, X2¼16.8; p<0.001). Systolic blood pressure (12313 vs. 11411 mm Hg, p¼0.019) was significantly higher in youth with type 2 diabetes, while percent of total body fat (34.07.6 vs. 39.66.0%; p¼0.005) was significantly lower in youth with type 2 diabetes, despite similar BMIs. However, the proportion of body fat composed of visceral adiposity appeared to be higher in youth with type 2 diabetes (not statistically significant). As expected, fasting glucose was higher in youth with type 2 diabetes compared to normoglycemic controls (6.70.5 vs. 4.70.1 mmol/L; p<0.001). Although insulin sensitivity was not significantly different in the 2 groups, both acute insulin response (33 [interquartile range (IQR): 0, 188] vs. 758 [IQR: 460, 1452]; p<0.001) and disposition index (DI: 493692 vs. 26121841; p<0.001) were significantly lower in those with type 2 diabetes (Table 1). Pancreatic lipid content was 2-fold higher in youth with type 2 diabetes compared to normoglycemic controls; however, this difference was not statistically significant (Table 1). Total pancreatic lipid content was significantly higher in Indigenous youth compared to non-Indigenous youth (2.60 [IQR: 1.06, 5.70] vs. 0.65 [IQR: 0.03, 5.95] %F/W, p¼0.034) (Table 2). Indigenous adolescents with type 2 diabetes who were carriers of the G319S polymorphism in the HNF-1alpha gene displayed a w4-fold higher total pancreatic triglyceride content than their wild-type or non Oji-Cree counterparts (7.45 [IQR: 2.85, 26.8] vs. 2.20 [IQR: 0.35, 3.30] %F/W, p¼0.03) (Table 4). In bivariate analysis, pancreatic fat content was not associated with either the acute insulin response (r¼e0.21; p¼0.13) or the disposition index (r¼e0.08; p¼0.55) adjusted for visceral adiposity. Hepatic lipid content was the only variable associated with pancreatic lipid content in the bivariate analysis (r¼0.31; p¼0.04), adjusted for age, sex, BMI z-score, level of insulin resistance, pubertal stage, ethnicity and visceral fat mass (r¼0.16; p¼0.016) (Table 3).
Table 2 Subgroup analysis of metabolic risk in youth matched for Indigenous ethnicity status Variable
Overweight/obese controls
Type 2 diabetes
n Age (years) Sex (male/female) Tanner stage (1e2/3e5) Adiposity BMI z score Waist circumference z score % body fat Visceral fat (cm2) Metabolic data Insulin sensitivity (mU kg1 min1) Disposition index (AIR Si) Acute insulin response* (mU/L) Total hepatic triglyceride (% F/W) Total pancreatic triglyceride (% F/W)*
11 15.1 (1.4) 9/2 1/10
20 15.9 (1.7) 8/12 1/19
2.06 (1.82e2.30) 7.26 (5.44e9.07) 40.89 (37.16e44.62) 87.2 (64.4e109.0) 3.03 (1.05e5.00) 2169 (1669e3735) 234 (132,870)
1.69 (1.39e1.98) 5.58 (4.40e6.76) 34.87 (31.16e38.58)z 112.4 (83.8e139.9) 4.97 (1.15e8.82) 330 (281e1009)y 97 (22 780)y
20.2 (4.2e23.3)
14.0 (0e30.5)
3.3 (1.7,6.6)
2.6 (1.2,5.0)
AIR, Acute insulin response; BMI, body mass index; F/W, fat/water; Si, insulin sensitivity. Data are means (95% CI). * Medians (25%, 75% quartiles) represent non-normally distributed variables. y p<0.001. z p<0.05 vs. overweight/obese.
Variable
Standardized beta
Triglyceride
p
Ethnicity BMI z score Insulin sensitivity Tanner stage Hepatic triglyceride
0.20 0.05 0.17 0.18 0.39
1.3 0.30 0.90 1.03 2.59
0.19 0.77 0.37 0.31 0.013
BMI, Body mass index.
Exploratory analyses To determine whether the presence of the G319S polymorphism was associated with differences in ectopic lipid content and beta cell function, we performed a series of exploratory cross-sectional comparisons between Indigenous adolescents who were carriers and noncarriers of the polymorphism. Adolescents with type 2 diabetes who were carriers of the G319S polymorphism in the HNF1alpha gene displayed a w4 fold higher total pancreatic triglyceride content than their wild-type or non Oji-Cree counterparts (7.45 [IQR: 2.85, 26.8] vs. 2.20 [IQR: 0.35, 3.30] %F/W; p¼0.03).
Discussion This study revealed that pancreatic triglyceride content was not associated with a diagnosis of type 2 diabetes in adolescents, nor was it related to pancreatic beta cell function in adolescents. In addition, we found that pancreatic fat content was higher in overweight Indigenous youth, irrespective of glycemic status and unaffected by sex, relative to non-Indigenous peers. Finally, we found that the presence of the G319S polymorphism in the HNF1alpha gene known to attenuate insulin secretory capacity was associated with a w4 fold increase in pancreatic triglyceride content despite similar levels of whole-body and visceral adiposity. It is possible that type 2 diabetes and the HNF-1alpha polymorphism may be associated with ectopic fat distribution in the pancreas, but the latter is not associated with beta cell dysfunction. Interest in the role of excessive pancreatic triglyceride content in the pathogenesis of type 2 diabetes has grown since the seminal work of Roger Unger in the late 1990s (35). Recently, the development of noninvasive imaging tools has made it possible to validate these studies in clinical populations (26,36). Previous studies in adolescents and young adults suggest that although the pancreatic fat fraction derived from computed tomography is associated with components of the metabolic syndrome, it is not associated with beta cell function (36). The data presented here support this observation and extend it by demonstrating that pancreatic triglyceride content, measured directly by proton magnetic resonance spectroscopy (1H-MRS), is not significantly elevated in youth with established type 2 diabetes and is not associated with glucosestimulated insulin secretion. Previous 1H-MRS studies of the pancreas found 2- to 3-fold higher triglyceride content in the pancreases of adults with impaired glucose tolerance or type 2 diabetes (14,26) compared to normoglycemic adults. None of these studies demonstrated an association between triglyceride content in the pancreas and any measure of beta cell function, and it was postulated that type 2 diabetes diagnosed in adults with the prolonged duration of hyperglycemia may be too late in the natural history to identify this early component of pathophysiology. Using a similar 1H-MRS technique, we found that pancreatic triglyceride content is not associated with measures of beta cell function in the early-onset, short-duration model of type 2 diabetes in adolescents. The data presented here suggest that pancreatic triglyceride
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content is not elevated, even in the early stages of beta cell dysfunction in adolescents, after adjusting for measures of adiposity. Similar to previous studies in adolescents (36), we found that lipid content in the pancreas is elevated in ethnic subgroups that are at particularly high risk for type 2 diabetes. Specifically, we found that pancreatic triglyceride content was w4-fold higher in Indigenous youth, who display a 3- to 5-fold greater risk for type 2 diabetes compared to Caucasian peers (37). This observation is consistent with previous studies by our group that showed that overweight Indigenous youth are more susceptible to ectopic triglyceride deposition (17). Ethnic disparity in ectopic lipid accumulation has been described by others (38,39). The ethnic differences in susceptibility may be attributed to the presence of genetic variants associated with increase lipid synthesis or uptake (40,41). It may also be attributed to differences in diet or physical activity patterns between ethnic groups (42e44). Regardless of the mechanisms, the consistent observation that ethnic groups with particularly high susceptibility to type 2 diabetes are characterized by excessive ectopic triglyceride suggests that clinical screening for steatosis in patients from these minority groups may provide insight into their risk for type 2 diabetes. The data presented here suggest that the G319S polymorphism in the HNF-1alpha gene may increase the susceptibility to excessive triglyceride deposition in the pancreas. Mutations in the HNF-1alpha gene are associated with monogenic maturity-onset diabetes in the young (45). The presence of the G319S polymorphism in the HNF-1alpha gene does not result in monogenic diabetes, despite a 50% reduction in insulin protein transcription Table 4 The presence of the G319S polymorphism of the HNF-1alpha gene is associated with elevated pancreatic triglyceride content in youth with type 2 diabetes Variable
G/G
S/G and S/S
n Age (years) Sex (male/ female) (n) Ethnicity (FN/Metis/ other) (n) Adiposity BMI z score Waist circumference z score Visceral fat (cm2) % body fat Cardiometabolic risk factors Systolic blood pressure (mm Hg) Fasting glucose (mmol/L) Total cholesterol (mmol/L) Serum triglycerides (mmol/L) HDL (mmol/L) LDL (mmol/L) AST (mmol/L) ALT (mmol/L) Metabolic profile Insulin sensitivity (mU kg1 min1) Disposition index AIR Total pancreatic triglyceride (% F/W)
15 15.0 (14.0, 17.0) 4/11
5 15.0 (15.0, 16.5) 3/2
11/2/2
5/0/0
1.89 (1.23, 2.27) 5.27 (3.62, 9.42)
1.79 (1.70, 1.98) 4.97 (4.23, 5.89)
114 (79.0, 144) 31.8 (28.6, 42.3)
85.3 (57.7, 148) 35.5 (25.2, 42.2)
121 (112, 128)
126 (111, 150)
6.3 (5.1, 9.1)
5.20 (4.85, 6.65)
4.0 (3.3, 4.9)
3.20 (3.15, 4.65)
1.5 (0.83, 2.6)
1.40 (1.10, 2.05)
1.0 2.1 19 21
1.00 1.70 16 13
(0.90, 1.2) (1.4, 3.2) (14, 29) (9.5, 41)
1.88 (0.919, 2.78) 35.59 (e57.93, 397.1) 12.81 (e98.48, 76.78) 2.20 (0.350, 3.30)
(1.00, 1.15) (1.45, 2.70) (15, 30) (12, 46)
6.89 (3.50, 8.60)* 1060 (162.6, 1694) 116.7 (16.56, 378.7) 7.45 (2.85, 26.8)*
AIR, Acute insulin response; ALT, alanine aminotransferase; AST, alternate site testing; FN, First Nations; F/W, fat/water; G/G, Wild Type; HDL, high-density lipoprotein; LDL, low-density lipoprotein; S/G, Heterozygote for G319S polymorphism; S/S, Homozygote for G319S polymorphism. Data are medians (25%, 75% quartiles). * p<0.05 vs. wild-type.
and insulin secretory capacity (20). Among heterozygotes, the decrement in beta cell reserve is mild and typically requires environmental pressures, such as increasing adiposity or exposure to gestational diabetes, to result in type 2 diabetes (46). However, among homozygotes, beta cell function is profoundly reduced, increasing the risk for type 2 diabetes 10-fold. Youth who are carriers of this polymorphism are characterized by less adiposity and less insulin resistance (19). Further studies are needed to determine whether the predisposition to pancreatic steatosis is evident in carriers of this polymorphism and to discover the potential mechanisms through which the HNF-1alpha gene regulate triglyceride metabolism in human tissues. Ectopic lipid accumulation is reversible with changes in lifestyle behaviours that lead to weight loss. A recent small study in adults with type 2 diabetes (n¼11) investigated the utility of dietary restriction on reversal of pancreatic fat and normalization of beta cell function (47). The authors reported that caloric restriction to 600 kcal per day in overweight individuals over a period of 8 weeks significantly reduced pancreatic (8.0%1.6% to 6.2%1.1%) and hepatic triglyceride content (12.8%2.4% to 2.9%0.2%). The reductions in these ectopic lipid deposits were associated with a near 2-fold improvement in hepatic insulin sensitivity and a 3-fold increase in the first-phase insulin response (0.190.02 to 0.460.07 nmol min1 m2) (47). We did not observe an association between any measures of lifestyle and the degree of pancreatic lipid content in this cohort of youth. It remains unclear whether lifestyle factors play roles in the loss of beta cell function or pancreatic steatosis in overweight youth. Youth with type 2 diabetes had lower percentages of body fat, but the distribution of body fat appeared to favour visceral fat and to be clinically recognized as higher waist circumference. There may be an important ethnic difference in body fat deposition and accumulation of metabolically active adipose. Additional research is needed to confirm that rapid changes in body weight are important in the regulation of ectopic lipid content and the insulin-glucose axis in adolescents with type 2 diabetes. Several limitations of this study require discussion. First, the cross-sectional design of the study precludes determination of a causal nature for the associations described. Differences in ethnicity existed between groups; however, including ethnicity as a covariate in a general linear model did not alter groupwise differences in pancreatic triglyceride content, insulin sensitivity or acutephase insulin secretion. We were unable to match for sex, age and BMI z-score for the subanalysis isolated to the Indigenous population of adolescents. Significant differences existed in the proportion of males within the 2 groups, which may contribute to the among-group differences identified within Indigenous subgroups. The use of an intravenous glucose tolerance test to assess beta cell function does have limitations, and its use in patients with type 2 diabetes has been questioned due to an inhibitory effect of hyperglycemia on glucose-stimulated insulin secretion (i.e. glucotoxicity). To reduce the effects of glucotoxicity on the measures of beta cell function, we recruited patients with good metabolic control (glycated hemoglobin 5.5% to 8.0%). In these studies, 95% of patients with type 2 diabetes presented with a fasting glucose <7.0 mmol/L. Because the inhibitory effects of hyperglycemia on glucose-stimulated insulin secretion become evident only with fasting glucose levels above 7 mmol/L, the use of this method to assess beta cell function was supported in our study cohort of subjects with type 2 diabetes (48). In conclusion, in our study, pancreatic triglyceride content was not associated with type 2 diabetes in adolescents or with pancreatic beta cell function. Our findings are in contrast to the theory of pancreatic lipotoxicity as a mechanism for beta cell failure in the setting of type 2 diabetes in adolescents. Pancreatic steatosis was evident in groups of youth at risk for type 2 diabetes and carriers of a polymorphism in the HNF-1alpha gene that is associated with beta cell dysfunction.
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These data suggest that pancreatic triglyceride accumulation occurs independent of beta cell failure and may be a marker of a more global defect in fatty-acid metabolism in high-risk cohorts of youth. Acknowledgements The authors are grateful for the clinical expertise provided by Dr. Heather Dean and Dr. Elizabeth Sellers (Pediatric Endocrinology, Diabetes Education Resource for Children, Winnipeg Children’s Hospital); Kristy Wittmeier (PhD, clinical investigator, Faculty of Medicine, University of Manitoba); Barry Clabbert (Clinical Chemistry, Faculty of Medicine, University of Manitoba); Lori Berard and Sherri Pockett (Clinical Research Unit, John Buhler Research Centre, Winnipeg, Manitoba); Quan Chung (National Research Council Institute for Biodiagnostics [NRC-IBD], Canada) Neil Pawlyshyn (MRI, St. Boniface Hospital, Winnipeg, Manitoba), Yao Fu (MR Spectroscopy, NRC-IBD, Winnipeg, Canada) and Anita Durksen (research associate, Manitoba Institute of Child Health). We are indebted to the parents and children who volunteered their time and effort for the completion of this study. Author Disclosures This work was supported by a Manitoba Medical Services Foundation grant to BAW; by MHRC, Lawson Foundation, Cosmopolitan Foundation and CIHR New Investigator Award to JMM; and by a Manitoba Health Research Council and Manitoba Institute of Child Health fellowship to BAW; ATRG and JND were supported by Manitoba Institute of Child Health Summer Studentships. Contributions of Authors BAW contributed to study design, researched data and wrote the manuscript; ATRG researched data and wrote the manuscript; JND researched data and contributed to discussion; NV and LNR contributed to the development of spectroscopy methods and analysis and to the discussion; JMM contributed to the concept and study design, researched data and edited the manuscript; BAW is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References 1. Stumvoll M, Goldstein BJ, van Haeften TW. Type 2 diabetes: Pathogenesis and treatment. Lancet 2008;371:2153e6. 2. Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin Invest 2006; 116:1802e12. 3. Robertson RP, Harmon J, Tran PO, et al. Beta-cell glucose toxicity, lipotoxicity, and chronic oxidative stress in type 2 diabetes. Diabetes 2004;53(Suppl 1): S119e24. 4. Bacha F, Lee S, Gungor N, et al. From pre-diabetes to type 2 diabetes in obese youth: Pathophysiological characteristics along the spectrum of glucose dysregulation. Diabetes Care 2010;33:2225e31. 5. Lee Y, Hirose H, Ohneda M, et al. Beta-cell lipotoxicity in the pathogenesis of non-insulin-dependent diabetes mellitus of obese rats: Impairment in adipocyte-beta-cell relationships. Proc Nat Acad Sci U S A 1994;91:10878e82. 6. Goh TT, Mason TM, Gupta N, et al. Lipid-induced beta-cell dysfunction in vivo in models of progressive beta-cell failure. Am J Physiol Endo Metab 2007;292: E549e60. 7. Lee Y, Lingvay I, Szczepaniak LS, et al. Pancreatic steatosis: Harbinger of type 2 diabetes in obese rodents. Int J Obes 2010;34:396e400. 8. Perman WH, Balci NC, Akduman I. Review of magnetic resonance spectroscopy in the liver and the pancreas. Top Magn Reson Imaging 2009;20:89e97. 9. Misra D, Gupta V, Sonkar AA, et al. Proton HR-MAS NMR spectroscopic characterization of metabolites in various human organ tissues: Pancreas, brain and liver from trauma cases. Physiol Chem Physics Med NMR 2008;40:67e88. 10. Esclassan J, Chemin-Thomas C, Creach Y, et al. High-resolution 1H NMR study of membrane phospholipids in rat pancreas stimulated by caerulein. Pancreas 1994;9:263e9. 11. Haaga JR. Magnetic resonance imaging of the pancreas. Radiol Clin North Am 1984;22:869e77.
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