Introduction

Type 2 diabetes generally develops when beta cells fail to maintain adequate insulin secretion for the corresponding insulin resistance, because of loss of function or apoptosis [1]. Dysfunction of beta cells occurs in islets susceptible to inherited abnormalities of function and insulin secretion, as opposed to islets capable of producing an adaptive response to insulin resistance [2]. Possible underlying mechanisms leading to beta cell dysfunction are abnormal mitochondrial function and oxidative stress, mainly due to glucose toxicity [3, 4] and lipotoxicity, along with elevated concentrations of NEFA [5, 6].

Lipotoxicity results from augmented adipose tissue lipolysis leading to triacylglycerol overflow and ectopic accumulation of lipid metabolites in non-adipose tissues. Previous research has focused mainly on the role of increased lipid accumulation in the liver [7] and skeletal muscle [8–10] for the development of insulin resistance and type 2 diabetes. However, recent data also show that obesity leads to an increase of pancreatic fat in animals [11–13] and humans [14–16], with evidence of triacylglycerol overflow inducing pancreatic steatosis and beta cell dysfunction [17–20]. Proton magnetic resonance spectroscopy (1H-MRS) is the current gold standard for non-invasive in vivo quantification of triacylglycerol content in liver and skeletal muscle, and has been validated against histological and biochemical quantification in human biopsies [7]. More recently, 1H-MRS [17–19] and magnetic resonance imaging (MRI) by fat/water imaging techniques [16, 21–23] have also been applied to determine pancreatic fat. These studies aimed not only to quantify the fat content of the pancreas, but also to examine the possible interaction of pancreatic fat with beta cell function and insulin secretion [18–20].

However, conflicting results were reported both with regard to the amount of pancreatic fat [16, 17, 19, 21] and the association with beta cell function in humans [18–21, 24]. These differences could be attributed to the various fat compartments found within the pancreas, and the variability they cause in invasive and non-invasive measurements. Similar to visceral adipose fat (VAT), peripancreatic, interlobular and intralobular adipose tissue [11, 14, 15] may alter beta cell function through the release of adipocytokines [6, 25]. On the other hand, lipid accumulation within exocrine or endocrine cells [12], or specifically in beta cells, could result in parenchymal or intracellular pancreatic steatosis [26, 27], activating cellular mechanisms in analogy to hepatic steatosis. Distinguishing between these compartments in vivo could therefore aid in the accurate assessment of the specific effects of individual fat depots on beta cell function.

Thus, this study aimed to apply different 1H-MRS and MRI techniques to non-invasively quantify pancreatic fat compartments in participants with normal glucose tolerance (NGT), impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT), and in patients with type 2 diabetes. Total pancreatic fat content was measured with a standard 1H-MRS technique [17, 19], intralobular fat content by a 1H-MRS method with localization via modified DIXON (mDIXON) [28] water images to avoid interlobular fat [28] and, finally, parenchymal fat accumulation was quantified by a validated [29] dual-echo mDIXON-MRI method. These measures of pancreatic fat compartments were then tested for correlation with beta cell function [30] derived from a frequent-sampling OGTT, as well as with VAT and hepatocellular fat content (HCL).

Methods

Participants

The study population comprised 56 white participants, 28 with NGT, 14 with IFG/IGT and 14 patients with type 2 diabetes of 5.8 ± 4.9 years (mean ± SD) known duration. Participants were excluded in the case of a medical history of pancreatitis, and/or alcohol consumption greater than 20 g per day for men or 10 g per day for women. Participants were also excluded if receiving treatment with thiazolidinediones, incretin mimetics or insulin, which interfere with ectopic fat deposition or beta cell function. The 1H-MRS/MRI protocol and OGTT were performed within 7 days, and patients withdrew from their oral glucose-lowering medication for at least 3 days before the metabolic test, as reported previously [31]. Type 2 diabetes patients were treated with metformin only (n = 6), metformin plus dipeptidyldipeptidase 4 (DPP4) inhibitor (n = 2), or sulfonylurea plus DPP4 inhibitor (n = 2). Four patients were using statin treatment. Finally, all participants gave written informed consent before inclusion in the study, which was approved by the institutional review ethics board and conducted in accordance with the Declaration of Helsinki.

OGTT

A 2 h OGTT (75 g) was performed after 3 days of carbohydrate-enriched diet and overnight fast. Blood was taken at 0, 30, 60 and 120 min for measuring glucose, insulin and C-peptide levels. Plasma glucose concentration was quantified with the EKF Biosen C-Line glucose analyser (EKF Diagnostic, Barleben, Germany) using the glucose oxidase method; serum insulin and C-peptide were measured by radioimmunoassay (Millipore, St Charles, MO, USA). The AUC was measured using the trapezoidal rule. The AUC of glucose (AUCGLUC) was used to quantify glucose tolerance on intervention. Insulin sensitivity was assessed from the oral glucose insulin sensitivity index (OGIS) [30, 32], which provides a measurement of insulin-mediated glucose clearance. Insulin secretion was assessed through C-peptide AUC (AUCCP), and the post-hepatic peripheral (systemic) appearance of insulin with AUCINS for the 2 h test. The interplay between insulin sensitivity and hormone secretion that describes the beta cell adaptive response to changes of insulin resistance [33] was determined by the adaptation index (OGIS × AUCCP). This variable was originally developed for the intravenous glucose test, but was validated as a reliable determinant of beta cell function derived from the OGTT [34, 35]. Finally, beta cell function, defined as hormone secretion relative to glucose stimulation, was derived from the ratios of total C-peptide to glucose (AUCCP/AUCGLUC), which is defined here as the insulinogenic index [30, 36, 37].

MRS and MRI

All measurements were performed using a whole-body 3.0-T Achieva scanner (Philips Healthcare, Best, the Netherlands). VAT was determined from the abdominal images from a T1-weighted turbo–spin–echo (TSE) sequence with 10 mm slice thickness [38]. Participants were then repositioned for the MRI/MRS protocol for HCL and pancreatic fat conducted with a 16 channel XL torso phased-array receiver coil (Philips Healthcare). HCL and pancreatic fat were determined by 1H-MRS (single voxel stimulated echo acquisition mode [STEAM], repetition time 4 s, echo time 10 ms, scans 32). Expiration respiratory triggering was used to avoid motion artifacts and VAT from outside the pancreas [19, 23]. The entire protocol was conducted within 45 min. 1H-MRS data were analysed with the software package NUTS (Acorn NMR, Freemont, CA, USA) and fat fraction was expressed as [fat / (water + fat)].

Total pancreatic fat

Standard measurements of 1H-MRS pancreatic fat served as a control, and were performed with a (20 × 10 × 10 mm3) voxel, with localization via navigator-triggered clinical T2-weighted TSE images (2,081 ms repetition time, 80 ms echo time, 4 mm slice thickness). VAT outside of the pancreas was avoided throughout all slices in coronal and transverse planes. The clinical T2-weighted images were not able to distinguish between adipose tissue infiltration and parenchymal tissue. Thus, results yielded a measure of total pancreatic fat consisting of peripancreatic, interlobular and intralobular adipose tissue infiltration and parenchymal fat.

Intralobular pancreatic fat

Transverse and coronal dual-echo mDIXON abdominal fat/water images were then acquired in separate 19 s breathholds, via a three-dimensional T1 fast field gradient echo pulse sequence (2 mm isotropic resolution, 5° flip angle, 5.0 ms repetition time, 1.2 ms echo time, 2.5 ms echo time) [29]. Single voxel (10 × 10 × 10 mm3) 1H-MRS was performed with voxel localization in the body or tail of the pancreas via the high-resolution transverse and coronal mDIXON water images. Measurements were conducted immediately after the mDIXON imaging to avoid the volunteer’s movement between pancreatic fat measurements. The voxel was placed not only to avoid VAT outside of the pancreas, but also regions of pure peripancreatic and interlobular fat. Thus, this method provided a measure of intralobular adipose tissue infiltration including parenchymal fat content. In all 1H-MRS pancreatic fat measurements, fat was calculated from the methylene signal at 1.3 ppm.

Parenchymal pancreas fat

The low-fat PRIDE software package (Philips Healthcare) was used in post-acquisition to reconstruct pixel by pixel fat-fraction maps from the mDIXON fat/water images. Fat-fraction measurements via mDIXON fat/water imaging with the low-fat PRIDE algorithm were previously validated against 1H-MRS in vivo HCL measurements [29], and found to have a CV of 4.3%. Measurements were performed after patient examination with the placement of 100 mm2 regions of interest (ROIs), which were defined to encompass the anterior and posterior walls of the pancreas in the transverse plane. Parenchymal fat measurements were represented as the average of four ROIs that avoided all regions of visible adipose tissue infiltration. Thus, this method yielded a measure of uniform pancreatic steatosis within parenchymal tissue.

1H-MRS of liver fat

Single voxel (30 × 30 × 20 mm3) 1H-MRS was used to measure liver fat using the above described STEAM sequence to assess water content and a variable power and optimization relaxation (VAPOR) water-suppressed STEAM sequence. Fat was expressed as the summation of the VAPOR water-suppressed fat peaks of methylene (1.3 ppm) and methyl (0.9 ppm). Measurements were T2 corrected to obtain values of proton density fat fraction, and corrected for per cent of total hepatic fat content [39].

Validation of 1H-MRS pancreas and liver fat measurements

The mean intra-individual CV between eight repeated hepatic and pancreatic measurements was conducted from the respective 1H-MRS measurements used in the present study. The CVs and Pearson correlation coefficient (r) were as follows (CV, r): 1H-MRS of liver fat (7.5%, 0.99), total pancreatic fat (7.5%, 0.95) and parenchymal pancreatic fat (9.5%, 0.94).

Statistical analysis

Results were expressed as mean ± SD or median (first quartile, third quartile) for cases of skewed distribution. Differences between groups were assessed using Wilcoxon–Mann–Whitney-tests, while differences between methods were assessed with a Wilcoxon signed-rank test. Associations of pancreatic fat and markers for obesity with ectopic fat distribution and beta cell function were assessed using Pearson correlations adjusted for age, sex and insulin sensitivity. Graphs were created using GraphPad PRISM V6 (GraphPad Software, La Jolla, CA, USA). All statistical analyses were performed using SAS for Windows Version 9.2 (SAS Institute, Cary, NC, USA). A p value ≤0.05 was considered to indicate a statistically significant difference.

Results

Participant characteristics

Patients with type 2 diabetes were older and had increased HbA1c compared with those with NGT and IFG/IGT, and had increased BMI and HCL compared with participants with NGT (Table 1). There were no differences in VAT and waist circumference between all participants. IFG/IGT participants had higher levels of fasting and 2 h glucose, increased insulin secretion (Fig. 1) and decreased insulin sensitivity and beta cell function compared with NGT. Furthermore, patients with type 2 diabetes demonstrated increased fasting and 2 h glucose levels compared with participants with NGT or IFG/IGT, along with decreased insulin sensitivity and beta cell function. However, 2 h insulin secretion results in participants with diabetes were similar to those of participants with NGT.

Table 1 The anthropometric characteristics of participants
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Fig. 1
figure 1

Circulating concentrations of (a) glucose, (b) insulin and (c) C-peptide during a 75 g OGTT. Data presented as mean ± SEM. Circles, NGT; squares, IFG/IGT; and triangles, type 2 diabetes

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Pancreatic fat content

Clinical T2-weighted imaging showed that the pancreas was clearly identifiable in NGT participants with low VAT (Fig. 2a–d). However, distinction of pancreatic parenchymal tissue from VAT for 1H-MRS localization was more difficult in participants with IFG/IGT or diabetes, due in part to irregularities in both the organ shape and size. The total pancreatic fat content was slightly lower in participants with NGT (1.95% [0.29%, 6.35%]) than in those with IFG/IGT (4.79% [1.55%, 8.27%]), in line with BMI and VAT measurements in the two groups. However, total pancreatic fat content and the variability of fat measurements were higher (p < 0.05) in patients with diabetes (8.35% [5.64%, 13.10%]).

Fig. 2
figure 2

Examples of pancreatic fat measurements and changes in pancreas (white circles) morphology found within the same NGT (a, e, i), IFG/IGT (b, f, j), and type 2 diabetes (c, g, k) participant. (a–d) Total pancreatic fat measurements with 2 cm3 1H-MRS voxel (white square) localization within clinical T2-weighted images (scale bar, 40 mm). (e–h) Intralobular pancreatic fat measurements with 1 cm3 voxel (white square) localization that avoided regions of peripancreatic and/or interlobular fat (black) visible in mDIXON water images. (i–l) Parenchymal fat measurements within fat-fraction maps reconstructed from mDIXON fat/water images to test for uniform pancreatic steatosis. Fat measurements range from 0% (blue) to 100% (red), with 100 mm2 ROIs (red circles). Box plots represent the median values of pancreatic fat, upper and lower limits represent the 25th and 75th percentiles, and whiskers the minimum and maximum. *p < 0.05 and **p < 0.001 (Wilcoxon–Mann–Whitney tests). T2DM, type 2 diabetes

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The high-resolution mDIXON water images clearly separated interlobular fat from parenchymal tissue, and accurately displayed the irregular shape of the pancreas in participants with IFG/IGT or type 2 diabetes (Fig. 2e–h). Within the pancreases of participants with NGT or IFG/IGT mDIXON water imaging often did not reveal any regions of pancreatic adipose tissue infiltration. However, in other cases, regions of pure adipose tissue infiltration were visible within the pancreas, and dissected large regions of parenchymal pancreatic tissue. Compared with total pancreatic fat measurements (p < 0.05), intralobular 1H-MRS measurements that avoided these regions of interlobular fat led to lower pancreatic fat values in participants with NGT (1.49% [0.01%, 2.30%]) and in those with IFG/IGT (0.67% [0.00, 5.07%]). Furthermore, mDIXON imaging within patients with type 2 diabetes found that increased adipose tissue infiltration led to difficulties in distinguishing between regions of intralobular or interlobular fat, due to either marked adipocyte infiltration or organ atrophy. This led to higher intralobular fat measurements in diabetic patients (6.20% [5.03%, 9.47%]) compared with participants with NGT (p < 0.001) or IFG/IGT (p < 0.05), in line with total pancreatic fat measurements.

Parenchymal pancreatic fat measurements revealed multiple regions of parenchymal tissue void of lipid accumulation in all participants, thereby providing evidence against uniform pancreatic steatosis (Fig. 2i–l). Parenchymal fat content in participants with NGT (0.14% [−0.10%, 0.36%]) or IFG/IGT (0.49% [0.14%, 0.88%]) was below the 2% sensitivity threshold of the fat quantification method, and were in line with intralobular fat measurements. However, the placement of ROIs during post-acquisition allowed for all regions of adipose tissue infiltration to be avoided, and also found no evidence of uniform parenchymal fat accumulation even in patients with type 2 diabetes (0.37% [−0.30%, 0.65%]). Further evidence against uniform pancreatic steatosis was found from the analysis of all 1H-MRS spectra obtained from total pancreatic and intralobular fat measurements. In all cases, the spectra exhibited a single broad fat peak in the region of 1.3 ppm that was signature of lipid accumulation within small volumes of pure adipose tissue, rather than the typical distribution of fat peaks obtained in intracellular HCL measurements (Fig. 3).

Fig. 3
figure 3

Comparison of 1H-MRS non-water-suppressed HCL and pancreatic fat spectra: (a) 20% HCL with clearly defined narrow fat peaks at 2.1 ppm, 1.3 ppm and 0.9 ppm; (b) 14% total pancreatic fat with single broad peak at 1.3 ppm; (c) 5% intralobular pancreatic fat with low amplitude broad peak at 1.3 ppm; (d) 11% pancreatic fat intentionally visceral-fat-doped voxel

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Fat distribution, insulin sensitivity and beta cell function

Prior to adjustments, total pancreatic fat (r = 0.477, p < 0.001), intralobular fat (r = 0.428, p < 0.001), and VAT 3(r = 0.282, p < 0.001) positively associated with age. On adjusting for sex and insulin sensitivity, total pancreatic fat (r = 0.385, p < 0.01) and intralobular fat (r = 0.310, p < 0.05) associated with age, but not VAT.

After adjusting for age, sex and insulin sensitivity, total pancreatic fat was positively associated with waist circumference (r = 0.326, p < 0.05), and intralobular fat was associated with HCL (r = 0.346, p < 0.05). However, no other associations were found between total, intralobular or parenchymal fat and fasting or 2 h glucose levels, VAT, BMI or HbA1c. Analysis further found no associations between total pancreatic fat, intralobular fat or parenchymal fat with insulin secretion or beta cell function in NGT participants: total insulin secretion (all p > 0.7), adaptation index (all p > 0.6) and insulinogenic index (all p > 0.2) (Fig. 4). Furthermore, no associations were found between any non-invasive pancreatic fat measurements and beta cell function in IFG/IGT participants—total insulin secretion (all p > 0.4), adaptation index (all p > 0.3), insulinogenic index (all p > 0.5)—or patients with type 2 diabetes—total insulin secretion (all p > 0.5), adaptation index (all p > 0.4) and insulinogenic index (all p > 0.3) (Fig. 5).

Fig. 4
figure 4

Association of total pancreatic fat, intralobular fat and parenchymal fat with (a–c) insulin AUC 120 min, (d–f) adaptation index (OGIS × AUCCP), and (g–i) insulinogenic index in participants with NGT. Data adjusted for age, sex and OGIS

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Fig. 5
figure 5

Individual analysis of the association of total pancreatic fat, intralobular fat and parenchymal fat with (a–c) insulin AUC 120 min, (d–f) adaptation index (OGIS × AUCCP), and (g–i) insulinogenic index within participants with IFG/IGT (squares) and type 2 diabetes (triangles). Data adjusted for age, sex and OGIS

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Discussion

This study found that pancreatic fat deposition in the region of the pancreas displayed an inhomogeneous distribution of adipose tissue infiltration that increased with decreasing glucose tolerance. On the other hand, MRI pancreatic fat measurements revealed large regions of parenchymal tissue void of relevant lipid accumulation in all participants, providing evidence against uniform pancreatic steatosis. Interestingly, despite positive correlation with age, no measure of pancreatic fat was associated with any variable of beta cell function, regardless of glucose tolerance status.

Values obtained from standard 1H-MRS pancreatic fat measurements were in line with previous reports [17, 24], and were highest in patients with type 2 diabetes [17, 19, 24]. It has been hypothesised through studies in humans [12, 17, 20] and Zucker fatty rats [13, 26, 40] that 1H-MRS measurements can therefore serve as a non-invasive biomarker of pancreatic steatosis, and a surrogate for triacylglycerol accumulation in beta cells. However, the present comparison of 1H-MRS techniques shows that non-invasive pancreatic fat measurements with localization via clinical T2-weighted images are not an accurate description of whole-organ fat, but are influenced by the inhomogeneous distribution of peripancreatic or interlobular fat that was visible in the high-resolution mDIXON fat/water images [28]. While this inhomogeneous distribution of fat is in contrast to previous MRI reports [16, 18], histological analyses of human pancreas tissue [11, 15, 41, 42] confirm the inhomogeneous distribution of pancreatic adipose tissue infiltration found in this study. This inhomogeneous distribution of adipose tissue could also explain the discrepancies in 1H-MRS [17, 19, 24] and MRI [16, 18, 21, 38] pancreatic fat studies, and suggests that non-invasive methods that assess whole-organ adipose tissue infiltration [14, 43] might provide more insight into the association between pancreas adipose tissue infiltration and beta cell function.

Analysis of all 1H-MRS fat spectra also revealed that total pancreatic fat and intralobular fat measurements were representative of lipid accumulation within adipose tissue, rather than parenchymal pancreatic triacylglycerol accumulation. Triacylglycerol overflow in the liver results in a distribution of narrow fat peaks representing different fatty acid chains, such as methyl (0.9 ppm) and methylene (1.3 ppm) [7, 39], whereas 1H-MRS pancreatic fat measurements revealed a single broad methylene peak even at high fat concentrations. This broad fat peak suggests that the fat is in a different chemical environment than the intracellular fat typically found in hepatocytes and myocytes [44], and thus experiences a different spin–spin relaxation. This hypothesis was verified by an in vivo pancreatic fat measurement intentionally doped with VAT; the results of the VAT-laden voxel matched a typical 1H-MRS pancreatic fat spectrum (Fig. 3).

Parenchymal pancreatic fat measurements further disprove the hypothesis of a uniform pancreatic steatosis [27] by showing, in all individuals, regions of parenchymal pancreatic tissue void of lipid accumulation. The fat-fraction algorithm within low-fat PRIDE reduced noise bias [45] at low-fat fractions, thereby improving the accuracy of parenchymal fat measurements [29, 38], but with an underestimation of results below 2%. Previous MRI methods used for pancreatic fat measurements either did not distinguish between parenchymal tissue and adipose tissue infiltration [18] or were hindered by low signal-to-noise ratio [21] and thick slices [16, 22]. Thus, results could also have been limited by the accuracy of the MRI method [16, 38, 46] for measuring low fat values or the method was not able to capture the irregular shape of the pancreas; higher pancreatic fat values could have reflected VAT infiltration. These findings are verified by histological reports of human pancreatic tissue that have clearly identified ectopic pancreatic fat as adipocyte infiltration visible between exocrine cells [11, 15, 41, 42]. Additionally, whole-organ autopsies have reported that pancreatic fat consists only of patchy areas of intralobular or perilobular fat [15], or pancreatic fat dissecting islands of pancreatic tissue [41]; to date, no human autopsy has reported evidence of intracellular pancreatic fat and beta cell dysfunction caused by pancreatic steatosis [11, 15, 42]. Taken together, the present data provide evidence that the accumulation of triacylglycerols within pancreatic parenchymal tissue is not a major contributor to the development of type 2 diabetes.

This study also confirmed reports that pancreatic adipose tissue infiltration is positively associated with age [14, 15, 24]. However, while adipose tissue infiltration increased with decreasing glucose tolerance, no associations were found with fasting or 2 h glucose, BMI, HCL or VAT. To date, the factors that lead to pancreatic adipose tissue infiltration are unknown as the associations of pancreas adipose tissue infiltration with BMI [18, 19], VAT [18, 21, 24] or HCL [19, 21, 24] are inconclusive. Previously, it has been reported that pancreatic fat infiltration is negatively associated with beta cell function in non-diabetic men [19] and prediabetic IFG/IGT patients [18], but not in patients with type 2 diabetes [19]. On the contrary, in this study we were unable to find any association between pancreatic adipose tissue infiltration and beta cell function regardless of glucose tolerance status. However, it has also been reported that pancreatic adipose tissue infiltration increases with age and BMI, but not type 2 diabetes [47]. Correspondingly, even participants with type 2 diabetes in this study had pancreases void of adipose tissue infiltration, and increased intralobular fat was found only in older participants with type 2 diabetes. Thus, this study also supports the hypothesis that adipose tissue rather reflects the replacement for parenchymal tissue loss [15, 48] that has been shown to occur with age [14] or type 2 diabetes [49], and only serves as a marker [24, 50] of beta cell dysfunction after the loss of parenchymal volume and corresponding loss of beta cell mass [49].

Among the limitations of this study was the rather small number of participants included in the study. Also, the older age of participants and patients with type 2 diabetes might have contributed to higher pancreatic fat values. Additionally, parenchymal fat measurements were not capable of detecting fat concentrations below 2% [29], or the accumulation of fat directly within islets. Last, the route of glucose administration in the OGTT compared with the intravenous glucose tolerance test could affect the assessment of beta cell function. However, the OGTT-derived beta cell variables have been validated against those obtained from intravenous glucose loading [36] and also more closely resemble physiological conditions of insulin secretion.

In conclusion, this study found that pancreatic fat consists of an inhomogeneous distribution of adipose tissue infiltration rather than uniform pancreatic steatosis. Adipose tissue infiltration increased with age and decreasing glucose tolerance. However, no associations were found with beta cell function regardless of glucose tolerance status. Taken together, the pancreas does not appear to be another target organ for abnormal endocrine function due to ectopic parenchymal fat storage.