Glucolipotoxicity age-dependently impairs beta cell function in rats despite a marked increase in beta cell mass
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Prolonged exposure of pancreatic beta cells to excessive levels of glucose and fatty acids, referred to as glucolipotoxicity, is postulated to contribute to impaired glucose homeostasis in patients with type 2 diabetes. However, the relative contribution of defective beta cell function vs diminished beta cell mass under glucolipotoxic conditions in vivo remains a subject of debate. We therefore sought to determine whether glucolipotoxicity in rats is due to impaired beta cell function and/or reduced beta cell mass, and whether older animals are more susceptible to glucolipotoxic condition.
Wistar rats (2 and 6 months old) received a 72 h infusion of glucose + intravenous fat emulsion or saline control. In vivo insulin secretion and sensitivity were assessed by hyperglycaemic clamps. Ex vivo insulin secretion, insulin biosynthesis and gene expression were measured in isolated islets. Beta cell mass and proliferation were examined by immunohistochemistry.
A 72 h infusion of glucose + intravenous fat emulsion in 2-month-old Wistar rats did not affect insulin sensitivity, insulin secretion or beta cell mass. In 6-month-old rats by contrast it led to insulin resistance and reduced insulin secretion in vivo, despite an increase in beta cell mass and proliferation. This was associated with: (1) diminished glucose-stimulated second-phase insulin secretion and proinsulin biosynthesis; (2) lower insulin content; and (3) reduced expression of beta cell genes in isolated islets.
In this in vivo model, glucolipotoxicity is characterised by an age-dependent impairment of glucose-regulated beta cell function despite a marked increase in beta cell mass.
KeywordsAgeing Biosynthesis Gene expression Glucose clamp Hyperglycaemia Hyperlipidaemia Insulin Islets of Langerhans
Intravenous fat emulsion
Type 2 diabetes occurs in genetically predisposed individuals when pancreatic beta cells fail to compensate for peripheral insulin resistance . Chronic hyperglycaemia , hyperlipidaemia  and a combination of both  have been proposed to contribute to beta cell failure. Specifically, the glucolipotoxicity hypothesis posits that chronically elevated levels of glucose and fatty acids synergistically contribute to beta cell dysfunction in type 2 diabetes [4, 5].
The mechanisms by which glucolipotoxicity impairs beta cell function in vivo remain poorly understood. While experiments performed in the Zucker diabetic fatty rat were instrumental in identifying some cellular mechanisms of glucolipotoxicity , the profound alterations in intracellular fatty acid metabolism resulting from disrupted leptin signalling in this model limit the relevance of these findings to the human condition. In non-genetic rodent models, prolonged elevations of circulating fatty acid levels generally lead to decreased insulin secretion in vivo [6, 7, 8] and ex vivo [8, 9], although conflicting results have been reported [10, 11]. Using alternate and cyclical infusions of glucose and intravenous fat emulsion (IF) over 72 h in 2-month-old Wistar rats, we previously observed reduced insulin gene expression and insulin content, whereas insulin secretion in hyperglycaemic clamps and in isolated islets remained unaltered . We hypothesise that the inconsistent effect of fatty acids on insulin secretion in vivo are due to: (1) incomplete replication of glucolipotoxic conditions by infusing lipids without glucose; and (2) the use of young animals, which are capable of mounting an adequate compensatory response to the insulin-resistant state induced by lipid infusion. In humans, age is known to represent an independent risk factor for beta cell failure .
To our knowledge, the question of whether simultaneously elevated levels of glucose and fatty acids impair insulin secretion and/or lead to decreased beta cell mass in normal rats in vivo has not been examined. However, this is an issue that is key to our understanding of the pathogenesis of glucolipotoxicity. We therefore sought to address the following questions. First, does glucolipotoxicity occur in vivo in normal rats? Second, is glucolipotoxicity due to defective beta cell function, loss of beta cell mass or both? Third, is age a susceptibility factor in the development of glucolipotoxicity?
Animals, infusions and hyperglycaemic clamps
All procedures were in accordance with the National Institutes of Health Principles of Laboratory Animal Care and were approved by the Institutional Committee for the Protection of Animals at the Centre Hospitalier de l’Université de Montréal. Male Wistar rats weighing 250 to 300 g (∼2 months old) and 500 to 600 g (∼6 months old; Charles River, St Constant, QC, Canada) were housed under controlled temperature on a 12-h light–dark cycle with unrestricted access to water and standard laboratory chow. Animals underwent catheterisation of the jugular vein and carotid artery, as described  and were enabled to recover for 5 days (Electronic supplementary material [ESM] Fig. 1). The animals were randomised into two groups, receiving either 0.9% saline (vol./vol.; Baxter, Mississauga, ON, Canada) or 70% glucose (wt/wt; McKesson, Montreal, QC, Canada) plus 20% IF (vol./vol.) (Intralipid; Fresenius Kabi, Uppsala, Sweden) with 20 U/ml heparin (Sandoz, Boucherville, QC, Canada; glucose + IF). Infusion was performed using infusion pumps (Pump 33; Harvard Apparatus Canada, Saint Laurent, QC, Canada) with independent operation of two syringes simultaneously. Initial glucose infusion rates were 3.3 and 2.8 ml kg−1 h−1 for glucose in 2- and 6-month-old rats, respectively, and were adjusted to maintain blood glucose levels within the target range of 13.8 to 16.7 mmol/l throughout the 72 h infusion period. The infusion rate of IF + heparin was 1.7 ml kg−1 h−1 and remained unchanged throughout the infusion. The infusion rate in the saline group was matched to that of the glucose + IF group. A first group of rats was subjected to one-step hyperglycaemic clamps followed by an arginine bolus prior to and at the end of the 72 h infusion to measure insulin secretion in vivo, as previously described . Briefly, a 50% dextrose (McKesson) solution was infused through the jugular vein to clamp plasma glucose at 13 to 14 mmol/l for 70 min and adjusted on the basis of instantaneous assessments using a glucose analyser (YSI Incorporated, Yellow Springs, OH, USA). A bolus of arginine (174 mg/kg) (Sandoz) was injected at 60 min. Plasma samples were collected from the carotid artery for insulin and C-peptide measurements at −30, 0, 5, 15, 30, 45, 60, 61 and 70 min. At the end of the post-infusion clamp the animals were killed and the pancreas removed for morphological analyses. The M/I index of insulin sensitivity was calculated by dividing the average glucose infusion rate during the second half of the glucose clamp (M expressed in μmol kg−1 min−1) by the average circulating insulin value (I expressed in pmol/l) during the same time period . The disposition index (DI) of insulin secretion corrected for insulin sensitivity was calculated by multiplying the M/I index by the average circulating C-peptide (in nmol/l) during the second half of the clamp . Insulin clearance was estimated by the C-peptide/insulin ratio .
Islet isolation and measurements of insulin secretion and proinsulin biosynthesis
In a second series of experiments, islets were isolated at the end of the infusion by collagenase digestion and dextran density gradient centrifugation as described . For perifusion experiments, batches of 100 islets each were placed in Swinnex chambers (Millipore, Nepean, ON, Canada) and perifused for 60 min with KRB buffer containing 2.8 mmol/l glucose. The glucose concentration was increased to 16.7 mmol/l at time 0 for a total of 70 min. At 60 min, arginine-potentiation of glucose-induced insulin secretion was measured by adding 10 mmol/l arginine to the KRB for 10 min. At 70 min the glucose concentration of the KRB was decreased to 2.8 mmol/l. Samples were collected at 1 min intervals throughout the perifusion for insulin determination. Intracellular insulin was extracted with acidified ethanol at the end of the perifusion to measure insulin content. For measurements of proinsulin biosynthesis, isolated islets were recovered for 1 h at 11 mmol/l glucose, after which batches of 100 islets each were washed, transferred to KRB containing 2.8 mmol/l glucose and incubated for 90 min at 37°C. After centrifugation, islets were resuspended for 30 min at 37°C in KRB with 2.8 or 16.7 mmol/l glucose. The medium was then replaced with KRB containing 2.8 or 16.7 mmol/l glucose and 3.7 × 106 Bq [3H]leucine (GE-Amersham Biosciences, Baie d’Urfé, QC, Canada), after which the islets were incubated for 30 min at 37°C. The islets were then washed three times with cold KRB and sonicated in 100 mmol/l HCl. The lysate was centrifuged at 17,100×g for 15 min and aliquots were precipitated with trichloroacetic acid for measurement of total protein synthesis. The remaining supernatant fraction was used for measurement of proinsulin biosynthesis by immunoprecipitation with anti-bovine insulin antisera (Millipore/Linco, Billerica, MA, USA) and alkaline urea-gel electrophoresis .
Plasma glucose and NEFA levels were assessed using a kit (NEFA C kit; Wako Chemical, Osaka, Japan). Insulin and C-peptide were assessed by radioimmunoassay (Linco Research, St Charles, MO, USA), and triacylglycerol by a kit (GPO Trinder kit; Sigma Aldrich, Saint Louis, MO, USA).
Total RNA was extracted from aliquots of 150 islets each and RT-PCR was carried out as described  using primers listed in ESM Table 1. Results are expressed as the ratio of target mRNA to cyclophilin mRNA.
Beta cell mass, proliferation and apoptosis
Pancreases were trimmed of fat, weighed, fixed in 4% buffered paraformaldehyde (wt/wt) and embedded in paraffin. Sections (5 μm) were mounted on glass slides for immunohistochemical and beta cell mass analyses after insulin immunostaining and haematoxylin counterstaining as described . Beta cell proliferation was measured immunohistochemically by staining for the nuclear marker, Ki67, and for insulin as previously detailed , counting 1,000 to 1,500 islet beta cells per animal. For determination of apoptotic beta cells, a modified TUNEL staining protocol was used .
Expression of data and statistics
Data are expressed as mean ± SEM. Statistical analyses were performed using Student’s t test or ANOVA, followed by two-by-two comparisons using the Tukey–Kramer honestly significant difference test or Bonferroni post hoc adjustments, as appropriate. A value of p < 0.05 was considered significant.
Infusion of glucose + IF induces insulin resistance and impairs insulin secretion in vivo in 6-month-old Wistar rats
Functional variables during hyperglycaemic clamps in 2- and 6-month-old Wistar rats before or following 72 h infusions with saline or glucose and IF
Glucose + IF
Glucose + IF
GIR (μmol kg−1 min−1)
346.4 ± 10.5
277.3 ± 12.0**
377.5 ± 13.9
329.0 ± 11.8*
274.5 ± 28.9
241.4 ± 22.3
Plasma insulin (pmol/l)
1,464.7 ± 141.3
1,392.1 ± 146.7
1,625.9 ± 188.1
1,658.5 ± 369.9
1,429.2 ± 247.5
2,650.4 ± 372.5*
M/I index (μmol kg−1 min−1 × pmol/l)
0.27 ± 0.03
0.25 ± 0.03
0.24 ± 0.02
0.26 ± 0.04
0.23 ± 0.03
0.11 ± 0.02*
Plasma C-peptide (pmol/l)
4,178.4 ± 278.2
4,654.4 ± 420.4
3,143.4 ± 153.4
3,322.3 ± 277.5
4,462.7 ± 531.9
5,603.6 ± 554.5
DI (C-peptide × M/I index)
1.10 ± 0.10
1.02 ± 0.09
0.76 ± 0.11
0.80 ± 0.10
0.98 ± 0.17
0.56 ± 0.09*
C-peptide:insulin (molar ratio)
3.19 ± 0.23
3.63 ± 0.25
2.03 ± 0.29
2.42 ± 0.30
4.03 ± 0.98
2.28 ± 0.24*
Metabolic variables and pancreatic weight of 2- and 6-month-old Wistar rats following 72 h infusions with saline or glucose + IF
Glucose + IF
Glucose + IF
6.6 ± 0.6
13.9 ± 1.5**
6.1 ± 0.3
16.2 ± 1.9**
341 ± 116.8
2900 ± 738.2*
181.2 ± 60.2
2195 ± 584.6**
0.3 ± 0.1
0.9 ± 0.2*
0.26 ± 0.07
0.8 ± 0.1**
0.5 ± 0.1
0.4 ± 0.1
1.0 ± 0.1***
0.6 ± 0.1
Pancreas weight (g)
1.44 ± 0.1
1.07 ± 0.0*
2.2 ± 0.1
1.73 ± 0.1**
In the 6-month-old group, circulating insulin levels were elevated at the end of the glucose + IF infusion (Fig. 2e) and, in contrast to 2-month-old rats, did not decrease during the 30 min period post-infusion and before the clamp was initiated (Fig. 2e vs Fig. 2b), although glucose levels returned to basal values (Fig. 2d). Insulin levels remained elevated throughout the steady-state period of the clamp compared with the saline group (Table 1). Insulin secretion in response to glucose during the initial phase of the clamp was markedly lower than in the saline group (Fig. 2e). In contrast, arginine potentiation of glucose-induced insulin secretion was of a similar magnitude in glucose + IF- and saline-infused animals (Fig. 2f). Despite higher circulating insulin levels during the clamp, the glucose infusion rate was not different in the two infusion groups, such that the M/I index was significantly lower in the glucose + IF-infused animals, indicative of insulin resistance (Table 1). Plasma C-peptide levels were not elevated in the glucose + IF group during the clamp; as a result the DI was significantly lower (Table 1), suggesting impaired insulin secretion. In addition, the C-peptide/insulin ratio was significantly lower in the glucose + IF than in the saline group (Table 1), suggesting reduced insulin clearance. Taken together, these data indicate that glucose + IF infusions induced insulin resistance and impaired insulin clearance in 6-month-old rats, these changes being associated with hyperinsulinaemia, but defective glucose-stimulated insulin release.
Infusion of glucose + IF in 6-month-old rats impairs second-phase insulin secretion in response to glucose, glucose-stimulated proinsulin biosynthesis and beta cell gene expression
Infusion of glucose + IF increases pancreatic beta cell mass and beta cell proliferation in 6-month-old rats
This study aimed to ascertain whether glucolipotoxicity in vivo in rats is due to a genuine defect in beta cell function, a loss of beta cell mass or both, and whether it is more pronounced in older animals. We report that continuous and combined infusions of glucose and IF for 72 h caused insulin resistance and impaired beta cell function, despite a marked increase in beta cell mass in 6-, but not in 2-month-old Wistar rats. The functional defect observed under these glucolipotoxic conditions involves coordinated inhibition of glucose-induced insulin secretion, proinsulin biosynthesis and insulin gene expression.
We have previously reported that 72 h of cyclical and alternating infusions of glucose and IF did not impair insulin secretion in 2-month-old Wistar rats . Our current results show that infusing the same overall amount of glucose and IF in a simultaneous and continuous fashion also failed to impair insulin secretion in young animals. Overall, these findings indicate that young Wistar rats are resistant to glucolipotoxicity, irrespective of the pattern of administration. These findings contrast with previous reports using glucose [11, 21, 22, 23, 24, 25] or IF [6, 7, 9, 10, 11], which showed enhanced [10, 11, 24, 25] or reduced [6, 7, 9, 21, 23] insulin secretion. We hypothesise that the lack of inhibition of insulin secretion by IF in young rats in this study, even in the concomitant presence of elevated glucose, is due to differences in strain, sex, age or infusion rates, as suggested by several studies [6, 7, 11].
Ageing is an independent risk factor for beta cell failure in humans , but the mechanisms by which age-related changes limit the beta cell’s ability to mount a compensatory response remain to be identified. In contrast to the lack of effect of the glucose + IF infusion in young rats, 6-month-old Wistar rats infused with glucose + IF exhibited reduced glucose-induced insulin secretion in vivo in hyperglycaemic clamps and ex vivo in perifused islets. The lack of susceptibility of young rats to glucolipotoxic conditions suggests that investigations pertaining to beta cell failure in rodents should be conducted in older animals, which more closely resemble the typical setting of type 2 diabetes in humans. Most studies examining the effects of prolonged fatty acid exposure on insulin secretion in humans have shown that in young, healthy individuals, infusion of fatty acids induces an adequate compensatory increase in insulin secretion [26, 27, 28], whereas this response is impaired in individuals who are obese  or have a family history of type 2 diabetes .
There are interesting similarities between the results of the present study and those of previous reports in humans. First, as observed here, impairment of insulin secretion resulting from prolonged lipid infusions in humans is specific to the response to glucose, as the response to arginine remains relatively normal . Second, the increase in DI observed in non-diabetic participants in response to a 24 h glucose infusion did not occur if lipids were infused simultaneously with glucose . Third, a recent study by Carpentier et al.  demonstrates a strong association between fasting plasma glucose and the susceptibility to lipid-induced beta cell dysfunction in humans. Therefore, the rat infusion protocol used in this study appears to represent a valuable model for studying the mechanisms of glucolipotoxicity, because it reproduces important features of this phenomenon in humans, i.e. a lack of detrimental effects in young, healthy individuals, but a decrease in DI in response to concomitant elevations of glucose and fatty acid levels in individuals susceptible to the effects of glucolipotoxicity because of age, genetic predisposition or pre-existing beta cell defects. It is important to note that the term glucolipotoxicity is used here in its broader sense, i.e. to describe the deleterious effects of simultaneously elevated glucose and fatty acids on beta cell function or mass. In fact, the functional defects in our model were not associated with significant cell death, which contrasts with previous in vitro studies [33, 34, 35, 36, 37, 38]. These differences may be due, in part, to the fact that the IF used (Intralipid) mainly contains unsaturated fatty acids, which have been shown to be protective against saturated fatty acid-induced apoptosis [33, 36].
To our knowledge, our results provide for the first time evidence that glucolipotoxicity in vivo affects three major aspects of glucose-regulated beta cell function, i.e. insulin gene expression, proinsulin biosynthesis and insulin secretion. The observed decrease in insulin gene expression in glucose + IF-infused 6-month-old rats is consistent with our previous observations using alternate infusions  and confirms our previous in vitro findings [39, 40, 41, 42] that glucolipotoxicity affects the insulin gene. In addition, our results show that glucose + IF infusion in 6-month-old rats impairs the ability of glucose to stimulate proinsulin biosynthesis, resulting in a marked decrease in insulin content. These observations are consistent with the inhibitory effect of oleate on proinsulin biosynthesis in isolated islets . It is important to acknowledge that these results do not necessarily indicate a defect in translation as such, but rather an altered response to the glucose signal. Because insulin secretion to a combined stimulation with arginine and glucose is considered in humans to be a measure of the total beta cell secretory reserve , the unaltered response to the arginine bolus at the end of the glucose clamp in glucose + IF-infused 6-month-old rats suggests that, despite a considerable reduction in insulin stores, the total secretory capacity remained intact in these animals.
Beta cell dysfunction in glucose + IF-infused 6-month-old rats occurred despite a doubling of beta cell mass. This was associated with a high percentage of proliferating beta cells in the absence of detectable apoptosis. Thus, the reported reduced beta cell proliferative capacity in older rodents [44, 45] does not appear to have played a role in this setting. Strikingly, however, the marked increase in beta cell mass was insufficient to provide adequate functional compensation. There are several examples in the literature of an apparent disconnect between increased beta cell mass and impaired beta cell function [22, 46, 47], and our observations further emphasise the concept that functional, rather than anatomical mass, is the physiologically relevant variable for insulin secretion . It is tempting to speculate that the rapid expansion in beta cell mass following experimental ablation [46, 48] or in response to increased metabolic demand (this study) produces functionally immature beta cells. This possibility is supported by the decreased expression of beta cell-specific genes (e.g. insulin, Pdx1, Glut2, glucokinase, Gpr40). Interestingly, similar decreases in expression of beta cell genes were observed in islets from Zucker fatty rats subjected to a 60% pancreatectomy . The marked increase in beta cell proliferation in the face of Pdx1 deficiency seems contradictory to previous studies showing the role of pancreas-duodenum homeobox-1 in beta cell replication , but is consistent with a recent report demonstrating that Pdx1 haploinsufficiency does not affect the beta cell proliferative capacity .
In conclusion, beta cell dysfunction in this model of glucolipotoxicity is characterised by a coordinated decrease in insulin gene expression and glucose-regulated proinsulin biosynthesis, leading to a dramatic reduction in insulin stores and a specific loss of second-phase insulin secretion in response to glucose, despite a marked increase in beta cell proliferation and mass, which occurs only in older animals. Our data support the notion that glucolipotoxicity, as defined by chronic excess of circulating glucose and fatty acids, can lead to functional beta cell failure in vivo without detectable cytotoxicity, providing an example of dysfunctional beta cell mass.
These studies were supported by: (1) the U.S. National Institutes of Health (R01DK58096 to V. Poitout; R01DK068329 to T. L. Jetton; R01DK50610 to C. J. Rhodes; and Ruth L. Kirschstein National Research Service Award to D. K. Hagman); (2) the Canadian Institutes of Health Research (MOP 77686 to V. Poitout and MOP 12653 to M. Prentki); and (3) the Canadian Diabetes Association (post-doctoral fellowship to G. Fontés and operating grant to M. Prentki). B. Zarrouki is supported by the Montreal Diabetes Research Center/Merck Frosst post-doctoral fellowship. V. Poitout holds the Canada Research Chair in Diabetes and Pancreatic Beta cell Function. M. Prentki holds the Canada Research Chair in Diabetes and Metabolism. We are grateful to G. Fergusson and M. Éthier (CRCHUM), and K. Herzer and J. Lausier (University of Vermont) for valuable technical assistance, and to S. Bonner-Weir (Joslin Diabetes Center) and É. Joly (CRCHUM) for fruitful discussions.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.