Beta cell compensation for insulin resistance in Zucker fatty rats: increased lipolysis and fatty acid signalling
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The aim of this study was to determine the role of fatty acid signalling in islet beta cell compensation for insulin resistance in the Zucker fatty fa/fa (ZF) rat, a genetic model of severe obesity, hyperlipidaemia and insulin resistance that does not develop diabetes.
Materials and methods
NEFA augmentation of insulin secretion and fatty acid metabolism were studied in isolated islets from ZF and Zucker lean (ZL) control rats.
Exogenous palmitate markedly potentiated glucose-stimulated insulin secretion (GSIS) in ZF islets, allowing robust secretion at physiological glucose levels (5–8 mmol/l). Exogenous palmitate also synergised with glucagon-like peptide-1 and the cyclic AMP-raising agent forskolin to enhance GSIS in ZF islets only. In assessing islet fatty acid metabolism, we found increased glucose-responsive palmitate esterification and lipolysis processes in ZF islets, suggestive of enhanced triglyceride–fatty acid cycling. Interruption of glucose-stimulated lipolysis by the lipase inhibitor Orlistat (tetrahydrolipstatin) blunted palmitate-augmented GSIS in ZF islets. Fatty acid oxidation was also higher at intermediate glucose levels in ZF islets and steatotic triglyceride accumulation was absent.
The results highlight the potential importance of NEFA and glucoincretin enhancement of insulin secretion in beta cell compensation for insulin resistance. We propose that coordinated glucose-responsive fatty acid esterification and lipolysis processes, suggestive of triglyceride–fatty acid cycling, play a role in the coupling mechanisms of glucose-induced insulin secretion as well as in beta cell compensation and the hypersecretion of insulin in obesity.
KeywordsFatty acid partitioning Fatty acid signalling Glucagon-like peptide 1 Insulin resistance Insulin secretion Islet beta cell compensation Lipolysis Triglyceride–fatty acid cycling Zucker fatty rat
acetyl-CoA carboxylase α
acetyl-CoA carboxylase β
Electronic Supplementary Material
free fatty acid receptor 1
fatty acid synthase
mitochondrial glycerol-3-phosphate acyltransferase
glucose-stimulated insulin secretion
long chain acyl-CoA
neutral cholesteryl ester hydrolase
cyclic AMP-dependent protein kinase
peroxisomal proliferator activator receptor-α
sterol-regulatory element binding factor 1c
Normoglycaemia is maintained in the majority of obese insulin-resistant individuals, as their islet beta cells compensate for insulin resistance with insulin hypersecretion [1, 2]. Obesity-associated type 2 diabetes develops when beta cell compensation processes fail [3, 4]. The mechanisms of islet beta cell compensation, however, are poorly understood. Particularly unclear is the role of excess circulating NEFA, which frequently coexists with insulin resistance. Acutely elevated NEFA are well known to enhance glucose-stimulated insulin secretion (GSIS) [3, 5]; however, some investigators believe that persistently elevated NEFA may be detrimental to beta cell function or viability [6, 7]. We have previously utilised the Zucker fa/fa fatty (ZF) rat model of insulin resistance in order to study beta cell compensation  and have done so again for the present work, which focuses on the role of NEFA. The ZF rat has a mutated leptin receptor and, as a consequence, is hyperphagic, obese, hyperlipidaemic and severely insulin-resistant [8, 9, 10]. Importantly, unlike the Zucker diabetic fatty rat, it does not develop diabetes [8, 9, 10], which makes it a good model for the investigation of islet beta cell compensation for insulin resistance.
In earlier work, we reported increased islet glucose utilisation and oxidation in ZF compared with control Zucker lean (ZL) islets . Increased glucose oxidation should contribute to the compensatory hyperinsulinaemia in ZF rats by increasing the activity of the KATP channel/Ca2+ pathway of nutrient–insulin secretion coupling . We also showed increased activities of enzymes and increased metabolites of the anaplerotic and malate–pyruvate cycling pathways . As anaplerosis is the pathway by which glucose increases malonyl-CoA levels, these results led us to hypothesise that enhanced glucose-induced increases in malonyl-CoA/long chain acyl-CoA (LC-CoA) signalling may also be involved in ZF islet compensation. This is based on the malonyl-CoA/LC-CoA model of lipid signalling for insulin secretion that we have described previously [12, 13]. Central to the model is that an increase in glucose, via increasing malonyl-CoA, which inhibits carnitine palmitoyltransferase-1 (CPT1), as was first described by McGarry and Brown , switches the partitioning of NEFA in the beta cell away from oxidation towards the accumulation of LC-CoA and fatty acid acylation and esterification products (e.g. diacylglycerol [3, 13, 14, 15]), which are proposed to have direct regulatory effects on insulin secretion [3, 12, 13].
Lipolysis is known to be active in the beta cell, and has been implicated in both lipid signalling for insulin secretion [16, 17, 18] and, if dysregulated, beta cell failure . However, little is known about its regulation and which lipolytic enzymes are operative. A question addressed in this study was whether activity of the lipolysis arm of beta cell lipid partitioning might be enhanced in situations of chronically increased malonyl-CoA/LC-CoA signalling as a way of protecting islets from excess lipid accumulation [3, 20].
The present study focused on the role of lipid signalling in beta cell compensation in ZF rats. The effects of NEFA in augmenting GSIS, alone and in combination with the incretin hormone glucagon-like peptide-1 (GLP-1), and the major pathways of intracellular fatty acid partitioning, including lipolysis, were studied in isolated islets from ZF and ZL control rats.
Materials and methods
Animals and measurement of blood and plasma parameters
Zucker fatty (fa/fa, ZF) and Zucker lean (fa/+ or +/+, ZL) rats (Harlan, Indianapolis, IN, USA) were fed standard laboratory chow and were all studied at 10 weeks of age. All protocols were approved by the Institutional Animal Care and Use Committee of the University of Vermont. Tail vein blood sampling was performed between 08.00 and 09.00 h from non-anaesthetised, fed rats for measurement of blood glucose using a glucose monitor (Therasense, Alameda, CA, USA), plasma insulin (Insulin RIA kit; Linco Research, St Charles, MS, USA), plasma triglyceride (TG) (GPO Trinder; Sigma-Aldrich, Saint Louis, MS, USA) and plasma NEFA (NEFA C kit; Wako Chemicals, Neuss, Germany).
Islet isolation and culture
Fed rats were anaesthetised between 08.00 and 09.00 h with sodium pentobarbital and then killed by exsanguination, and pancreatic islets were isolated as described previously . Prior to initiating experiments or collecting islets for analysis, islets were rested for 1 h in regular RPMI 1640 medium supplemented with 10% fetal calf serum, 10 mmol/l HEPES (pH 7.4), 1 mmol/l sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin (RPMI complete) at 11 mmol/l glucose at 37°C in a humidified atmosphere containing 5% CO2.
Measurement of islet insulin secretion
Insulin secretion was measured in freshly isolated islets from ZL and ZF rats at various concentrations of glucose (3–16 mmol/l) and palmitate (0–0.4 mmol/l) in the absence or presence of 10 nmol/l GLP-1, 5 μmol/l forskolin or 0.2 mmol/l Orlistat (tetrahydrolipstatin), as described in detail in the Electronic Supplementary Material (ESM). Total insulin content of the islets was measured following extraction with acid-ethanol (0.2 mmol/l HCl in 75% ethanol).
Determination of islet DNA, protein and triglyceride content
Batches of 15 freshly isolated islets were used for determination of DNA and protein as described previously , and batches of 75–100 freshly isolated islets were used for determination of TG .
Oxidation and esterification of islet fatty acids
Fatty acid oxidation and esterification were measured in isolated islets of ZL and ZF rats using radiolabelled tracers, as described in detail in the ESM.
Measurement of islet lipolysis
Lipolysis was measured in freshly isolated islets from ZL and ZF rats as the rate of glycerol release at 3 and 16 mmol/l glucose in the presence or absence of 10 nmol/l GLP-1, 5 μmol/l forskolin or 0.2 mmol/l Orlistat, as described in detail in the ESM.
Assays for activities of triglyceride lipase and neutral cholesteryl ester hydrolase
Triglyceride lipase (TGL) and neutral cholesteryl ester hydrolase (NCEH) enzyme activities were determined in islet homogenates from ZL and ZF islets, as described in the ESM.
RNA extraction and RT-PCR analysis
All results are expressed as mean±SEM. Statistical significance was calculated with Student’s t-test or, for multiple comparisons, two-way ANOVA with Bonferroni post hoc testing as indicated. A p value of <0.05 was considered significant. Prism v.4 (GraphPad Software, San Diego, CA, USA) was used to perform the analyses.
Body weight, fed blood chemistry at 09.00 h and islet parameters
Augmentation of insulin secretion by exogenous NEFA is enhanced in ZF islets
In the absence of exogenous palmitate, GSIS was increased in ZF compared with ZL islets ∼5-fold at both 8 and 16 mmol/l glucose (p<0.001) (Fig. 1j). Exogenous palmitate at 0.2 mmol/l augmented GSIS at 16 mmol/l glucose by 38% in ZF, but had no effect in ZL islets (Fig. 1j). Exogenous palmitate at the higher concentration of 0.4 mmol/l augmented GSIS at 5, 8 and 16 mmol/l glucose (8-, 4- and 3-fold, respectively) in ZF islets, but in ZL islets increased GSIS by 3-fold at 16 mmol/l glucose only (Fig. 1j). Figure 1k magnifies the insulin secretion data at 5 mmol/l glucose shown in Fig. 1j. Clearly, exogenous palmitate allowed very robust insulin secretion at a physiologically low (5 mmol/l) glucose concentration in ZF islets only. Hence, ZF compared with ZL islets are more sensitive to the effect of exogenous NEFA in augmenting GSIS. Furthermore, the glucose set-point for insulin secretion is lowered by exogenous NEFA in ZF, but not ZL, islets.
GLP-1 and forskolin synergise with NEFA to augment GSIS in ZF rats
Fatty acid partitioning into oxidation and esterification pathways is increased in ZF islets
Glucose-stimulated lipolysis is enhanced in ZF islets
Lipolysis inhibition by Orlistat abolishes glucose-stimulated lipolysis and markedly reduces insulin secretion in ZF islets
Expression of key transcription factors and enzymes of lipid metabolism in ZL and ZF islets
The findings of this study support an important role for lipid signalling (activation of the NEFA receptor FFAR1 and the production of lipid-derived signals, such as diacylglycerol, signalling phospholipids or fatty acyl-CoA, that are known to modulate biological effectors such as ion channels and enzymes) in beta-cell compensation for insulin resistance in ZF rats. A key observation in this regard was that the normal augmentation of GSIS by exogenous palmitate was markedly enhanced in ZF compared with ZL islets, and this effect was particularly apparent within the physiological range of plasma glucose (5–8 mmol/l). Also, the incretin effect of GLP-1 and the insulin response to the cyclic AMP-enhancing agent forskolin were both further augmented by exogenous palmitate at 8 mmol/l glucose in ZF islets. In investigating the underlying mechanism of this NEFA effect, we found that all pathways of fatty acid partitioning (fatty acid oxidation, esterification and lipolysis) were enhanced in ZF islets. Importantly, the lipase inhibitor Orlistat reversed the enhanced GSIS in ZF islets. Also, our results provide potential insight into the regulation of lipolysis in ZF islets by showing increased activities of NCEH and TGL. Together, these findings support enhancement in ZF islets of the normal cycle of TG production and lipolysis as a mechanism of their insulin hypersecretion. In addition, enhanced lipolysis may prevent lipid-induced islet damage by minimising the accumulation of toxic lipids. Our results thus support a beneficial role for the in vivo hyperlipidaemia in ZF rats in the beta cell compensation for insulin resistance, as opposed to the belief of some that chronic excess of NEFA, even in the absence of hyperglycaemia, invariably causes beta cell dysfunction or death [6, 7].
The finding that NEFA dramatically synergised with GLP-1 and the cyclic AMP-increasing agent forskolin in augmenting GSIS in ZF islets is interesting with respect to beta cell compensation. This finding is in keeping with the previous observation that NEFA augment insulin secretion in response not only to glucose but also to all fuel and non-fuel secretagogues, including GLP-1 . The present findings reinforce the concept that NEFA are not only essential for insulin secretion  but are also involved in enhancement of secretion in response to a wide variety of secretagogues. Consequently, NEFA and circulating TG within lipoproteins, which are often elevated in obesity-associated insulin resistance states , are prime candidates for factors that link visceral adiposity and beta cell compensation for insulin resistance . Interestingly, GLP-1 levels were reported to be increased in the high-fat-fed dog model of insulin resistance , also underscoring the potential role of GLP-1 and NEFA synergy in hyperinsulinaemic compensation mechanisms.
Increased glucose metabolism and increased availability of NEFA, resulting in increased TG synthesis, are almost certainly contributors to the proposed TG–FA cycle in ZF islets (Fig. 7). The regulatory factors of cycling at the level of lipase enzymes in both normal and compensating islets, however, are yet to be determined. ZF islet extracts were shown to have greater than 2-fold increased TGL activity, which could be a result of increased expression of lipase enzymes or increased lipase intrinsic activity. There was a non-significant 38% increase in Hsl mRNA levels. Interestingly, we have now analysed islet Hsl mRNA levels by real-time PCR in a new series of ZL and ZF animals that had a sham laparotomy for another study protocol. In that study, Hsl mRNA was significantly increased in ZF islets (1.49±0.08 compared with 0.78±0.11 relative units; ZF vs ZL, p<0.01). Therefore, it seems likely that the expression of Hsl is higher in ZF islets, which is consistent with the enzyme activity data. The role of HSL in islet beta cell function is unclear, however, as insulin secretion has been shown to be little or not affected in Hsl −/− animals [18, 31]. We showed impaired GSIS in only male fasted Hsl −/− mice, and this was completely reversed by the provision of exogenous NEFA . The expression of HSL in beta cells, however, has been shown to be upregulated by a prolonged high glucose concentration  and HSL has been shown to be co-localised with insulin granules . There has been particular interest in the role of non-HSL lipases in adipose tissue metabolism [34, 35]. The roles of these alternative lipases in the islet also warrant investigation.
There is increasing evidence that islet beta cell lipolysis is increased by elevated glucose, as shown previously in normal rat islets  and in both wild-type and Hsl −/− mouse islets . We have also shown glucose-stimulated lipolysis to be tightly correlated with GSIS in INS832/13 beta cells (C. J. Nolan and M. Prentki, unpublished observations). It may be that the effect of glucose on lipolysis results from its effect in increasing TG synthesis, perhaps into a labile intracellular pool, thus providing more substrate for lipase enzymes. Another possibility is that elevated glucose might activate lipase enzyme activity. Of relevance, we recently showed that LC-CoA, which is thought to increase in the cytoplasm in response to elevated glucose [3, 13], activates lipolysis in vitro in islet extracts from control and Hsl −/− mice . It is an attractive hypothesis, therefore, that activation of lipolysis by LC-CoA could also be involved in the enhanced TG–FA cycling that was observed in ZF islets.
In fat cells, hormonal stimulation of lipolysis is by activation of HSL via PKA phosphorylation. While GLP-1 and forskolin, both activators of PKA, have been shown to increase lipolysis in clonal pancreatic beta cells (HIT cells) , neither agent enhanced lipolysis in ZF or ZL islets. This finding is in agreement with our study of Hsl wild-type and knockout mice . Glucose activation of lipolysis was noted in the HSL-deficient mice but no significant effect of GLP-1 on lipolysis was observed . Thus, it seems that glucose acts via a mechanism other than PKA, possibly via an enzyme other than HSL . The results also indicate that the synergistic effect of GLP-1 or forskolin with NEFA in augmenting GSIS is not due to an effect of GLP-1 or forskolin in accelerating lipolysis.
At first glance, it seems odd that increased malonyl-CoA/LC-CoA signalling and increased fatty acid oxidation at a physiologically relevant glucose level should occur together in ZF islets. Interestingly, the expression of important enzymes of both pathways, ACACB (synthesises malonyl-CoA) and CPT1 (the rate-limiting step in mitochondrial fatty acid oxidation), were increased in ZF islets. We suspect that the increased fatty acid oxidation is maintained, despite more inhibition of CPT1 by malonyl-CoA, because of the maintenance of higher levels of LC-CoA (substrate for fatty acid oxidation) by accelerated TG–FA cycling. Previously it has been shown that inhibition of fatty acid oxidation by an inhibitor of CPT1 reduces basal hypersecretion in ZDF rats, such that the maintenance of fatty acid oxidation may also be important in compensation processes . Islets of the ZF rat displayed none of the well-described features of lipotoxicity [7, 39, 40, 41, 42, 43]. The TG content was mildly decreased rather than dramatically increased [7, 40]. The mRNA expression of genes that promote lipogenesis and fatty acid esterification (Srebf1c, Acaca, Fasn and Gpam) was not altered in ZF islets. Thus, the question arises as to how ZF islets escape lipotoxicity. As illustrated in Fig. 7, enhanced TG–FA cycling provides an attractive mechanism of protection from lipid toxicity in the face of obesity-associated hyperlipidaemia. The cycle prevents steatosis due to the high rates of TG lipolysis and by allowing the maintenance of LC-CoA as a substrate for detoxification via fatty acid oxidation. In addition, TG–FA cycling is futile, in that fatty acid activation to LC-CoA and its metabolism back to NEFA via TG expend ATP energy.
In conclusion, our results document very active fatty acid glucose-responsive esterification/lipolysis processes in pancreatic beta cells, which are further enhanced in the islets of ZF rats. We [16, 18] and co-workers  have provided genetic (Hsl knockout studies) and pharmacological (Orlistat, dimethylpyrazole) evidence that lipolysis plays a role in GSIS. This study expands that concept by providing evidence that enhanced lipolysis and, most probably, TG–FA cycling in ZF islets appear to be a mechanism for the enhanced insulin secretion that characterises beta cell compensation for their insulin resistance.
We wish to thank Y. Long and J. Morin for expert technical assistance and B. E. Corkey for thoughtful discussion of the work. This work was supported by grants from the American Diabetes Association (J. L. Leahy), the National Institute of Health (J. L. Leahy, DK56818; M. Prentki, DK-63356), Genome Quebec, Canada (M. Prentki) and the Canadian Institute of Health Research (CIHR) (M. Prentki). C. Guay is supported by a graduate studentship from the Fonds de Recherche en Santé du Québec.