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Endocrinology, doi:10.1210/en.2003-1673
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Endocrinology Vol. 145, No. 8 3746-3753
Copyright © 2004 by The Endocrine Society

Hormone-Sensitive Lipase Deficiency in Mouse Islets Abolishes Neutral Cholesterol Ester Hydrolase Activity but Leaves Lipolysis, Acylglycerides, Fat Oxidation, and Insulin Secretion Intact

Malin Fex, Charlotta S. Olofsson, Ulrika Fransson, Karl Bacos, Håkan Lindvall, Maria Sörhede-Winzell, Patrik Rorsman, Cecilia Holm and Hindrik Mulder

Department of Cell and Molecular Biology (M.F., U.F., K.B., H.L., C.H., H.M.) and Departments of Medicine (M.S.-W.) and Physiological Sciences (C.S.O., P.R.) at Lund University, Biomedical Center, SE-221 84, Sweden; and The Oxford Center for Diabetes, Endocrinology and Metabolism (P.R.), Churchill Hospital, Oxford OX3 7LJ, United Kingdom

Address all correspondence and requests for reprints to: Hindrik Mulder, Section for Molecular Signaling, Department of Cell and Molecular Biology, Lund University, BMC C11, SE-221 84, Lund Sweden. E-mail: hindrik.mulder{at}medkem.lu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lipids are thought to serve as coupling factors in insulin secretion. Hormone-sensitive lipase (HSL) is expressed in pancreatic ß-cells and could potentially regulate insulin secretion via mobilization of stored triglycerides. Here, we examined the impact of HSL deficiency on fuel metabolism and insulin secretion in mouse islets. Lack of HSL resulted in abrogation of neutral cholesterol ester hydrolase activity, whereas diglyceride lipase activity remained intact. Although glucose stimulates lipolysis in rat islets, elevation of glucose with or without addition of cAMP failed to increase lipolysis in mouse islets regardless of genotype, as indicated by release of glycerol from islets. Storage of lipids, assayed as total acylglycerides, was unaltered in HSL null islets, and oxidation of fatty acids or glucose was not different. The intracellular rise in Ca2+ triggered by glucose and its subsequent oscillations was unaffected in HSL null islets. Accordingly, insulin secretion in static incubations of islets, in response to fuel- and nonfuel secretagogues, was in no instance significantly different between wild-type and HSL null mice. The lacking impact of HSL deficiency on insulin secretion may be attributed to the failure of insulin secretagogues to stimulate lipolysis. Consequently, a regulatory function of lipid mobilization in insulin secretion in the mouse appears unlikely.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PANCREATIC ß-CELLS are known to store triglycerides (1). The role of these stored lipids is not entirely clear, but evidence suggests that it can be both beneficial and adverse. On one hand, lipids are required for glucose-stimulated insulin secretion (GSIS). For example, when triglycerides in pancreatic islets are depleted by hyperleptinemia, the pancreatic ß-cells fail to release insulin (2). Moreover, it has been shown that the perfused pancreas from fasted rats is unresponsive to a rise in glucose (3). Importantly, in both these cases, GSIS is reinstated by addition of exogenous fatty acids.

On the other hand, another line of research points out that overstorage of triglycerides causes ß-cell failure and dia-betes. This process has been termed lipotoxicity (4). In the Zucker diabetic fatty rat, the failure of ß-cells is preceded by a rise in circulating free fatty acids and accumulation of triglycerides in islets (1). It has been suggested that the accumulation of lipids exceeds the capacity for fat oxidation. This may allow lipids to enter harmful pathways, e.g. ceramide formation, which via induction of nitric oxide production causes ß-cell apoptosis (5). Some features of lipotoxicity have been replicated in normal islets and animals (1, 6). For instance, chronic infusion of lipids in rats blunts GSIS (7), whereas acute exposure to lipids potentiates insulin secretion. Recently, it has been shown that increased formation of triglycerides is associated with impaired GSIS (8).

In view of both the beneficial and harmful effects of lipids in ß-cells, it is important to understand precisely how lipids are metabolized in these cells. To this end, we have demonstrated that hormone-sensitive lipase (HSL) is expressed and active in pancreatic ß-cells (9). The lipase has a broad substrate specificity, hydrolyzing acylglycerides as well as retinyl- and cholesteryl esters (10). By serving a similar role in ß-cells, HSL could provide lipids for metabolic signaling in GSIS. Impaired function of the lipase, however, could lead to accumulation of acylglycerides and cause ß-cell failure. To investigate these possibilities, mice with a disruption of the HSL gene could be helpful. In fact, a number of such lines exist and have been subject to studies of glucose homeostasis (11, 12, 13). One line exhibits glucose intolerance caused mainly by impaired insulin secretion (11); isolated islets from these HSL null mice are unresponsive to glucose. In contrast, the HSL null line developed in our laboratory is insulin resistant and moderately hyperglycemic, which results in hypersecretion of insulin (12). In the third line, details on insulin secretion in vivo and in vitro have not been reported (13). Interestingly, these HSL null mice show no signs of whole-body insulin resistance, but their livers are more sensitive to insulin.

To more fully understand glucose homeostasis in HSL null mice, we have here examined fuel metabolism and insulin secretion in islets from these mice. We show that, whereas neutral cholesterol ester hydrolase (NCEH) activity is completely abolished, other aspects of fuel metabolism in HSL null islets remain unaffected.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and animals
All reagents were from Sigma (St. Louis, MO) unless otherwise stated. Palmitate for the secretion experiments was prepared as previously described (12). HSL null mice were generated by targeted disruption of the HSL gene in 129SV-derived embryonic stem cells by standard procedures (14), as previously reported (12, 15). In brief, the cDNA encoding the Aequorea victoria green fluorescent protein was inserted in-frame into exon 5 of the HSL gene, followed by a neomycin resistance gene, thereby disrupting the catalytic domain. The herpes simplex thymidine kinase gene was inserted at the 3'-end of the construct. As a result, in mice homozygous for the mutated allele, HSL protein was absent from all tissues examined (12); and NCEH activity, which most specifically reflects the activity of HSL (16), was virtually lacking from white adipose tissue and every other tissue examined (12). The genotype of the mice in the present study was determined by PCR on DNA extracted from tail biopsies. All mice in the studies were littermates, derived from breeding of mice heterozygous for the HSL null allele. Unless otherwise stated, experiments were performed in female adult mice given a normal chow diet. The studies were approved by the Animal Ethics Committee in Lund, Sweden.

Isolation and batch incubation of islets
Islets were isolated by standard collagenase digestion and handpicked under a stereomicroscope. Islets were first allowed to recover in RPMI 1640 medium, containing 10% fetal calf serum and 11.1 mM glucose, in an incubator at 37 C with humidified atmosphere and 5% CO2 for 2–3 h. This was followed by incubation for 60 min at 37 C in HEPES balanced salt solution (HBSS; 114 mM NaCl, 4.7 mmol KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 20 mM HEPES, 2.5 mM CaCl2, 25.5 mM NaHCO3, 0.2% BSA; pH 7.2) containing 2.8 mM glucose. For each condition, batches (n = 8) of three size-matched islets were transferred to a 96-well plate kept on ice and containing 200 µl HBSS per well with the addition of the respective secretagogue. After transfer of all islets, the plate was again placed in an incubator at 37 C. At 60 min, a sample from the buffer was removed for measurement of insulin by RIA (Linco Research Inc., St. Charles, MO).

Determination of islet DNA and protein contents
Aliqouts of 80–120 islets were sonicated 3 x 10 sec on ice in 0.25 M sucrose, 1 mM EDTA (pH 7.0), 1 mM dithioerythritol, 20 µg/ml leupeptin, 20 µg/ml antipain, and 1 µg/ml pepstatin A. Quantitation of DNA and protein in samples from the prepared islet lysates were done, using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Eugene, OR) and BCA Protein Assay Kit (Pierce, Rockford, IL), respectively. Analyses were performed according to the manufacturers’ instructions.

RT-PCR
Aliquots of 200–400 islets were frozen in liquid N2, and total RNA was extracted according to the procedure described by Chomczynski and Sacchi (17). The extracted RNA was used as a template for RT-PCR, employing the Advantage RT-for-PCR kit (BD Biosciences, Franklin Lakes, NJ). The forward primer was 5'-TCT CCA TCG ACT ACT CCC TGG C-3', and the reverse primer was 5'-AAG GAG TTG AGC CAT GAG GAG GC-3'. These primers have previously been described (9) and generate a PCR-product of 540 bp, which are within the sequence in mouse HSL cDNA that has been deleted in the HSL null mouse, i.e. exon 5–7 (12). The PCRs were run for 35 cycles at 95 C (40 sec), 55 C (60 sec), and 72 C (90 sec). The PCR products were resolved by agarose gel electrophoresis.

Western blot analysis
Islets were sonicated in buffer (see above), after which proteins were resolved by SDS-PAGE, and electroblotted to nitrocellulose membranes. Western blot analysis was performed by the ECL system (SuperSignal ULTRA, Pierce), using an affinity-purified chicken antirat HSL primary antibody (9).

Lipolysis in islets
Batches of 100 islets were kept in 150 µl HBSS, containing 2.8 or 16.7 mM glucose with or without 8-bromoadenosine cAMP (8-Br-cAMP), a cAMP analog with increased stability. Islets were kept for 4 h in an incubator at 37 C with humidified atmosphere and 5% CO2. A limited experiment was also performed with forskolin, as another means to raise cAMP levels in islets, and potentially stimulate lipolysis. Forskolin (2.5 µM) was added at 0 and 120 min during the 4-h incubation. After this time, the buffer was removed and assayed for basal and stimulated lipolysis. Glycerol released into the buffer was used as an index of lipolysis and was measured as described (18).

Enzyme activity and acylglyceride assays
Islets were sonicated in buffer (see above) to allow assays of enzyme activities, as previously described in detail. For diglyceride lipase activity, the diacylglycerol analog mono-oleoyl-2-O-mono-oleylglycerol was used as substrate (19). NCEH activity was determined as previously described (20). Acylglycerides were extracted from the homogenates, using methanol/chloroform according to the method of Folch (21), and hydrolyzed by KOH; acylglyceride levels were determined as glycerol, using a bioluminescence assay (18).

Palmitate and glucose oxidation
After isolation, islets were kept in HBSS containing 2.8 mM glucose for 60 min at 37 C. Batches of size-matched islets (n = 30) were transferred to a cup (Kimble-Kontes, Vineland, NJ) suspended from a rubber sleeve stopper (Fisher, Pittsburgh, PA), which was inserted into a glass scintillation vial. For palmitate oxidation, a reaction mixture consisting of 0.5 mM palmitic acid complexed to 1% BSA (essentially fatty acid free), with approximately 10 dpm/pmol [1-14C]-palmitic acid (NEN Life Science Products, Boston, MA) as tracer, 0.8 mM L-carnitine, and glucose, at a final concentration of 2.8 or 16.7 mM, was added; and the vials were sealed. For glucose oxidation, a reaction mixture containing approximately 2 dpm/pmol [14C]-glucose was added. The reaction was terminated after 2 and 1 h, for palmitate and glucose oxidation, respectively, by injection of 100 µl 7% perchloric acid into the suspended cup. The rate of [1-14C]-palmitate or [14C]-glucose oxidation was measured, as released 14CO2, which was trapped by adding 300 µl benzethonium hydroxide to the bottom of the sealed vials, followed by an additional 2-h incubation at 37 C. After discarding the cups and rubber stoppers, 14CO2 production was determined by scintillation counting.

Measurements of intracellular Ca2+ concentration ([Ca2+]i)
Dual-wavelength microfluorimetry (22) was used to record [Ca2+]i. Intact islets were loaded with 3 µM fura 2 in the presence of 0.007% wt/vol pluronic acid (Molecular Probes, Leiden, The Netherlands) for 30 min at 37 C. During the experiments, the islets were held in place by a heat-polished glass pipette and continuously superfused with a solution containing (in mM): 140 NaCl, 3.6 KCl, 2 NaHCO3, 0.5 MgSO4, 5 HEPES (pH 7.4 with NaOH), 2.6 CaCl2, and glucose as indicated. The measurements were carried out using a microfluorimeter system (D104, PTI, Monmouth Junction, NJ); islet cells were studied in an optical plane at the lower surface of the islets. The fluorophore was excited alternatively at 350 and 380 nm, and emitted light was collected at 510 nm. The fluorescence ratio F350/F380 was determined at a ratio frequency of 10 Hz. The [Ca2+]i was calculated using the equation given previously (22) and a dissociation constant of 224 nM. The maximum ratio was achieved by addition of 60 µM ionomycin at the end of each experiment, and background subtraction was performed after quenching of the fura 2 signal with 1 mM MnCl2. Measurements were carried out at 33 C.

Statistical measures
Mean ± SEM are given. Data were compared with a two-tailed unpaired t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HSL expression in islets
Using RT-PCR on RNA extracted from wild-type islets, the primers hybridizing to exon 5 and 7 generated a product with the expected size of 540 bp (Fig. 1AGo); an identical product was amplified from adipose cDNA derived from an unrelated mouse strain. In contrast, no such product was observed in the reaction involving RNA from the HSL null mouse.



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  		FIG. 1. Expression of HSL. RT-PCR analysis of RNA extracted from wild-type and HSL null islets (A). The arrow indicates the 600 bp band of the ladder. The primers used amplify a 540-bp DNA fragment when HSL mRNA is expressed. Western blot analysis of homogenates of isolated islets from HSL null and wild-type mice, and recombinant HSL protein, probed with anti-HSL antibodies (B). Note that HSL from islets is slightly larger (89 kDa) than the recombinant protein (84 kDa), which is the adipocyte isoform (9 23 ).

 
Western blot analysis of proteins extracted from wild-type islets confirmed expression of HSL also at the protein level (Fig. 1BGo). Again, such a band was lacking in proteins extracted from HSL null islets. However, the HSL-immunoreactive band in wild-type islets was slightly larger (~89 kDa) than the recombinant rat adipocyte form of HSL (~84 kDa). This agrees with our previous findings of a slightly larger isoform of HSL in ß-cells (12, 23). Thus, the present results confirm that the disruption of the HSL gene has resulted in lacking expression of HSL mRNA and protein.

Protein and DNA content in islets
We have previously reported that our HSL null line adapts to insulin resistance by increasing ß-cell mass by approximately 2-fold (12). To further corroborate this finding, we sonicated islets and measured total protein and DNA content. The data are given in Table 1Go and indeed demonstrate a significant increase in total protein content by 50% (P < 0.01). There was a trend toward an increase also in DNA content (15%), which did not reach statistical significance.


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  		TABLE 1. Total protein and DNA content per islet was analysed as described in Materials and Methods

 
Lipase activities in islets
A high NCEH activity of HSL is a unique property among mammalian lipases (24). Furthermore, findings in another strain of HSL null mice suggest that NCEH activity in many cells may largely be attributed to HSL (16). Therefore, we proceeded to determine NCEH activity in islets from the mutant mice. In accordance with the absence of the HSL protein, NCEH activity was virtually absent from extracts of isolated HSL null islets, whereas it was readily detectable in wild-type islets (Fig. 2AGo).



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  		FIG. 2. Lipid metabolism in islets. NCEH activity (A), diglyceride lipase activity (B), acylglyceride levels (C), and lipolysis (D) were measured in HSL null and wild-type islets as described in Materials and Methods; for experiments in A–C, extracts from whole islet homogenates were used, whereas lipolysis (D) was determined by glycerol release from islets kept in buffer. Batches of approximately 100 islets were used; n = 3–6 for each experiment and condition. cAMP in D denotes 1 mM 8-Br-cAMP.

 
Next, we determined lipase activity toward a synthetic diglyceride substrate. In this case, diglyceride lipase activity remained intact in HSL null islets (Fig. 2BGo). This is in sharp contrast to the situation in white adipose cells in our HSL null line, in which HSL-deficiency results in a decrease of di-glyceride lipase activity by 25-fold (12).

Lipid storage in islets
We then examined the consequences of the altered profile of lipase activity in HSL null mice for storage of lipids. Although lipid extracts of islets from both genotypes exhibited measurable levels of acylglycerides, they were similar in wild-type and HSL null islets (Fig. 2CGo).

Lipolysis in islets
Although the experiments so far have been performed in cell homogenates, we next assessed the consequences of the lack of HSL for hydrolysis of acylglycerides in intact islets; this was accomplished by determination of glycerol release into the assay buffer. There was no difference in lipolysis at 2.8 mM glucose in islets from HSL null mice compared with wild-type littermates. (Fig. 2DGo). Surprisingly, maintaining islets from either wild-type or HSL null mice at 16.7 mM glucose failed to increase glycerol release from the islets. Moreover, under conditions where a potential maximal stimulation of lipolysis was achieved by use of 8-Br-cAMP, a cAMP-analog that activates protein kinase A (PKA), which is responsible for HSL activation (10), there was still no increase in glycerol release from islets of either genotype (Fig. 2DGo). Finally, using forskolin (2.5 µM), an activator of adenylate cyclase that generates cAMP, glycerol release from wild-type and HSL null islets was similar (1.60 ± 0.25 vs. 1.64 ± 0.22 pmol/islet), and there was no increase compared with basal glycerol release.

Oxidation of palmitate and glucose in islets
We next asked whether the oxidation of fuels was altered in HSL null islets. To this end, islets were incubated in the presence of radiolabeled palmitate, and the full oxidation to CO2 was assayed. In both wild-type and HSL null islets, palmitate oxidation was reduced by an increase in glucose from 2.8 to 16.7 mM (Fig. 3AGo). However, at neither low nor high glucose was there any difference in the rate of palmitate oxidation between the two genotypes. It should be borne in mind that the analysis reveals oxidation of exogenous free fatty acids. Thus, the possibility remains that oxidation of lipids stored in islet cells is changed.



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  		FIG. 3. Palmitate and glucose oxidation. Oxidation of palmitate (A; 2 h) or glucose (B; 1 h) at 2.8 and 16.7 mM glucose in isolated islets from HSL null and wild-type islets. Triplicate batches of 30 islets were used; three independent experiments were performed.

 
Next, we assessed oxidation of glucose. When glucose was raised from 2.8 to 16.7 mM (3B), the rate of oxidation of the hexose increased 3-fold in both wild-type and HSL null islets. Again, there was no difference in the rate of glucose oxidation between the two genotypes at low or high glucose.

Insulin secretion in isolated islets
First, insulin secretion from freshly isolated islets in response to a low, intermediate, and high concentration of glucose was examined. The islets were isolated from 4-month female mice, which in in vivo experiments were shown to exhibit the most profound changes in insulin secretion (12). Thus, insulin secretion was not significantly different between the genotypes at any of the glucose concentrations examined (Fig. 4AGo). If anything, the fold-response at 16.7 mM glucose appeared to be greater in HSL null mice (15-fold vs. 6-fold), mainly due to a slightly lower basal insulin secretion at 2.8 mM glucose.



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  		FIG. 4. Static incubation of islets. Insulin secretion in 1-h static incubations of isolated islets from HSL null and wild-type islets from female, fed, 4-month-old mice. Eight replicates of batches of three islets were used for each condition; three independent experiments were performed; 100 nM GLP-1 and 100 µM carbacholine (Carb) were used. PA, Palmitic acid; KIC, {alpha}-ketoisocaproic acid.

 
Given the fact that lipids, perhaps in the form of long-chain acyl-coenzyme A (LC-CoA), have been thought to mediate ATP-sensitive K+ channel (KATP)-independent glucose sensing (25), we also examined insulin secretion when closure of the KATP-channel was blocked. However, in the presence of 250 µM diazoxide and 35 mM KCl, insulin release was similar in islets from either genotype at both low and high glucose (Fig. 4BGo). Again, the fold-response to glucose appeared greater in HSL null mice (10-fold vs. 5.4-fold).

Next, a number of fuel and hormonal secretagogues were tested for their capacity to stimulate and/or potentiate insulin secretion. Addition of 1 mM palmitate, a known potentiator of GSIS (26), to 16.7 mM glucose, amplified insulin secretion similarly in wild-type and HSL null islets (Fig 4CGo). {alpha}-Ketoisocaproic acid, a precursor of leucine and an established stimulator of mitochondrial metabolism, also provoked insulin secretion in islets from both wild-type and mutant mice to a similar extent (Fig. 4CGo).

Insulinotropic agents that stimulate insulin secretion via a rise in cAMP and subsequent activation of PKA have been suggested to exert their effects through HSL-mediated mobilization of lipids from stored triglycerides (27). Nevertheless, glucagon-like peptide-1 (GLP-1) (28), an incretin that stimulates cAMP production in ß-cells, was equally efficient in islets of both genotypes to potentiate insulin secretion. Finally, carbacholine, a muscarinic agonist that activates the phospholipase C pathway, resulting in formation of diacylglycerol and inositol trisphosphates, was as potent in wild-type as in HSL null islets to stimulate insulin secretion (Fig. 4DGo).

To exclude the possibility that a secretory pertubation is evident only at a particular age, all the experiments described above were also performed in female mice at 4 wk (Fig. 5Go) and 7.5 months of age (data not shown). Again, no significant difference in insulin secretion between the two genotypes was observed in response to any secretagogue tested. To ensure that gender plays no role in these processes, islets from male mice aged 7.5 months were examined with respect to insulin secretion in response to 2.8 and 16.7 mM glucose, and 16.7 mM glucose with the addition of 1 mM palmitate; insulin secretion in either freshly isolated or overnight-incubated islets was assayed. Islets of both genotypes responded with a 10-fold increase to glucose alone, and a further 2- to 3-fold potentiation by the lipid (data not shown). Again, no significant difference in insulin secretion between the two genotypes was observed. Finally, to examine a potential impact of nutritional status on insulin secretion, female HSL null mice were either freely fed or subjected to an overnight fast before isolation of islets for secretion experiments. Insulin secretion, however, from these freshly isolated islets in response to glucose was similar in the different genotypes under both conditions (data not shown).



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  		FIG. 5. Static incubation of islets. Insulin secretion in 1-h static incubations of isolated islets from HSL null and wild-type islets from female, fed, 4-wk-old mice. Eight replicates of batches of three islets were used for each condition; three independent experiments were performed; 100 nM GLP-1 and 100 µM carbacholine were used.

 
[Ca2+]i in intact islets
We correlated our insulin release experiments with measurements of [Ca2+]i in intact islets; data are given in Table 2Go. This analysis will also rule out that increases in ß-cell mass and/or insulin content, which we have previously noted (12), compensate for a functional impairment. Thus, upon increasing the glucose concentration from 5 to 15 mM, [Ca2+]i rose similarly from a basal low level to a first discrete peak, followed by sustained oscillations in both wild-type and HSL null islets (Fig. 6Go). The levels were not significantly different between HSL null and wild-type islets (n = 8 and 7), which further support that there are no apparent functional differences between ß-cells from the two genotypes in our line of HSL null mice.


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  		TABLE 2. [Ca2+]i in fura 2-preloaded islets superfused by glucose increasing from 5 to 15 mM

 


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  		FIG. 6. Intracellular Ca2+ in islets. Changes in [Ca2+]i in a fura 2 preloaded islet from wild-type (A) and HSL null (B) mice after an increase of the glucose concentration from 5 to 15 mM. Note the biphasic response in [Ca2+]i. This recording is representative of recordings from seven to eight islets for each genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is now a substantial body of work addressing the role of lipids in ß-cell stimulus-secretion coupling. The idea of a lipid-derived signal essential for GSIS originates from a number of studies (2, 3, 29). This signal is thought to amplify GSIS, by complementing the coupling signal resulting from glucose metabolism, i.e. the increase in the ATP:ADP ratio that triggers insulin secretion in response to glucose (30). As such, this amplifying signal has been suggested to be responsible for the so-called KATP-independent pathway of glucose sensing (25, 31). A key issue is the identity of this lipid-derived signal. LC-CoAs are candidates for this signal (25, 29); they have been shown to stimulate insulin secretion in permeabilized clonal ß-cells (32), perhaps via activation of atypical protein kinase C (33), and are likely to rise as a consequence of glucose metabolism in ß-cells (29). Recently, the role of malonyl-CoA and a subsequent rise in LC-CoA have been challenged by studies in INS-1-derived ß-cells; here, GSIS remains intact despite a total block of the glucose-induced increase of malonyl-CoA, or inhibition of long-chain acyl-CoA ester synthesis (34). Yet another candidate for lipid signaling is diacylglycerol; this lipid is a known activator of protein kinase C (35), which in turn may activate exocytosis in ß-cells.

All of these putative lipid-derived signals can be generated by the action of HSL. Therefore, acylglyceride stores in ß-cells could be a source of the lipid-derived coupling signal. Along these lines, we have shown that HSL is expressed in mouse and rat islets and in clonal ß-cells (9). In adipocytes, the lipase is regulated by catecholamines and insulin through changes in cAMP levels and subsequent activation and inactivation, respectively, of PKA (10); this kinase is responsible for reversible serine phosphorylations of HSL, which activate the lipase (36). We also found that different preparations of ß-cells exhibit lipase activity (9, 37), raising the possibility that HSL mobilizes cellular lipids from acylglycerides in ß-cells. Furthermore, it has been shown that lipolysis in HIT-T15 cells is stimulated by GLP-1 (27), a hormone that raises cAMP (28), and it was proposed by the authors that the increase in lipolysis was mediated by HSL. A critical role for HSL in GSIS was even more strongly suggested by studies in another line of HSL null mice (38), which is glucose-intolerant mainly due to an impairment of insulin secretion (11). In vitro, isolated HSL-deficient islets fail to respond to glucose, whereas the response to KCl remains intact, suggesting a specific impairment of stimulus-secretion coupling in ß-cells.

In the present work, we have described that genetic inactivation of HSL only affects the NCEH activity in islets. Despite this, there were no changes in storage of lipids, measured as total acylglycerides. This could perhaps be explained by the unaltered diglyceride lipase activity that we observed in HSL null islets. A similar situation exists in skeletal muscle in our line of HSL null mice (12); here, NCEH activity is abolished, whereas diglyceride lipase activity as well as acylglyceride storage are unchanged. The diglyceride lipase activity in islets is, as of yet, not fully characterized; but our present results suggest the existence of additional enzymes possessing hydrolase/esterase activity (39). That such lipases exist normally concurs with our previous finding that diglyceride lipase activity is partially, but not totally, inhibited by the addition of HSL antibodies to these assays in different preparations of clonal ß-cells (9). In contrast, total inhibition of diglyceride lipase activity in white adipocytes results from addition of such antibodies. The redundancy of these unknown enzymes may account for the adaptation to HSL-deficiency. However, it should also be borne in mind that lipase activity assays are performed in a cellular extract. Consequently, compartmentation of proteins is lost, and any enzyme that can hydrolyze lipid esters may contribute to lipase activity that is assayed toward a given substrate.

For assays of lipolysis, the cells are intact, circumventing the problem of unknown enzymes. Interestingly, whereas we were able to detect glycerol release from islets, this release was not stimulated by elevation of glucose and cAMP. This is contrary to the situation in rat islets, where we observe a stimulatory effect of glucose on lipolysis, and this effect is further amplified by the addition of 8-Br-cAMP (40). Moreover, addition of a pan-lipase inhibitor, orlistat, profoundly blocks glucose- and forskolin-stimulated insulin secretion in parallel with inhibition of lipase activity and lipolysis in rat islets (40). Previously, use of another lipase inhibitor, 3,5-dimethylpyrazole, has yielded similar results (41). Based on these observations and the fact that the expression of HSL is stimulated by glucose in ß-cells (37), we hypothesized that HSL, which is expressed and active in ß-cells (9), controls lipolysis in ß-cells. However, our present findings suggest that lipolysis in ß-cells plays little, if any, role in the regulation of GSIS in the mouse. One possible explanation could be activation of insulin signaling by insulin retained in the medium during the 4-h static incubation; it is well known that insulin activates antilipolysis in white adipocytes (42), and the same signaling components are expressed in ß-cells (43, 44, 45). Thus, a signaling cascade culminating in activation of phosphodiesterase 3B would decrease cAMP levels (42), and thereby activation of HSL by PKA is abrogated (10). Because of the low levels of glycerol release from islets, accumulation of glycerol in a fixed, but small, volume of buffer is required, and therefore we have been unable to test whether insulin is responsible for the lacking stimulation of lipolysis in mouse islets.

Surprisingly, whereas our line of HSL null mice shares most metabolic features of the other lines reported, glucose homeostasis in our mice is quite different. The consistent finding in our line is moderate hyperglycemia and insulin resistance (12), which elicit hyperinsulinemia. In contrast, the line created by the Zechner laboratory (13) exhibits increased insulin sensitivity at the level of the liver. Although also the line created by the Mitchell laboratory (38) is insulin resistant, no adaptive hyperinsulinemia occurs (11). These apparent discrepancies are even more pronounced in vitro, where islets from our line of HSL null mice exhibit no discernible impairment of insulin secretion (12), whereas those of the Mitchell laboratory (11) exhibit a specific impairment of GSIS. The reasons for this discrepancy are unclear at this time, particularly because little data were reported on lipid metabolism in islets in the other HSL null line (11). However, it may relate to differences in genetic background and/or breeding of the mice. The mutated HSL allele in the study by Roduit et al. was transferred from BALB/c mice (38) to C57/BL/6J mice (11). Our mice, on the other hand, are SV129/C57/BL/6J hybrid mice. It is conceivable that the different genetic backgrounds have either allowed or disallowed a genetic redundancy involving lipases in ß-cells. Nevertheless, also in inbred C57/BL/6J mice, we were unable to see activation of lipolysis by glucose (46), implying that the diminutive role of lipolysis in GSIS may relate more to differences in species than in strain. Clearly, the complex role of lipid mobilization in regulation of GSIS requires further investigation.


   Acknowledgments
 
The authors thank Ann-Helen Thorén, Birgitta Danielsson, and Sara Larsson for technical assistance.


   Footnotes
 
This work was supported by the Swedish Research Council (HM 14196, CH 11284), a Center of Excellence Grant from the Juvenile Diabetes Foundation, USA, and the Knut and Alice Wallenberg Foundation, Sweden (CH), the Juvenile Diabetes Research Foundation International (HM, 10-2000-676), the Swedish Diabetes Association; the Novo Nordisk, Crafoord, Thelma Zoega, Ingrid and Fredrik Thuring, Åke Wiberg and Albert Påhlsson Foundations, the Krapperup Foundation and the Medical Faculty at Lund University.

Abbreviations: 8-Br-cAMP, 8-Bromoadenosine cAMP; [Ca2+]i, intracellular Ca2+ concentration; GLP-1, glucagon-like peptide-1; GSIS, glucose-stimulated insulin secretion; HBSS, HEPES balanced salt solution; HSL, hormone-sensitive lipase; KATP, ATP-sensitive K+ channel; LC-CoA, long-chain acyl-coenzyme A; NCEH, neutral cholesterol ester hydrolase; PKA, protein kinase A.

Received December 9, 2003.

Accepted for publication May 5, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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