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Originally published In Press as doi:10.1074/jbc.M213032200 on June 30, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36380-36388, September 19, 2003
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Hormone-sensitive Lipase Null Mice Exhibit Signs of Impaired Insulin Sensitivity whereas Insulin Secretion Is Intact*

Hindrik Mulder {ddagger} §, Maria Sörhede-Winzell {ddagger}, Juan Antonio Contreras {ddagger}, Malin Fex {ddagger}, Kristoffer Ström {ddagger}, Thorkil Ploug ¶, Henrik Galbo ¶, Peter Arner ||, Cecilia Lundberg **, Frank Sundler **, Bo Ahrén {ddagger}{ddagger} and Cecilia Holm {ddagger}

From the Departments of {ddagger}Cell and Molecular Biology, **Physiological Sciences, and {ddagger}{ddagger}Medicine, Lund University, SE-221 84 Lund, Sweden, ||Department of Medicine, Huddinge University Hospital, SE-141 86 Stockholm, Sweden, and Copenhagen Muscle Research Center, Panum Institute, Copenhagen DK-2200, Denmark

Received for publication, December 20, 2002 , and in revised form, June 2, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic {beta}-cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of {beta}-cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Perturbation of plasma lipids is a hallmark of Type 2 diabetes mellitus (1). In fact, circulating free fatty acids (FFA)1 and triglycerides are elevated in many, if not most, individuals with impaired insulin sensitivity, obese individuals in particular. Therefore, much attention has been drawn to the possibility that hyperlipidemia is a cause, as well as a consequence, of insulin resistance (2). For instance, in skeletal muscle, infusion of lipids has been shown to interfere with glucose disposal (3). On a molecular level, it was proposed that lipids activate I{kappa}B kinase-{beta} (4) and/or atypical protein kinase C species in skeletal muscle (5); these kinases may attenuate insulin signaling by inappropriately phosphorylating serine residues of insulin receptor substrate proteins. Once these processes have commenced, insulin resistance will result in an inability of insulin to block mobilization of lipids from adipose tissue (6), i.e. lipolysis. Hyperlipidemia will be aggravated, fat deposition will ultimately also occur in non-adipocyte cells, and insulin sensitivity will be further impaired. Indeed, patients with Type 2 Diabetes Mellitus accumulate triglycerides in muscle fibers (7), an event correlating inversely with insulin sensitivity (8), whereas treatment of insulin resistance by thiazolidinediones depletes these accumulations and enhances insulin sensitivity (9).

Given the potential pathogenetic role of both circulating and stored lipids, the hormone-sensitive lipase (HSL) may play an important role in the development of Type 2 Diabetes Mellitus. The lipase is the key regulator of lipid mobilization from white adipose tissue (10), catalyzing the hydrolysis of tri- and diglycerides. HSL is unique among lipases in that it is regulated by hormones (10); thus, between meals HSL provides circulating FFA, a process stimulated by cAMP-raising hormones, such as glucagon and catecholamines. cAMP activates protein kinase A, which turns on the lipase by reversible phosphorylation of three serine residues (11). By contrast, in the post-prandial state, insulin signaling in adipocytes activates phosphodiesterase 3B, which hydrolyzes cAMP (6), hereby preventing activation of HSL and lipolysis. These processes may also be in operation in cells other than adipocytes. In fact, we demonstrated recently that HSL is expressed and active in skeletal muscle (12) and pancreatic {beta}-cells (13). This raises the possibility that perturbed function of HSL could lead to accumulation of acylglycerides in non-adipocytes, such as skeletal muscle and {beta}-cells, subsequently impairing insulin sensitivity and {beta}-cell function (14).

To address the role of HSL in these events, generation of mice with a disruption of the HSL gene should be helpful. A number of such lines have been created (15–18), all exhibiting quite similar phenotypes. Unexpectedly, HSL null mice maintain normal body weight, and accumulation of triglycerides is not apparent. Instead, increased levels of diglycerides are detected in some tissues (18), suggesting that in vivo HSL primarily acts as a diglyceride lipase and that other, as yet unknown, lipases are responsible for hydrolysis of acylglycerides. In one line (17), glucose intolerance was observed, which could be accounted for by a combination of insulin resistance and impaired glucose-stimulated insulin secretion (GSIS). Here, we demonstrate in an independent HSL null mouse line that lack of HSL is associated with a moderate impairment of insulin sensitivity whereas insulin secretion remains intact. Impaired insulin sensitivity is most apparent in liver, but reduced lipogenesis in white adipose tissue and glucose uptake in skeletal muscle observed in vitro likely contribute to the phenotype. The findings emphasize the essential metabolic role of a regulated mobilization of cellular lipids by HSL in normal glucose homeostasis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Inactivation of the HSL Gene—HSL null mice were generated by targeted disruption of the HSL gene in 129SV-derived embryonic stem cells by standard procedures (19), as described elsewhere (20). 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, hereby disrupting the catalytic domain. Two recombinant embryonic stem cell colonies were used for generation of two independent HSL null mouse lines. Preliminary experiments showed that both HSL null lines were similar with regard to glucose homeostasis and in that diglyceride hydrolysis in white adipocytes was virtually absent (data not shown); therefore, mice from both HSL null lines were pooled for the experiments in this work. The studies were approved by the Animal Ethics Committee at Lund University.

Western Blot Analysis—Soleus muscle and white adipose tissue were homogenized 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. For homogenates of adipose tissue, fat-free infranatants were prepared by centrifugation at 110,000 x g for 45 min at 4 °C. Proteins were resolved by SDS-PAGE and electroblotted to nitrocellulose membranes. Western blot analysis was performed by an enhanced chemiluminescence system (SuperSignal ULTRA; Pierce, Rockford, IL), using an affinity-purified chicken anti-rat HSL primary antibody.

Enzyme Activity and Acylglyceride Assays—Infranatants of white adipose tissue and homogenates of skeletal muscle (see above) and liver were assayed for diglyceride lipase activity; the diglyceride analogue mono-oleoyl-2-O-mono-oleylglycerol was used as substrate, as described elsewhere (21, 22). Neutral cholesteryl ester hydrolase (NCEH) activity was determined as described previously (22). Acylglycerides were extracted from the homogenates using methanol/chloroform according to the method of Folch et al. (23) and hydrolyzed by KOH; acylglyceride levels were determined as glycerol, using a bioluminescence assay (24).

Lipolysis in Isolated White Adipose Cells—Parametrial adipose tissue was excised, cut into small pieces, and incubated in Krebs-Ringer solution supplemented with 3.5% bovine serum albumin, 2 mM glucose, and collagenase (0.6 mg/ml; Sigma) in a shaking incubator for 90–100 min according to a modification (25) of the Rodbell method (26). The digested tissue was filtered, and the isolated cells were washed. The cells were suspended at a final concentration of 5% (v/v) in Krebs-Ringer buffer with 1% bovine serum albumin, 200 nM adenosine, and 2 mM glucose and aliquoted (100 µl/well) into microtiter filterplates (Millipore). Varying concentrations of isoproterenol were added, and after incubation in a shaking incubator for 1 h at 36.5 °C, the cell medium was collected using an Eppendorf 5 Prime device. Glycerol in the medium was determined using a bioluminescence assay (24), and FFA were determined using the NEFA-C kit (Wako Chemicals, Neuss, Germany).

Immunocytochemistry and Morphometry—Pancreatic tissue was prepared for immunocytochemistry as described previously (27), and sections were cut on a cryostat (15 µm). An affinity-purified rabbit anti-human HSL antibody (diluted 1:640) and antibodies to insulin (diluted 1:1280; code 9003; Euro-Diagnostica, Malmö, Sweden) and glucose transporter-2 (GLUT2; diluted 1:640; code 1342; Chemicon, Temecula, CA) were used for indirect immunofluorescence, as described previously (27). Islet number and {beta}-cell volume were determined in an unbiased procedure, according to the principles of stereological morphometry. To this end, serial sections (n = 100–120) were prepared and immunostained for insulin, using the peroxidase-anti-peroxidase method as described (27). Islet numbers were determined by dissector analysis (28), employing adjacent sections as reference and look-up sections, respectively. Total {beta}-cell volume was determined in serial sections using point-counting and Cavalieri's principle (29). As an index of {beta}-cell mass, mean islet {beta}-cell volume was calculated by dividing total islet {beta}-cell volume by total number of islets in the sections; these numbers were compared statistically. The investigator was unaware of the identity of the sections during analysis.

Glucose and Insulin Tolerance Tests—For the intravenous glucose tolerance test, D-glucose (1g/kg) was injected into the tail vein of anesthetized mice (midazolam (0.4 mg/mouse; Dormicum®; Hoffmann-La Roche) and a combination of fluanison (0.9 mg/mouse) and fentanyl (0.02 mg/mouse; Hypnorm®; Janssen, Beerse, Belgium)). In an additional experiment, 5 mg of arginine (Sigma) were injected intravenously to elicit insulin and glucagon secretion. In the oral glucose tolerance test, 150 mg of D-glucose were administered by oral gavage to the anesthetized mice after a 12-h fast. In the insulin tolerance test, 0.75 milliunits/g human insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) were given intraperitoneally to non-fasted anesthetized mice. In all in vivo investigations, plasma glucose and hormone levels were determined in retro-orbital blood samples collected at the time points indicated in the figures. For fasting samples, mice were deprived of food at 8:00 a.m., and retro-orbital blood was drawn from anesthetized mice at 2:00 p.m.

Euglycemic Hyperinsulinemic Clamp—A euglycemic hyperinsulinemic clamp was performed in HSL null (n = 5) and wild type mice (n = 7); the mice were anesthetized as described above. The clamp was carried out as described previously (30), with the addition of an infusion of [3H]glucose to enable measurements of glucose turnover rate. Briefly, the right jugular vein and the left carotid artery were catheterized. The venous catheter was used for infusion of glucose and insulin (Actrapid); the arterial catheter was used for sampling. Thirty min after introduction of the catheters (t = –100 min), infusion of [3H]glucose (Amersham Biosciences) was started. At t = 0 min, insulin was infused at a rate of 60 milliunits·kg–1·min–1 for 1 min, followed by a constant infusion of 20 milliunits·kg–1·min–1. Blood glucose levels were determined at 5-min intervals for 90 min. A variable rate of glucose infusion kept blood glucose at 6 mM. A blood sample was taken at t = 0, t = 60, and t = 90 min for determination of plasma insulin. Insulin sensitivity was calculated as the glucose infusion rate (GIR) during the second hour (M) divided by the mean insulin concentration at 60, 90, and 120 min (I). At t = 0 and t = 90 min, a blood sample was drawn for measurement of [3H]glucose specific activity by liquid scintillation counting. Glucose rate of appearance (Ra) was calculated by dividing the rate of infusion of [3H]glucose by the plasma glucose specific activity. Endogenous glucose output (EGO), which represents the glucose production from mainly hepatic, but to a slight extent also from renal sources, equals Ra before insulin infusion. During the insulin infusion, EGO was calculated as the difference between Ra and GIR. In cases in which Ra was less than GIR, EGO was considered to be zero. Finally, we also calculated the glucose clearance rate by dividing glucose disposal rate (being the highest of Ra and GIR) divided by the plasma glucose concentration.

Plasma Analyses—Insulin and glucagon were measured radioimmunochemically (Linco Research, St. Louis, MO). Leptin was determined by enzyme-linked immunosorbent assay (Chrystal Chemistry, Chicago, IL); glucose was determined by the glucose oxidase method (Sigma). Plasma FFA and triglycerides were determined according to the manufacturer's specifications (Wako Chemicals and Sigma, respectively).

Isolation and Perifusion of Islets—Islets were isolated by standard collagenase digestion and hand-picked under a stereo microscope. Insulin content in islets was measured radioimmunochemically (Linco Research) after acid ethanol extraction of the hormone. Perifusion of islets was performed as described previously in detail (31). In brief, islets were pre-incubated in HEPES balanced salt solution (125 mM NaCl, 5.9 mmol KCl, 1.2 mM MgCl2, 25 mM HEPES, 1.28 mM CaCl2, 0.1% human serum albumin, pH 7.36) containing 2.8 mM glucose for 60 min. Then, batches of 20 islets were sandwiched between two layers of gel (Bio-Gel P4; Bio-Rad). The HEPES balanced salt solution was kept at 37 °C, and islets were perifused at a flow rate of 0.5 ml/min; fractions were collected at 1-min intervals. Insulin was measured by enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden).

Glucose Uptake into Skeletal Muscle in Vitro—Glucose uptake into soleus muscle was determined as described previously (32). Briefly, the soleus muscle in anesthetized mice (see above) was gently dissected free with intact tendons at both ends and incubated for up to 4 h in test tubes at 29 °C in Krebs-Henseleit bicarbonate buffer containing 8 mM glucose, 1 mM pyruvic acid, and 0.2% bovine serum albumin. The medium was continuously gassed with 95% O2 and 5% CO2. Muscles were then placed in glucose-free Krebs-Henseleit buffer containing 2 mM pyruvic acid and 0.2% bovine serum albumin with or without 1000 microunit/ml of insulin (Actrapid). After 30 min, glucose transport was measured as [3H]-2-deoxy-D-glucose uptake with [14C]sucrose as extracellular marker. Isotopes and unlabeled sugars were added to the incubation medium to yield final concentrations of 0.81 µCi of [3H]-2-deoxy-D-glucose and 0.64 µCi of [14C]-sucrose per ml and 1 mM of both unlabeled 2-deoxy-D-glucose and sucrose. After a 10-min exposure to isotopes, muscles were briefly blotted on filter paper and immediately frozen in liquid N2 and stored at –80 °C until analyzed.

Lipogenesis in White Adipose Cells in Vitro—Lipogenesis was determined as described (33) with some modifications. Briefly, 1-ml aliquots of 2% (v/v) cell suspension of adipocytes, prepared as described above, and suspended in Krebs-Ringer solution with 25 mM HEPES, 3.5% bovine serum albumin, and 0.55 mM glucose, were added to vials containing D-[6-14C]glucose (0.4 µCi/ml), 30 nM isoproterenol, and varying concentrations of insulin. A low concentration of isoproterenol, in the absence of adenosine deaminase, has been shown to increase the sensitivity of the lipogenesis assay by reducing basal lipogenesis (no insulin) while leaving insulin-stimulated lipogenesis unaffacted.2 Incubations at 37 °C lasted for 3 h with shaking. Reactions were terminated by addition of 8 ml of a toluol-based scintillation mixture, containing 0.3 mg/ml 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene and 5 mg/ml 2,5-diphenyloxazole (Sigma). De novo synthesis of lipids was determined by liquid scintillation counting of radioactivity incorporated into total cellular lipids. D-[6-14C]Glucose is superior to several other glucose analogs in reflecting fatty acid synthesis and thus lipogenic status of the cell (34).

Statistical Analysis—Means ± S.E. were given. Data were analyzed statistically with a two-tailed unpaired t test with the following exceptions: for analysis of total insulin secretion during glucose challenges, area under curve (AUC) was calculated by use of the trapezoid rule; because the null hypothesis in this case concerned rejection of a decreased insulin secretion, AUC was compared with a one-tailed t test. In the lipogenesis experiments, data were compared with a paired one-tailed Wilcoxon's singed rank test, because arbitrary values were used (-fold changes), and pairing was made, because absolute values varied between experiments. Again, a one-tailed t test was used, because the null hypothesis in this case concerned rejection of an increase in lipogenesis, based on the previous observation of decreased glucose uptake in skeletal muscle. Numerical p values rather than cut-off values are given in most instances.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
General Characteristics of the HSL Null Mice—Male HSL null mice were sterile because of oligo- and azospermia (data not shown), which is in agreement with previous reports on other lines of HSL null mice (15, 16). For this reason, HSL null mice were generated by mating male HSL+/– mice from the F2 generation with female HSL+/– mice. Offspring, which are SV129/C57/BL/6J hybrids, were born in the expected Mendelian frequency. HSL null mice appeared grossly normal. Body weight (Table I) and food intake (data not shown) were similar in HSL null and wild type mice.


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  		TABLE I
Metabolic characteristics of HSL wild type and null mice

Data are from fed, anesthetized mice, unless otherwise stated. Mean ± S.E. are given; data were compared with a two-tailed unpaired t test.

 

HSL Expression in Adipocytes and Skeletal Muscle—Using Western blot analysis of extracts from white adipose tissue and soleus muscle, HSL was detected in HSL wild type, but not null, tissues (see Fig. 1A and Fig. 7A), confirming that the disruption of the HSL gene has resulted in mice that do not express HSL protein.



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  		FIG. 1.
Lipolysis in white adipose cells. Lipolysis was determined as release of glycerol (A) and FFA (B) into the assay buffer. A typical experiment on isolated white adipose cells from one-two mice for each genotype is shown. All experiments were performed in quadruplicate. Results at each dose of isoproterenol were compared with a two-tailed unpaired t test. *, p < 0.05; **, p < 0.01. Inset in A shows a Western blot of a homogenate of white adipose tissue (40 µg of protein resolved on the gel) probed with anti-HSL antibodies.

 


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  		FIG. 7.
In vitro glucose uptake in soleus muscle and lipogenesis in white adipose cells. The soleus muscle was excised in its entirety, and [3H]2-deoxy-D-glucose uptake was determined in the presence and absence of 1000 microunit/ml insulin (A), as described under "Experimental Procedures." The inset in A shows a Western blot of a homogenate of soleus muscle (80 µg of protein resolved on the gel) probed with anti-HSL antibodies. *, p = 0.025; two-tailed unpaired t test. For lipogenesis, white adipocytes were incubated in the presence of increasing concentrations of insulin, and incorporation of 14C into total cellular lipids was determined by scintillation counting (B). Data were compared with a paired one-tailed Wilcoxon's singed rank test, because arbitrary values were used (-fold changes); four independent experiments were performed in triplicate; *, p = 0.034.

 

Lipolysis in Isolated White Adipose Cells—To assess the consequences of the lack of HSL for catecholamine-stimulated lipolysis, we performed lipolysis experiments in isolated paragonadal adipocytes, using the non-selective {beta}-adrenergic agonist isoproterenol. We found that adipocytes of HSL null mice exhibited a completely blunted catecholamine-stimulated release of glycerol (Fig. 1A) and a reduced release of FFA (Fig. 1B) into the medium. These findings are in agreement with results from another line of HSL null mice (18). Concurrent with results in this strain, we also observe an accumulation of diglycerides in HSL null white adipocytes (data not shown). This suggests that HSL is the sole lipase in these cells capable of hydrolyzing diglycerides, whereas an additional lipase may hydrolyze triglycerides to diglycerides. Also, this would explain the moderate release of FFA from HSL null adipocytes, whereas glycerol remains within cells as diglycerides. However, the isoproterenol-induced increase in FFA release into the medium from HSL null adipocytes does not necessarily prove the existence of an alternative activable lipase. Rather, it may be the result of perilipin phosphorylation (35), with the consequent loss of the barrier function of this protein, allowing alternative triglyceride lipases access to the lipid droplet. The nature of these triglyceride lipases remains to be established (36).

Lipase Activity and Lipid Metabolism in Adipocytes, Skeletal Muscle, and Liver—Next, we examined the consequences of lacking HSL for lipase activity in adipocytes and skeletal muscle. In white adipose cells, HSL deficiency resulted in a decrease of diglyceride lipase activity by 27-fold (Fig. 2A). This is in sharp contrast to the situation in soleus muscle; here, lipase activity toward the synthetic diglyceride substrate was similar in both genotypes (Fig. 2B). Furthermore, also in liver extracts, diglyceride lipase activity was unaffected by the absence of HSL (Fig. 2C). It should be noted, however, that diglyceride lipase activity in soleus muscle and liver is 50–100-fold lower than that in wild type adipocytes. The lacking impact of the HSL ablation on diglyceride lipase activity in skeletal muscle and liver may be explained by the occurrence of additional, as of yet unknown hydrolases/esterases (36).



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  		FIG. 2.
Diglyceride lipase activity and NCEH activity in white adipose cells (A), soleus muscle (B), and liver (C). Determinations shown are as described under "Experimental Procedures." Isolated white adipose cells (WAT) from two-three mice for each genotype were used for diglyceride (DAG) lipase and NCEH activity. Seven and nine soleus muscles were used for diglyceride lipase activity determinations in wild type and HSL null mice, respectively; for NCEH activity in soleus muscle n = 3 for each genotype. Livers from three mice of each genotype were used for the enzyme activity assays. All experiments were performed in triplicate.

 

A high NCEH activity of HSL is a unique property among mammalian lipases (37). Along such lines, findings in another strain of HSL null mice suggest that NCEH activity in many cells may largely be attributed to HSL (15). Therefore, we proceeded to determine NCEH activity in white adipose cells, skeletal muscle, and liver from the mutant mice. In accord with the absence of HSL, NCEH activity was exceedingly low in extracts of HSL null white adipocytes, soleus muscle, and liver, while being readily demonstrable in wild type cells (Fig. 2).

Next, we examined the consequences of the altered profile of lipase activity in HSL null mice for storage of lipids. We found that acylglyceride levels, measured in the fed state, were not significantly altered in soleus muscle from HSL null mice compared with wild type mice (68.5 ± 5.6 versus 72.4 ± 11.3 nmol/mg; n = 3 for each genotype), whereas in liver from HSL null mice there was a small but significant increase in acylglyceride levels compared with wild type mice (1003 ± 140 versus 703 ± 149 nmol/mg; n = 5 for each genotype; p = 0.011; two-tailed unpaired t test). Lipid analyses revealed that the increase in acylglyceride levels in the liver of null mice was accounted for by an increase in triglycerides (data not shown); in agreement with results from an independent strain of HSL null mice (18) there were no signs of diglyceride accumulation in the liver of HSL null mice.

Metabolic Characterization of the HSL Null Mice—Metabolic data from the two mouse strains are shown in Table I. Plasma glucose levels were slightly but significantly elevated in the fasted (Table I), but not the fed (data not shown), state in HSL null mice compared with wild type mice. Accordingly, insulin levels were significantly increased in HSL null mice in both the fasted (Table I) and fed state (data not shown). This suggests that lack of HSL leads to impaired insulin sensitivity in mice. Random fed levels of FFA and triglycerides, as well as leptin, were similar in the two strains, which is in agreement with previous reports on HSL null mice (15, 16, 18). Glucagon levels in the fasted state were similar in both strains of mice.

Glucose Tolerance Tests—To further reveal a perturbation of glucose homeostasis, the mice were injected intravenously with D-glucose (1 g/kg), and plasma glucose and insulin levels were determined at the time points indicated in Fig. 3. Glucose disposal was similar in HSL null and wild type mice (Fig. 3A). Concomitant determinations of insulin, however, revealed that the glucose-stimulated insulin response in the HSL null mice was consistently increased during the whole challenge; thus, insulin secretion at 0, 1, and 5 min after glucose injection was elevated by 116, 247, and 360%, respectively, in the HSL null mice compared with wild type mice (p = 0.043, 0.042, and 0.067, respectively; two-tailed unpaired t test; see Fig. 3B). Insulin release during the whole test, calculated as AUC, was elevated by 75% (p = 0.07; one-tailed unpaired t test).



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  		FIG. 3.
Glucose tolerance tests in anesthetized mice. Plasma glucose (A and C) and insulin levels (B and D) were analyzed at the indicated time points (mean ± S.E. are given). Six female mice (age: 6 months) of each genotype were used for the intravenous tolerance tests (A and B); the intravenous test was done twice in the same mice. For the oral glucose tolerance test (C and D), eight HSL wild type and 11 HSL null mice were examined once. Insulin levels were significantly different (p < 0.05) at 0 and 1 min after glucose injection (B; two-tailed unpaired t test). AUC (inset in B and D) was increased in HSL null mice (p = 0.07 and 0.04, respectively; one-tailed unpaired t test).

 

It has been suggested that HSL plays a role in the incretin-mediated potentiation of insulin secretion (38); incretins such as glucagon-like peptide 1 raise intracellular cAMP, which could potentially activate HSL via protein kinase A. Given this possibility, we assessed whether the insulin response upon oral administration of glucose would be altered. Again, glucose disposal was similar in both strains of mice, whereas HSL null mice appeared to release exaggerated amounts of insulin (Fig. 3, C and D). Although insulin release was consistently increased during the whole challenge, as evidenced by an increase in AUC by 76% (p = 0.04; one-tailed unpaired t test), differences at each individual time point, however, did not reach statistical significance. There was no gender difference in HSL null mice with regard to glucose disposal and insulin secretion (data not shown). Thus, together the data further suggest that the lack of HSL causes a moderate impairment of peripheral insulin sensitivity, which is overcome by increased secretion of the hormone.

Arginine Test—The amino acid arginine is known to be taken up into islet cells, which subsequently are depolarized. Hence, an index of the hormone secretory capacity of the islets cells can be obtained. As shown in Fig. 4A, plasma glucose levels were similarly affected in HSL null and wild type mice upon an intravenous injection of 5 mg of arginine; insulin secretion rose 10-fold at 1 min after injection in both mouse strains (Fig. 4B). In contrast, glucagon secretion was differentially stimulated in the mice (Fig. 4C); in HSL null mice, glucagon levels rose only 3.5-fold compared with 6.8-fold in wild type mice (p = 0.014; two-tailed unpaired t test).



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  		FIG. 4.
Arginine (A–C) and insulin tolerance test (D). Five mg of arginine were injected intravenously, and plasma glucose (A), insulin (B), and glucagon (C) were analyzed at the time point shown in the graphs. Experiments were performed once in five-eight female mice of each genotype. Glucagon secretion was significantly lower at 1 min in HSL null mice (p = 0.014; two-tailed unpaired t test). For the insulin tolerance test (D) 0.75 kg/kg were injected intraperitoneally into fed, anesthetized female mice; plasma glucose was analyzed at the indicated time points (mean ± S.D. are given). Eight wild type and 10 HSL null female mice (age: 9 months) were used. Glucose levels were significantly different (p < 0.05) at 15 through 90 min after insulin injection (two-tailed unpaired t test). AUC (inset) was increased in HSL null mice (p = 0.04; two-tailed unpaired t test).

 

Insulin Tolerance Tests—To further establish that the HSL null mice exhibit impaired insulin sensitivity, mice were given an intraperitoneal injection of insulin, and plasma glucose was determined at the time points shown in Fig. 4D. As seen, the HSL null mice eliminated glucose at a slower rate than the wild type mice; glucose, measured as AUC, was elevated by 54% (p = 0.04; two-tailed unpaired t test). This indicates that exogenous insulin is less effective in promoting disposal of the sugar in peripheral tissues, again in agreement with impaired insulin sensitivity in HSL null mice.

Euglycemic Hyperinsulinemic Clamp—To further characterize impaired insulin sensitivity in the HSL null mice, euglycemic hyperinsulinemic clamps were performed. During the clamp, glucose was infused to maintain a target level of 6 mM. The data obtained during the clamp are given in Table II. Although baseline glucose levels did not differ between the groups, the baseline insulin levels were elevated by 59% in HSL null mice (p = 0.027; two-tailed unpaired t test). During the clamp, mean plasma insulin levels were similar in HSL null and wild type mice. Whole body insulin sensitivity was calculated based on the amount of glucose that was infused into the mice and corrected for the amount of insulin given during the clamp. Although there was a trend toward decreased whole body insulin sensitivity in HSL null mice, the difference was not statistically significant (p = 0.36).


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  		TABLE II
Data from a euglycemic hyperinsulinemic clamp in HSL wild type (n = 7) and null mice (n = 5-6)

Mean ± S.E. are given; data were compared with a two-tailed unpaired t test.

 

Next, we evaluated the role of liver on one side, and peripheral tissues on the other, for glucose homeostasis. Already prior to the clamp, EGO, which mainly reflects hepatic glucose production, was elevated, albeit not statistically significant, by 49% in HSL null mice (p = 0.125). At the end of the clamp, however, EGO was 4.5-fold higher in the mutant mice (p = 0.027; two-tailed unpaired t test). The glucose clearance rate was unaltered in HSL null mice.

Insulin Secretion from Perfused Islets—At this point, we examined insulin secretion from islets in vitro, using a perifusion system allowing dynamic characterization of insulin secretion. Basal insulin secretion was similar at 2.8 mM glucose in both strains of mice (Fig. 5). Upon an increase in glucose to 16.7 mM, there was no significant difference in insulin release from HSL null islets (AUC = 236 ± 49 versus 121 ± 31 in HSL null and wild type mice, respectively; p = 0.16; two-tailed unpaired t test). Acute addition of FFA to islets is known to potentiate GSIS (39). When 1 mM palmitate was added to 16.7 mM glucose, insulin secretion seemed to rise more rapidly in HSL null mice, but again overall insulin release in response to the fat was not different between the two mouse strains (AUC = 387 ± 59 versus 332 ± 66 in HSL null and wild type mice, respectively). Finally, 35 mM KCl was added to the perfusate, and this resulted in release of insulin from HSL null and wild type islets to a similar extent.



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  		FIG. 5.
Perifusion of isolated islets. Islets from female mice (age: 4 months) were perifused during 1 h, and insulin was determined in fractions collected at 1-min intervals. At t = 10 min, glucose (Glc) was raised from 2.8 to 16.7 mM; at t = 35 min, 1 mM palmitate (PA) was added to the perfusate containing 16.7 mM glucose; at t = 55 min, 35 mM KCl was added to the perforate. Means ± S.E. from five independent experiments for each genotype are shown.

 

Immunochemical and Morphological Characterization of Islets—In view of islet hypertrophy followed by {beta}-cell apoptosis as lipotoxic manifestations in the Zucker diabetic fatty rat (40, 41), we examined islet morphology in the HSL null mice. HSL immunofluorescence was readily detected in wild type islets, while lacking in HSL null islets (6A, B). Judging from immunocytochemistry, HSL null islets appeared hypertrophic compared with wild type islets (Fig. 6, D and E). Indeed, stereological determinations revealed that mean islet {beta}-cell volume was 2-fold increased in HSL null mice (0.96 ± 0.44 x 105 versus 1.98 ± 0.15 x 105 µm3; p = 0.02; two-tailed unpaired t test). Moreover, insulin content was 3-fold higher in freshly isolated islets from HSL null mice (3.6 ± 1.0 versus 11 ± 2.7 ng/islet; p = 0.041; two-tailed unpaired t test). The increases in both {beta}-cell mass and insulin content in the HSL null mice are likely to be adaptations to insulin resistance.



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  		FIG. 6.
Immunofluorescence in pancreatic sections from wild type (A and D) and HSL null mice (B, C, and E). Antibodies to human HSL (A and B), insulin (C), and GLUT2 (D and E) were employed. The green V in B and C indicates a blood vessel in two adjacent sections to aid orientation; the islet immunofluorescent for insulin in C has been indicated in the adjacent section B, in which the islet lacks immunoreactivity. The scale bar is 10 µm.

 

GLUT2, the protein responsible for facilitated transport of glucose into the {beta}-cell, is normally found in association with the plasma membrane in rodent islets. In the Zucker diabetic fatty rat, an early sign of cellular dysfunction following triglyceride accumulation is a disrupted, cytoplasmic, localization of GLUT2 (42). Immunofluorescence, however, for GLUT2 in HSL null and wild type islets was indistinguishable (Fig. 6, D and E), displaying the normal plasma membrane localization of the transporter. Collectively, our morphological findings do not support a lipotoxic condition in HSL null islets. Instead, the findings are in accord with islet alterations commonly observed in states of insulin resistance.

Glucose Uptake into Skeletal Muscle—To assess insulin action in muscle, [3H]2-deoxy-D-glucose uptake into excised soleus muscle was measured in the presence and absence of insulin. Although insulin stimulated glucose uptake 6-fold in wild type soleus muscle, such uptake was decreased by 17% in HSL null mice (p = 0.025; two-tailed unpaired t test), thus suggesting one site of insulin resistance (Fig. 7A). Basal glucose uptake, however, was unchanged in HSL null mice.

Lipogenesis in White Adipose Cells—Next, to assess insulin action in paragonadal white adipose tissue, synthesis of lipids in the presence and absence of insulin was determined. Because HSL null adipose tissue has a heterogeneous appearance, with large and small sized adipocytes (data not shown) (15), the data are expressed as -fold changes over lipogenesis in the absence of insulin. As shown in Fig. 7B, insulin robustly induced lipogenesis in wild type adipocytes; such induction was impaired in HSL null adipose cells at 10 and 100 nM insulin. Thus, both skeletal muscle and white adipose tissue in HSL null mice show signs of impaired insulin sensitivity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies demonstrate that an abrogation of regulated mobilization of lipids from cellular triglyceride stores, via disruption of HSL, results in impaired insulin sensitivity. A moderate impairment of sensitivity to the hormone was evident in all target tissues of insulin examined in the study, i.e. liver, white adipose tissue, and skeletal muscle, and is presumably driving the increase in basal levels of circulating insulin, as well as the exaggerated response to glucose observed in the tolerance tests. In liver, glucose production was trended toward an increase already in the basal state, and during the euglycemic hyperinsulinemic clamp, insulin failed to sufficiently block glucose production by the liver. In vitro, insulin-stimulated glucose uptake into soleus muscle and lipogenesis in white adipose cells were moderately, but significantly, impaired in HSL null mice, suggesting two additional sites that contribute to the observed insulin resistance. Despite the observed perturbations in glucose production and uptake, whole body insulin sensitivity, as assessed by the euglycemic hyperinsulinemic clamp, was not significantly reduced (–30%; p = 0.36). It is possible that the high insulin levels infused during the clamp surpass a threshold so that resistance to the hormone is no longer evident. Also, it should be kept in mind that in mouse ~70% of glucose disposal is insulin-independent (30). Nevertheless, glucose disposal in the HSL null mice was significantly retarded when exogenous insulin was injected. In sum, our investigations show that lack of HSL impairs peripheral insulin sensitivity.

Glucose homeostasis has been examined previously in another line of HSL null mice (17), which exhibits a more pronounced glucose intolerance. In contrast to our line, glucose disposal was retarded, which could be explained by a perturbation of GSIS, in addition to insulin resistance. Interestingly, also in vitro, isolated HSL null islets were unresponsive to glucose but not to KCl (17), suggesting a specific perturbation of fuel-stimulated insulin secretion. Based on the fact that HSL is expressed in pancreatic {beta}-cells (13), the authors suggest that HSL could play a role as provider of a lipid-derived signal in {beta}-cell stimulus-secretion coupling (17). Although our present studies confirm that lack of HSL results in impaired insulin sensitivity, we find no evidence for perturbed insulin secretion in our line of HSL null mice. Instead, islets adapted appropriately to insulin resistance by hypertrophy and increased storage of insulin, which are likely to underlie basal hyperinsulinemia and the exaggerated in vivo insulin response to glucose. Interestingly, perifused isolated islets from our HSL null mice appeared to retain some of the hyper-responsiveness to glucose seen in vivo, whereas no signs of a perturbed insulin secretion were evident. We have also performed extensive investigations of insulin secretion in static incubations of isolated islets, in response to a variety of secretagogues, at different ages, and in both genders, but in no instance was any secretory perturbation found in our current breed of HSL null mice.3 Although the reason for this discrepancy is unclear, it may relate to genetic background and/or breeding of the mice. The HSL mutation in the mice in the study by Roduit et al. (17) was transferred from BALB/c mice (16) to C57/BL/6J mice (17), whereas our mice were SV129/C57/BL/6J hybrid mice. It is possible that the different genetic backgrounds have either allowed/disallowed an appropriate adaptation to insulin resistance or a genetic compensation for the lack of HSL in terms of insulin secretion. Interestingly, early in our breeding of HSL null mice, we also observed perturbed GSIS in vitro despite a full compensatory response to insulin resistance in vivo. With subsequent breeding, this phenotype has been lost, and impaired insulin sensitivity is the outstanding and consistent finding in our HSL null line. Nevertheless, it must be borne in mind that the genetic manipulation, such as an inactivating mutation in a general knock out, is present from the gastrula stage. This potentially sets the stage for a number of compensatory mechanisms that may blur the phenotype of a null mutation. Thus, the lacking impact of HSL deficiency on {beta}-cell function in our model does not rule out a role for the lipase in {beta}-cell stimulus-secretion coupling in normal mice.

A number of possible mechanisms for insulin resistance in HSL-deficient mice exist. It has been reported previously (18) that HSL null mice accumulate diglycerides, but not triglycerides, in a number of cells, including skeletal and cardiac muscle and white and brown adipose cells, whereas triglycerides remain essentially unchanged. This suggests that in vivo HSL primarily acts as a diglyceride lipase, whereas an unidentified lipase(s) accounts for hydrolysis of triacylglycerides. Whether this reflects normal physiology or occurs only in HSL null mice remains to be clarified. Nevertheless, diglycerides are known to be potent intracellular signals and may, via activation of e.g. atypical protein kinase C species (5) and/or I{kappa}B kinase-{beta} (4), attenuate insulin signaling. It is also possible that accumulation of these lipids affects transcriptional activity in target tissues of insulin, perhaps via peroxisome proliferator activator receptors, hereby inducing chronic changes in gene expression. Studies addressing these issues are currently ongoing. An alternative mechanism, although more speculative, is that impaired insulin sensitivity in HSL null mice is caused by abrogated formation of a lipid from acylglycerides, which serves as a signaling molecule. This lipid would normally be provided by the action of HSL and putatively activate some part of the signaling cascade culminating in glucose uptake or exert transcriptional effects on pertinent genes. Such signaling has been proposed for stimulus-secretion coupling (43) and transcription (44) in pancreatic {beta}-cells.

Given that liver normally expresses HSL at a very low level (45–47), if at all, the pivotal role of this organ in insulin resistance in the HSL null mice is surprising. Moreover, it was reported that HSL null mice exhibit unaltered liver triglyceride levels in the fed state (47), fail to raise these levels in the fasted state, and show no significant accumulations of diglycerides in the fasted state (18). In the present study, liver diglyceride lipase and NCEH activities, albeit at low levels, were detected in wild type mice. The latter activity was abolished in the HSL null liver, strongly suggesting that HSL is indeed expressed in mouse liver and has been disrupted in our null line. As in soleus muscle, diglyceride lipase activity was unaltered. A rise in tissue acylglyceride storage is what was initially anticipated in the HSL-deficient mouse strains, presumably leading to obesity and cellular dysfunction. Surprisingly, so far no signs of obesity have been reported in HSL null mice, and only islets (17) and liver (this study) exhibit a modest, but significant, rise in acylglyceride levels. The mechanism of the increased acylglyceride levels in liver is unclear, particularly in light of the unaltered diglyceride lipase activity; we have also assayed triglyceride lipase activity in liver, which was not different in HSL null mice (data not shown). If there is redundancy of an additional unidentified lipase, it appears to be unable to sufficiently control liver storage of acylglycerides. Moreover, our observations do not agree with what was found in a previously reported HSL null line (18); the reason for this is also unclear, but we have consistently found a modest increase in tissue acylglycerides in HSL null mice of different ages (data not shown). Given the unaltered lipase activities, it is surprising that an accumulation, albeit a modest one, of acylglycerides was observed. Nevertheless, the accumulation of acylglycerides in liver may account for the impairment of insulin sensitivity that we observe, because increased hepatic glucose production is associated with an increase in storage of hepatocellular acylglycerides (48). To speculate, the fact that glucagon secretion, which normally stimulates glucose production in liver, was diminished in HSL null mice may be a an endocrine compensation to the increased hepatic glucose production. Thus, contrary to what was believed previously, our data suggest that HSL does play a role in liver under normal conditions. At any rate, the HSL-deficient mouse model is unique in the sense that insulin resistance is coupled to an inability to release appropriate amounts of FFA during fasting (18) and is further proof of the essential link between adipose tissue and other target tissues of insulin in maintaining glucose homeostasis (49). The absence of HSL from a number of tissues and its consequences have revealed a complexity of lipid metabolism that was not anticipated.


   FOOTNOTES
 
* This work was supported in part by the Swedish Research Council Grants 14196 (to H. M.), 11284 (to C. H.), and 6834 (to B. A.), by A Center of Excellence Grant from the Juvenile Diabetes Foundation, United States of America, by the Knut and Alice Wallenberg Foundation, Sweden (to C. H. and P. A.), by Juvenile Diabetes Research Foundation International Grant 10-2000-676 (to H. M.), and by the Swedish Diabetes Association, Novo Nordisk Fond, the Crafoord, Thelma Zoega, Ingrid and Fredrik Thuring, Åke Wiberg and Albert Påhlsson Foundations, and the Medical Faculty at Lund University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Section for Molecular Signaling, Dept. of Cell and Molecular Biology, Lund University, BMC C11, SE-221 84 Lund, Sweden. Tel.: 46-46-222-0473; Fax: 46-46-222-4022; E-mail: hindrik.mulder{at}medkem.lu.se.

1 The abbreviations used are: FFA, free fatty acids; AUC, area under curve; EGO, endogenous glucose output; GIR, glucose infusion rate; GSIS, glucose-stimulated insulin secretion; Ra, glucose rate of appearance; GLUT2, glucose transporter-2; HSL, hormone-sensitive lipase; NCEH, neutral cholesteryl ester hydrolase; WT, wild type. Back

2 Eva Degerman, personal communication. Back

3 M. Fex, M. Sörhede-Winzell, C. Holm, and H. Mulder, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Ahnders Franzén, Department of Cell and Molecular Biology, and Reinhard Fässler, Department of Experimental Pathology, both at Lund University, for advice and materials for the embryonic stem cell work herein. Also, Eva Degerman, Department of Cell and Molecular Biology, Lund University, advised on the optimization of the lipogenesis assay, and Hans Tornqvist at Novo Nordisk A/S helped with the micro-titer-based lipolysis assay. We thank Lena Kvist, Lillian Bengtsson, Ann-Helen Thorén, Sara Larsson, Birgitta Danielsson, Kristina Göransson, Åsa Nordfeldt, and Doris Persson for technical assistance.



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 ABSTRACT
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 DISCUSSION
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