Biochemical and Biophysical Research Communications 387 (2009) 511–515

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Hormone-sensitive lipase deﬁciency suppresses insulin secretionfrom pancreatic islets of Lepob/ob mice
Motohiro Sekiya a, Naoya Yahagi a,d,*, Yoshiaki Tamura a, Hiroaki Okazaki a, Masaki Igarashi a, Keisuke Ohta a,
Mikio Takanashi a, Masayoshi Kumagai a, Satoru Takase a, Makiko Nishi a, Yoshinori Takeuchi a,
Yoshihiko Izumida a, Midori Kubota a, Ken Ohashi a, Yoko Iizuka a, Hiroaki Yagyu f, Takanari Gotoda b,
Ryozo Nagai c, Hitoshi Shimano e, Nobuhiro Yamada e, Takashi Kadowaki a,d, Shun Ishibashi f,
Jun-ichi Osuga aaDepartment of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan
Department of Nephrology and Endocrinology, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japanc
Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, JapandLaboratory of Molecular Physiology on Energy Metabolism, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, JapaneAdvanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaragi 305-8575, Japanf
Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical University, Tochigi 329-0498, Japanba r t i c l e

i n f oArticle history:
Received 25 June 2009
Available online 18 July 2009
Keywords:
Hormone-sensitive lipase
Insulin secretion
Pancreatic islets
Lipotoxicity

a b s t r a c t
It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis inpancreatic b-cells can affect insulin secretion through the alteration of lipotoxicity. We generated micelacking both leptin and HSL (Lepob/ob/HSL / ) and explored the role of HSL in pancreatic b-cells in the setting of obesity. Lepob/ob/HSL / developed elevated blood glucose levels and reduced plasma insulin levels
compared with Lepob/ob/HSL+/+ in a fed state, while the deﬁciency of HSL did not affect glucose homeostasis in Lep+/+ background. The deﬁciency of HSL exacerbated the accumulation of triglycerides in Lepob/ob
islets, leading to reduced glucose-stimulated insulin secretion. The deﬁciency of HSL also diminishedthe islet mass in Lepob/ob mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.
Ó 2009 Elsevier Inc. All rights reserved.

Introduction
Obesity and type 2 diabetes represent a growing threat to thehealth of the population of almost every country in the world.
Accumulation of lipids in non-adipose tissues can lead to cell dysfunction, a phenomenon known as lipotoxicity as a molecular link
between obesity and glucose homeostasis dysregulation [1].
Pancreatic b-cells are known to be highly susceptible to lipotoxicity, and both exogenous and endogenous sources of free fatty acids(FFAs) are believed to be involved in insulin secretory machinery[2,3]. The liberation of FFA by intracellular hydrolysis of acylglycerols, which is referred to as lipolysis, is observed in the pancreatic
islets [4,5], and the lipolytic reaction supplies the endogenous FFA.
Hormone-sensitive lipase (HSL) is an intracellular neutral lipasethat is capable of hydrolyzing triglycerides (TGs), diglycerides,
monoglycerides, and cholesterol esters as well as other lipids* Corresponding author. Address: Department of Metabolic Diseases, Graduate
School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655,
Japan. Fax: +81 3 5802 2955.
E-mail address: nyahagi-tky@umin.ac.jp (N. Yahagi).
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.07.078[6–9]. Although HSL was initially identiﬁed as an adipose-speciﬁc
lipase, it has been clariﬁed that HSL is also expressed and functions
in a wide variety of organs and cells, including heart, skeletal muscle, adrenal glands, testes, ovaries, intestines and liver [7,8,10,11].
HSL was also identiﬁed in pancreatic b-cells [12], and several laboratories reported the relationship between the HSL-mediatedlipolysis and insulin secretory machinery using rodent models
without coming to consensus [13–17].
We reported the phenotypes caused by the combined deﬁciencyof leptin and HSL (Lepob/ob/HSL / ) to explore the role of HSL in thesetting of obesity [18]. Here we further explored the potential roleof HSL in the insulin secretion using the leptin-deﬁcient model.
Lepob/ob/HSL / showed elevated plasma glucose levels anddecreased plasma insulin levels compared with Lepob/ob/HSL+/+ in a
fed state, while the deﬁciency of HSL did not affect both plasma glucose levels and plasma insulin levels in a fasted state. In the Lep+/+
background, the glucose homeostasis was not affected by HSL. The
impaired insulin secretion of Lepob/ob/HSL / was further supported
by glucose-stimulated insulin secretion (GSIS) from isolated islets.
In addition, the deﬁciency of HSL decreased the enlarged islet massin Lepob/ob mice due to decreased cell proliferation.

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M. Sekiya et al. / Biochemical and Biophysical Research Communications 387 (2009) 511–515

Materials and methods

Results and discussion

Animals. Lep+/+/HSL / and Lepob/ob/HSL / mice were generated,and genotyping was performed as described previously [18,19].
Mice were housed in a temperature-controlled environment witha 12-h light/dark cycle and allowed free access to water and a standard chow diet (Oriental MF, Oriental Yeast, Tokyo, Japan). Mice
were euthanized at 16 weeks after a 6-h fast. Blood was collectedfrom the retro-orbital venous plexus after a 6-h fast (fasted state,
at 8 and 16 weeks of age) or at the beginning of the light cycle(fed state, at 9 and 15 weeks of age). Plasma glucose was measured
by ANTSENSE II (Bayer Medical, Tokyo, Japan), and plasma insulin
was measured by the mouse insulin ELISA kit (Morinaga, Tokyo,
Japan).
Pancreatic islets isolation and insulin secretion experiment. Islet
isolation was carried out using the collagenase technique from
16-week-old non-fasted mice. In brief, 2.5 ml of collagenase (Type{I, Sigma, St. Louis, MO) solution at 4 mg/ml were introduced into
the common bile duct after occlusion of the distal end just proximal to the duodenum. The distended pancreas was excised andthe digestion was performed in a water bath at 37 °C for 3.5 min.
The islets were washed and puriﬁed with a hand pick-up. Glucose
stimulation tests were performed on isolated pancreatic islets
essentially according to Sutton et al. [20]. Six groups (each group
comprising 8 islets) were prepared from each mouse and threegroups were used for low glucose (2.8 mM) stimulation and theother three were for high glucose (20 mM) stimulation. After preincubation at 37 °C for 30 min in KRBH buffer (130 mM NaCl,
5.2 mM KCl, 1.3 mM KH2PO4, 2.7 mM CaCl2, 1.3 mM MgSO4,
24.8 mM NaHCO3, and 10 mM Hepes at pH 7.4) containing
2.8 mM glucose and 0.5% bovine serum albumin (BSA; Fraction
V; Sigma, St. Louis, MO), groups of islets were incubated at 37 °C
in KRBH containing 0.5% bovine serum albumin and either
2.8 mM glucose or 20 mM glucose for 30 min, then the media werewithdrawn for insulin measurement. Lipids were extracted from100 islets/each mouse by chloroform–methanol method and TG
content was measured by a kit (GPO-Trinder, Sigma, St. Louis,MO) (n = 8–11). Lipid extracted islets were dissolved in phosphatebuffered saline by sonication. DNA content was measured by ﬂuorescent method with Hoechst 33258 [21]. The size of islets underthese experiments was controlled visually to be similar between
HSL+/+ and HSL / group.
Histology and quantiﬁcation of islet mass. Sixteen-week-old
mice were euthanized, and the pancreases were excised, ﬁxed
in 10% neutral buffered formalin, embedded in parafﬁn, andstained with hematoxylin–eosin. Three non-sequential sections
from each pancreas were scanned (three mice for each genotype)by NIH image. At least 50 islets from each pancreas were quantiﬁed and total number of islets scanned was from 195 to 260
for each genotype.
Proliferating cells were detected by bromodeoxyuridine (BrdU)(Sigma, St. Louis, MO) immunostaining. Two hours after administration of BrdU intraperitoneally at 50 mg/kg body weight in phosphate buffered saline, mice were euthanized, and the pancreases
were excised and embedded in parafﬁn. Immunostaining was performed by a BrdU in situ detection kit (PharMingen, San Diego, CA)
and counterstained with Mayer’s hematoxylin (Wako Pure Chemicals). Apoptotic cells were detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) using anin situ apoptosis detection kit (Takara Biomedicals, Otsu, Japan)
according to the manufacturer’s instructions, with counterstaining
with methyl green (Wako Pure Chemicals).
Statistics. Statistical differences between groups were analyzed
by the Student’s t-test or the one-way analysis of variance and apost hoc Tukey–Kramer test, unless otherwise stated.

Levels of plasma insulin and glucose suggested the impaired insulin
secretion in Lepob/ob/HSL / mice
To gain insight into the physiological roles of HSL especially inthe setting of obesity, we generated Lepob/ob/HSL / mice. In thisstudy, we attempted to characterize the roles of HSL in the pancreatic b-cells in the setting of obesity. In these mice, fasting plasma
glucose and insulin levels were not affected by the HSL deﬁciency
in both Lep+/+ and Lepob/ob backgrounds (Fig. 1). The results suggested the limited contribution of HSL to the whole-body insulin
sensitivity, and in support of this ﬁnding, intraperitoneal insulin
tolerance test was not affected by the HSL deﬁciency (data notshown). On the other hand, Lepob/ob/HSL / mice showed elevatedplasma glucose levels (26% increase for males and 72% increase
for females) and decreased plasma insulin levels (45% decrease
for males and 53% decrease for females) compared with Lepob/ob/
HSL+/+ mice under fed conditions (Fig. 1), which suggested impaired insulin secretion in Lepob/ob/HSL / mice. In the Lep+/+ background, there was no signiﬁcant difference between Lep+/+/HSL+/+and Lep+/+/HSL / (Fig. 1). Since it was reported that the impaired
insulin secretion of HSL / mice was age- and gender-dependent[15], we examined both gender at two time points (8 weeks and
16 weeks for fasted conditions, and 9 weeks and 15 weeks for fed
conditions. Data shown are representative of the two time points.
It is also known that almost all the gender differences disappear in
Lepob/ob mice). The Lepob/ob/HSL / had defective insulin secretion
even at an earlier age. We previously reported that the food intake
was reduced in Lepob/ob/HSL / mice [18], and the impaired insulin
secretion of Lepob/ob/HSL / might be underestimated under freefeeding conditions. In accordance with the impaired insulin secretion, the hepatic expression of gluconeogenetic enzymes such as
phospho-enol-pyruvate carboxykinase (PEPCK) was reduced in Lepob/ob/HSL / (data not shown).
Islets isolated from Lepob/ob/HSL / mice displayed a decreased GSIS
response
To eliminate the indirect effect, we isolated islets from eachgenotype, and GSIS assay was performed. In Lepob/ob background,
the response of Lepob/ob/HSL / islets to high concentration of
glucose was signiﬁcantly reduced by 36% compared with that of
Lepob/ob/HSL+/+ islets (Fig. 2). In Lep+/+ background, the deﬁciencyof HSL caused a milder decrease in GSIS, which did not reach statistical signiﬁcance. The discrepancy between the earlier report[13] and our results needs to be studied further.
The deﬁciency of HSL led to marked accumulation of TG in Lepob/ob
islets
Leptin deﬁciency may confer vulnerability to the lipotoxicity,
because leptin causes TG depletion in islets via stimulating b-oxidation [22]. Furthermore leptin-treatment of Lepob/ob mice is
known to reverse their glucose intolerance and is reported to
upregulate HSL expression in islets [23], which imply protectivefunction of HSL against lipotoxicity. Therefore, we examined
whether and to what extent the deﬁciency of HSL inﬂuenced theislet TG content. There was a striking increase (78%) in islet TG content in Lepob/ob/HSL / (Fig. 3A and C). The size of islets used for the
GSIS experiment and lipid extraction was controlled visually to be
similar between HSL+/+ and HSL / group. To assure the precision,
islet DNA content was measured ﬂuorometrically (Fig. 3B), andthe marked accumulation of islet TG in Lepob/ob/HSL / was also
supported by the data normalized with respect to DNA content

M. Sekiya et al. / Biochemical and Biophysical Research Communications 387 (2009) 511–515

male

fastedfemale300

glucose(mg/dl)

glucose(mg/dl)

300
200
100

200
100
0
20

insulin(ng/ml)

020

insulin(ng/ml)

513

10

0

10

0

fed
500

*

400

glucose(mg/dl)

glucose(mg/dl)

500

300
200
100

300
200
100
0
200

**
100

0

insulin(ng/ml)

insulin(ng/ml)

0
200

**

400

**

100

0

HSL +/+ +/- -/- +/+ +/- -/-HSL +/+ +/- -/- +/+ +/- -/-

Lep

Lep

+/+

ob/ob

+/+

ob/ob

Fig. 1. Lepob/ob/HSL / mice exhibited impaired insulin secretion in vivo. Blood samples were collected from either fasted (after a 6-h fast, at the age of 8 weeks, displayed in
the upper four panels) or fed (at the beginning of light cycle, at the age of 15 weeks, displayed in the lower four panels) mice (n = 10). Each value represents the mean ± SE.
*And **denote signiﬁcance at P < 0.05 and P < 0.01, respectively, vs HSL+/+ mice in the same Lep background determined by ANOVA followed by the Tukey–Kramer post hoc
test. Similar pattern was observed in both younger (at the age of 8–9 weeks) and adult (at the age of 15–16 weeks) mice, and data shown are representative of the two time
points.

(Fig. 3C). On the other hand, there was no signiﬁcant difference between Lep+/+/HSL+/+ and Lep+/+/HSL / , which is inconsistent withprevious report [13]. Although it is necessary to further elucidate
whether or not there might be a difference that was undetectableby our methods between Lep+/+/HSL+/+ and Lep+/+/HSL / , the contribution of HSL to islet TG store was certainly clariﬁed by the coablation of the leptin gene. Since excessive TG accumulation in islets is reported to require hyperglycemic background [24], HSL
deﬁciency might induce lipotoxic impairment of insulin secretion
only in the Lepob/ob background.

mice showed no speciﬁc data regarding islet mass [13,15], while
Holm and her colleagues reported the increased islet mass in HSL
deﬁcient mice accompanied by the whole-body insulin resistance[16]. Taken together with the ﬁndings in previous reports, the roleof HSL in islet function would be limited, and the experiments
using HSL deﬁcient mice could sometimes produce confusing results. Our investigation using co-ablation of leptin gene would
facilitate our understanding of the role of HSL in pancreatic b-cells.
The combined effects of the reduction of insulin secretion per same
size islet and reduced islet mass in Lepob/ob/HSL / would impair
insulin secretion in vivo.

Islet mass was signiﬁcantly reduced in Lepob/ob/HSL /Examination of pancreatic sections revealed that the islet mass
was reduced by 25% in Lepob/ob/HSL / compared with Lepob/ob/
HSL+/+, whereas there was no signiﬁcant difference between
Lep+/+/HSL+/+ and Lep+/+/HSL / (Fig. 4A). Previous reports which addressed the reduced insulin secretory capacity in HSL deﬁcient

Reduction in islet mass in Lepob/ob/HSL / occurred via defective cellproliferation
To determine whether the decreased islet mass is due todefective proliferation or increased apoptosis, pancreatic sections
were analyzed by immunohistochemistry for apoptosis by TUNELM. Sekiya et al. / Biochemical and Biophysical Research Communications 387 (2009) 511–515

insulin (ng/islet/30min)

4

*

LG
HG

3
2
1

0

HSL
Lep

+/+
-/+/+

+/+
-/ob/ob

Fig. 2. Glucose-stimulated insulin secretion (GSIS) from Lepob/ob/HSL / islets was
impaired. Six groups (each group comprising 8 islets) were prepared from eachmouse and three groups were used for low glucose (LG, 2.8 mM) stimulation andthe other three were used for high glucose (HG, 20 mM) stimulation (n = 5 for eachgenotype). After preincubation at 37 °C for 30 min in KRBH containing 2.8 mM
glucose, groups of islets were incubated at 37 °C in KRBH containing either 2.8 mM
glucose or 20 mM glucose for 30 min, then the media were withdrawn for insulin
measurement. Each value represents the mean ± SE. *Denotes signiﬁcance at
P < 0.05, vs HSL+/+ mice in the same Lep background determined by the Student’s
t-test.

(Fig. 4B) and for cell proliferation by BrdU incorporation(Fig. 4C). No TUNEL positive nuclei were detected in pancreaticsections of all genotypes. On the other hand, BrdU incorporation
study revealed that increased proliferative activity of Lepob/ob/
HSL+/+ islets completely disappeared in Lepob/ob/HSL / islets.
The mechanism of upregulated proliferation of islet cells of Lepob/ob
mice is not established [25], but this is believed to be due to theadaptation to the increased insulin demand [26]. The promoted

TG content (ng/islet)

A 30

**

20

10

0

HSL
Lep

+/+
-/+/+

TG content/DNA (ng/ug)

C 500

+/+
-/ob/ob

islet cell proliferation in Lepob/ob/HSL+/+ was completely abolishedand no BrdU positive cells were detected in the sections ofLepob/ob/HSL / as well as Lep+/+/HSL+/+ and Lep+/+/HSL / . Although
the precise mechanism should be elucidated, cAMP/cAMPdependent protein kinase A (PKA) pathway might be involved.
Glucagon-like peptide (GLP)-1 increases cell survival via cAMPdependent stimulation of cAMP response element binding protein(CREB) activity, and subsequent enhancement of the insulin receptor substrate (IRS)-2 regulated pathway [27]. The decreased
feeding behavior in Lepob/ob/HSL / might reduce the serum
Glucagon-like peptide (GLP)-1 levels.
The long-term exposure to FFA leads to disruption of GSIS [28],and genetic deletion of HSL decreases plasma FFA levels mobilized
from adipose tissue in both Lep+/+ and Lepob/ob background. Sincedeﬁciency of HSL raises numerable changes caused from phenotype of adipose tissue or the other tissues, tissue-speciﬁc genetic
deletion will be required to further determine the precise role of
HSL on pancreatic b-cells.
It was reported that lipids generated by acylglycerol hydrolysis
play a critical role in insulin secretion using a chemical inhibitionof b-cell lipolysis [4]. However, it still remained to be determined
whether the lipase(s) responsible for the regulation of insulin
secretion would be HSL or the other lipase(s). It was reported recently that adipose triglyceride lipase is another promising candidate [29]. In the present study, we demonstrated that HSL is atleast one of the key molecules involved in the insulin secretorymachinery.
In summary, we clariﬁed the role of HSL in pancreatic b-cells by
the co-ablation of leptin gene. It was demonstrated that the deﬁciency of HSL in Lepob/ob mice causes impaired insulin secretion
and reduces the islet mass due to the defective cell proliferation.
These ﬁndings provide the basis for understanding the pathophysiology of obesity and diabetes.

B 0.12
DNA content (ug/islet)

514

0.10
0.08
0.06
0.04
0.02
0

HSL
Lep

+/+
-/+/+

+/+
-/ob/ob

**

400
300
200
100
0

HSL
Lep

+/+
-/+/+

+/+
-/ob/ob

Fig. 3. Excessive triglyceride accumulation was observed in Lepob/ob/HSL / islets (n = 9–11). (A) Lipids were extracted from 100 islets/each mouse by the chloroform–
methanol method. (B) Lipid extracted islets were dissolved in phosphate buffered saline by sonication, and the DNA content was measured by ﬂuorescent method. (C) TG
content of islets was normalized with respect to DNA content. Each value represents the mean ± SE. **Denotes signiﬁcance at P < 0.01, vs HSL+/+ mice in the same Lep
background determined by the Student’s t-test.

M. Sekiya et al. / Biochemical and Biophysical Research Communications 387 (2009) 511–515

islet area (µm 2 )A 50000

**

40000
30000
20000
10000
0

HSL +/+
-/Lep
+/+

B

+/+ -/ob/obC

Lep+/+/HSL+/+

Lep+/+/HSL-/-

Lepob/ob/HSL+/+

Lepob/ob/HSL-/-

Fig. 4. The deﬁciency of HSL reduced the pancreatic islet mass of Lepob/ob mice. (A)Islet mass was quantiﬁed using NIH image. At least 50 islets from each pancreas
were quantiﬁed and total number of islets scanned was 195–260 for each genotype(n = 3). Each value represents the mean ± SE. **Denotes signiﬁcance at P < 0.01,
respectively, vs HSL+/+ mice in the same Lep background determined by theStudent’s t-test. (B, C) Immunohistochemical analysis of islets. (B) TUNEL staining
and (C) BrdU labeling. Arrows point to the labeled cells (C).

Acknowledgments
This work was partially supported by Grants-in-Aid from Japan
Health Science Foundation and Research Fellowship of the Japan
Society for the Promotion of Science for Young Scientists (to M.
Sekiya). This work was also supported by a Grant-in-Aid for Scientiﬁc Research from the Ministry of Education, Science and by
Culture the Program for Promotion of Fundamental Studies in
Health Science of the National Institute of Biomedical Innovation(NIBIO).
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