Relative Hypoglycemia and Hyperinsulinemia in Mice with Heterozygous Lipoprotein Lipase (LPL) Deficiency

Relative Hypoglycemia and Hyperinsulinemia in Mice with Heterozygous Lipoprotein Lipase (LPL) DeficiencyRelative Hypoglycemia and Hyperinsulinemia in Mice with Heterozygous Lipoprotein Lipase (LPL) Deficiency ISLET LPL REGULATES INSULIN SECRETION*
(Received for publication, March 25, 1999, and in revised form, July 13, 1999)
Bess A. Marshall‡§, Karen Tordjman‡, Helen H. Host, Nancy J. Ensor, Guim Kwon, Connie A. Marshall, Trey Coleman, Michael L. McDaniel, and Clay F. Semenkovich¶
From the Departments of Medicine, Pediatrics, Pathology, and Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL1/2). LPL1/2 mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL1/1 littermates. Despite having lower glucose levels, LPL1/2 mice had fasting insulin levels that were twice those of 1/1 mice. Hyperinsulinemic clamp experiments showed no effect of genotype on ba- sal or insulin-stimulated glucose utilization. LPL mes- sage was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, 1/2 islets ex- pressed half the enzyme activity of 1/1 islets. Islets iso- lated from 1/1 mice secreted less insulin in vitro than 1/2 and 2/2 islets, suggesting that LPL suppresses in- sulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing hu- man LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-b-galactosid- ase-treated cells. INS-1 cells transfected with an anti- sense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They fur- ther suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.
Lipoprotein lipase (LPL)1 catalyzes the rate-limiting step for clearance of triglycerides from the blood. Hydrolysis of lipopro-
tein-associated triglycerides in the capillary beds of peripheral tissues such as muscle and adipose tissue produces free fatty acids that are available for local uptake (1). LPL enzyme ac- tivity is probably the major factor controlling movement of exogenous fatty acids into peripheral tissues. The overexpres- sion of LPL in mouse muscle (2) increases tissue lipid as well as mitochondria and peroxisomes, the sites of fatty acid metabo- lism. Mice with homozygous LPL deficiency (LPL2/2) (3, 4) die soon after birth with minimal tissue lipid. Mice deficient in adipose tissue LPL develop adipose tissue lipid stores but only by inducing de novo fatty acid biosynthesis from glucose (5). These results suggest that tissue lipid content plays important roles in normal physiology and that LPL is essential for the acquisition of exogenous fatty acids by tissues.
Fatty acids and glucose compete as respiratory substrates in many tissues (6). In muscle, fatty acids inhibit glucose utiliza- tion and oxidation. In liver, fatty acids inhibit glucose oxidation and promote gluconeogenesis. In the pancreatic beta cell (7), fatty acids have complex effects that differ depending on the duration of exposure. Since LPL is the dominant provider of fatty acids to tissues and fatty acids alter insulin secretion and glucose utilization, defects in the LPL gene could affect glucose metabolism.
Recent reports in mice and humans suggest that LPL geno- type affects glucose metabolism. The reason for the death of LPL2/2 mice soon after birth (3, 4) is unknown. Merkel and colleagues (8) reported that LPL2/2 mice are profoundly hy- poglycemic (mean glucose of 15 mg/dl), although the underlying mechanisms are unknown. LPL2/2 humans are rare, but hu- man heterozygous LPL deficiency (LPL1/2) occurs in about 3% of unselected subjects of various ethnic backgrounds (9, 10). These individuals have elevated triglycerides and decreased high density lipoprotein cholesterol (11). It is not yet clear whether such individuals are at increased risk for atheroscle- rosis, ischemic heart disease, or diabetes. Recently, Nordest- gaard and colleagues (12) screened Danish subjects for muta- tions in the LPL gene. As expected, LPL1/2 humans had higher triglycerides and lower high density lipoprotein choles- terol. Unexpectedly, these unrelated LPL1/2 humans had re- duced plasma glucose concentrations compared with LPL1/1 humans. Two previous studies of related LPL1/2 humans found no effect on glucose levels (13, 14).
We tested the hypothesis that LPL has a direct effect on glucose metabolism in mice. Our data show that LPL1/2 mice are relatively hypoglycemic. Since LPL provides fatty acids to muscle (the major site of insulin-stimulated glucose disposal) and fatty acids compete with glucose as substrates, we ex-
* This work was supported in part by National Institutes of Health Grants HL58427, DK53198, DK02339, and DK06181, the Hardison Family Foundation, and the Washington University Diabetes Research and Training Center Grant DK20579. This work was presented in part at the 1998 Annual Scientific Sessions of the American Diabetes Asso- ciation (Marshall, B. A. and Semenkovich, C. F. (1998) Diabetes 47, S1, A27). 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.
‡ These authors contributed equally to this study. § Scholar of the Child Health Research Center of Excellence in
Developmental Biology at Washington University and supported by National Institutes of Health Grant HD33688.
¶ To whom correspondence should be addressed: Washington Univer- sity School of Medicine, 660 South Euclid Ave., Box 8046, St. Louis, MO 63110. Tel.: 314-362-4454; Fax: 314-747-4477; E-mail: semenkov@im. wustl.edu.
1 The abbreviations used are: LPL, lipoprotein lipase; BSA, bovine serum albumin; RT-PCR, reverse transcriptase-polymerase chain reac- tion; NEFA, non-esterified fatty acids; AAV, adeno-associated virus.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 39, Issue of September 24, pp. 27426–27432, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org27426
This is an Open Access article under the CC BY license.
EXPERIMENTAL PROCEDURES
Animals—The mice used in the studies were animals that carry one disrupted allele of the LPL gene as described by Coleman et al. (3) and their unaffected littermates. These animals (originally C57BL/6J- 129Sv hybrids) have been continually mated with C57BL/6J mice; N6 and N7 generation descendants from this cross into the C57BL/6J background were used to compare glucose metabolism in LPL1/2 and LPL1/1 mice. For comparison of insulin secretion by isolated mouse islets, experiments also included islets from mice lacking LPL in all tissues except muscle. LO-MCK mice, deficient in native mouse LPL but expressing human LPL driven by the mouse MCK promoter (5, 15), were a gift from Jan L. Breslow (New York).
Mice were housed on a 12-h light/dark cycle at 23 °C with free access to food and water. The chow diet was PicoLab Rodent Diet 20 (number 5053, PMI Nutrition International, Richmond, IN). Some animals were fed a high fat, high simple carbohydrate diet (F3282, BioServe, French- town, NJ) consisting primarily of lard, sucrose, and casein: 20% protein, 36.5% fat, 36.6% carbohydrate (67% mono- and disaccharides). All experimental protocols were approved in advance by the Washington University Animal Studies Committee.
Glucose Tolerance Tests and Fasting Glucose Measurements—Mice were accustomed to handling for 1 week prior to study. Mice were placed in clean cages with no food but with free access to water at 8:30 a.m. After a 4-h fast, the mice were weighed. The tip of the tail was then clipped to obtain blood for glucose measurement. When a glucose tol- erance test was to be performed, mice were injected intraperitoneally with 10% dextrose (1 mg/g body weight). Blood (5 ml) was taken from the tail tip at 0, 30, 60, 90, 120, and 150 min for measurement of glucose.
A 4-h fast was chosen in part based on preliminary experiments. Mice were fasted for 0, 2, 5, or 18 h, and then the stomach, small intestines, and large intestines were removed and the contents weighed. Stomach contents were 260 6 48 mg (n 5 8) in the fed state, fell to 91 6 19 mg at 2 h of fasting, and did not significantly change at later time points. The contents of the small intestines decreased by 63% by 2 h of fasting and did not change subsequently. In addition, fasting for longer than 4 h in mice has been shown to elevate paradoxically triglyceride levels (16) and produce substantial, probably non-physio- logical changes in glucose metabolism (17, 18).
Hyperinsulinemic Clamp—Clamp experiments were carried out as described previously (19, 20) with the following modifications. After placement of the infusion catheter, an infusion of high pressure liquid chromatography-purified 3-[3H]glucose (NEN Life Science Products) at 0.04 mCi/min (240 ml/h) with an initial priming dose of 1.25 mCi (125 ml) was begun for measurement of the rate of appearance of glucose. The infusion was continued during a 1-h control period, and 20 ml of blood was taken from the tail for determination of glucose-specific activity at 45, 52.5, and 60 min.
After 60 min, an infusion of insulin (regular human, Lilly) was begun and continued for at least 90 min for each experimental period. A dextrose infusion (25%) was started with the insulin infusion. The dextrose infusion rate varied in order to maintain the blood glucose at approximately 170 mg/dl, the average blood glucose in a freely feeding, wild-type conscious mouse of this strain in our hands (19). The infusion of 3-[3H]glucose tracer was maintained during the insulin infusion period. In addition, the tracer was added to the 25% dextrose infusion to approximate the glucose-specific activity in the blood at the end of the control period. This approximation was based on measurement of spe- cific activity during identical conditions in the same type of mice in previous experiments. Blood samples for determination of specific ac- tivity were collected 10 and 20 min prior to and at the end of the experimental period. The glucose infusion rate was not changed for at least 20 min prior to the first determination of specific activity. Both the blood glucose and the glucose-specific activity were in steady state during these 20-min sampling periods. Blood for insulin determination was obtained by cardiac puncture at the conclusion of the experiment.
The rate of appearance of glucose (Ra), which equals the rate of total body glucose utilization (Rd) when blood glucose is in steady state, was calculated by dividing the infusion rate of 3-[3H]glucose by the specific activity at the same time. Glucose production was calculated by sub- tracting the cold glucose infusion rate from Ra.
Mouse Islet Manipulation—Islets were isolated by collagenase diges- tion and Ficoll step-density gradient separation and then selected with a microscope to exclude any contaminating tissues (21). Mouse islets were counted and aliquoted for insulin secretion studies (20 islets per aliquot), analysis of triglyceride content (10 islets per aliquot), RNA preparation (2000 islets per aliquot), or assay of LPL enzyme activity (see below). There were no morphological differences in islets isolated from LPL1/1 and LPL1/2 mice. There was no difference in RNA recovery for the two genotypes.
For insulin secretion studies, islets were washed in Krebs-Ringer bicarbonate buffer (KRBB, 115 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 24 mM NaHCO3, and 25 mM Hepes, pH 7.4) containing 3 mM
glucose and 0.1% BSA. Islets were placed in 10 3 75-mm siliconized borosilicate tubes in 200 ml of KRBB with 3 mM glucose, 0.1% BSA and incubated for 30 min. Buffer was then replaced with KRBB containing 3 mM glucose, 0.1% BSA and incubated for 30 min. The buffer was then removed and assayed for insulin content.
For analysis of triglyceride content, islets were placed in glass tubes, and lipids were extracted with 2:1 (v/v) chloroform:methanol. The or- ganic phase was taken to dryness under N2. Following this procedure, a clearly visible lipid film was present at the bottom of the tube despite the fact that each tube contained only 10 mouse islets. This film was carefully resuspended, which required several minutes for each tube, in reaction mixtures provided in kit form for the determination of triglyc- eride content (see below). Each individual assay for islet triglyceride included a glycerol blank exactly as described by the manufacturer of the assay reagents.
For RNA preparation, islets were counted by hand using a micro- scope to ensure that samples were not contaminated by acinar tissue. Total RNA was prepared using guanidinium isothiocyanate and sedi- mentation in cesium chloride as described (22). RT-PCR was performed using AMV RT for first strand synthesis, Taq polymerase for the PCR step, and primers as described in Table II.
Human Islet Manipulation—Human pancreatic islets were obtained from the Islet Core of the Washington University Diabetes Research and Training Center, which has approval from the Human Studies Committee for these procedures. Cultured islets were counted with a microscope and aliquoted to tubes for determination of LPL enzyme activity (100 islets per tube) or used for preparation of total RNA exactly as described above for mouse islets.
LPL Enzyme Activity—Islets were assayed for LPL enzyme activity in two ways. To determine if islets secrete LPL activity, mouse islets (30 islets per aliquot) and human islets (100 islets per aliquot) were washed with KRBB containing 3 mM glucose and 0.1% BSA and then incubated in the same buffer for 30 min as described above for insulin secretion studies. Islets were centrifuged at 300 3 g and then the buffer was assayed for secreted LPL enzyme activity, determined as the salt- inhibitable capacity of samples to hydrolyze radiolabeled fatty acids from a phospholipid-stabilized triolein emulsion as described (23). To determine the effect of LPL genotype on islet LPL activity, islets from LPL1/1 and 1/2 mice (100 islets per aliquot) were directly homoge- nized in sample assay buffer and activity assayed.
Adeno-associated Virus Overexpression of LPL in INS-1 Cells—A recombinant adeno-associated virus (AAV) containing the human LPL cDNA driven by the cytomegalovirus immediate early promoter was generated by Avigen Corp. (Alameda, CA) using techniques described for other recombinant AAV vectors (24). In preliminary experiments, transfection of AAV-LPL into both C2C12 and COS cells resulted in the dose-dependent expression of LPL enzyme activity. The generation of AAV-b-galactosidase, also provided by Avigen, has been described (24).
INS-1 cells, a rat insulinoma cell line established by Asfari et al. (25), were a gift from Christopher B. Newgard (Dallas, TX). Cells were cultured at a glucose concentration of 11 mM in 10% fetal bovine serum as described previously (26), and at early passages exhibited glucose- stimulated insulin secretion. Cells were seeded at a density of 2 3 104
cells/cm2 in multiwell clusters, and medium was changed every other day. At 80% confluence, cells were infected with AAV-LPL or AAV-b- galactosidase at 1011 plaque-forming units/ml. Virus-containing me- dium was replaced with fresh medium after 48 h. Cells were assayed for LPL enzyme activity and insulin secretion 4 or 5 days after infection. At no point did cells exhibit cytopathic effects. For LPL enzyme activity, cells were grown in 6-well clusters and homogenized in 250 ml of sample assay buffer. For insulin secretion studies, cells were plated on 12-well
Glucose Metabolism in LPL Knockout Mice 27427
clusters. Five days following infection, cells were rinsed then incubated in 0.5 ml of glucose-free modified Krebs-Ringer Hepes buffer (KRHB, 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.0 mM
CaCl2, 10 mM Hepes, 0.5% BSA) for 2 h. Buffer was then replaced with KRHB. One hour later, the insulin content of the buffer was assayed by radioimmunoassay. Data were normalized for cellular DNA content that was measured fluorometrically (26).
Antisense Oligonucleotide-mediated Suppression of LPL Activity in INS-1 Cells—Phosphorothioate-modified oligonucleotides were synthe- sized on an ABI model 394-08 DNA synthesizer. An antisense oligonu- cleotide encompassing the LPL translation initiation site had the fol- lowing sequence: 59 GCT CTC CAT CTC GGC GCG. A scrambled oligonucleotide with the identical base composition (59 CCC GAT CGT CGT CCT GCG) was used as control. INS-1 cells at 50–75% confluence were transfected with the antisense or control oligonucleotide at 20 mM
using LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer’s recommendations. Oligonucleotides were replenished at 24 h. After a total incubation time of 48 h, cells were processed for LPL activity and insulin secretion as detailed above.
Analytical Procedures—Blood glucose was measured using 5 ml of whole blood in the Hemocue (Mission Viejo, CA) blood glucose meter. Glucose, triglycerides, and cholesterol were measured using reagents provided by Sigma. The triglyceride reagent was product number 339- 10. NEFA in serum were assayed using reagents provided by Wako Chemicals (Richmond, VA). For clamp experiments (in which human insulin was infused), serum insulin was measured by double-antibody radioimmunoassay using human standards (Lilly). Otherwise, insulin was assayed by radioimmunoassay using rat standards (Linco, St. Charles, MO). The specific activity of glucose in whole blood was deter- mined by aqueous scintillation counting of 20 ml of blood that was deproteinized with barium hydroxide (0.3 N) and zinc sulfate (0.3 N). The supernatant resulting from deproteinization was dried at 70 °C to remove tritiated water prior to resuspension and counting.
RESULTS
Serum chemistries for LPL1/1 and 1/2 mice between the ages of 2 and 4 months are shown in Table I. LPL1/2 mice in two separate experiments had fasting serum glucose values that were 15–22% (28–39 mg/dl, p 5 0.0145 and 0.0021) lower than LPL1/1 mice. As expected, fasting triglycerides were 52–83% (p 5 0.0108 and 0.0035) higher in LPL1/2 mice, but there were no significant differences in NEFA, body weight, or cholesterol.
More detailed characterization of glucose metabolism was carried out in mice over the age of 12 months since the larger caliber of the tail vein at this age simplifies glucose tolerance testing. Blood glucose values in chow-fed mice over the age of 12 months were 181 6 3 mg/dl for LPL1/1 (n 5 39) versus 170 6 3 mg/dl for LPL1/2 (n 5 34) (p 5 0.0137). In two
additional groups over the age of 12 months matched for weight and sex, the blood glucose after a 4-h fast was as follows: group 1, 187 6 5 for 1/1 versus 163 6 3 mg/dl for 1/2 (p 5 0.0003); group 2, 191 6 9 for 1/1 versus 169 6 4 for 1/2 (p 5 0.036). These mice underwent glucose tolerance testing after a 4-h fast. Data for group 1 are shown in Fig. 1. Similar results were seen for group 2. Both genotypes had similar glucose excur- sions although values in the 1/2 mice tended to be lower throughout the test and returned to significantly lower levels in LPL1/2 mice at 150 min after the glucose injection.
Insulin levels were higher in LPL1/2 compared with 1/1 mice (Fig. 2). In chow-fed mice between the ages of 2 and 4 months (left side of figure), fasting serum insulin levels were 0.81 6 0.21 for 1/1 versus 1.90 6 0.44 for 1/2 (Mann-Whitney two-tailed p 5 0.0251). High fat feeding is known to elevate insulin levels in mice. When mice were fed a high fat diet for 6 weeks, insulin levels were elevated in both genotypes but re- mained higher in LPL1/2mice (right side of figure). In a large group of mice with the same mean weight by genotype, insulin levels were 2.09 6 0.37 (n 5 30) for 1/1 mice versus 4.38 6 1.03 (n 5 25) for 1/2 mice (Mann-Whitney two-tailed p 5 0.0286). Thus, heterozygous LPL deficiency is associated…