From the Hexos, Inc., Woodinville, Washington 98072
Received for publication, March 19, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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The current studies were initiated to examine the hypothesis that the
glucose-mediated up-regulation of lipogenic enzymes in isolated adipocytes is
mediated by glucose flux through the hexosamine biosynthesis pathway (HBP) and
the subsequent regulation of lipogenic enzyme mRNA levels. The rationale for
exploring this hypothesis is based on the 1991 discovery that glucose-induced
desensitization of the glucose transport system is linked to hexosamine
biosynthesis (5,
6). At that time, it was
proposed that the HBP serves as a glucose sensor coupled to a metabolic
transducer that regulates the insulin-responsive glucose transport system.
Subsequent studies have expanded upon the idea of hexosamine-mediated
regulation by implicating the HBP in the regulation of genes for pyruvate
kinase, leptin, transforming growth factor (TGF
), and
transforming growth factor
(TGF
)
(714).
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EXPERIMENTAL PROCEDURES |
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Preparation of Sterile Isolated AdipocytesIsolated adipocytes were obtained from the epididymal fat pads of male Sprague-Dawley rats (180225 g) by collagenase digestion (15) as described previously (16). Briefly, minced tissue (12 g) in 4 ml of DMEM containing collagenase (1 mg/ml) and albumin (40 mg/ml) was shaken in 4-oz sterile polypropylene containers at 37 °C for 45 min. At the end of the digestion period, cells were filtered through nylon mesh (1000 µm) and then washed three times in Hepes-buffered balanced saline solution (HBSS). HBSS contains 25 mM Hepes, 120 mM NaCl, 0.8 mM MgSO4, 2 mM CaCl2, 5.4 mM KCl, 1 mM NaH2PO4, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 1% BSA, pH 7.6. After washing, adipocytes were resuspended as a 10% (w/v) solution in HBSS (final concentration about 5 x 105 cells/ml).
Primary Culture of Adipocytes and Extraction of Total
RNAAdipocytes were maintained in primary culture as described
previously (16). Briefly,
adipocytes were added to sterile 250-ml polypropylene bottles or 50-ml
polypropylene tubes and diluted to a final volume of 20 or 60 ml (2
x 105 cells/ml) in sterile incubation medium consisting of
glucose-free DMEM supplemented with 1 mM pyruvate and 1% BSA (SIM0)
or HBSS. Cells were then incubated at 37 °C for 18 h with various
combinations of glucose (20 mM), insulin (25 ng/ml), glutamine (16
mM), or glucosamine (2 mM) unless otherwise indicated.
After treatment, adipocytes were washed three times with HBSS. During the
final wash, the infranatant was aspirated, and total RNA was extracted from
adipocytes by adding 500 µl of RNAzol B, 200 µl of chloroform, and 200
µl of 10 mM Tris pH 7.4. After centrifugation for 15 min at 4
°C, the upper aqueous phase containing the RNA was precipitated at
20 °C for 90 min with 0.3 M sodium acetate and
isopropanol. The RNA pellets were washed with 75% ethanol and then resuspended
in 10 mM Tris pH 7.4. RNA was quantified by measuring
A260 and deemed pure
(A260:A280
2). RNA was stored at
80 °C prior to use in the ribonuclease protection assay.
PlasmidsS2 Ribosomal protein (S2) cDNA from bp 121 to 906 (GenBankTM accession number X57432 [GenBank] ) was amplified from rat adipocyte cDNA using GeneAmp PCR kit with amplitaq (PerkinElmer Life Sciences) and primers (Keystone BIOSOURCE, Foster City, CA) designed to facilitate subsequent cloning into the AscI/PacI sites of pJMR1 (17). The sequences of the primers used were 5'-actggcgcgcccttaggggccgcggtcgtgg-3' and 5'-aaattaattaattatgtggtagccactgctggagcct-3'. Cytosolic glycerophosphate dehydrogenase (GPDH; EC 1.1.1.8 [EC] ) cDNA from bp 1 to 1050 (GenBankTM accession number AB002558 [GenBank] ) was similarly cloned. The sequences of the GPDH primers were 5'-cagggcgcgccatggctggcaagaaagtctgcat-3' and 5'-attttaattaatcacatgtgttccgggtggttctgc-3'. The hormone-sensitive lipase (HSL; EC 3.1.1.3 [EC] ) plasmid was a gift from Dr. Allan Green (Bassett Research Institute, Cooperstown, NY). The pCRII-FAS and pCRII-ACC plasmids were gifts from Sankyo Ltd. (Shinagawa, Japan).
Generation of Nucleic Acid ProbesPortions of the various
cDNAs (above) were subcloned into either pT7/T3-18 vector (Ambion, Austin, TX)
or pJMR1. The resulting plasmids were subsequently linearized, gel-purified
with GENECLEAN (Bio 101, La Jolla, CA), and then used as a template for T7 RNA
polymerase-mediated in vitro transcription. The MAXIscript in
vitro transcription kit (Ambion, Austin, TX) and
[-32P]UTP (PerkinElmer Life Sciences) were used to prepare
antisense RNA probes that protect S2 mRNA (bases 734906 of
GenBankTM accession number X57432
[GenBank]
), FAS (bases 73407612 of
GenBankTM accession number X14175), GPDH (bases 1299 of
GenBankTM accession number AB002558
[GenBank]
), ACC (bases 68277004 of
GenBankTM accession number J03808
[GenBank]
), and HSL (bases 1337 of
GenBankTM accession number X51415
[GenBank]
). RNA probes were purified over a
NucTrap push column (Stratagene, La Jolla, CA) before use in the ribonuclease
protection assay.
Ribonuclease Protection AssayThe ribonuclease protection assay (RPA) was performed using the Ambion RPA II kit (Ambion, Austin, TX) following the manufacturer's instructions. Multiplexed antisense RNA probes (2550,000 cpm/probe) were hybridized with 58 µg of total adipocyte RNA for 16 h at 42 °C. After RNase digestion, the protected probes were resolved on a 6% polyacrylamide-urea gel in Tris-Borate-EDTA buffer and then quantified using the Storm 840 phosphorimaging system (Molecular Dynamics, Piscataway, NJ).
Northern Analysis20 µg of total RNA was separated on a 1% (w/v) agarose-formaldehyde gel. RNA was transferred to nylon membranes (Schleicher & Schuell) and then cross-linked using the UV stratalinker 1800 (Stratagene). Hybridizations were performed with radiolabeled cDNA probe for5hat60 °C in ExpressHyb buffer (Clontech) containing 100 µg/ml salmon sperm DNA. After washing the blot, hybridization signals were visualized using the Storm 840 phosphorimaging system. Each blot was stripped and reprobed with an S2 cDNA probe to control for equal loading of RNA.
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RESULTS |
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Maximal changes in mRNA levels for FAS, GPDH, and ACC were observed between 12 and 24 h with little glucose regulation seen at 6 h (data not shown). Based on these preliminary studies, we decided to quantify mRNA levels at 18 h in all subsequent experiments. As can be seen by Northern analysis in Fig. 1A, glucose treatment resulted in up-regulation of mRNA levels for both FAS and GPDH. Since S2 mRNA levels were unchanged with glucose treatment, it can serve as an internal control. The ribonuclease protection assay was used as a quantitative method to measure FAS, GPDH, and S2 mRNA levels (Fig. 1B). Radiolabeled antisense probes for GPDH (299 bases), FAS (273 bases), and S2 (172 bases) were prepared using in vitro transcription and were then multiplexed in a hybridization reaction with RNA from control or glucose-treated adipocytes. After normalization to S2 mRNA levels, glucose treatment for 18 h resulted in a 241% increase in FAS mRNA levels and a 576% increase in GPDH mRNA levels. It is important to note that the addition of the transcription inhibitor actinomycin D (200 nM) completely blocked glucose-induced up-regulation of mRNA levels (data not shown). All subsequent measurements of mRNA levels were performed using the RPA.
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Fig. 1, C and D, depicts experiments in which adipocytes were treated for 18 h with 25 ng/ml insulin plus various sugars (all at a concentration of 20 mM). D-glucose effectively increased both FAS (Fig. 1C) and GPDH mRNA levels (Fig. 1D), whereas L-glucose had no effect. This indicates sugar specificity and eliminates osmolarity changes as a causative factor. Mannose was nearly as effective as glucose in elevating mRNA levels of FAS and GPDH, whereas fructose and galactose had much smaller effects. The differential effects of the various sugars on FAS and GPDH mRNA levels are most likely due to differences in uptake and metabolism of these sugars in isolated adipocytes.
To better quantify glucose-induced up-regulation of mRNA levels, we incubated adipocytes for 18 h in glucose-free DMEM containing insulin and various concentrations of glucose (Fig. 2). Glucose treatment resulted in a dose-dependent increase in mRNA levels for both FAS (ED50 15.5 mM) and GPDH (ED50 7.5 mM). Maximal concentrations of glucose (40 mM) increased FAS mRNA >3-fold and elevated GPDH mRNA about 9-fold. The fact that FAS and GPDH mRNA levels were relatively unchanged in the hypoglycemic to euglycemic range (15 mM glucose) indicates that the glucose regulation of mRNA levels is not a consequence of glucose deprivation but rather is a result of hyperglycemia. We also examined the effect of xylitol on FAS and GPDH mRNA levels (Fig. 2 insets) because glucose flux into the pentose phosphate shunt has been postulated to mediate up-regulation of various glucose-responsive genes (3, 4). Xylitol is known to directly enter the pentose phosphate shunt through the intracellular formation of xyulose-5-phosphate. Because xylitol treatment had no effect on either FAS or GPDH mRNA levels in insulin-treated cells, we conclude that the pentose phosphate shunt does not contribute significantly to glucose up-regulation of FAS and GPDH mRNA in isolated adipocytes.
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Facilitative Role of Insulin in Glucose-induced Up-regulation of mRNA LevelsGlucose regulation of gene expression can be either insulin-dependent or insulin-independent. Examples of insulin-dependent regulation include glucose-mediated up-regulation of FAS mRNA in liver (through activation of glucokinase); insulin-independent regulation is typified by glucose regulation of stearoyl-CoA desaturase mRNA in 3T3-L1 adipocytes in the absence of insulin (1, 18). To examine the role of insulin in glucose-induced up-regulation of GPDH and FAS mRNA in isolated adipocytes, we treated cells for 18 h in HBSS in the absence or presence of 25 ng/ml insulin (Fig. 3). When compared with controls (no additions), insulin alone down-regulated FAS and GPDH mRNA levels. In contrast, treatment with insulin and glucose resulted in a glucose dose-dependent up-regulation of FAS and GPDH mRNA levels (Fig. 3). Since insulin is known to stimulate glucose transport in adipocytes by >10-fold, we believe that insulin facilitates glucose action by enhancing glucose uptake into cells.
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Glutamine Potentiates Glucose-induced Up-regulation of FAS and GPDH mRNA LevelsGlucose-induced insulin resistance in isolated adipocytes is mediated by glucose flux through the HBP (5). Evidence supporting this conclusion includes the finding that desensitization requires the presence of three components in the medium, glutamine, glucose, and insulin (5, 6). Glutamine was necessary because it serves as an essential amide donor for the conversion of glucose to hexosamine products. Specifically, glutamine-fructose-6-P amidotransferase (the first and rate-limiting enzyme of the hexosamine pathway) requires glutamine for conversion of fructose-6-P to glucosamine-6-P.
To investigate whether the HBP mediates glucose-induced up-regulation of FAS and GPDH mRNA, we incubated adipocytes with 25 ng/ml insulin and 20 mM glucose in the absence or presence of 16 mM glutamine. As shown in Fig. 3, inclusion of glutamine potentiated the glucose-induced increase of FAS and GPDH mRNA. This indicates that formation of hexosamine products underlies glucose regulation of FAS and GPDH mRNA levels.
Ability of Glucosamine to Up-regulate FAS and GPDH mRNA
LevelsTo obtain additional evidence for hexosamine-mediated
regulation of FAS and GPDH mRNA, we treated adipocytes for 18 h with 25 ng/ml
insulin and various concentrations of glucosamine. Glucosamine was used
because it is readily transported into adipocytes through the glucose
transport system where it directly enters the hexosamine pathway at the level
of glucosamine-6-phosphate (5).
The data depicted in Fig. 4
show that glucosamine treatment resulted in a dose-dependent up-regulation of
mRNA levels for FAS (ED50 0.45 mM) and GPDH
(ED50
0.75 mM). Glucosamine was
1530
times more potent than glucose in inducing FAS and GPDH mRNA levels.
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Regulation of Acetyl-CoA Carboxylase mRNA Levels by Glucose and GlucosamineTo explore whether the HBP may control other mRNAs involved in lipid metabolism, we evaluated the regulation of mRNA for the lipogenic enzyme ACC and the lipolytic enzyme HSL. As depicted in Fig. 5A, ACC mRNA levels were up-regulated by co-treatment with insulin and glucose, but the extent of up-regulation was significantly enhanced by the inclusion of glutamine. Treatment with insulin and glucosamine up-regulated ACC mRNA levels to a greater extent than glucose, suggesting that ACC represents another lipogenic enzyme under the control of the HBP. Considered together, these data suggest that ACC mRNA levels are also regulated through the HBP. As shown in Fig. 5, B and C, HSL mRNA levels were not up-regulated by glucose, glutamine, or glucosamine treatment. In fact, HSL mRNA levels were actually down-regulated with insulin treatment. Since HSL is a lipolytic enzyme, these data highlight the specificity of hexosamine action on lipogenic enzymes in isolated rat adipocytes.
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DISCUSSION |
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Although previous studies have established that intracellular glucose metabolism results in up-regulation of lipogenic enzyme mRNA levels, the cascade of metabolic events leading to up-regulation remains obscure. Formation of glucose-6-phosphate has been postulated to regulate lipogenic mRNA levels (13); however, there is no mechanistic rationale for how Glc-6-P could modify mRNA levels (1). Another idea is that xyulose-5-phosphate functions as a metabolite regulator of mRNA levels (13) through activation of protein phosphatase 2a and dephosphorylation of transcription factors mediating the glucose response (3, 21, 22). To better understand the mechanism(s) underlying glucose-mediated regulation of lipogenic enzyme mRNA levels, we used isolated adipocytes maintained in primary culture as our model system so that we could investigate mRNA regulation under defined in vitro conditions.
When adipocytes were treated for 18 h with 25 ng/ml insulin and various
concentrations of D-glucose, we observed increases in mRNA levels
for FAS (280%), ACC (93%), and GPDH (633%). The glucose ED50 values
were 15 mM for FAS and
7 mM for GPDH. Sugar
specificity studies revealed that mannose elevated mRNA levels almost as
effectively as glucose, whereas galactose and fructose were only partially
effective. L-glucose, which is a non-metabolizable analog of
D-glucose, had no effect on mRNA levels. Insulin was required for
the expression of glucose-induced up-regulation of mRNA levels. However,
insulin itself appears to play no direct role in mRNA up-regulation since
treatment of adipocytes with insulin alone (in the absence of glucose)
resulted in an actual decrease in FAS and GPDH mRNA levels
(Fig. 3). Since insulin
enhances the rate of glucose uptake in isolated adipocytes by >10-fold, it
is likely that insulin is required to facilitate glucose entry into cells.
Two lines of evidence support the hypothesis that mRNA regulation of lipogenic enzymes is coordinately regulated by enhanced glucose flux through the HBP. First, we found that glutamine significantly augmented the stimulatory effect of glucose on mRNA expression of FAS and GPDH (Fig. 3). This is consistent with the idea that intracellular formation of hexosamine products requires a supply of both glucose (in the form of Fru-6-P) and glutamine (as a cofactor for glutamine:fructose-6-phosphate amidotransferase in the transfer of an amide group to Fru-6-P). The primary role of insulin in this scheme is to facilitate the uptake of glucose (about 1020-fold). The second line of evidence for hexosamine involvement entailed the use of glucosamine, which has previously been shown to enter adipocytes through the glucose transport system (5) where it directly enters the hexosamine pathway distal to glutamine: fructose-6-phosphate amidotransferase (through formation of GlcN-6-P). In insulin-treated adipocytes, glucosamine was 1530 times more potent than glucose in up-regulating FAS and GPDH mRNA levels. Considered together, these studies lead to the conclusion that the HBP plays an integral role in lipid metabolism by up-regulating mRNA levels for various lipogenic enzymes. In contrast, mRNA levels of the lipolytic enzyme HSL were unaffected by treatment of isolated rat adipocytes with glucose or glucosamine for 18 h. It should be mentioned that in human adipocytes and cultured 3T3-F442A adipocytes, prolonged glucose treatment for 48 h has been shown to culminate in up-regulation of HSL (23, 24). The reason for the discrepancy between primary cultured rat adipocytes and other adipocyte model systems remains unclear.
Up-regulation of mRNA levels can result from transcriptional activation
and/or changes in mRNA stability. Although the exact mechanism(s) by which
glucose regulates lipogenic mRNAs in primary cultured adipocytes remains to be
elucidated, changes in both transcription rates and mRNA stability may be
involved (25,
26). A glucose-inducible
mRNA-binding protein has previously been shown to stabilize FAS mRNA levels in
HepG2 cells (27).
Whether the hexosamine pathway regulates mRNA-binding proteins in rat
adipocytes is unclear. Since we found that the transcription inhibitor
actinomycin D completely inhibits the glucose-induced up-regulation of
lipogenic mRNA levels in primary cultured adipocytes, this suggests that
transcriptional regulation plays a prominent role in regulating mRNA levels.
This conclusion fits well with previous studies implicating the hexosamine
pathway in the transcriptional regulation of pyruvate kinase
(7),
glutamine:fructose-6-phosphate amidotransferase
(13), leptin
(8,
9), TGF
(9,
11,
12), and TGF
(13,
14). Hexosamine-mediated
regulation of lipogenic enzymes at the level of transcription becomes even
more compelling given that glucosamine was also found to be nearly six times
more potent than glucose at inducing the glucose response element of
TGF
(10).
The model depicted in Fig. 6 schematically integrates the current data on regulation of mRNA levels with previous studies on glucose-induced insulin resistance (28, 29). Upon entering adipocytes, glucose is phosphorylated to Glc-6-P and rapidly converted to Fru-6-P. From these two metabolites, glucose fluxes into various pathways involved in glucose storage and utilization. These include the glycogen biosynthesis pathway, the pentose phosphate shunt, and the glycolysis/lipogenesis pathway. Although these three pathways represent the major routes traversed by the vast majority of glucose, a small percentage of incoming glucose (about 12%) is routed into the HBP. It was originally proposed that the HBP serves as a glucose sensor coupled to a biological transduction system that functions to reduce glucose uptake as the rate of glucose transport exceeds the capacity of the major glucose-utilizing pathways (5). In other words, the enhanced flux of glucose through the HBP culminates in insulin resistance of the glucose transport system. Based on the current study, it appears likely that the regulatory role of the HBP also encompasses control of lipid storage through gene regulation of lipogenesis enzyme mRNA levels. This coordinated response to hyperglycemia makes sense in that overall glucose uptake would be reduced (by the development of insulin resistance), and excess incoming glucose would be stored as triglycerides (through the enhancement of lipogenesis).
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Under hyperglycemic conditions, as occurs after eating, the body mounts a normal, adaptive response to re-establish glucose homeostasis. However, under prolonged hyperglycemic conditions, as occurs in diabetes mellitus, cellular adaptation to excessive glucose uptake may lead to many of the pathophysiological consequences of diabetes, including insulin resistance, impaired glucose metabolism, and dyslipidemia. Accordingly, it can be hypothesized that prolonged hyperglycemia and excessive glucose flux through the HBP may play a role in the etiology and pathogenesis of diabetes. Therefore, pharmacological intervention targeting the HBP may ameliorate hyperglycemia and/or hyperlipidemia and possibly prevent or delay the onset of diabetic complications that accompany disease progression.
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FOOTNOTES |
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Present address: Bassett Research Institute Cooperstown, NY 13326.
To whom correspondence should be addressed: Hexos, Inc., 18304 NE 153rd St.,
Woodinville, WA 98072. Tel.: 425-844-2527; E-mail:
Hexos{at}comcast.net.
1 The abbreviations used are: FAS, fatty acid synthase; ACC, acetyl-CoA
carboxylase, GPDH, glycerol-3-phosphate dehydrogenase; HSL, hormone-sensitive
lipase; RPA, ribonuclease protection assay; HBP, hexosamine biosynthesis
pathway; TGF, transforming growth factor; DMEM, Dulbecco's modified Eagle's
medium; HBSS, Hepes-buffered balanced saline solution; Glc-6-P,
glucose-6-phosphate; Fru-6-P, fructose-6-phosphate.
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REFERENCES |
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