Intrinsic Gluconeogenesis Is Enhanced in Renal Proximal Tubules of Zucker Diabetic Fatty Rats
Assaad Eid,
Sophie Bodin,
Bernard Ferrier,
Hélène Delage,
Michelle Boghossian,
Mireille Martin,
Gabriel Baverel and
Agnès Conjard
Laboratory of Metabolic and Renal Pathophysiology, National Institute of Health and Medical Research and Claude Bernard-Lyon 1 University, UMR 499, Faculty of Medicine R.T.H. Laennec, Lyon, France
Address correspondence to: Dr. Gabriel Baverel, Laboratoire de Physiopathologie Métabolique et Rénale, INSERM UMR 499, Faculté de Médecine R.T.H. Laennec, rue G. Paradin, 69372 Lyon Cedex 08, France. Phone: 33-4-78-77-86-65; Fax: 33-4-78-77-87-39; baverel{at}sante.univ-lyon1.fr
Received for publication July 19, 2005.
Accepted for publication November 25, 2005.
Recent studies indicate that renal gluconeogenesis is substantiallystimulated in patients with type 2 diabetes, but the mechanismthat is responsible for such stimulation remains unknown. Therefore,this study tested the hypothesis that renal gluconeogenesisis intrinsically elevated in the Zucker diabetic fatty rat,which is considered to be an excellent model of type 2 diabetes.For this, isolated renal proximal tubules from diabetic ratsand from their lean nondiabetic littermates were incubated inthe presence of physiologic gluconeogenic precursors. Althoughthere was no increase in substrate removal and despite a reducedcellular ATP level, a marked stimulation of gluconeogenesiswas observed in diabetic relative to nondiabetic rats, withnear-physiologic concentrations of lactate (38%), glutamine(51%) and glycerol (66%). This stimulation was caused by a changein the fate of the substrate carbon skeletons resulting froman increase in the activities and mRNA levels of the key gluconeogenicenzymes that are common to lactate, glutamine, and glycerolmetabolism, i.e., mainly of phosphoenolpyruvate carboxykinaseand, to a lesser extent, of glucose-6-phosphatase and fructose-1,6-bisphosphatase.Experimental evidence suggests that glucocorticoids and cAMPwere two factors that were responsible for the long-term stimulationof renal gluconeogenesis observed in the diabetic rats. Thesedata provide the first demonstration in an animal model thatrenal gluconeogenesis is upregulated by a long-term mechanismduring type 2 diabetes. Together with the increased renal mass(38%) observed, they lend support to the view so far based onlyon in vivo studies performed in humans that renal gluconeogenesismay be stimulated by and crucially contribute to the hyperglycemiaof type 2 diabetes.
Type 2 diabetes is a heterogeneous metabolic disorder that isbecoming a global epidemic with major health consequences. Itis characterized by defects in both insulin secretion and tissuesensitivity to insulin (1,2), leading to hyperglycemia. It iswell accepted that part of this hyperglycemia results from theabnormal persistence of hepatic glucose production during theabsorptive state and by an increase to excessive levels of hepaticglucose production in the postabsorptive state (3). Thus, anelevated glucose production in type 2 diabetes has until recentlybe generally attributed exclusively to the liver. This is notnecessarily justified because it is well established that, ona gram-for-gram basis, the kidney can synthesize glucose atrates several times higher than those observed in the liver(4). In line with this, studies that were performed in vivoduring the past decade suggested that the synthesis of glucoseby the kidneys of postabsorptive humans may explain 5 to 25%of the total glucose formed by gluconeogenesis in the body (5–8).Human renal gluconeogenesis has also been demonstrated to bealmost equal to hepatic gluconeogenesis in obese patients whoundergo a prolonged fasting (9); moreover, this metabolic processhas been shown to be stimulated in patients with type 2 diabetes(10,11) and inhibited by insulin (8,13,14). Despite the latterobservations and the potential pathophysiologic importance ofrenal gluconeogenesis in type 2 diabetes, an intrinsic stimulationof renal gluconeogenesis to our knowledge has not been reportedin any animal model of type 2 diabetes. This lack of an appropriatemodel that spontaneously displays increased renal gluconeogenesisso far has represented a significant limitation in the understandingof the mechanisms by which the kidney might be involved in thehyperglycemia observed in type 2 diabetes.
Therefore, to test the hypothesis that intrinsic renal gluconeogenesisis stimulated in type 2 diabetes, we conducted a study in whichrenal gluconeogenesis in Zucker diabetic fatty (ZDF) rats, anexcellent model for the study of type 2 diabetes (15,16), wascompared with that in lean nondiabetic Zucker rats. For this,isolated renal proximal tubules, the exclusive nephron segmentsthat contain the key gluconeogenic enzymes (17,18), were isolatedand incubated with lactate or glutamine or glycerol, three physiologicsubstrates that are taken up by the human and rat kidney invivo (19–21). We also measured the activities of the keygluconeogenic enzymes that are common to lactate, glutamine,and glycerol gluconeogenesis and the expression of the genesthat code for these enzymes. In addition, we attempted to identifyfactors that are responsible for the changes observed.
Reagents
l-Glutamine, glycerol, and glutaminase (grade V) were from Sigma(Saint Quentin-Fallavier, France). l-Lactate, other enzymes,coenzymes, and oligo dT were purchased from Roche (Meylan, France).Superscript II reverse transcriptase (RT), platinum Taqpolymerase,and dNTP were obtained from Invitrogen (Pontoise, France). ThemRNA extraction kit was purchased from Dynal (Oslo, Norway),and primers were obtained from Genset SA (Paris, France). L-[U-14C]lactate(5.62 GBq/mmol) was from Amersham (Little Chalfont, UK).
Rats
All experiments were approved by the Institutional Animal Careand Use Committee of the Lyon 1 University. Male ZDF rats (ZDF/Gmi:fa/fa) and lean control rats (ZDF/Gmi-lean:fa/+ or +/+) wereobtained from Charles River (Saint Germain sur l’Arbresle,France) at 13 wk of age. They were given a Purina 5008 Chow(IPS Product Supplies Inc., London, England) ad libitum andhad free access to water. For testing their possible role inregulating renal gluconeogenic enzyme activities in diabeticrats, either dexamethasone or cAMP (25 or 50 mg/kg body wt,respectively) was injected intraperitoneally in nondiabeticrats either 6 or 18 h before the preparation of isolated kidneytubules. The control, nondiabetic rats were treated intraperitoneallywith the vehicle (tricaprylin in the case of dexamethasone andsaline in the case of cAMP).
Preparation of Renal Proximal Tubules and Incubations
At 14 to 17 wk of age, the fed rats were anesthetized with sodiumpentobarbital (35 mg/kg intraperitoneally). After catheterizationof a carotid artery, blood was withdrawn for measurement ofblood acid-base parameters with a blood microsystem acid baseanalyzer (model BMS 3; Radiometer, Copenhagen, Denmark) andblood ketone-body levels as described previously (21); plasmawas also separated by centrifugation at 4°C for measurementof cAMP and corticosterone levels. Then, the kidneys were removedand weighed and placed in ice-cold Krebs-Henseleit medium. Renalproximal tubules were prepared by collagenase treatment of renalcortex slices as described by Baverel et al. (22). Incubationswere performed for 60 min in 25-ml stoppered Erlenmeyer flasksin an atmosphere of O2/CO2 (19:1). Tubules were incubated in4 ml of Krebs-Henseleit medium (24) with or without 1 and 2mM lactate or glutamine and 0.5 and 2 mM glycerol. The flaskswere prepared in duplicate for all experimental conditions.Incubation was stopped by adding perchloric acid (3% [vol/vol]final concentration) to each flask. In all experiments, zero-timeflasks with and without substrates were prepared by adding perchloricacid before the tubules. After removal of the denatured proteinby centrifugation, the supernatant was neutralized with 20%(wt/vol) KOH for metabolite determination. For testing theirinfluence, insulin and glucose were added to the medium of tubulesfrom diabetic and nondiabetic rats incubated for 60 min with1 mM [U-14C]lactate (approximately 2000 Bq/flask); in theseexperiments, incubation and collection of the 14CO2 that wasformed were carried out as described previously (24).
Metabolite Assays
All of the metabolites studied were determined by the methodsdescribed by Passonneau and Lowry (25). The 14C-glucose thatwas formed from [U-14C]lactate was isolated as described byKatz et al. (26) and counted by liquid scintillation. The dryweight of the tubules that were added to each flask and bloodmetabolites and acid-base parameters were determined as describedpreviously (21,22). Plasma cAMP and corticosterone levels weremeasured by competitive immunoassay kits provided by R&DSystems Europe (Lille, France).
Measurement of Enzyme Activities
Preparation of renal proximal tubule homogenates and measurementof phosphoenolpyruvate carboxykinase (EC 4.1.1.32), fructose-1,6-bisphosphatase(EC 3.1.3.11), and glucose-6-phosphatase (EC 3.1.3.9) were conductedby methods previously described in detail (27).
Semiquantitative Analysis of mRNA Expression
To determine the glucose-6-phosphatase, phosphoenolpyruvatecarboxykinase, and fructose-1,6-bisphosphatase mRNA levels inthe kidney cortex of diabetic rats and their control littermates,we performed semiquantitative RT-PCR as described previously(27). Levels of each mRNA of interest were related to thoseof the housekeeping -actin gene transcripts. Gene-specific oligonucleotideprimers (20 nucleotides) were selected from the published cDNAsequences of mouse and rat glucose-6-phosphatase, phosphoenolpyruvatecarboxykinase, and fructose-1,6-bisphosphatase. As an internalcontrol, a primer pair was selected from the cDNA sequence ofrat -actin. Forward and reverse primers that were chosen forglucose-6-phosphatase and phosphoenolpyruvate carboxykinasewere those that were used for the mouse kidney in a previousstudy (27). The fructose-1,6-bisphosphatase (accession no. M86240)primers were as sense 5'-TGTTTTGATCCCCTCGATGG-3' and antisense5'-TCCAGCATGAAGCAGTTGAC-3'. The -actin (accession no. NM_031144)primers were sense 5'-GAAGTGTGACGTTGACATCC-3' and antisense5'-AATCTCCTTCTGCATCCTGT-3', giving a PCR product of 103 bp.The -actin was amplified for 24 cycles with glucose-6-phosphatase,23 cycles with phosphoenolpyruvate carboxykinase, and 22 cycleswith fructose-1,6-bisphosphatase, each cycle using the followingparameters: 94°C for 30 s, 60°C for 45 s, and 72°Cfor 60 s. The linearity of amplification was verified in eachexperiment. PCR products were separated electrophoreticallyon a 2% (wt/vol) agarose gel (Invitrogen, Paisley, Scotland)for glucose-6-phosphatase and fructose-1,6-bisphosphatase oron a 2% (wt/vol) agarose –1000 gel for phosphoenolpyruvatecarboxykinase (Invitrogen) and stained with SYBR Green I (MolecularProbes Europe, Leiden, The Netherlands). The gel then was scannedusing a fluorescence laser scanner (Molecular Dynamics, Sunnydale,CA). Band intensities were quantified using the Image Quantsoftware. mRNA levels are reported relative to -actin.
Statistical Analyses
Net substrate utilization and product formation by kidney tubuleswere calculated as the difference between the total contentsof the flask (tissue + medium) at the start (zero-time incubations)and after 1 h of incubation. The metabolic rates are expressedin nanomoles of substance removed or produced per hour per milligramof tubule dry weight, and the enzyme activities are reportedas micromoles of substrate used or product formed per milligramof tubule protein per hour; they are given as means ±SEM. The conversion of [U-14C]lactate into 14C-glucose was calculatedby dividing the radioactivity in glucose by the specific radioactivityof 14C-lactate. The results were analyzed by the t test forunpaired data, comparing values obtained in diabetic with thosein nondiabetic rats.
As shown in Table 1, the body weight of the nondiabetic ratswas slightly but significantly lower than that of the diabeticanimals. Striking, the weight of the two kidneys of the diabeticrats exceeded by 38% that of the nondiabetic rats. Table 1 alsoshows that there was no metabolic acidosis in the diabetic ratsdespite their significantly higher blood concentrations of both-hydroxybutyrate and acetoacetate. It is interesting that theplasma cAMP and corticosterone levels were markedly elevated(80 and 84%, respectively) in diabetic rats when compared withnondiabetic rats.
Table 1. Baseline characteristics of nondiabetic lean Zucker rats and ZDF rats
Metabolism of Lactate, Glutamine, and Glycerol in Renal Proximal Tubules from Lean Zucker Rats and ZDF Rats Table 2 shows that the removal of lactate but not glutamineand glycerol was inhibited in tubules from diabetic rats whencompared with that in tubules from control rats. With lactate,glutamine, and glycerol as substrate, there was no change inthe accumulation of pyruvate, glutamate, and lactate, respectively.Striking, mean glucose synthesis was stimulated by 38% fromlactate, 51% from glutamine, and 66% from glycerol, despitea fall of cellular ATP levels. Accumulation of intermediatesof neither the tricarboxylic acid cycle nor aspartate, alanine,3-glycerophosphate, or glycogen was observed in any of the experimentalconditions used. Assuming that all of the products that werefound to accumulate arose from added substrates, the completeoxidation of lactate, glutamine, and glycerol, which can beestimated by carbon balance, was greatly reduced—evensuppressed in the case of glycerol—in tubules from diabeticrats.
Table 2. Metabolism of 1 mM lactate and glutamine and of 0.5 mM glycerol in isolated renal proximal tubules from nondiabetic lean Zucker rats and ZDF ratsa
At zero-time, the tubules from five control rats and those fromfour diabetic rats contained 1.5 ± 1.1 and 3.8 ±0.7 nmol/mg dry wt glycogen (in glycosyl equivalents), respectively(NS). After 60 min of incubation in the presence of 1 mM lactate,the glycogen content was 2.2 ± 1.1 and 3.5 ± 0.7nmol/mg dry wt, respectively (NS). Glucose synthesis in theabsence of exogenous substrate was 6 ± 1 and 15 ±1 nmol/mg dry wt per h (114%; P < 0.01) in tubules from control(n = 16) and diabetic (n = 15) rats, respectively. After 60min of incubation without any exogenous substrate, the glycogencontent of these tubules was 1.1 ± 0.6 and 2.4 ±0.5 nmol/mg dry wt, respectively (NS).
In tubules from diabetic rats when compared with those fromnondiabetic rats, glucose synthesis from 2 mM lactate, glutamine,and glycerol was stimulated by 69, 56, and 50%, respectively.The corresponding values were 91 ± 10 (n = 10) versus54 ± 4 (n = 11) nmol/mg dry wt per h (P < 0.05), 81± 8 (n = 10) versus 52 ± 5 (n = 11) nmol/mg drywt per h (P < 0.05), and 78 ± 1 (n = 5) versus 52± 3 (n = 5) nmol/mg dry wt per h (P < 0.05).
Effect of Insulin and Glucose on Conversion of 14C-Lactate into 14C-Glucose Table 3 shows that addition of 100 nM insulin or 5 or 25 mMglucose did not alter the conversion of [U-14C]lactate into14C-glucose in tubules either from nondiabetic or from diabeticrats. It also shows that, irrespective of the experimental condition,such conversion was higher in tubules from diabetic than intubules from nondiabetic rats. As seen in Table 2, glucose productionwas also higher in tubules from diabetic than in those fromnondiabetic rats (Table 3). In the presence of 1 mM [U-14C]lactateplus 5 or 25 mM glucose, it was not possible to measure enzymaticallythe glucose synthesized from lactate in a reliable manner becauseof the high glucose concentration. For the same reason, no substantialglucose removal could be measured enzymatically with 5 and 25mM glucose as sole substrate, and lactate accumulation was negligible(results not shown).
Table 3. Effect of insulin and glucose on the metabolism of 1 mM [U-14C]lactate in isolated renal proximal tubules from nondiabetic lean Zucker rats and ZDF ratsa
Activities of Phosphoenolpyruvate Carboxykinase, Fructose-1,6-Bisphosphatase, and Glucose-6-Phosphatase in Renal Proximal Tubules from Lean Zucker Rats and ZDF Rats Table 4 shows that diabetes stimulated in a statistically significantmanner the activity of the three key gluconeogenic enzymes thatare common to lactate and glutamine gluconeogenesis; it greatlystimulated the activity of phosphoenolpyruvate carboxykinase(2.2-fold) and, to a much lesser extent, those of glucose-6-phosphatase(1.4-fold) and of fructose-1,6-bisphosphatase (1.2-fold).
Table 4. Activity of key gluconeogenic enzymes that are common to lactate and glutamine gluconeogenesis in renal proximal tubules from nondiabetic lean Zucker and ZDF ratsa
Phosphoenolpyruvate Carboxykinase, Fructose-1,6-Bisphosphatase, and Glucose-6-Phosphatase mRNA Levels in the Renal Cortex from Lean Zucker Rats and ZDF Rats Figure 1 clearly shows that a statistically significant elevationof the renal cortical cellular levels of phosphoenolpyruvatecarboxykinase (3.0-fold increase), glucose-6-phosphatase (2.3-foldincrease), and fructose-1,6-bisphosphatase (1.5-fold increase)mRNA occurred in diabetic rats when compared with those in controlanimals.
Figure 1. mRNA levels of glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and fructose-1,6-bisphosphatase (FBPase) in diabetic rats and their nondiabetic littermates. The mRNA levels were analyzed by semiquantitative reverse transcription–PCR analysis, as described in Materials and Methods. The amplified cDNA were separated by agarose-gel electrophoresis, as shown in the lower part of the figure. Band intensities were quantified and are reported relative to the -actin band. The values are means ± SEM for four diabetic rats and four nondiabetic rats. The statistical difference between diabetic and lean rats was measured by the unpaired t test: *P < 0.05.
Effect of Dexamethasone and Dibutyryl-cAMP Administration on Activity of Key Gluconeogenic Enzymes in Renal Proximal Tubules from Nondiabetic Lean Zucker Rats Table 5 shows that the intraperitoneal administration of 25mg/kg body wt dexamethasone 18 h earlier stimulated the activityof phosphoenolpyruvate carboxykinase (63%) and, to a much lesserextent, of glucose-6-phosphatase (12%) but not that of fructose-1,6-bisphosphatase.Therefore, at least one other factor was responsible for thestimulation of the activity of the last enzyme. It is interestingthat the activities of fructose-1,6-bisphosphatase and glucose-6-phosphatasewere increased by 21 and 19%, respectively, in renal proximaltubules of lean rats at the same time point (18 h) after anintraperitoneal injection with 50 mg/kg body wt dibutyryl cAMP(Table 5); under the same conditions, the activity of phosphoenolpyruvatecarboxykinase was only slightly (9%) but in a statisticallysignificant manner stimulated in these tubules (Table 5). Givenits weak effect on phosphoenolpyruvate carboxykinase activity,the effect of cAMP was studied 6 h after its injection; thisresulted in a much more pronounced stimulation of the threeenzyme activities (Table 5).
Table 5. Effect of dexamethasone and dibutyryl-cAMP on the activity of key gluconeogenic enzymes in renal proximal tubules from nondiabetic lean Zucker rats
Unlike type-2 diabetic patients in vivo (10,11), no animal modelof type 2 diabetes has been reported until now to display anincreased renal gluconeogenesis. To our knowledge, this studyis the first to demonstrate such an elevation in vitro fromthree physiologic substrates that are taken up by the humanand rat kidney in vivo (19–21).
Biochemical and Molecular Mechanisms for Long-Term Stimulation of Renal Gluconeogenesis in Diabetic Zucker Rats
Increased renal gluconeogenesis in diabetic rats was due toa change in the fate of lactate, glutamine, and glycerol carbonskeleton; indeed, gluconeogenesis was favored at the expenseof complete oxidation (Table 2). It should be emphasized thatincreased glucose synthesis in tubules from diabetic rats wasconfirmed by using radioactive lactate as substrate even inthe presence of a high glucose concentration (Table 3). Notethat the distribution of lactate between glucose synthesis andoxidation that was observed in this work was similar to thatfound by Krebs et al. (28) in one study but different from whatthey reported in another study (29). It is also important tounderline that lactate, glutamine, and glycerol gluconeogenesiswas greater in tubules from diabetic rats despite a lower concentrationof ATP in these tubules when compared with those of controlrats. Because ATP is a key compound in gluconeogenesis, itslower availability was compensated by other mechanisms thatled to a stimulation of glucose synthesis. Note that the fallin ATP concentration, which can be only a consequence but notthe cause of the increased gluconeogenesis observed in thesetubules, is in agreement with the diminution of substrate oxidation.
In agreement with the elevation of renal gluconeogenesis inour diabetic rats, we found a stimulation of the three key gluconeogenicenzymes that are common to gluconeogenesis from both lactateand glutamine. In keeping with the stimulation of the activitiesof fructose-1,6-bisphosphatase and glucose-6-phosphatase, thesynthesis of glucose from glycerol was also found to be greatlystimulated. It is interesting that the elevations of the correspondingmRNA levels in the renal cortex of diabetic rats establish thatthe stimulation of the activities of the three enzymes mentionedabove occurred by a long-term regulation involving the upregulationof the genes that code for these enzymes. Note here that theexpression of the -actin gene, which we used as a referencefor the expression of the genes for key gluconeogenic enzymes,has been shown to remain unaltered in the kidney of diabeticrats (30).
Factors Responsible for Long-Term Stimulation of Renal Gluconeogenesis in Diabetic Zucker Rats Metabolic Acidosis, Insulin, and Leptin.
Because our diabetic rats were not acidotic, the role of metabolicacidosis, which is responsible for the stimulation of gluconeogenesisin renal cortical slices prepared from rats that have type 1diabetes (31–33), can be ruled out. Although insulin hasbeen shown repeatedly to inhibit gluconeogenesis in the humankidney in vivo (7,9,13,14) and in one study in the rat kidneyin vivo (34), it is unlikely that it did so by a direct mechanismthat altered gene expression because experiments that have beenperformed in the rat kidney indicate that insulin has no directeffect on the activity or synthesis of phosphoenolpyruvate carboxykinasein the kidney of diabetic rats (32,35). Moreover, our studyshows that insulin does not inhibit glucose synthesis from lactatein isolated kidney tubules (Table 3). Therefore, disappearancein vivo of a possible direct inhibitory effect of insulin onrenal gluconeogenesis in our diabetic rats, which are knownto be insulinopenic at 14 to 17 wk of age, was probably notinvolved in the intrinsic stimulation of in vitro renal gluconeogenesisthat we observed in these rats. It also should be pointed outthat leptin resistance, which is characteristic of our diabeticrats, could not be involved in the effects observed becauseleptin receptors in the rat kidneys are localized exclusivelyin the renal inner medulla (36). Similarly, glucagon, whoseplasma levels have been shown to be elevated in patients withtype 2 diabetes (37), was probably not involved directly inthe stimulation of renal gluconeogenesis, because this hormonedoes not stimulate the production of cAMP in the renal proximaltubule (38).
Glucocorticoids and cAMP.
By contrast, it is conceivable that corticosterone, whose circulatingconcentration was elevated in our diabetic Zucker rats likein a previous study by other authors (39), was responsible,at least in part, for the stimulation of renal glucose synthesisin our diabetic rats. Indeed, glucocorticoids have been shownto upregulate (1) the expression of the phosphoenolpyruvatecarboxykinase gene (33,40,41), (2) the enzymatic activitiesof phosphoenolpyruvate carboxykinase and glucose-6-phosphatase(35,42–44), and (3) the capacity of rat renal corticalslices to synthesize glucose from pyruvate and succinate (45)but not the expression of the glutaminase gene (41) or the activityof fructose-1,6-bisphosphatase (43) in the rat kidney. In agreementwith the last observations, the activities of phosphoenolpyruvatecarboxykinase and glucose-6-phosphatase but not that of fructose-1,6-bisphosphatasewere stimulated in renal proximal tubules of lean Zucker ratsthat were treated with dexamethasone (Table 5). The absenceof effect of dexamethasone on the activity of fructose-1,6-bisphosphatase,in agreement with the absence of a glucocorticoid response elementin the corresponding gene (46), suggests, therefore, that atleast another factor was responsible for the upregulation ofthe expression of the fructose-1,6-biphosphate gene. In thisrespect, our data presented in Table 5 strongly suggest thatcAMP, which has been shown to stimulate the transcription ofthe fructose-1,6-bisphosphatase gene in liver (46), had thesame effect in the kidney of our diabetic rats, which containsthe same fructose-1,6-bisphosphatase subunit as the rat liver(47). In agreement with our findings, cAMP stimulates the transcriptionof the phosphoenolpyruvate carboxykinase gene (41,43) but notthat of glutaminase in the rat kidney (41). Note that it isconceivable that the diabetic hyperglucagonemia, resulting inpart from the disappearance of the insulin-induced inhibitionof glucagon secretion, via the elevation of circulating cAMPlevels, was indirectly responsible in vivo for the stimulationof intrinsic renal gluconeogenesis that we observed.
Thus, our results strongly suggest that, in our diabetic rats,at least circulating glucocorticoids and cAMP acted in conjunctionto stimulate renal gluconeogenesis. In agreement with this viewand our finding that plasma cAMP levels were increased by 80%in our diabetic rats is the demonstration by Nakae et al. (48)that dexamethasone plus cAMP stimulated the expression of thephosphoenolpyruvate carboxykinase and glucose-6-phosphatasegenes in LLCPK1-FBPase+ cells.
Renal proximal tubules from ZDF rats synthesized much more glucosefrom lactate, glutamine, and glycerol than tubules from theirlean nondiabetic littermates. This stimulation correlates withthe increased activities and mRNA level of key gluconeogenicenzymes. These data, together with the observation that theweight of the kidneys of our diabetic rats was augmented by38% when compared with that of the nondiabetic animals, stronglysuggest that renal gluconeogenesis may contribute cruciallyto the hyperglycemia and the elevated systemic glucose productionobserved in these animals (49). Among the factors that potentiallyare involved in the probably multifactorial long-term stimulationof renal gluconeogenesis that we observed, our results suggestthat both glucocorticoids and cAMP might play a substantialrole by augmenting the expression of key gluconeogenic genes
Acknowledgments
A.E. was supported by a fellowship from the National Centerfor Scientific Research (Lebanon). This work has benefited fromthe CeCIL facilities.
We thank Claire Morel for secretarial assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-actin gene transcripts. Gene-specific oligonucleotide primers (20 nucleotides) were selected from the published cDNA sequences of mouse and rat glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase. As an internal control, a primer pair was selected from the cDNA sequence of rat 
