International Journal of Obesity (1998) 22, 667±672 ß 1998 Stockton Press All rights reserved 0307±0565/98 $12.00 http://www.stockton-press.co.uk/ijo Effect of starvation on gene expression of regulatory of = in genetically obese (fa=fa) Zucker rats

JX PeÂrez, A Manzano, A Tauler and R Bartrons

Unitat de BioquõÂmica, Departament de CieÁncies FisioloÁgiques Humanes i de la NutricioÂ, Campus de Bellvitge, Universitat de Barcelona, Spain

OBJECTIVE: To study the mechanism that controls fructose-2,6-bisphosphate (Fru-2,6-P2) accumulation, as well as the mRNAs levels of the glycolytic=gluconeogenic regulatory enzymes in the livers of fed and starved lean ( fa=-) and obese ( fa=fa) Zucker rats. DESIGN: Rats were fed a standard chow or deprived of food for 24 h. SUBJECTS: Male lean (fa=-) and genetically obese ( fa=fa) rats (nine weeks old). MEASUREMENTS: Fru-2,6-P2 concentration, 6-phosphofructo-2- (PFK-2), (GK), pyruvate kinase (PK) activities and the mRNA levels of GK, PFK-2, L-type pyruvate kinase, fructose-1,6-bisphosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PEPCK) were analyzed. RESULTS: PFK-2=FBPase-2 mRNA decreased during starvation in both fa=- and fa=fa animals. Although PFK- 2=FBPase-2 mRNA levels were similar in fed lean and obese rats, PFK-2 concentration and activity were higher in fed obese than in fed lean animals, which might explain the high concentration of Fru-2,6-P2 observed in obese animals. During starvation, PFK-2 protein concentration decreased, correlating with the enzymatic activity and Fru- 2,6-P2 levels. The activities of GK and L-pyruvate kinase (L-PK) also increased in fed obese (fa=fa) rats compared with fed lean (fa=-) animals, but decreased during starvation. The mRNA levels of glycolytic enzymes in fed obese rats were similar (PFK-2) or higher than (GK, L-PK) in fed lean animals. During starvation, they decreased in lean and obese rats with one important exception, GK mRNA remained high in obese animals. The mRNA of gluconeogenic enzymes remained constant (FBPase-1) or increased (PEPCK) during fasting. CONCLUSION: The changes observed might be explained by the hyperinsulinaemia observed in the liver of obese rats, which might lead to the stimulation of glycolysis and lipogenesis.

Keywords: obese; gene expression; glycolysis; gluconeogenesis; starvation

Introduction expenditure, severe resistance, and genetic background-dependent diabetes.4 Enhanced lipogenesis has been observed in livers of Obese ( fa=fa) Zucker rats have a recessive gene obese rats.5,6 This might be one of the mechanisms mutation, localized in chromosome 5 producing total responsible for the increased fat deposition which inability to respond to leptin.1 The fa locus, linked to occurs in these animals. Liver glycolysis provides obesity in the rat, has been mapped to a region C3 units for the synthesis of lipids and is an syntenic with the mouse db locus,2,3 implying that important component of the control of lipogenesis. the fa mutation lies within the rat ob-receptor. The hepatic gluconeogenic=glycolytic pathway is The products of the ob and db genes, constitute a regulated by allosteric modulators, and phosphoryla- hormone-receptor pair (leptin and leptin receptor, tion=dephosphorylation and control of gene expres- respectively) that provides molecular identity to one sion of several regulatory enzymes.7,8 These enzymes system, through which the status of energy stores is control hepatic production and utilization signalled to the brain. Total inability to produce leptin through regulation of three major substrate cycles: (ob=ob) or to respond to it (db=db) result in early glucose=glucose 6-, fructose 6-phosphate= obesity with excessive food intake, a decreased energy fructose-1,6-bisphosphate and phosphoenolpyruvate= pyruvate. The fructose 6-phosphate=fructose-1,6-bis- phosphate substrate cycle is also regulated by a Correspondence: Ramon Bartrons, Unitat de BioquõÂmica, Facultat subcycle in which the amount of the regulatory d'Odontologia, Universitat de Barcelona, Campus de Bellvitge, 08907-Hospitalet, Spain. molecule fructose-2,6-bisphosphate (Fru-2,6-P2)is Received 15 December 1997; accepted 23 February 1998 controlled by the bifunctional 6-phospho- Gene expression in obese (fa=fa) Zucker rats JX PeÂrez et al 668 fructo-2-kinase=fructose-2,6-bisphosphatase (PFK-2= EcoRI fragment from cDNA for PFK-2=FBPase-2;17 a FBPase-2).7±10 0.65 kb EcoRI fragment from cDNA for fructose 2,6- 11±14 18 It has been shown that the Fru-2,6-P2 concen- biphosphate (FBPase-1); a 2.8 kb PstI fragment tration and the PFK-2 activity in livers of genetically from cDNA clone (pPCK10) for phosphoenolpyruvate obese ( fa=fa) rats are greater than in livers of control carboxykinase (PEPCK);19 a 2.4 kb EcoRI fragment 20 lean animals. Fru-2,6-P2 stimulates phosphofructo- from cDNA for GK; and a 1.8 kb PstI fragment from kinase7±10 and since that activity cDNA clone (G4) for L-pyruvate kinase (L-PK).21 A is increased,11 this may contribute to keeping glyco- cDNA for 18S ribosomal RNA was also used as a lysis active. Since the activities of other glycolytic probe.22 All DNA probes were generated by labelling enzymes, such as glucokinase (GK) and L-pyruvate with [a-32P]dCTP to a speci®c radioactivity of kinase, have also been found to have increased,11±13 it 1.5Â109 cpm=mg of DNA by random priming with was our purpose to study the mechanism that controls Klenow DNA . The levels of mRNAs were Fru-2,6-P2 accumulation, as well as the mRNAs levels measured by densitometric scanning of the autoradio- of the glycolytic=gluconeogenic regulatory enzymes grams with a Vilbert Lourmat densitometer and cor- in the livers of fed and starved lean ( fa=-) and obese rected for the amount of 18S rRNA that was used as a ( fa=fa) Zucker rats. control.

Materials and methods Metabolite and enzyme assays Fru-2,6-P2 was extracted and measured as described by Van Schaftingen et al.23 Total and active PFK-2 Materials 32 activities were measured as described by Bartrons et [a- P]dCTP (3000 Ci=mmol) was from Amersham al.24 In the conditions of the assay, the active form (London, UK). The random primed DNA labelling corresponds to the activity of the non-phosphorylated kit and restriction endonucleases were from Boehrin- form of the enzyme measured.24 L-PK activity was ger Mannheim (Mannheim, Germany). N-hybond determined at saturating concentrations of phospho- membranes and ECL kit were from Amersham. Nitro- enolpyruvate (5 mM), as previously described by cellulose membranes were from Millipore Corpora- FelõÂu et al25 and GK as described by Davidson and tion (Bedford, MA). Rat albumin antiserum was from Arion.26 The protein concentration was determined Nordic (Tilburg, The Netherlands). Anti-rabbit anti- according to Bradford,27 using bovine serum albumin body was from DAKO A=S, (Glostrup, Denmark). as standard. Other enzymes and biochemical reagents were either from Boehringer Mannheim (Mannheim, Germany) or Sigma (St Louis, MO). All chemicals were of analy- tical grade. Western blot analysis Immunoblot analysis was performed by a modi®cation of the method described by Burnette.28 Previous to Animals and dietary manipulations Western blot analysis, the concentration of protein Male lean ( fa=-) and genetically obese ( fa=fa) rats was determined by the method of Bradford.27 For were obtained from Iffa Credo EspanÄa SA (Barcelona, PFK-2=FBPase-2 analysis we used a 1:2000 dilution Spain). The rats were nine weeks old at the time of the of polyclonal antibody raised against rat liver pro- experiments. They were fed a standard chow tein.29 Albumin, using a 1:2000 dilution of polyclonal (PANLAB A04, Barcelona, Spain) and water ad antibody raised against rat albumin. After Western libitum. Animals were subjected to a 12 h light=12 h blots with the anti-PFK-2=FBPase-2 antibody, mem- dark cycle (light starting at 08.00 h). Starved rats were branes were dehybridizated with stripping buffer con- deprived of food for 24 h. All the animals were killed taining 100 mM 2-mercaptoethanol, 4% SDS, 125 mM at 10.00 h by decapitation and the livers were freeze- TrisHCl pH 6.8. After dehybridization, the same clamped in liquid and stored at 780C prior membranes were used with the antibody against to extraction of Fru-2,6-P2, enzymes or RNA. albumin, that was used to correct. Bound antibody was detected by the ECL (enhanced chemilumines- RNA analyses and DNA-hybridation probes cence) method. The levels of protein were measured Total RNA was extracted from frozen rat tissues by by densitometric scanning of the autoradiograms with the LiCl=urea method.15 Previous to Northern blot a Vilbert Lourmat densitometer and corrected for the analysis, the concentration of the RNA was deter- amount of albumin that was used as a control. mined by measuring the OD260 of an aliquot of the ®nal preparation.16 The integrity of the RNA was veri®ed by observing the rRNA bands in the ethidium Statistical analysis bromide gel under uv irradiation. Northern blot ana- Statistical comparisons were performed using analysis lyses were performed by standard procedures.16 The of variance (ANOVA) with a post-hoc test (Fischer following fragments were used as probes: a 1.4 kb PLSD). Gene expression in obese (fa=fa) Zucker rats JX PeÂrez et al 669 Results

Effect of starvation on Fru-2,6-P2 levels Fed genetically obese ( fa=fa) Zucker rats contained higher levels of Fru-2,6-P2 than fed lean animals (32 and 14 nmol=g liver, respectively). A signi®cantly lower concentration was observed in starved (24 h) lean ( fa=-) and obese ( fa=fa) animals, compared to that measured in their fed littermates. Starvation caused a fall in Fru-2,6-P2 concentration from 14 to 4 nmol=g in lean ( fa=-) and from 32 to 19 nmol=gin obese ( fa=fa) rats (Table 1). These results are in concordance with previous reports11 and con®rm the starvation of the rats.

Figure 1 Effect of starvation on hepatic 6-phosphofructo-2- kinase=fructose-2,6-bisphosphatase (PFK-2=FBPase-2) enzyme Effect of starvation on key regulatory enzymes of abundance in obese Zucker ( fa=fa) rats. Total protein (20 mg=lane) glycolysis=gluconeogenesis activities obtained from livers of fed and starved (24 h) rats were trans- ferred to nylon membranes after electrophoresis in SDS=PAGE In order to determine the mechanism by which the (10%) and hybridized with speci®c antibodies as described in Fru-2,6-P2 levels were higher in obese animals, total Materials and methods. The level of PFK-2 was measured by and active (non phosphorylated form) PFK-2 activities densitometric scanning of the autoradiograms and corrected for the amount of albumin. The values represent meansÆ s.e.m. of were determined. Both PFK-2 activities increased 3±5 Western blots of different liver extracts from three lean and (20%) in the obese group. After 24 h starvation, total ®ve obese animals. Representative Western blots are shown. and active PFK-2 activities decreased in lean ( fa=-) Statistically signi®cant differences are indicated by: *P < 0.05, with respect to lean (fa=-) fed; {{{ P < 0.001, obese (fa=fa) starved and obese ( fa=fa) rats, although the differences with respect to obese (fa=fa) fed. became more marked in the active PFK-2 form of lean starved animals, indicating a higher phosphory- lated enzyme in this group of animals. The activities of GK and L-PK were also signi®cantly higher in Effect of starvation on regulatory enzymes of obese animals. Starvation caused a decrease in both glycolysis=gluconeogenesis mRNA abundance groups of animals, being the levels of starved obese In order to determine whether the changes in enzyme rats very similar to the fed lean rats (Table 1). activities found correlated with mRNA levels, North- ern blot analyses were performed on hepatic RNA extracted from fed and starved lean ( fa=-) and obese Effect of starvation on PFK-2=FBPase-2 abundance ( fa=fa) rats. Values were corrected for the amount of To ascertain whether starvation affected the abun- 18S rRNA that was used as control. As shown in dance of the bifunctional enzyme, Western blot ana- Figure 2, mRNA levels of PEPCK increased four-fold lyses were performed as described in Materials and over control at 24 h after starvation in both groups. methods. Fed obese rats contained 30% more of the FBPase-1 mRNA was increased slightly during star- enzyme than the fed lean animals. Starvation caused a vation in both fa=- and fa=fa, although the differences fall of 30% and 39% in lean and obese rats, respec- were not signi®cant. Concerning the glycolytic tively, compared with their fed littermates (Figure 1). enzymes, PFK-2 mRNAs were similar in lean and

Table 1 Fructose-2,6-bisphosphate (Fru-2,6-P2) concentration and activity of key glycolytic enzymes in fed and starved lean (fa=-) and obese (fa=fa) rat livers

Lean rats Obese rats

Fed Starved Fed Starved

Fru-2,6-P2 (nmol=g liver) 14Æ 2 3.6Æ 2** 32Æ 4*** 19Æ 3*{{{{{{ PFK-2 total (mU=mg protein) 35Æ 226Æ 1* 42Æ 330Æ 2{{ PFK-2 active (mU=mg protein) 25Æ 213Æ 2** 37Æ 3*** 29Æ 3{{{{{ Glucokinase (U=g liver) 2.5Æ 0.1 1.9Æ 0.1* 2.9Æ 0.2* 2.4Æ 0.2{{{ PK (U=g liver) 55Æ 436Æ 2* 111Æ 7** 74Æ 8{{{{

Values are the meanÆ s.d. of 3±5 experiments. Fru-2,6-P2 and enzyme activities were measured in samples of liver as indicated in Materials and methods. Statistically signi®cant differences are indicated by: * P<0.05, ** P<0.01 and *** P<0.001 with respect to lean (fa=-) fed; { P<0.05, {{ P<0.01 and {{{ P<0.001, obese (fa=fa) starved with respect to obese (fa=fa) fed; { P < 0.05, {{ P<0.01 and {{{ P<0.001, obese (fa=fa) starved with respect to lean (fa=-) starved. PFK-2 ˆ 6-phosphofructo-2-kinase; PK, pyruvate kinase. Gene expression in obese (fa=fa) Zucker rats JX PeÂrez et al 670

Figure 2 Gene expression of regulatory enzymes of glycolysis=gluconeogenesis in obese Zucker (fa=fa) rats. a) Total RNA (20 mg=lane) extracted from livers of fed and starved (24 h) rats were transferred to nylon membranes after electrophoresis in 1.5% agarose and hybridized with 6-phosphofructo-2-kinase=fructose-2,6-bisphosphatase (PFK-2=FBPase-2), glucokinase (GK), L-pyruvate kinase (L-PK), fructose-1,6-bisphosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PEPCK) cDNAs as it is described in Materials and methods. Representative Northern blots are shown in Figure 2a. b) The level of these mRNAs was measured by densitometric scanning of the autoradiograms and corrected for the amount of 18S rRNA. The values represent meansÆ s.e.m. of different liver extracts from three lean and ®ve obese animals. Statistically signi®cant differences are indicated by: * P < 0.05, with respect to lean (fa=-) fed.

obese animals and decreased to 65% in lean starved and 3-phosphate,31,32 and may simulta- rats and to 15% in obese rats. The levels of GK neously reduce gluconeogenesis. mRNA, which were signi®cantly higher in obese In good agreement with the hypothesis that hepatic rats (70%), decreased to only 20% in starved lean glycolysis is favoured in obese Zucker rats,11±13,33,34 rats, but remained at similar levels in obese animals. and con®rming previous reports,11,13,34±36 GK, PFK-2 L-pyruvate kinase mRNA levels also increased sig- and L-PK activities were all signi®cantly higher in ni®cantly in obese rats and decreased after starvation livers of obese ( fa=fa) rats compared to lean animals, to very low levels in both fa=- and fa=fa rats. however in livers of starved obese rats, the activities of these enzymes were similar to those found in fed lean animals. Furthermore, our data indicate that Discussion during starvation, PFK-2 is mainly phosphorylated in lean animals, as indicated by the decrease in the active non-phosphorylated form of PFK-2, whereas Enhanced lipogenesis has been observed in livers of there are no signi®cant differences between the active obese ( fa=fa) animals.5,6 In contrast with lean con- and total PFK-2 forms in obese starved animals. trols, isolated hepatocytes of obese fa=fa rats are Artefactual cAMP-dependent changes observed in known to glycolyze and to synthesize fatty acids, conscious rats killed by decapitation have been even after a period of starvation.30,31 This might be observed in starved animals.37 This effect could be, one of the mechanisms responsible for the fat deposi- in part, responsible for the decrease of the `active' tion. Liver glycolysis provides C3 units for the synth- PFK-2 activity and Fru-2,6-P2 concentration in esis of lipids and it is thus an important component of starved lean rats. However, in starved obese animals, the control of lipogenesis. In liver, one of the main the mechanism seems to be different, as there are no glycolytic regulatory metabolites is Fru-2,6-P2. Its differences between `total' and `active' PFK-2 activ- concentration is increased in livers of obese animals, ities and, also, low cAMP concentrations have been 11±13 11,37 even after starvation. Fru-2,6-P2 can contribute found in the livers of these animals. Moreover, the to keep an active glycolysis and, thus increase lipo- data obtained with Western blot analysis con®rm the genesis by providing an increased supply of pyruvate increased levels of PFK-2 in fed obese animals, which Gene expression in obese (fa=fa) Zucker rats JX PeÂrez et al 671 also show, in agreement with the total PFK-2 activ- synergistic=antagonistic action of multiple factors. ities, that during starvation, the levels are similar in Glucocorticoids stimulate L-type mRNA PFK-2 tran- lean and obese rats. All these data favour the PFK-2 scription in rat liver,8,39,45 whereas inhibits kinase activity in obese animals and provide a plau- transcription and destabilizes the L-mRNA.46 In sible explanation, in addition to other metabolic starved or diabetic rats, refeeding or insulin treatment 11,13,31 17,47,48 changes observed, for the high Fru-2,6-P2 con- increases, whereas starvation causes a decrease centration. in the PFK-2 mRNA levels, both in lean and obese In order to explain the changes in enzyme activities, rats. The effects of insulin on L-promoter, depend on we studied the levels of their mRNAs by Northern the hormonal context. Lemaigre et al 49 have reported blot analysis. The mRNA levels of glycolytic enzymes that insulin inhibits and reverses the glucocorticoid- were similar (PFK-2) or increased (GK, L-PK) in fed induced stimulation of transcription of the L-type obese with respect to lean animals. During star- mRNA. This antagonistic action may explain the vation, they decreased in lean and obese rats with higher PFK-2 decrease in the obese animals, bearing one important exception, GK mRNA remains at in mind the increased levels of both hormones in high levels in obese animals. The mRNAs of gluco- ( fa=fa) rats. neogenic enzymes remained constant (FPBase-1) or increased (PEPCK) during fasting . Gluconeogenesis has been reported to be lower in Conclusion hepatocytes from obese Zucker rats,13,14,30,31 in spite of the fact that phosphoenolpyruvate carboxykinase mRNA increases at similar levels than in lean ani- The data presented here might be explained by the mals. These results agree with the transcriptional hyperinsulinaemia observed in obese animals,5,6 activation of this gene reported previously.38,39 How- which lead to the stimulation of glycolysis and lipo- ever, although increases in FBPase-1 activity have genesis. been found during starvation in obese animals, we did not ®nd signi®cant changes in its mRNA levels. This Acknowledgements fact indicates the main role of PEPCK in the regula- We are grateful to Dr S. Ambrosio and Dr J. Gil for tion of gluconeogenesis ¯ux during this process. their help and for much valuable advice during the Insulin is increased in obese ( fa=fa) animals,40,41 40 course of this work. The skillful technical assistance even during fasting. This can explain the high of C. OrtunÄo is also acknowledged. JXP is the mRNA levels of GK and L-PK in fed animals. Insulin recipient of a research fellowship from Fundacio Pi i stimulates transcription of GK.38,39 This stimulation 42 Sunyer (Campus de Bellvitge). AM is the recipient of does not depend on extracellular glucose. Glucagon, a research fellowship from the Fondo de Investiga- acting via cyclic AMP, inhibits the stimulation by 42 ciones Sanitarias (FIS). This work has been supported insulin. The mechanism is unknown, but the dom- by the Fondo de Investigaciones Sanitarias de la inance of glucagon over insulin suggests that insulin 39 Seguridad Social (FIS 95=0286) and by Generalitat stimulation may consist of derepression. The de Catalunya (1995 SGR=00427). increase in GK mRNA levels in obese rats during starvation could be explained, in addition to other factors, by the hyperinsulinaemia40 and the low cyclic References AMP levels.11 It has been reported previously,43 that 1 Phillips MS, Liu Q, Hammond HA, Dugan V, Hey PJ, Caskey starvation causes increased GK mRNA levels in the CJ, Hess JF. Leptin receptor missense mutation in the fatty Zucker rats. Nat Genet 1996; 13: 18±19. livers of obese-hyperglycaemic Wistar fatty rats, 2 Truett G, Bahary N, Friedman JM, Leibel RL. Rat obesity whereas in an insulin de®cient state, caused by strep- gene fatty (fa) maps to chromosome 5: evidence for homology tozotocin, the levels of GK mRNA were low during with the mouse gene diabetes (db). Proc Natl Acad Sci USA starvation. 1991; 88: 7806±7809. Hormones and diet have an important role in the 3 Chua SC, Chung WK, Wu-Peng S, Zhang Y, Liu S, Tartaglia 38,39,44 L, Leibel RL. Phenotypes of mouse diabetes and rat fatty due regulation of L-PK gene expression. Glucose to mutations in the OB (Leptin) receptor. Science 1996; 271: and insulin are required together, neither of them 994±996. being active alone in order to stimulate transcrip- 4 Spiegelman BM, Flier JS. Adipogenesis and Obesity: Round- tion.44 In contrast with GK, L-PK mRNA levels ing out of the Big Picture. Cell 1996; 87: 377±389. were very low in the liver of both fatty and lean rats 5 Bray GA. The Zucker-fatty rat. Fed Proc 1977; 36: 148±153. after starvation. Similar results have been reported in 6 Freedman MR, Horwitz BA, Stern JS. Effect of adrenalectomy 43 and glucocorticoid replacement on development of obesity. obese rats. Am J Physiol 1986; 250: R595±R607. The mRNA levels of PFK-2 were not signi®cantly 7 Hers HG, Hue L. Gluconeogenesis and related aspects of different between the two types of rats, both decreased glycolysis. Ann Rev Biochem 1983; 52: 617±653. during starvation. However, the decrease in the 8 Pilkis SJ, Claus TH, Kurland IJ, Lange AJ. 6-phosphofructo- 2-kinase=fructose-2,6-bisphosphatase: a metabolic signaling mRNA of PFK-2 is more apparent in the obese rats enzyme. Annu Rev Biochem 1995; 64: 799±835. than in the lean controls. The different behaviour of 9 Van Schaftingen E. Fructose 2,6-bisphosphate. Adv Enzymol this bifunctional enzyme could be explained by the 1987; 59: 315±395. Gene expression in obese (fa=fa) Zucker rats JX PeÂrez et al 672 10 Pilkis SJ, El-Maghrabi MR, Claus TH. Fructose-2,6-bis- 29 El-Maghrabi MR, Correia JJ, Heil PJ, Pate TM, Cobb C, Pilkis phos-phate in control of hepatic gluconeogenesis. From meta- SJ. Tissue distribution, immunoreactivity and physical proper- bolites to molecular genetics. Diabetes Care 1990; 13: ties of 6-phosphofructo-2-kinase=fructose-2,6-bisphosphatase. 582±599. Proc Natl Acad Sci USA 1986; 83: 5005±5009. 11 Hue L, Van de Werve G, Jeanrenaud B. Fructose-2,6-bisphos- 30 McCune SA, Durant PJ, Jenkins PA, Harris RA. Comparative phate in livers of genetically obese rats. Biochem J 1983; 214: studies on fatty acid synthesis, glycogen , and 1019±1022. gluconeogenesis by hepatocytes isolated from lean and obese 12 Van de Werve G, Jeanrenaud B. The onset of liver glycogen Zucker rats. Metabolism 1981; 30: 1170±1178. synthesis in fasted-refed lean and genetically obese ( fa=fa) 31 Terrettaz J, Jeanrenaud B. Contribution of glycerol and rats. Diabetologia 1987; 30: 169±174. to basal hepatic glucose production in the genetically obese 13 SaÂnchez-GutieÂrrez JC, SaÂnchez-Arias JA, Lechuga CG, Valle ( fa=fa) rat. Biochem J 1990; 270: 803±807. JC, Samper B, FelõÂu JE. Decreased responsiveness of basal 32 Carb N, LoÂpez-Soriano F, ArgileÂs JM. Glucose handling by gluconeogenesis to insulin action in hepatocytes isolated from hepatocytes from obese Zucker rats. Biosci Rep1991; 11: 285± genetically obese ( fa=fa) Zucker rats. Endocrinology 1994; 292. 134: 1868±1873. 33 Jeanrenaud B. Neuroendocrine and metabolic basis of type II 14 SaÂnchez-GutieÂrrez JC, Lechuga CG, SaÂnchez-Arias JA, diabetes as studied in animal models. Diabetes Metab Rev Samper B, FelõÂu JE. Impairment of the modulation by glucose 1988; 4 603±614. of hepatic gluconeogenesis in the genetically obese ( fa=fa) 34 Bloxham DP, York DA. Metabolic ¯ux through phosphofruc- Zucker rats. Endocrinology 1995; 136: 1877±1884. tokinase and fructose 1,6-diphosphatase and its relation to 15 Auffray C, Rougeon F. Puri®cation of mouse immunoglobulin lipogenesis in genetically obese rats. Biochem Soc Transac heavy-chain messenger RNAs from total myeloma tumor 1976; 4: 989±993. RNA. Eur J Biochem 1980; 107: 303±314. 35 Martin RJ, Cahagan J. Serum hormone levels and tissue 16 Sambrook J, Fristsch EF, Maniatis T. Molecular Cloning: A metabolism in pair-fed lean and obese Zucker rats. Horm Laboratory Manual (2nd edn). Cold Spring Harbor Laboratory Metab Res 1977; 9: 181±186. Press: Cold Spring Harbor, NY, 1989. 36 Spydevold SO, Greenbaum AL, Baquer NZ, McLean P. 17 Colosia AD, Marker AJ, Lange AJ, El-Maghrabi MR, Granner Adaptative responses of enzymes of carbohydrate and lipid DK, Tauler A, Pilkis J, Pilkis SJ. Induction of rat liver 6- metabolism to dietary alteration in genetically obese Zucker phosphofructo-2-kinase=fructose-2,6-bisphosphatase mRNA rats (fa=fa). Eur J Biochem1978; 89: 329±339. by refeeding and insulin. J Biol Chem 1988; 263: 18 669± 37 van de Werve G, Jeanrenaud B. Synthase activation is not a 18 667. prerequisite for glycogen synthesis in the starved liver. Am J 18 El-Maghrabi MR, Pilkis J, Marker AJ, Colosia AD, D'Angelo G, Physiol 1984; 247: E271±E275. Fraser BA, Pilkis SJ. cDNA sequence of rat liver fructose 1,6- 38 Granner D, Pilkis SJ. The genes of hepatic glucose metabol- bisphosphatase and evidence for down regulation of its mRNA ism. J Biol Chem 1990; 265: 10173±10176. by insulin. Proc Natl Acad Sci USA 1988; 85: 8430±8434. 39 Lemaigre FP, Rousseau GG. Transcriptional control of genes 19 Yoo-Warren H, Monohan JE, Short J, Short H, Bruzel A, that regulate glycolysis and gluconeogenesis in adult liver. Wynshaw-Boris A, Meisner HM, Samols D, Hanson RW. Biochem J 1994; 303: 1±14. Isolation and characterization of the gene coding for cytosolic 40 van de Werve G. Fasting enhances glycogen synthase activa- phosphoenolpyruvate carboxykinase (GTP) from the rat. Proc tion in hepatocytes from insulin-resistant genetically obese Natl Acad Sci USA 1983; 80: 3656±3660. ( fa=fa) rats. Biochem J 1990; 269: 789±794. 20 Andreone TL, Printz RL, Pilkis SJ, Magnuson MA, Granner 41 Terrettaz J, Jeanrenaud B. In vivo hepatic and peripheral DK. The amino acid sequence of rat liver glucokinase deduced insulin resistance in genetically obese (fa=fa) rats. Endocri- from cloned cDNA. J Biol Chem 1989; 264: 363±369. nology 1983; 112: 1346±1351. 21 Lone YCh, Simon MP, Kahn A, Marie J. Complete nucleotide 42 Iynedjian PB, Jotterand D, Nouspikel T, Asfari M, Pilot PR. and deduced amino acid sequences of rat L-type pyruvate Transcriptional induction of glucokinase gene by insulin in kinase. FEBS Lett 1986; 195: 97±100. cultured liver cells and its repression by the glucagon-cAMP 22 Gerlach WL, Bedbrook JR. Cloning and characterization of system. J Biol Chem 1989; 264: 21 824±21 829. ribosomal RNA genes from wheat and barley. Nucleic Acid 43 Noguchi T, Matsuda T, Tomari Y, Yamada K, Imai E, Wan Z, Res 1979; 7: 1869±1885. Ikeda H, Tanaka T. The regulation of gene expression by 23 Van Schaftingen E, Lederer B, Bartrons R, Hers HG. A kinetic insulin is differentially impaired in the liver of the genetically study of phosphate: fructose-6-phosphate obese-hyperglycemic wistar fatty rat. FEBS Lett 1993; 328: from potato tubers. Application to a microassay of fructose 145±148. 2,6-bisphosphate. Eur J Biochem 1982; 129: 191±195. 44 Vaulont S, Kahn A. Transcriptional control of metabolic 24 Bartrons R, Hue L, Van Schaftingen E, Hers HG. Hormonal regulation genes by carbohydrates. FASEB J 1994; 8: 28±35. control of fructose 2,6-bisphosphate concentration in isolated 45 Marker AJ, Colosia AD, Tauler A, Solomon DH, Cayre Y, Lange rat hepatocytes. Biochem J 1983; 214: 829±837. AJ, El-Maghrabi MR, Pilkis SJ. Glucocorticoid regulation 25 FelõÂu JE, Hue L, Hers HG. Regulation in vitro and in vivo of hepatic 6-phosphofructo-2-kinase=fructose-2,6-bisphos- of adenosine 30:50 monophosphate-dependent inactivation of phatase gene expression. J Biol Chem 1989; 264: 7000±7004. rat-liver pyruvate kinase type L. Eur J Biochem 1977; 81: 46 Rosa JL, Ventura F, Tauler A, Bartrons R. Regulation of 609±617. 6-phosphofructo-2-kinase=fructose-2,6-bisphosphatase gene ex- 26 Davidson AL, Arion WJ. Factors underlying signi®cant under- pression by glucagon. J Biol Chem 1993; 268: 22 540±22 545. estimations of glucokinase activity in crude liver extracts; 47 Crepin KM, Darville MI, Hue L, Rousseau GG. Starvation or physiological implications of higher cellular activity. Arch diabetes decreases the content but not the mRNA of 6-phospho- Biochem Biophys 1987; 253: 156±167. fructo-2-kinase in rat livers. FEBS Lett 1988; 227: 136±140. 27 Bradford M. A rapid and sensitive method for the quantitation 48 Miralpeix M, Carballo E, Bartrons R, Crepin K, Hue L, of microgram quantities of protein utilizing the principle of Rousseau GG. Oral administration of vanadate to diabetic protein-dye binding. Anal Biochem 1976; 72: 248±254. rats restores liver 6-phosphofructo-2-kinase content and 28 Burnette WN. `Western blotting': electrophoretic transfer of mRNA. Diabetologia 1992; 35: 243±248. proteins from sodium dodecyl sulfate-polyacrilamide gels to 49 Lemaigre FP, Lause P, Rousseau GG. Insulin inhibits gluco- unmodi®ed nitrocellulose and radiographic detection with corticoid-induced stimulation of liver 6-phosphofructo-2- antibody and radioiodinated protein A. Anal Biochem 1981; kinase=fructose-2,6-bisphosphatase gene transcription. FEBS 112: 195±203. Lett 1994; 340: 221±225.