Liver Glycogen Reduces Food Intake and Attenuates Obesity ...

Iliana López-Soldado,1,2 Delia Zafra,1,2 Jordi Duran,1,2 Anna Adrover,1 Joaquim Calbó,1,2 andJoan J. Guinovart1,2,3

Liver Glycogen Reduces FoodIntake and Attenuates Obesityin a High-Fat Diet–Fed MouseModelDiabetes 2015;64:796–807 | DOI: 10.2337/db14-0728

We generated mice that overexpress protein targetingto glycogen (PTG) in the liver (PTGOE), which results inan increase in liver glycogen. When fed a high-fat diet(HFD), these animals reduced their food intake. Theresulting effect was a lower body weight, decreasedfat mass, and reduced leptin levels. Furthermore, PTGoverexpression reversed the glucose intolerance andhyperinsulinemia caused by the HFD and protectedagainst HFD-induced hepatic steatosis. Of note, whenfed an HFD, PTGOE mice did not show the decrease inhepatic ATP content observed in control animals andhad lower expression of neuropeptide Y and higher ex-pression of proopiomelanocortin in the hypothalamus.Additionally, after an overnight fast, PTGOE animals pre-sented high liver glycogen content, lower liver triacylglyc-erol content, and lower serum concentrations of fattyacids and b-hydroxybutyrate than control mice, regard-less of whether they were fed an HFD or a standard diet.In conclusion, liver glycogen accumulation caused a re-duced food intake, protected against the deleteriouseffects of an HFD, and diminished the metabolic impactof fasting. Therefore, we propose that hepatic glycogencontent be considered a potential target for the pharma-cological manipulation of diabetes and obesity.

Liver glycogen acts as an energy store in times of nutritionalsufficiency for use in times of need. The metabolism of thispolysaccharide in the liver is controlled by the activities oftwo key enzymes: glycogen synthase (GS) and glycogenphosphorylase (GP) (1). GS is phosphorylated at multiplesites, which induces its inactivation, whereas GP is activated

by phosphorylation at a single site. Both enzymes are alsoregulated allosterically (2,3).

Glycogen-targeting subunits bind to glycogen and pro-tein phosphatase 1 (PP1) and facilitate the dephosphory-lation of GS and GP, thus activating the former andinactivating the latter. Six genes encode glycogen-targetingsubunits (4). Among these, protein targeting to glycogen(PTG) (PPP1R3C or PPP1R5), which is expressed in manytissues, has been shown to control glycogen stores invarious animal models (5–7).

Adenoviral PTG overexpression in the liver of normalrats increases glycogen and improves glucose tolerancewithout perturbing lipid metabolism (8). In a diabetic-focused approach, Yang and Newgard (9) showed thatadenoviral expression of PTG in the liver of STZ-diabeticrats increased glycogen content and reversed hyperglyce-mia and hyperphagia. Through a different approach, wereported that hepatic adenoviral expression of an activeform of liver GS (LGS), which also increases glycogencontent, in STZ-diabetic rats reduced food intake andhyperglycemia (10).

Russek (11) was the first to propose a hepatostatictheory of food intake, which was further redefined asa glycogenostatic model by Flatt (12). This model predictsthat individuals consume food to a level that maintainsglycogen levels in the body (12). In fact, many lines ofexperimental evidence establish a correlation between thesize of liver glycogen stores and food intake (13,14); how-ever, other results have argued against this hypothesis(15–17). The results in Yang and Newgard (9) and Roset al. (10) support the notion that liver glycogen is a factor

1Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain2CIBERDEM, Madrid, Spain3Department of Biochemistry and Molecular Biology, University of Barcelona,Barcelona, Spain

Corresponding author: Joan J. Guinovart, [email protected].

Received 7 May 2014 and accepted 27 September 2014.

© 2015 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

796 Diabetes Volume 64, March 2015

METABOLISM

Dow

nloaded from http://diabetesjournals.org/diabetes/article-pdf/64/3/796/415374/db140728.pdf by guest on 20 January 2022

http://crossmark.crossref.org/dialog/?doi=10.2337/db14-0728&domain=pdf&date_stamp=2015-02-11

mailto:[email protected]

controlling food intake in hyperphagic type 1 diabeticanimals. However, these studies had the constraint ofusing adenovirus transduction in animal models, whichlimit the experimental period to 1 week. In the presentstudy, we examined whether a sustained increase inliver glycogen stores regulates food intake. For this pur-pose, we generated mice that overexpress PTG specifi-cally in the liver (PTGOE), which results in a sustainedincrease in hepatic glycogen, and fed them either a stan-dard diet or a high-fat diet (HFD). The HFD-fed animalis a suitable model for studying impaired glucose toler-ance and type 2 diabetes (18), and prolonged ingestionof an HFD is associated with overconsumption andobesity (19).

We show that when fed an HFD, PTGOE animals hada reduced food intake and presented a lower body weightand fat mass than control animals. These results identifyliver glycogen stores as a regulator of food intake ina model of hyperphagia and obesity and suggest thatstrategies to increase liver glycogen may provide a treat-ment option for diabetes and obesity.

RESEARCH DESIGN AND METHODS

MiceTo generate mice that overexpress PTG, targeting-vectorconstruction and a site-directed transgenic strategy weredesigned and performed by genOway (Lyon, France).Briefly, the PTG cDNA under the control of the ubiquitousCAG promoter (cytomegalovirus immediate early enhancer/chicken b-actin promoter fusion) was introduced into aninnocuous locus by homologous recombination. A loxP-flanked transcription stop cassette was included betweenthe CAG promoter and the PTG cDNA to allow the ex-pression to depend on the action of a Cre recombinase.The resulting mouse line was bred with an albumin pro-moter Cre recombinase–expressing animal (The JacksonLaboratory), which drove the expression of PTG specif-ically to the liver. All mice studied were littermates.Mice were maintained on a 12:12-h light-dark cyclewith free access to water and fed a standard diet (HarlanLaboratories) or an HFD (45% kcal fat; catalog #D12451;Research Diets) for 16 weeks, starting at 6 weeks of age.

Blood and Liver Biochemical AnalysisLiver glycogen content was determined by an amyloglucosidase-based assay, as described elsewhere (20). LGS activity wasdetermined as previously described in the presence or absenceof Glc-6-P (10). GS activity measured in the presence of sat-urating Glc-6-P [(+) Glc-6-P] corresponds to the total amountof enzyme, whereas measurement in its absence [(2) Glc-6-P]is an indication of the active (unphosphorylated) GS form.The (2) Glc-6-P/(+) Glc-6-P activity ratio (GS activity ratio)is an estimation of the activation state of the enzyme.The intracellular concentration of ATP was measuredfrom perchloric acid extracts of livers using a previouslydescribed fluorimetric method (21). Triglycerides in liver

were quantified using a triacylglyceride kit (Sigma-Aldrich) in 3 mol/L KOH and 65% ethanol extracts basedon the method described by Salmon and Flatt (22) forliver saponification. Blood glucose levels were measuredusing a glucometer (Ascensia Breeze2; Bayer HealthCare).Serum concentrations of b-hydroxybutyrate (Sigma-Aldrich),triacylglycerides (Sigma-Aldrich), and nonesterified fattyacids (Wako) were measured spectrophotometrically.Plasma insulin and leptin were analyzed by ELISA (Crys-tal Chem).

Glucose and Insulin Tolerance TestsFor glucose tolerance tests (GTTs), overnight-fasted(16 h) mice were injected with glucose 2 g/kg i.p. Wholeblood was drawn from the tail tip for glucose measure-ments. In vivo glucose-stimulated insulin secretion wasdetermined in separate experiments. For insulin toler-ance tests (ITTs), mice fasted for 6 h were injected withinsulin 0.75 units/kg i.p., and glycemia was measuredfrom tail blood taken at the indicated times afterinjection.

RNA Preparations and Quantitative RT-PCRTissue preparation, RNA extraction, RT-PCR, and quantita-tive real-time PCR analyses were performed as described(23). The following TaqMan primer/probe sets (Applied Bio-systems, Madrid, Spain) were used for quantitative RT-PCR:PTG (Mm01204084_m1), Hprt (Mm00446968_m1), pyru-vate kinase (Pklr) (Mm00443090_m1), fatty acid synthase(Fasn) (Mm00662319_m1), acetyl CoA carboxylase (Acc1a)(Mm01304257_m1), SREBP1 (Mm00550338_m1), peroxi-some proliferator–activated receptor (PPAR) g (Mm01184322_g1), monoacylglycerol O-acyltransferase 1 (MGAT1)(Mm00503358_m1), glucokinase (GK) (Mm00439129),Ucp1 (Mm01244861_m1), Ucp2 (Mm00495907_g1), Ucp3(Mm00494077_m1), PPARa (Mm00440939_m1), Fgf21(Mm00840165_g1), neuropeptide Y (NPY) (Mm03048253),proopiomelanocortin (POMC) (Mm 00435874_m1), car-nitine palmitoyltransferase 1a (Cpt1a) (Mm01231183_m1),acyl-CoA oxidase (Acox1) (Mm01246834_m1), and Ppia(Mm02342429_g1). Ppia was used as a housekeeping genein the liver. Hprt was used as a housekeeping gene in thehypothalamus and brown adipose tissue.

Indirect Calorimetry, Food Intake, and BodyTemperatureIndirect calorimetry was performed using an eight-chamberOxymax system (Columbus Instruments) to measure heatproduction, which was calculated from oxygen consumptionand CO2 production. Mice were allowed to acclimate to thecages for 2 days before one or two cycles of 24-h measure-ments. Energy expenditure was calculated as (3.185 + 1.2323RER) 3 VO2 (24), where the RER (respiratory exchange ra-tio) = VCO2/VO2. Glucose oxidation (in g/min/kg0.75 =[(4.545 3 VCO2) 2 (3.205 3 VO2)] / 1,000) and lipid ox-idation (in g/min/kg0.75 = [1.6723 (VO2 2 VCO2)] / 1,000)were calculated. Ambulatory and total locomotor activitywas monitored by an infrared photocell beam interruption

diabetes.diabetesjournals.org López-Soldado and Associates 797

Dow


method. Body temperature was determined using an animalrectal probe thermometer (Cibertec). To monitor food intake,mice were housed individually and acclimatized for 1 weekbefore study. Food intake was measured daily for 5 consecu-tive days. Epididymal adipose tissue was removed and pre-pared in paraffin after fixation in 10% phosphate-bufferedformalin. Hematoxylin-eosin stains were then performed.To measure the size of the adipocytes, total adipocyte areawas traced manually and analyzed. White adipocyte areaswere measured in $100 cells per mouse in each group.

Oil Red O StainingLiver tissues were embedded in optimal cutting temperaturecompound (Sakura Finetek) and frozen. Frozen tissues werecut into 5-mm-thick cryosections and stained with Oil Red O(Sigma-Aldrich).

StatisticsData are expressed as mean 6 SEM. P values were calcu-lated using unpaired Student t test, two-way ANOVA, orthree-way ANOVA with post hoc Tukey tests as appropri-ate. P , 0.05 was considered significant.

Study ApprovalAll procedures were approved by Barcelona SciencePark’s Animal Experimentation Committee and were car-ried out in accordance with the European Community

Council Directive and the National Institutes ofHealth guidelines for the care and use of laboratoryanimals.

RESULTS

Generation and Characterization of Mice With Liver-Specific PTG OverexpressionTo increase the accumulation of glycogen in the liver, wegenerated mice that overexpress PTG specifically in theliver (PTGOE) (see RESEARCH DESIGN AND METHODS). ThemRNA level of PTG in the livers of these mice was 12-fold greater than that of control animals (Fig. 1A). Asexpected, LGS was activated because PTG, by targetingPP1 to the glycogen particle, maintains GS and GP ina dephosphorylated state (7). As expected, LGS was acti-vated in these mice (Fig. 1B).

Mice were fed a standard diet or an HFD. When fed,the PTGOE mice showed an approximately twofold in-crease in liver glycogen content compared with controlanimals (Fig. 1C) regardless of whether they receiveda standard diet or an HFD (Fig. 1C).

After an overnight fast, PTGOE mice showed decreasedhepatic glycogen content compared with the fed state. Thisobservation indicates net mobilization of liver glycogen,although this glycogen was not depleted to the same extent

Figure 1—Characterization of mice with liver-specific PTG overexpression fed a standard diet or an HFD. Control and liver PTGOE miceaged 6 weeks were fed a standard diet or an HFD for 16 weeks. Fed and 16-h fasted mice were killed. A: Relative mRNA of PTG in the liverunder fed conditions. B: Liver GS activity expressed as the ratio of (2) Glc-6-P/(+) Glc-6-P under fed conditions. C: Liver glycogen contentunder fed conditions or a 16-h fast. D: Quadriceps muscle glycogen content under fed conditions. Data are mean 6 SEM. n = 5–8/group.*P < 0.05 between control and PTGOE mice fed a standard diet or an HFD; ^P < 0.05 between control fed mice and control 16-h fastedmice fed a standard diet or an HFD; #P < 0.05 between fed PTGOE mice and fasted PTGOE mice; §P < 0.05 between control fasted micefed a standard diet or an HFD. SD, standard diet.

798 Liver Glycogen Reduces Food Intake Diabetes Volume 64, March 2015

Dow


as in control mice (Fig. 1C). Moreover, when fasted, thecontrol mice receiving the HFD showed a higher hepaticglycogen content than the fasted control animals fed thestandard diet, as previously reported (25) (Fig. 1C).Skeletal muscle glycogen content was similar betweenthe groups and, thus, consistent with the idea that gly-cogen synthesis in nonhepatic tissues was not altered(Fig. 1D).

HFD-Fed PTGOE Mice Have a Lower Food Intake andReduced Obesity

Mice fed an HFD present hyperphagia and obesity (26,27).In the current experiments, mice of both genotypes feda standard diet had similar body weights (Fig. 2A). Thoseon an HFD became obese compared with their counter-parts fed a standard diet (Fig. 2A). However, the increasein body weight in PTGOE animals fed an HFD was smaller

Figure 2—HFD-fed PTGOE mice have a lower food intake and reduced obesity. Control and liver PTGOE mice aged 6 weeks were feda standard diet or an HFD for 16 weeks. Fed and 16-h fasted mice were killed. A: Growth curve. Body weights were measured every otherweek. B: Epididymal adipose tissue. C: Subcutaneous adipose tissue. D: Hematoxylin-eosin staining of epididymal adipose tissue (scalebar = 200 mm) and adipocyte size from epididymal adipose tissue. E: Fasting serum leptin. F: Food intake. G: Quantitative real-time PCRshowing relative mRNA level of POMC and NPY in the hypothalamus of mice fed an HFD. H: Relative liver ATP content of mice feda standard diet or an HFD. Data are mean 6 SEM. n = 5–20/group. *P < 0.05 between control and PTGOE mice fed a standard diet or anHFD; #P < 0.05 between standard diet and HFD. SD, standard diet.


Dow


than that detected in control mice on the same diet (Fig.2A). After 16 weeks on an HFD, PTGOE animals showeda reduction in epididymal and subcutaneous fat weight,whereas when fed a standard diet, they had a similar fatweight as their control littermates (Fig. 2B and C). Controlanimals on an HFD had larger adipocytes than PTGOE miceon the same diet (Fig. 2D). Consistent with the reducedadiposity of PTGOE mice fed an HFD, serum leptin concen-tration was significantly lower in these animals comparedwith control mice on the same diet, whereas no differences

in serum leptin were found between the two genotypeswhen fed a standard diet (Fig. 2E). The decreased adiposityand reduced HFD-induced obesity could have resulted fromreduced food intake, increased energy expenditure, or fatmalabsorption. PTGOE mice on an HFD showed a reduceddaily food intake of ;20% compared with control mice fedthe same diet (Fig. 2F). The hypothalamus is the masterregulator of energy intake, energy expenditure, andbody weight (28). In the hypothalamus of PTGOE micefed an HFD, the expression of the satiating neuropeptide

Figure 3—Energy expenditure in control and PTGOE animals fed a standard diet or an HFD. Control and liver PTGOE mice aged 6 weeks were feda standard diet or an HFD for 16 weeks. A: Resting oxygen consumption during the light and dark phases and total animals fed a standard diet. B:Resting oxygen consumption during the light and dark phases and total animals fed an HFD. C: RER during the light and dark phases and totalanimals fed a standard diet. D: RER during the light and dark phases and total animals fed an HFD. E: Locomotor activity (ambulation) during the lightand dark phases and total activity of animals fed a standard diet. F: Locomotor activity (ambulation) during the light and dark phases and total activityof animals fed an HFD. G: Locomotor activity (total counts) during the light and dark phases and total activity of animals fed a standard diet. H:Locomotor activity (total counts) during the light and dark phases and total activity of animals fed an HFD. Data are mean6 SEM. n = 6–8/group. SD,standard diet.


Dow


POMC was higher, whereas that of the orexigenic peptideNPY was lower than in control mice fed the same diet(Fig. 2G).

We also determined the total daily energy expenditure inthe two genotypes by measuring VO2 and VCO2. When feda standard diet, control and PTGOE mice had a similar oxygenconsumption (Fig. 3A), energy expenditure (standardized forbody weight) (Fig. 3C), locomotor activity (Fig. 3E and G),and RER (Fig. 4A). No significant difference in oxygen

consumption (Fig. 4B), energy expenditure (Fig. 4D), orlocomotor activity (Fig. 3B and D) was observed betweenthe HFD-fed groups. To examine the influence of liverglycogen content on thermogenesis, mRNA expression ofUcp1, a marker of thermogenesis, was analyzed in brownadipose tissue. No difference was found in the levels ofUcp1 mRNA between control and PTGOE mice fed anHFD (Fig. 4G). Other genes involved in energy expenditure,such as Ucp2 and Ucp3, were also analyzed, and no

Figure 4—RER, glucose, and lipid oxidation. Control and liver PTGOE mice aged 6 weeks were fed a standard diet or an HFD for 16 weeks.A: RER during the light and dark phases and total animals fed a standard diet. B: RER during the light and dark phases and total animals fedan HFD. C: Glucose oxidation during the light and dark phases and total animals fed a standard diet. D: Glucose oxidation during the lightand dark phases and total animals fed an HFD. E: Lipid oxidation during the light and dark phases and total animals fed a standard diet. F:Lipid oxidation of animals during the light and dark phases and total animals fed an HFD. G: Quantitative real-time PCR showing relativemRNA levels of Ucp1, Ucp2, and Ucp3 in the brown adipose tissue of animals fed an HFD. H: Core body temperature of animals fed anHFD. Data are mean 6 SEM. n = 6–8/group. *P < 0.05 between control and liver PTGOE mice fed a standard diet or an HFD. BAT, brownadipose tissue; SD, standard diet.


Dow


differences were found between the groups (Fig. 4G). Nochanges in core body temperature were detected in the twogenotypes (Fig. 4H). Moreover, there were no differencesin stool lipid content (data not shown).

During the feeding period (dark phase), the RER wasslightly increased in HFD-fed PTGOE mice, thereby indicat-ing that these animals used more carbohydrates as an en-ergy source than the control group at night (Fig. 4B). Theseresults were confirmed by calculating the amount of glucoseoxidized, which was increased in PTGOE mice fed an HFD(Fig. 4D). However, PTGOE fed a standard diet oxidized thesame amount of glucose as control mice (Fig. 4C). No changein lipid oxidation was found in PTGOE mice fed a standarddiet (Fig. 4E) or an HFD (Fig. 4F). Because glucose oxidationwas higher in PTGOE fed an HFD, we addressed hepatic ATPcontent. It is known that this parameter is reduced in thelivers of HFD-induced diabetic mice (29). The ATP content inthe livers of HFD-fed mice was significantly reduced, andPTG overexpression resulted in an ATP content similar tothat of mice fed a standard diet (Fig. 2H).

Effects of Liver PTG Overexpression on Blood Glucose,Insulin Levels, Glucose Tolerance, and InsulinSensitivityThe liver plays a key role in the clearance of blood glucosein the postprandial state (30). Fed PTGOE animals hadlower blood glucose levels than control littermates, regard-less of the diet received (Fig. 5A). Blood glucose levels andplasma insulin concentration decreased in control animalswhen they were deprived of food for 16 h (Fig. 5A and B).However, fasted PTGOE mice had similar glucose and in-sulin levels as fed PTGOE mice. This effect was observedregardless of the diet given (Fig. 5A and B). Moreover,HFD-fed PTGOE mice showed lower levels of insulin inthe fed state compared with control mice on the samediet (Fig. 5B).

We next performed an intraperitoneal GTT on all fourexperimental groups. PTGOE mice fed a standard diet hadbetter glucose tolerance, with a 40% decrease in the areaunder the curve (AUC) compared with control mice. Whensubjected to an HFD, these mice also showed reduced glu-cose intolerance and presented a 25% decrease in the AUCcompared with control littermates (Fig. 5C). Remarkably,when fed an HFD, PTGOE mice presented a glucose toler-ance curve analogous to that of control animals fed a stan-dard diet (Fig. 5C).

We also measured insulin secretion during the intra-peritoneal GTT. In response to an HFD, PTGOE mice pre-sented a reduction in glucose-stimulated insulin releasecompared with control mice (Fig. 5D). Of note, the insulinrelease in the former animals was similar to that of PTGOE

animals fed a standard diet (Fig. 5D).Next, an ITT was performed after a 6-h fast. In these

conditions, PTGOE mice fed an HFD already had signifi-cantly lower blood glucose concentration than the HFD-fed control mice (14 6 1.7 vs. 10 6 0.3 mmol/L), whichmade the analysis of the results of the ITT difficult to

compare (Fig. 5E). However, when the ITT was expressedas the percentage of the initial values, the curve for HFD-fed animals was similar in both genotypes (Fig. 5F). Ofnote, PTGOE mice fed a standard diet presented higherblood glucose levels 60 min after the insulin injection.This finding suggests that these animals had a faster re-covery from the hypoglycemia induced by insulin thantheir control littermates (Fig. 5E and F).

Liver PTG Overexpression Reduces HFD-InducedHepatic SteatosisWe also analyzed the effect of PTG overexpression on thestorage of liver triacylglycerides. When fed a standard diet,PTGOE mice presented a similar liver triacylglycerol contentas their control littermates (Fig. 6A and B), suggesting thatPTG is not associated with lipid metabolism under thesecircumstances. Moreover, the expression of genes relatedto de novo lipogenesis were not modified in PTGOE animalsfed a standard diet (Fig. 6C). However, when fed an HFD,these animals showed a lower hepatic triglyceride content(Fig. 6A and B), which was associated with the downregu-lation of SREBP1, GK, PPARg, and MGAT1 gene expression(Fig. 6C and D). As has been previously described (31), weconfirmed that PPARg and MGAT1 expression was verylow in normal liver but was highly express in fatty liver(Fig. 6D). There were no statistically significant differencesin the expression of lipogenic genes, such as Pklr, Fasn, andAcc1a, between the HFD-fed groups (Fig. 6C). Furthermore,the expression of genes related to lipid oxidation was eval-uated. No differences between genotypes were found in theexpression of PPARa, Cpt1a, or Acox1 in the liver (Fig. 6E).

Liver PTG Overexpression Diminishes the MetabolicImpact of FastingMany metabolic changes take place during fasting. Aspreviously mentioned, PTGOE mice fed either a standarddiet or an HFD did not show reduced levels of blood glu-cose or insulin after an overnight fast (Fig. 5A and B).Moreover, serum nonesterified fatty acids (Fig. 7A) andb-hydroxybutyrate (Fig. 7B) were lower in fasted PTGOE

animals. It is well-known that after an overnight fast,hepatic triacylglycerol content increases in mice (32,33).However, PTGOE mice presented a lower fasting liver tri-acylglycerol content compared with control mice (Fig. 7Cand D). During fasting, the expression of the hepatokineFgf21 increased in the liver, but no differences were foundin the expression of Fgf21 between genotypes (data notshown).

DISCUSSION

Using mice that overexpress PTG specifically in the liver,we examined the impact of liver glycogen on food intake.The overexpression of this protein caused an increase inhepatic glycogen stores in mice. When fed an HFD, theseanimals decreased their food intake and had a lower bodyweight and decreased fat mass. Changes in key regulatorsof food intake in the hypothalamus support the decrease


Dow


in appetite observed in these animals. Expression ofPOMC, an anorexigenic signal, increased, whereas thatof orexigenic NPY decreased. These data support the ideathat liver glycogen stores regulate food intake, thusreinforcing the glycogenostatic theory (12). However, inthe present study, this effect was limited to hyperphagicconditions, such as HFD. Friedman (34) proposed thatchanges in glycogen stores do not necessarily signal

changes in food intake; rather, the partitioning of carbo-hydrates in and out of glycogen affects eating behavior byaltering fuel fluxes, and, by analogy to fat fuels, shiftsbetween oxidation and storage of carbohydrate fuels in-fluence food intake (34). PTGOE mice used more carbohy-drates as an energy source than control animals duringthe dark phase, which is when mice typically eat more.This increase in carbohydrate oxidation was observed in

Figure 5—Effects of liver PTG overexpression on blood glucose, insulin levels, glucose tolerance, and insulin sensitivity. Control and liverPTGOE mice aged 6 weeks were fed a standard diet or an HFD for 16 weeks. A: Blood glucose concentration in fed, 6-h fasted, and 16-hfasted conditions. B: Plasma insulin concentration in fed and 16-h fasted conditions. For GTTs, mice were fasted for 16 h and injected with2 g glucose/kg body weight i.p. C: GTT curve for glucose 12 weeks after being put on a standard diet or an HFD and glucose AUC. D: GTTcurve for insulin 12 weeks after being put on a standard diet or an HFD and insulin AUC. For ITTs, mice were fasted for 6 h and injected with0.75 units insulin/kg body weight i.p. E: ITT 14 weeks after being put on a standard diet or an HFD, expressed as absolute glucose. F: ITT 14weeks after being put on a standard diet or an HFD, expressed as the percentage of initial values. Data are mean 6 SEM. n = 5–14/group.*P < 0.05 between control and PTGOE mice fed a standard diet or an HFD; ^P < 0.05 between control fed mice and control 16-h fastedmice fed a standard diet or an HFD; #P < 0.05 between a standard diet and an HFD. SD, standard diet.


Dow


animals fed an HFD but not a standard diet. These datahighlight the role of liver glycogen stores in modulatingenergy substrate utilization in response to an HFD. ATP isa final product of the oxidation of glucose and fatty acids.It has been proposed that a decrease in the amount ofhepatic ATP is a metabolic stimulus that triggers feedingbehavior (35,36). We found that consumption of an HFDdecreases hepatic ATP levels. Of note, PTGOE mice main-tained hepatic ATP content when fed an HFD. We pro-pose that increased liver glycogen stores, through the

maintenance of liver energy status, contribute to de-creased appetite and adiposity. This effect is probablytriggered by signals from the liver that are carried tothe brain by vagal sensory neurons, as previously reported(37,38).

In addition, PTGOE mice fed a standard diet showedimproved glucose tolerance. Consistent with this notion,overexpression of PTG induced by adenovirus in normalrats resulted in a modest improvement of glucose toler-ance; however, these animals failed to show lower

Figure 6—Liver PTG overexpression reduces HFD-induced hepatic steatosis. Control and liver PTGOE mice aged 6 weeks were feda standard diet or an HFD for 16 weeks. Fed mice were killed. A: Fed liver triacylglycerol content. B: Lipid deposition as indicated by OilRed O staining in liver sections from fed mice in the standard diet and HFD groups. Original magnification 310. C: Quantitative real-timePCR showing relative mRNA levels of SREBP1, GK, Fasn, Acc1a, and Pklr from the liver of mice fed a standard diet or an HFD.D: Quantitative real-time PCR showing relative mRNA level of PPARg2 and MGAT1 in the livers of mice fed a standard diet or anHFD. E: Quantitative real-time PCR showing relative mRNA level of PPARa, Cpt1a, and Acox in the livers of mice fed a standard dietor an HFD. Data are mean 6 SEM. n = 5–10/group. *P < 0.05 between control and PTGOE mice fed a standard diet or an HFD; #P < 0.05between the standard diet and HFD. SD, standard diet.


Dow


glycogen levels in response to fasting (8). In our model ofliver PTGOE mice, the animals degraded glycogen in re-sponse to fasting, although they were not able to com-pletely deplete the stores of this polysaccharide aftera 16-h fasting period. More importantly, PTG overexpres-sion reversed HFD-induced glucose intolerance and hyper-insulinemia. Similar studies showed that expression—usingadenovirus—of other targeting subunit isoforms of PP1,such as the truncated version of muscle isoform termed“GMDC,” ameliorated glucose intolerance in rats fed anHFD but did not reduce the high fasting insulin levelsof these animals (39).

Also noteworthy was the effect of PTG overexpressionin decreasing hepatic steatosis induced by HFD. Thereduction in feeding observed in PTGOE mice fed an HFDmay account for the lower levels of hepatic triacylglycerolin the fed condition. This decrease in hepatic steatosiswas associated with a decrease in the expression of PPARgand MGAT1. PPARg is a transcriptional factor that par-ticipates in hepatic steatosis in rodents (40,41), andPPARg-regulated MGAT1 expression has been describedas responsible for lipid accumulation in diet-induced he-patic steatosis (31). SREBP1 and GK expression weredownregulated in the HFD-fed PTGOE group. SREBP1and GK expression is stimulated by insulin (42–44), andboth genes contribute to hepatic steatosis (45,46).

Because PTG overexpression reversed HFD-inducedhyperinsulinemia, the lower insulin levels in PTGOE micemay account for the downregulation of SREBP1 and GK.The expression of some of the main lipogenic genes reg-ulated by SREBP1, such as Acc1a and Fasn (47), as well asgenes related to lipid oxidation was not statistically dif-ferent between the genotypes. We therefore conclude thatneither changes in de novo lipogenesis nor changes inlipid oxidation contributed to the decrease in hepatic lipidcontent in the HFD-fed PTGOE mouse group.

A recent study revealed that glycogen shortage in theliver triggers the liver-brain-adipose tissue neural axisindependently of glucose and insulin/glucagon levels, thusplaying a key role in switching the fuel source fromglycogen to triglycerides under prolonged fasting con-ditions (48). The present results support these observa-tions because overnight-fasted PTGOE mice had higherglycogen levels than control mice. Consequently, manyof the changes in metabolism that occur during fastingwhen the glycogen supply dwindles were attenuated inthese animals. PTGOE mice showed a markedly lower he-patic lipid content during fasting, which could be attribut-able to a lower flux of fatty acids arriving from adiposetissue to the liver. These animals also showed lower levelsof fatty acids and b-hydroxybutyrate in serum under fast-ing conditions, suggesting that lipolysis in adipose tissue

Figure 7—Fasting metabolic changes were attenuated in liver PTGOE animals. Control and liver PTGOE mice aged 6 weeks were feda standard diet or an HFD for 16 weeks. Mice fasted for 16 h were killed. A: Fasting serum nonesterified fatty acids. B: Fasting serumb-hydroxybutyrate. C: Fasting liver triacylglycerol content. D: Lipid deposition as indicated by Oil Red O staining in liver sections from mice inthe standard diet and HFD groups. Original magnification310. Data are mean6 SEM. n = 5–8/group. *P< 0.05 between control and PTGOE

mice fed a standard diet or an HFD; #P < 0.05 between the standard diet and HFD. NEFA, nonesterified fatty acid; SD, standard diet.


Dow


is attenuated, as is ketogenesis in the liver. Although theimpact of increased PTG or increased hepatic glycogen isconsistent throughout the literature, the effects of de-creased PTG appear to be more complex. In a first report(49), it was shown that heterozygous deletion of PTG inmice reduced glycogen levels and induced glucose intoler-ance and insulin resistance with age. These observationsagree with other studies of animal models where glycogenstores are decreased by other means, such as depletion ofLGS (50). However, a recent article reported that totalablation of PTG reduced fasting glucose and insulin levelsin obese mice while improving insulin sensitivity andblocking hepatic steatosis during fasting in HFD-fedmice (25). Changes in the background of the animalsused may explain these differences and may indicatethat the consequences of decreased PTG are greatly influ-enced by other factors. It is also important to stress thatin that study (25), homozygous deletion of PTG was con-stitutive, and the effects caused by PTG depletion werenot circumscribed to the liver. Therefore, PTG depletionin other tissues, such as adipose tissue, may affect thewhole picture. In contrast, we generated mice with aliver-specific overexpression of PTG to avoid interferenceswith the effect of PTG in other tissues.

The present results demonstrate that liver glycogenaccumulation prevents HFD-induced glucose intolerance,decreases food intake, and lowers body weight. In conclu-sion, the results point to hepatic glycogen content asa potential target for the pharmacological manipulation ofdiabetes and obesity.

Acknowledgments. The authors thank Ramon Gomis, Rosa Gasa, MarcSchneeberger, and Marc Claret, Institut d’Investigacions Biomèdiques August Pi iSunyer (IDIBAPS) (Barcelona, Spain), for useful suggestions. They also thank thefollowing members of IRB Barcelona: Mar García Rocha for help and advice;Manuel Gris, Emma Veza, Natalia Plana, and Nuno Vasconcelos for technicalassistance; Antonio Berenguer for advice on the statistical analysis of the data;and Tanya Yates for correcting the English version of the manuscript.Funding. This study was supported by grants from the Spanish Ministry ofEconomy and Competitiveness (BFU2011-30554) and the CIBERDEM (Instituto deSalud Carlos III, Ministerio de Ciencia e Innovación).None of the supporting agencies had any role in establishing the work or inwriting the manuscript.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. I.L.-S. contributed to the study design, data re-search, and writing and revision of the manuscript. D.Z. contributed to the studydesign, data research, and revision of the manuscript. J.D. designed the PTGOE

mice. A.A. contributed to the data research and revision of the manuscript. J.C.contributed to the study design and revision of the manuscript. J.J.G. contributedto the study design and writing and revision of the manuscript. J.J.G. is theguarantor of this work and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and the accuracy of the dataanalysis.

References1. Roach PJ, Depaoli-Roach AA, Hurley TD, Tagliabracci VS. Glycogen and itsmetabolism: some new developments and old themes. Biochem J 2012;441:763–787

2. Gomis RR, Ferrer JC, Guinovart JJ. Shared control of hepatic glycogensynthesis by glycogen synthase and glucokinase. Biochem J 2000;351:811–8163. von Wilamowitz-Moellendorff A, Hunter RW, García-Rocha M, et al. Glucose-6-phosphate-mediated activation of liver glycogen synthase plays a key role inhepatic glycogen synthesis. Diabetes 2013;62:4070–40824. Newgard CB, Brady MJ, O’Doherty RM, Saltiel AR. Organizing glucosedisposal: emerging roles of the glycogen targeting subunits of protein phos-phatase-1. Diabetes 2000;49:1967–19775. Printen JA, Brady MJ, Saltiel AR. PTG, a protein phosphatase 1-bindingprotein with a role in glycogen metabolism. Science 1997;275:1475–14786. Brady MJ, Printen JA, Mastick CC, Saltiel AR. Role of protein targeting toglycogen (PTG) in the regulation of protein phosphatase-1 activity. J Biol Chem1997;272:20198–202047. Berman HK, O’Doherty RM, Anderson P, Newgard CB. Overexpression ofprotein targeting to glycogen (PTG) in rat hepatocytes causes profound activationof glycogen synthesis independent of normal hormone- and substrate-mediatedregulatory mechanisms. J Biol Chem 1998;273:26421–264258. O’Doherty RM, Jensen PB, Anderson P, et al. Activation of direct and in-direct pathways of glycogen synthesis by hepatic overexpression of proteintargeting to glycogen. J Clin Invest 2000;105:479–4889. Yang R, Newgard CB. Hepatic expression of a targeting subunit of proteinphosphatase-1 in streptozotocin-diabetic rats reverses hyperglycemia and hy-perphagia despite depressed glucokinase expression. J Biol Chem 2003;278:23418–2342510. Ros S, García-Rocha M, Calbó J, Guinovart JJ. Restoration of hepatic gly-cogen deposition reduces hyperglycaemia, hyperphagia and gluconeogenic en-zymes in a streptozotocin-induced model of diabetes in rats. Diabetologia 2011;54:2639–264811. Russek M. Participation of hepatic glucoreceptors in the control of intake offood. Nature 1963;197:79–8012. Flatt JP. Carbohydrate balance and body-weight regulation. Proc Nutr Soc1996;55:449–46513. Emery PW, Carpenter TT, Obeid OA. Alterations in postprandial glycogenand lipid synthesis in cachectic tumor-bearing rats. Nutr Cancer 1993;20:231–24014. Obeid OA, Powell-Tuck J, Emery PW. The postprandial rates of glycogenand lipid synthesis of lean and obese female Zucker rats. Int J Obes Relat MetabDisord 2000;24:508–51315. Shetty PS, Prentice AM, Goldberg GR, et al. Alterations in fuel selection andvoluntary food intake in response to isoenergetic manipulation of glycogen storesin humans. Am J Clin Nutr 1994;60:534–54316. Snitker S, Larson DE, Tataranni PA, Ravussin E. Ad libitum food intake inhumans after manipulation of glycogen stores. Am J Clin Nutr 1997;65:941–94617. Ji H, Friedman MI. Compensatory hyperphagia after fasting tracks recoveryof liver energy status. Physiol Behav 1999;68:181–18618. Winzell MS, Ahrén B. The high-fat diet-fed mouse: a model for studyingmechanisms and treatment of impaired glucose tolerance and type 2 diabetes.Diabetes 2004;53(Suppl. 3):S215–S21919. Woods SC, Seeley RJ, Rushing PA, D’Alessio D, Tso P. A controlled high-fatdiet induces an obese syndrome in rats. J Nutr 2003;133:1081–108720. García-Rocha M, Roca A, De La Iglesia N, et al. Intracellular distribution ofglycogen synthase and glycogen in primary cultured rat hepatocytes. BiochemJ 2001;357:17–2421. Goodman MN, Berger M, Ruderman NB. Glucose metabolism in rat skeletalmuscle at rest. Effect of starvation, diabetes, ketone bodies and free fatty acids.Diabetes 1974;23:881–88822. Salmon DM, Flatt JP. Effect of dietary fat content on the incidence of obesityamong ad libitum fed mice. Int J Obes 1985;9:443–44923. Ros S, Zafra D, Valles-Ortega J, et al. Hepatic overexpression of a consti-tutively active form of liver glycogen synthase improves glucose homeostasis.J Biol Chem 2010;285:37170–37177


Dow


24. Chen Y, Heiman ML. Increased weight gain after ovariectomy is nota consequence of leptin resistance. Am J Physiol Endocrinol Metab 2001;280:E315–E32225. Lu B, Bridges D, Yang Y, et al. Metabolic crosstalk: molecular links betweenglycogen and lipid metabolism in obesity. Diabetes 2014;63:2935–294826. Savastano DM, Covasa M. Adaptation to a high-fat diet leads to hyperphagiaand diminished sensitivity to cholecystokinin in rats. J Nutr 2005;135:1953–195927. Nefti W, Chaumontet C, Fromentin G, Tomé D, Darcel N. A high-fat dietattenuates the central response to within-meal satiation signals and modifies thereceptor expression of vagal afferents in mice. Am J Physiol Regul Integr CompPhysiol 2009;296:R1681–R168628. Schwartz MW, Porte D Jr. Diabetes, obesity, and the brain. Science 2005;307:375–37929. Wang C, Chen Z, Li S, et al. Hepatic overexpression of ATP synthase b

subunit activates PI3K/Akt pathway to ameliorate hyperglycemia of diabetic mice.Diabetes 2014;63:947–95930. Cherrington AD. Banting Lecture 1997. Control of glucose uptake and re-lease by the liver in vivo. Diabetes 1999;48:1198–121431. Lee YJ, Ko EH, Kim JE, et al. Nuclear receptor PPARg-regulated mono-acylglycerol O-acyltransferase 1 (MGAT1) expression is responsible for the lipidaccumulation in diet-induced hepatic steatosis. Proc Natl Acad Sci U S A 2012;109:13656–1366132. Kersten S, Seydoux J, Peters JM, Gonzalez FJ, Desvergne B, Wahli W.Peroxisome proliferator-activated receptor alpha mediates the adaptive responseto fasting. J Clin Invest 1999;103:1489–149833. Hashimoto T, Cook WS, Qi C, Yeldandi AV, Reddy JK, Rao MS. Defect inperoxisome proliferator-activated receptor alpha-inducible fatty acid oxidationdetermines the severity of hepatic steatosis in response to fasting. J Biol Chem2000;275:28918–2892834. Friedman MI. Fuel partitioning and food intake. Am J Clin Nutr 1998;67(Suppl.):513S–518S35. Friedman MI. Control of energy intake by energy metabolism. Am J Clin Nutr1995;62(Suppl.):1096S–1100S36. Langhans W, Scharrer E. Metabolic control of eating. World Rev Nutr Diet1992;70:1–6737. Grill HJ, Friedman MI, Norgren R, Scalera G, Seeley R. Parabrachial nucleuslesions impair feeding response elicited by 2,5-anhydro-D-mannitol. AmJ Physiol 1995;268:R676–R682

38. Ritter S, Dinh TT, Friedman MI. Induction of Fos-like immunoreactivity(Fos-li) and stimulation of feeding by 2,5-anhydro-D-mannitol (2,5-AM) requirethe vagus nerve. Brain Res 1994;646:53–6439. Gasa R, Clark C, Yang R, DePaoli-Roach AA, Newgard CB. Reversal of diet-induced glucose intolerance by hepatic expression of a variant glycogen-targetingsubunit of protein phosphatase-1. J Biol Chem 2002;277:1524–153040. Yamauchi T, Waki H, Kamon J, et al. Inhibition of RXR and PPARgammaameliorates diet-induced obesity and type 2 diabetes. J Clin Invest 2001;108:1001–101341. Vidal-Puig A, Jimenez-Liñan M, Lowell BB, et al. Regulation of PPAR gammagene expression by nutrition and obesity in rodents. J Clin Invest 1996;97:2553–256142. Horton JD, Bashmakov Y, Shimomura I, Shimano H. Regulation of sterolregulatory element binding proteins in livers of fasted and refed mice. Proc NatlAcad Sci U S A 1998;95:5987–599243. Azzout-Marniche D, Bécard D, Guichard C, Foretz M, Ferré P, Foufelle F.Insulin effects on sterol regulatory-element-binding protein-1c (SREBP-1c)transcriptional activity in rat hepatocytes. Biochem J 2000;350:389–39344. Iynedjian PB, Pilot PR, Nouspikel T, et al. Differential expression and reg-ulation of the glucokinase gene in liver and islets of Langerhans. Proc Natl AcadSci U S A 1989;86:7838–784245. Peter A, Stefan N, Cegan A, et al. Hepatic glucokinase expression is as-sociated with lipogenesis and fatty liver in humans. J Clin Endocrinol Metab2011;96:E1126–E113046. Shimomura I, Bashmakov Y, Horton JD. Increased levels of nuclear SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J BiolChem 1999;274:30028–3003247. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the completeprogram of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125–113148. Izumida Y, Yahagi N, Takeuchi Y, et al. Glycogen shortage during fastingtriggers liver-brain-adipose neurocircuitry to facilitate fat utilization. Nat Commun2013;4:231649. Crosson SM, Khan A, Printen J, Pessin JE, Saltiel AR. PTG gene deletioncauses impaired glycogen synthesis and developmental insulin resistance. J ClinInvest 2003;111:1423–143250. Irimia JM, Meyer CM, Peper CL, et al. Impaired glucose tolerance andpredisposition to the fasted state in liver glycogen synthase knock-out mice.J Biol Chem 2010;285:12851–12861


Dow


Liver Glycogen Reduces Food Intake and Attenuates Obesity ...

Documents

Transcript of Liver Glycogen Reduces Food Intake and Attenuates Obesity ...