Regulation of de novo hepatic lipogenesis by insulin infusion in rainbow trout fed a high-carbohydrate diet

S. Polakof, F. Médale, L. Larroquet, C. Vachot, G. Corraze and S. Panserathigh-carbohydrate diet

Regulation of de novo hepatic lipogenesis by insulin infusion in rainbow trout fed a

published online May 13, 2011J ANIM SCI

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1

Regulation of de novo hepatic lipogenesis by insulin infusion in rainbow trout

fed a high-carbohydrate diet1

S. Polakof*#1, F. Médale*, L. Larroquet*, C. Vachot*, G. Corraze,* and S. Panserat*

* INRA, UR1067 Nutrition Metabolism Aquaculture, F-64310 Saint-Pée-sur-Nivelle, France

#Laboratorio de Fisioloxía Animal, Departamento de Bioloxía Funcional e Ciencias da Saúde,

Facultade de Bioloxía, Universidade de Vigo, E-36310 Vigo, Spain.

1Acknowledgements

This study was supported by research grants from Agence Nationale de la Recherche

(ANR-08-JCJC-0025-01) and INRA Animal Physiology – Livestock Systems (PHASE)

Department. S. Polakof was the recipient of a postdoctoral fellowship from the Xunta de Galicia

(Program Ángeles Alvariño). We thank the technical staff (Y. Hontang, F. Sandres, and F.

Terrier) of the INRA experimental fish farm of Donzacq for supplying the experimental animals.

2Corresponding author:

Dr. Sergio Polakof

INRA, UR1067 Nutrition Metabolism Aquaculture, Pôle d’hydrobiologie, CD918, F-64310 St-

Pée-sur-Nivelle, France

Tel: (33) 5 59 51 59 60; Fax: (33) 5 59 54 51 52; e-mail: [email protected]

Present adress: INRA, UMR 1019, UNH, CRNH Auvergne, Clermont-Ferrand, France;

Clermont Université, Université d’Auvergne, Unité de Nutrition Humaine, Clermont-Ferrand,

France

Published Online First on May 13, 2011 as doi:10.2527/jas.2010-3733 by guest on May 28, 2012jas.fass.orgDownloaded from


2

Abstract

Carbohydrate energy intake in excess of total energy expenditure is converted to fat. In

fish, the liver is considered to be the main lipogenic tissue. Its regulation by insulin is not fully

understood and some of the available in vivo findings are contradictory. In this study, bovine

insulin was infused for 5 d into rainbow trout fed a high-carbohydrate diet and parameters of de

novo hepatic lipogenesis were measured. We found that hepatic lipogenesis in trout is stimulated

by insulin, reflected in enhanced mRNA and protein levels and enzyme activity of ATP-citrate

lyase, acetyl-CoA carboxylase, and fatty acid synthase. These results were further supported by

parallel changes in enzymes acting as NADPH donors, especially those participating in the

pentose phosphate pathway. This is the first time that the main enzymes involved in de novo

hepatic lipogenesis have been studied at molecular, protein, and activity levels in fish. We

hypothesize that some of the delayed changes found in the different levels of regulation were

probably related to the insulin resistance achieved by the trout liver after 5 d of insulin infusion.

We assessed enzyme activity and mRNA levels of lipid oxidation-related enzymes in the livers

of insulin-infused fish in which paradoxically increased β-oxidation potential was found. The

insulin-stimulated de novo hepatic lipogenesis in carbohydrate-fed trout reinforces the

hypothesis that this pathway may act as an important sink for an excess of glucose, which could

ultimately contribute to improved glucose homeostasis in this carnivorous and ‘glucose-

intolerant’ species when fed high-carbohydrate diets.



3

Key Words: dietary carbohydrate, fish, insulin, lipogenesis, liver



4

INTRODUCTION

As in rainbow trout (Oncorhynchus mykiss), de novo hepatic lipogenesis (DNL) in

salmonids, is regulated by several factors, including nutritional status (Lin et al., 1977a), energy

content in the diet (Kolditz et al., 2008), genetic background (Skiba-Cassy et al., 2009), anti-

diabetic drugs (Panserat et al., 2009; Polakof et al., 2011b), macronutrients (Álvarez et al., 2000),

migration processes (Sheridan et al., 1985), and hormones (Cowley and Sheridan, 1993; Higgs et

al., 2009).

Insulin stimulates synthesis of fatty acids and triacylglycerols and enhances

triacylglycerol storage in the liver through long-term effects on the expression of lipogenic

genes, and this is often accompanied by inhibition of fatty acid oxidation in mammals (Kersten,

2001). The picture is unclear in fish, where the insulin effects reported are contradictory and the

mechanisms of insulin regulation have not been fully elucidated (Machado et al., 1988; Pérez-

Sánchez, 1988; Plagnes-Juan et al., 2008; Polakof et al., 2010b; Polakof et al., 2009). Recent

findings suggest a major role of DNL in glucose homeostasis, including a better glycemic profile

in trout treated with metformin (Panserat et al., 2009; Polakof et al., 2011b) and in genetically

selected lines (Skiba-Cassy et al., 2009). To obtain greater understanding of the effects of insulin

on hepatic DNL under physiological conditions, we infused rainbow trout with insulin for 5 d.

Due to the anabolic action of this hormone (Navarro et al., 2006), we hypothesized that insulin at

physiological doses would stimulate DNL in order to store the excess circulating glucose. We

assessed the activity, protein and mRNA levels of key enzymes involved in lipogenesis as well

as the main enzymes acting as NADPH donors. Tissue insulin sensitivity was estimated on the

basis of the phosphorylation status of Akt/PKB, a critical node in the insulin signaling pathway



5

in both fish (Seiliez et al., 2008) and mammals (Taniguchi et al., 2006). Insulin-regulated lipid

metabolism was examined through measurement of representative lipid oxidation enzymes.

MATERIALS AND METHODS

The experiments were conducted following the Guidelines of the National Legislation on

Animal Care of the French Ministry of Research (Decret Nº 2001-464, May 29, 2001) and were

approved by the Ethics Committee of INRA (according to INRA 2002-36, April 14, 2002).

Fish

Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from the INRA

experimental fish farm facilities of Donzacq (Landes, France). Fish were maintained in tanks

kept in open recirculated circuits with 17°C well-aerated water and controlled photoperiod

(LD12:12), and fed a standard trout commercial diet during the acclimation period (T-3P classic,

Trouw, France; crude protein = 49.8% dry matter, crude fat = 13.8% dry matter; gross energy =

22 kJ/g dry matter). Mean fish weight was 200 ± 10 g.

Experimental Protocols

For sustained hormone infusions, fish were feed-deprived for 48 h and then implanted

with 1003D Alzet mini-osmotic pumps (ALZED Osmotic Pumps, Cupertino, CA) containing

either saline (control, n = 12) or bovine insulin (Sigma Chemical Co., St. Louis, MO) solution (n



6

= 12) at 2 doses. Fish were anesthetized, BW measured, and pumps inserted into the peritoneal

cavity through a 1.0-cm incision made in the ventral midline at ca. 2.0 cm rostral of the pelvic

fins. The incision was closed with 1 stitch and an antibiotic gel was applied topically to the

incision area. Pumps were implanted in the morning and fish were allowed to recover for 24 h.

Fish were then hand-fed once daily at 2% BW with a diet containing a high level of carbohydrate

(30% dextrin, 57% fish meal, and 10% fish oil) for 5 d and sampled 6 h after their last meal.

Pump flow rate was established to be 0.39 L·h-1, which at 17ºC should provide sustained

release of 0.35 or 0.7 insulin IU·kg-1·d-1. The doses and heterologous insulin used in the present

study were selected on the basis of previous studies (Plisetskaya et al., 1993; Polakof et al.,

2009).

Tissue and Blood Sampling

Trout were sacrificed by a sharp blow on the head. Blood was removed from the caudal

vessel and centrifuged (3,000 × g, 5 min, 4ºC). Plasma was frozen at -20ºC pending analyses.

The gut contents of each fish were systematically checked to confirm that the fish being sampled

had consumed the diet. Livers were collected and frozen in liquid nitrogen and stored at -80°C

pending analyses, except for 1 piece of each liver that was immediately homogenized to assess

carnitine palmitoyltransferase (CPT1) activity (see below).

Molecular and Biochemical Analyses

Plasma glucose (Biomérieux, Marcy l'Etoile, France), triglycerides (Biomérieux), and

NEFA (NEFA C from Wako Chemicals GmbH, Neuss, Germany) levels were determined using



7

commercial kits adapted to a microplate format (Polakof et al., 2010a, 2011b). Bovine insulin

levels were measured in trout plasma using a bovine-specific commercial ELISA kit (Mercodia,

Sweden) according to Polakof et al. (2009).

Tissue mRNA levels encoding proteins involved in lipid and energy metabolism were

determined by realtime quantitative RT-PCR (qPCR; Polakof et al., 2010a). Total RNA was

extracted from livers using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the

manufacturer's recommendations. One microgram of the resulting total RNA was reverse

transcribed into cDNA using the SuperScript III RNaseH- Reverse Transcriptase kit (Invitrogen)

and random primers (Promega, Charbonnières, France) according to the manufacturer’s

instructions. Realtime RT-PCR was carried out with an iCycler iQ realtime PCR detection

system (BIO-RAD, Hercules, CA) using iQ SYBR Green Supermix. The transcripts assessed

were acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), ATP-citrate lyase (ACLY),

glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH),

malic enzyme (ME), and isocitrate dehydrogenase (ICDH). Primers were designed to overlap an

intron where possible (Primer3 v. 0.4.0 software; http://frodo.wi.mit.edu/primer3/#disclaimer;

Rozen and Skaletsky, 2000) using known sequences found in trout nucleotide databases

(Genbank and INRA-Sigenae) as previously described (Ducasse-Cabanot et al., 2007; Polakof et

al., 2010a, 2011a). The transcript level of elongation factor 1α (EF1) was stably expressed in

this study. Relative quantification of the target gene transcript with the EF1 reference gene

transcript was made following the Pfaffl method (Pfaffl, 2001). The relative expression ratio (R)

of a target gene was calculated on the basis of realtime PCR efficiency (E) and the CT deviation

(ΔCT) of the unknown sample compared to a control sample and expressed in comparison to the

EF1 reference gene: R = [(Etarget gene)ΔCt target gene (mean control-mean sample)/(Eef1α)

ΔCt ef1α(mean control-mean



8

sample)]. Efficiency (E) of PCR was measured by the slope of a standard curve using serial

dilutions of cDNA.

Western blotting (20 µg protein extracted from the liver) was undertaken using anti-

phospho-Akt Ser473 (Cell Signaling Technology, Ozyme, St Quentin-en-Yvelines, France), anti-

FAS (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ACLY (Cell Signaling Technology),

anti-phospho-ACC Ser79 (Upstate, Euromedex, Munolsheim, France) and anti-β-tubulin (Cell

Signaling Technology). All antibodies were raised against human protein, except for Akt/PKB,

which was raised against mouse protein. Briefly, frozen livers (300 mg) were homogenized on

ice with an Ultraturrax homogenizer in a buffer containing 150 mM NaCl, 10 mM Tris , 1 mM

EGTA, 1 mM EDTA (pH 7.4), 100 mM sodium fluoride, 4 mM sodium pyrophosphate, 2 mM

sodium orthovanadate, 1% Triton X-100, 0.5% NP-40-IGEPAL, and

cOmplete,ULTRA,Mini,EDTA-free,EASYpack Protease Inhibitor Cocktail (Roche, Basel,

Switzerland; 1 tablet / 5mL) (4-(2-Aminoethyl)-benzenesulfonyl fluoridehydrochloride,

polyethylene glycol, polyvinylpyrrolidone, N-alpha-p-Tosyl-L-lysine chloromethyl ketone

hydrochloride, benzethonium chloride, D-Mannitol, phenylmethylsulfonyl fluoride).

Homogenates were centrifuged for 15 min at 12,000 × g, 4ºC and the resulting supernatants

stored at -80ºC. Protein concentrations were determined using the Bio-Rad protein assay kit

(BIO-RAD). Protein lysates (20 μg) were subjected to SDS-PAGE and Western blotting using

the appropriate antibody. After washing, membranes were incubated with an IRDye infrared

secondary antibody (LI-COR Biotechnology, Lincoln, NE). Bands were visualized by infrared

fluorescence using the Odyssey imaging system (LI-COR Biotechnology) and quantified by

Odyssey infrared imaging system software (version 1.2). The signal of each protein was

normalized to the β-tubulin signal. Validation of Akt/PKB and FAS mammalian antibodies was



9

previously demonstrated in the trout (Seiliez et al., 2008; Polakof et al., 2010a). The trout ACLY

(GAY7CUQ01A40MK.s.om.10, CA349411.s.om.10) AA sequence is 90% homologous to

human ACLY (NP_001087.2), while the trout FAS (Sigenae database, GAY7CUQ02F6SW9)

AA sequence is 88% homologous to human FAS (NP_004095.4). As in the rat ACC

(NP_071529.1), a serine was also found in the trout ACC protein (Sigenae database,

FYV3OTN01B4R4N.s.om.10).

Livers used to assess enzyme activities were homogenized with ice-cold buffer (10

wt/vols; 20 mM Tris, pH 7.4, 250 mM sucrose, 2 mM EDTA, 10 mM β-mercaptoethanol, 100

mM NaF, 0.5 mM EDTA). The homogenate was centrifuged for 20 min at 17,000 × g, 4ºC and

the supernatant used immediately for enzyme assays. Hydroxyacyl-CoA dehydrogenase (HOAD)

and ACC were assessed as in Kolditz et al. (2008), while G6PDH and FAS were assessed

following the method described by Figueiredo-Silva et al. (2010) adapted to trout tissues. Levels

of enzyme activity of ME and 6PGDH were assessed as in Mommsen et al. (2003) adapting the

substrate liver concentration: 0.5 mM phosphogluconic acid for 6PGDH and 0.08 mM malic acid

for ME. Enzyme activity of ACLY was determined as in Álvarez et al. (2000). Total CPT1

activity was assessed on fresh tissue as in Gutieres et al (2003). Enzyme activity levels are

expressed in terms of mg protein. Protein concentrations were determined using a Bradford

protein assay kit (Bio-Rad, Germany) with BSA as standard (Bradford, 1976).

.

Statistical Analysis

Results are expressed as means ± SEM. (n = 6). Data were analyzed by one-way

ANOVA. When necessary, data were log-transformed to fulfill the conditions of the analysis of



10

variance. Post-hoc comparisons were made using a Student-Newman-Keuls test, and differences

were considered statistically significant at P < 0.05.

RESULTS

Plasma glucose levels in the group receiving the lower insulin dose were less (P = 0.029)

than in the other 2 groups (control and higher insulin dose), while in the higher dose group (P >

0.10) glycemia was similar to the saline-infused group. Plasma triglyceride levels were

unaffected (P > 0.10) by insulin infusion when compared with the saline-infused group. In

contrast, free fatty acid levels in plasma were reduced after insulin infusion (P = 0.011 and 0.046

respectively). Bovine insulin levels were 2-fold higher in the animals infused with the 0.7 IU·kg-

1·d-1 insulin dose than in those receiving 0.35 IU·kg-1·d-1 insulin (P = 0.024).

The phosphorylation status of liver Akt/PKB Ser473 was unchanged (P > 0.10) by insulin

infusion (Figure 1).

Liver ACLY, ACC, and FAS enzyme activities, protein, and mRNA transcript levels are

shown in Figure 2. No changes were observed (P > 0.10) in ACLY activity irrespective of the

insulin dose. Low dose insulin increased (P = 0.016) ACLY mRNA 2-fold compared to controls.

High-dose insulin increased (P = 0.033) ACLY protein compared to saline controls. Both protein

(P = 0.039) and activity levels (P = 0.013) of ACC were upregulated in fish receiving 0.7 IU·kg-

1·d-1 insulin. No changes (P > 0.10) in ACC mRNA levels were noted in the control group. The

levels of FAS mRNA levels were upregulated (P = 0.048) by insulin, only at the 0.35 IU·kg-1·d-1

dose. The protein and activity levels of FAS were unaffected (P > 0.10) by the low dose of

insulin treatment, and increased (P = 0.034) by the higher-insulin dose.



11

Changes in enzyme activity and mRNA levels of proteins acting as NADPH donors in the

liver are shown in Figure 3. Levels of enzyme activity of 6PGDH, G6PDH, and ME were

increased (6PGDH, P = 0.009; G6PDH, P = 0.001; ME, P = 0.05) by insulin. Transcript levels

for 6PGDH were upregulated (Ins0.35, P < 0.001; Ins0.7, P = 0.009) in a dose-dependent

manner.

Changes in activity levels of enzymes involved in lipid oxidation pathways in the liver

are shown in Table 2. The level of activity of HOAD was increased in trout infused with the

hormone independently of the insulin dose (P = 0.048 for 0.35; P = 0.021 for 0.7), whereas no

changes in mRNA levels were noted (P > 0.10). No changes in levels of CPT1 activity were

noted in any of the insulin-infused groups (P > 0.10), although mRNA levels increased with the

higher-insulin dose for CPT1A (P = 0.025) and increased with both insulin doses for CPT1B (P =

0.017 for 0.35; P = 0.025 for 0.7).

DISCUSSION

Insulin-stimulated DNL Potential in Rainbow Trout Liver

Although the role of adipose tissue in DNL remains to be elucidated in fish (Polakof et

al., 2011a), the liver has been traditionally considered to be the main lipogenic organ in many

fish species, including salmonids (Henderson and Sargent, 1985). In the study presented here, we

analyzed important enzymes involved in DNL, including ACLY, ACC, and FAS. This is the first

global assessment of insulin regulated-DNL in the trout liver to date.



12

Despite the lack of changes in the relative phosphorylation status of Akt/PKB in our

study, significant changes were noted in DNL-related enzymes. The fact that Akt/PKB was

unaffected by the insulin infusion 6 h after feeding does not mean that the effects observed here

were Akt/PKB-independent, since earlier phosphorylation of this protein has been reported in the

postprandial state (Seiliez et al., 2008; Skiba-Cassy et al., 2009). It should be borne in mind that,

despite the presence of insulin receptors, the liver is not a highly insulin-sensitive tissue

(Gutiérrez et al., 2006; Navarro et al., 2006). We cannot rule out that some degree of insulin

resistance may be induced by insulin infusion in this species (Polakof et al., 2010c) or that there

is insufficient stimulation of the trout Akt/PKB systyem by heterologous insulin. This may

explain the lack of hyperglycemia with the higher insulin dose and some of the differences

between the molecular and biochemical regulation of the enzymes studied.

The primary enzyme responsible for the synthesis of cytosolic acetyl-CoA is ACLY.

Although its activity and gene expression have already been described for fish liver, this is the

first time that insulin regulation of the enzyme has been assessed in vivo. We found enhanced

ACLY mRNA levels in fish receiving the lower-insulin dose, while protein levels were

upregulated with the higher-insulin dose. These differences between mRNA and protein levels

may be due to delayed protein synthesis after increased transcript production, although we

cannot eliminate the possibility of an insulin-resistance effect with the higher-hormone dose.

Further support for this phenomenon could be found in the different regulation of ACLY mRNA

levels (unchanged; Lansard et al., 2010) and activity (increased; Álvarez et al., 2000) in trout

hepatocytes stimulated with both insulin and glucose. The lack of changes in activity in the

present study may have been due to increased citrate levels from the tricarboxylic acid cycle or

changes in the phosphorylation status of the protein (Potapova et al., 2000). Our results also



13

agree with the regulation described for this enzyme in mammals, where ACLY is stimulated in

the presence of insulin and glucose (Fukuda and Iritani, 1999).

The formation of malonyl-CoA, an essential substrate for FAS and fatty acid chain

elongation in DNL, is catalyzed by ACC. We observed complex regulation of ACC in the livers

of rainbow trout, based upon gene expression analysis, protein phosphorylation profiles, and

enzyme activity measurements. The findings confirm the presence of a functional ACC in the

trout liver, supporting previous studies in which ACC activity was increased in trout fed high

carbohydrate (Rollin et al., 2003) or low energy (Kolditz et al., 2008) diets. As stated above,

insulin stimulates the hepatic lipogenic pathway in mammals. While no changes in gene

expression were noted, both the phosphorylation on the Ser79 and activity were clearly

upregulated in those trout receiving the higher-insulin dose. It is possible that a transient increase

in ACC transcription occurred over the 5-d infusion period that could account for the elevated

enzyme activity. Although ACC activity in trout is increased in glucose and insulin-stimulated

hepatocytes (Álvarez et al., 2000), no information is available regarding the phosphoregulation

of ACC in fish. In mammals, AMP-kinase phosphorylation of ACC on Ser79 leads to inhibition

of enzyme activity, and insulin-stimulated dephosphorylation of the serine causes an increase in

liver ACC activity (Witters and Kemp, 1992). The fact that in the present study insulin seems to

increase ACC activity despite the enhanced phosphorylation status on Ser79 suggests that the

regulation of this enzyme in trout is complex and probably different from the mammalian model.

In mammals and birds, FAS gene expression, protein, and activity levels are upregulated

in the presence of insulin (Griffin and Sul, 2004; Radenne et al., 2008). We observed that FAS

activity is also upregulated by insulin, suggesting that the regulation by both insulin and glucose

is similar in the trout to that reported in other vertebrates. These results agree with previous



14

findings obtained in fed trout subjected to similar treatments (Polakof et al., 2011b). The amount

of FAS mRNA (Lansard et al., 2010) and activity (Álvarez et al., 2000) are upregulated in

glucose- and insulin-stimulated trout hepatocytes.

Insulin Regulates NAPDH Donors in Rainbow Trout Liver

The DNL in the cytoplasm of vertebrate cells requires a carbon source (acetyl-CoA) and

reducing equivalents (NADPH) produced by 1 or more of 4 cytoplasmic dehydrogenases

(G6PDH, 6PGDH, ICDH, and ME). All these enzymes have been characterized in the liver of

different fish species, including salmonids (Baldwin and Reed, 1976; Walton and Cowey, 1982).

The mRNA and levels of enzyme activity of the 4 NADPH donors in the trout liver were

measured. Activity levels were in the same range as reported by others (Lin et al., 1977b; Kolditz

et al., 2008), with ME exhibiting the lowest level of activity. Insulin enhanced the activity of all

the enzymes acting as NADPH donors, except for ICDH, which remained unaffected. The fact

that these enzymes are mainly affected at similar activity levels as the lipogenic ACLY, ACC,

and FAS suggests a minor molecular regulation by insulin for these enzymes and confirms the

importance of the NADPH donors for DNL in fish liver (Lin et al., 1977b; Aster and Moon,

1981). The higher levels of activity of G6PDH and 6PGDH compared to ME also suggest that

the major NADPH donors in trout liver are those involved in the pentose phosphate pathway and

that insulin acts as a stimulator of such a metabolic route (Novello et al., 1969).

Paradoxical regulation of β-oxidation Potential in Trout Liver by Insulin



15

A paradoxical imbalance in the regulation of DNL and lipid oxidation pathways seems to

exist when insulin is infused. The CPT1 activity remained unchanged and even upregulated at

the molecular level, especially with the higher insulin dose. The absence of inhibition of CPT1

activity in our conditions could be due to the presence of long-chain n-3 fatty acids provided by

fish oil in the diet. Liver CPT1 activity is increased by n-3 fatty acids in mammals (Ide et al.,

2000). Surprisingly, we also found increased levels of HOAD activity in fed fish infused with

insulin. It was previously reported that high glucose levels in fish, both in vivo (ip injection;

Harmon et al., 1991) and in vitro (Harmon and Sheridan, 1992), stimulate lipolysis rates through

increased hepatic lipase activity. Moreover, in mammals increased levels of HOAD activity were

found in sand rats (Psammomys obesus) subjected to hyperglycemia and hyperinsulinemia

(Nakai et al., 1997). We therefore suggest that β-oxidation in trout liver is highly influenced by

diet composition (high levels of fish oil and carbohydrates), which could act as the major

regulator of CPT1 and HOAD activity, irrespective of the insulin dose.

Conclusions and perspective

Our findings indicate that DNL in rainbow trout is regulated by insulin, which increases

the 3 major enzymes involved in this pathway at molecular, protein, and enzyme activity levels.

This is further supported by the concomitant upregulation of some enzymes acting as NADPH

donors, especially those participating in the pentose phosphate pathway. The fact that insulin

also stimulates this pathway in high carbohydrate-fed rats (Nepokroeff et al., 1974) and in

hyperinsulinemic patients fed low-fat/high-carbohydrate diets (Schwarz et al., 2003), supports

the idea of a strongly conserved regulatory role of insulin in hepatic DNL. On the other hand,

although some studies in the 1970 to 1980s were contradictory concerning the role of insulin in



16

high-carbohydrate-fed catfish (Warman III and Bottino, 1978; Machado et al., 1988), later

studies in vitro confirmed that insulin stimulates DNL in trout (Cowley and Sheridan, 1993).

This is relevant in carnivorous fish species fed high-carbohydrate diets, since their natural

carbohydrate intake is about 1%. Despite this apparent paradox, the incorporation of the excess

glucose from the diet into hepatic lipogenesis seems to be a common feature in this species

(Hung and Storebakken, 1994). In our study, part of the hypoglycemia observed in fish infused

with the lower insulin dose (data not shown) could then be due to this increased DNL potential.



17

LITERATURE CITED

Álvarez, M. J., A. Díez, C. López-Bote, M. Gallego, and J. M. Bautista. 2000. Short-term

modulation of lipogenesis by macronutrients in rainbow trout (Oncorhynchus mykiss)

hepatocytes. Br. J. Nutr. 84:619-628.

Aster, P. L., and T. W. Moon. 1981. Influence of fasting and diet on lipogenic enzymes in the

american eel, Anguilla rostrata LeSueur. J. Nutr. 111:346-354.

Baker, P. W., and G. F. Gibbons. 2000. Effect of dietary fish oil on the sensitivity of hepatic lipid

metabolism to regulation by insulin. J. Lipid Res. 41:719-726.

Baldwin, J., and K. C. Reed. 1976. Cytoplasmic sources of NADPH for fat synthesis in rainbow

trout liver: effect of thermal acclimation on enzyme activities. Comp. Biochem. Physiol. B

54:527-529.

Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities

of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.

Cowley, D. J., and M. A. Sheridan. 1993. Insulin stimulates hepatic lipogenesis in rainbow trout,

Oncorhynchus mykiss. Fish Physiol. Biochem. 11:421-428.

Ducasse-Cabanot, S., J. Zambonino-Infante, N. Richard, F. Médale, G. Corraze, M. Mambrini, J.

Robin, C. Cahu, S. Kaushik, and S. Panserat. 2007. Reduced lipid intake leads to changes in

digestive enzymes in the intestine but has minor effect on key enzymes of hepatic

intermediary metabolism in rainbow trout (Oncorhynchus mykiss). Animal 1:1272-1282.

Figueiredo-Silva, A. C., G. Corraze, P. Borges, and L. M. P. Valente. 2010. Dietary protein/lipid

level and protein source effects on growth, tissue composition and lipid metabolism of

blackspot seabream (Pagellus bogaraveo). Aquac. Nutr. 16:173-187.



18

Fukuda, H., and N. Iritani. 1999. Regulation of ATP citrate-lyase gene expression in hepatocytes

and adipocytes in normal and genetically obese rats. J. Biochem. 126:437-444.

Griffin, M. J., and H. S. Sul. 2004. Insulin regulation of fatty acid synthase gene

transcription:roles of USF and SREBP-1c. IUBMB Life 56:595-600.

Gutieres, S., M. Damon, S. Panserat, S. Kaushik, and F. Medale. 2003. Cloning and tissue

distribution of a carnitine palmitoyltransferase I gene in rainbow trout (Oncorhynchus

mykiss). Comp. Biochem. Physiol. B-Biochem. Mol. Biol. 135:139-151.

Gutiérrez, J., I. Navarro, J. V. Planas, N. Montserrat, P. Rojas, J. Castillo, O. V. Chystiakova, J.

C. Gabillard, A. Smith, S. J. Chan, and B. N. Leibush. 2006. Insulin and IGF receptors in

fish. In: M. Reinecke, G. Zaccone and B. G. Kapoor (eds.) Fish Endocrinology. p 131-165.

Science Publishers, Enfield.

Harmon, J. S., C. D. Eilertson, M. A. Sheridan, and E. M. Plisetskaya. 1991. Insulin suppression

is associated with hypersomatostatinemia and hyperglucagonemia in glucose-injected

rainbow trout. Am. J. Physiol. Regul. Integr. Comp. Physiol. 261:R609-R613.

Harmon, J. S., and M. A. Sheridan. 1992. Glucose stimulated lipolysis in rainbow trout,

Oncorhynchus mykiss, liver. Fish Physiol. Biochem. 10:189-199.

Henderson, R. J., and J. R. Sargent. 1985. Fatty acid metabolism in fish. In: C. B. Cowey (ed.)

Nutrition and Feeding in Fish. Academic Press, New York.

Higgs, D. A., J. N. Sutton, H. Kim, J. D. Oakes, J. Smith, C. Biagi, M. Rowshandeli, and R. H.

Devlin. 2009. Influence of dietary concentrations of protein, lipid and carbohydrate on

growth, protein and energy utilization, body composition, and plasma titres of growth

hormone and insulin-like growth factor-1 in non-transgenic and growth hormone transgenic

coho salmon, Oncorhynchus kisutch (Walbaum). Aquaculture 286:127-137.



19

Hung, S. S. O., and T. Storebakken. 1994. Carbohydrate utilization by rainbow trout is affected

by feeding strategy. J. Nutr. 124:223-230.

Ide, T., H. Kobayashi, L. Ashakumary, I. A. Rouyer, Y. Takahashi, T. Aoyama, T. Hashimoto,

and M. Mizugaki. 2000. Comparative effects of perilla and fish oils on the activity and gene

expression of fatty acid oxidation enzymes in rat liver. Biochim. Biophys. Acta 1485:23-35.

Kersten, S. 2001. Mechanisms of nutritional and hormonal regulation of lipogenesis. EMBO

Rep. 2:282-286.

Kolditz, C., M. Borthaire, N. Richard, G. Corraze, S. Panserat, C. Vachot, F. Lefevre, and F.

Medale. 2008. Liver and muscle metabolic changes induced by dietary energy content and

genetic selection in rainbow trout (Oncorhynchus mykiss). Am J Physiol Regul Integr Comp

Physiol 294:R1154-R1164.

Lansard, M., S. Panserat, E. Plagnes-Juan, I. Seiliez, and S. Skiba-Cassy. 2010. Integration of

insulin and amino acid signals that regulate hepatic metabolism-related gene expression in

rainbow trout: role of TOR. Amino Acids 39:801-810.

Lin, H., D. R. Romsos, P. I. Tack, and G. A. Leveille. 1977a. Effects of fasting and feeding

various diets on hepatic lipogenic enzyme activities in coho salmon (Oncorhynchus kisutch

(Walbaum)). J. Nutr. 107:1477-1483.

Lin, H., D. R. Romsos, P. I. Tack, and G. A. Leveille. 1977b. Influence of dietary lipid on

lipogenic enzyme activities in coho salmon, Oncorhynchus kisutch (Walbaum). J. Nutr.

107:846-854.

Machado, C. R., M. a. R. Garofalo, J. E. S. Roselino, I. C. Kettelhut, and R. H. Migliorini. 1988.

Effects of starvation, refeeding, and insulin on energy-linked metabolic processes in catfish

(Rhamdia hilarii) adapted to a carbohydrate-rich diet. Gen. Comp. Endocrinol. 71:429-437.



20

Mommsen, T. P., H. L. Osachoff, and M. E. Elliott. 2003. Metabolic zonation in teleost

gastrointestinal tract. Effects of fasting and cortisol in tilapia. J. Comp. Physiol. B-Biochem.

Syst. Environ. Physiol. 173:409-418.

Nakai, N., G. R. Collier, Y. Sato, Y. Oshida, N. Fujitsuka, and Y. Shimomura. 1997. Activities

of liver pyruvate dehydrogenase complex and 3-hydroxyacyl-CoA dehydrogenase in sand rat

(Psammomys obesus). Life Sci. 60:51-55.

Navarro, I., E. Capilla, A. Castillo, A. Albalat, M. Díaz, M. A. Gallardo, J. Blasco, J. V. Planas,

and J. Gutiérrez. 2006. Insulin metabolic effects in fish tissues. In: M. Reinecke, G. Zaccone

and B. G. Kapoor (eds.) Fish endocrinology. p 15-48. Science Publishers, Inc., Enfield.

Nepokroeff, C. M., M. R. Lakshmanan, G. C. Ness, R. A. Muesing, D. A. Kleinsek, and J. W.

Porter. 1974. Coordinate control of rat liver lipogenic enzymes by insulin. Arch. Biochem.

Biophys. 162:340-344.

Novello, F., J. A. Gumaa, and P. Mclean. 1969. The pentose phosphate pathway of glucose

metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the

cycle in liver. Biochem. J. 111:713-725.

Panserat, S., S. Skiba-Cassy, I. Seiliez, M. Lansard, E. Plagnes-Juan, C. Vachot, P. Aguirre, L.

Larroquet, G. Chavergnac, F. Médale, G. Corraze, S. Kaushik, and T. W. Moon. Metformin

improves postprandial glucose homeostasis in rainbow trout fed dietary carbohydrates: a link

with the induction of hepatic lipogenic capacities? Am. J. Physiol. Regul. Integr. Comp.

Physiol. 293:707-715.

Pérez-Sánchez, J. 1988. Metabolismo intermediario en juveniles de lubina: hormonas

pancreáticas, control de la glucemia y efecto "sparing". PhD diss. Instituto de Acuicultura de

Torre la Sal (CSIC), Barcelona.



21

Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR.

Nucleic Acids Res. 29:e45.

Plagnes-Juan, E., M. Lansard, I. Seiliez, F. Medale, G. Corraze, S. Kaushik, S. Panserat, and S.

Skiba-Cassy. 2008. Insulin regulates the expression of several metabolism-related genes in

the liver and primary hepatocytes of rainbow trout (Oncorhynchus mykiss). J. Exp. Biol.

211:2510-2518.

Plisetskaya, E. M., S. Duguay, M. P. Schrgibman, C. E. Scanes, and P. Pang. 1993. Pancreatic

hormones and metabolism in ectotherm vertebrates: current views. p 266-287. Academic

Press, New York.

Polakof, S., F. Medale, L. Larroquet, C. Vachot, G. Corraze, and S. Panserat. 2011a. Insulin

stimulates lipogenesis and attenuates b-oxidation in white adipose tissue of fed rainbow trout.

Lipids 46:189-199.

Polakof, S., F. Medale, S. Skiba-Cassy, G. Corraze, and S. Panserat. 2010a. Molecular regulation

of lipid metabolism in liver and muscle of rainbow trout subjected to acute and chronic

insulin treatments. Domest. Anim. Endocrinol. 39:26-33.

Polakof, S., T. W. Moon, P. Aguirre, S. Skiba-Cassy, and S. Panserat. 2010b. Effects of insulin

infusion on glucose homeostasis and glucose metabolism in rainbow trout fed a high-

carbohydrate diet. J. Exp. Biol. 213:4151-4157.

Polakof, S., T. W. Moon, P. Aguirre, S. Skiba-Cassy, and S. Panserat. 2011b. Glucose

homeostasis in rainbow trout fed a high carbohydrate diet: metformin and insulin interact in a

tissue-dependent manner. Am. J. Physiol. Regul. Integr. Comp. Physiol. 300:166-174.



22

Polakof, S., S. Skiba-Cassy, G. Choubert, and S. Panserat. 2010c. Insulin-induced

hypoglycaemia is co-ordinately regulated by liver and muscle during acute and chronic

insulin stimulation in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 213:1443-1452.

Polakof, S., S. Skiba-Cassy, and S. Panserat. 2009. Glucose homeostasis is impaired by a

paradoxical interaction between metformin and insulin in carnivorous rainbow trout. Am. J.

Physiol. Regul. Integr. Comp. Physiol. 297:1769-1776.

Potapova, I. A., M. R. El-Maghrabi, S. V. Doronin, and W. B. Benjamin. 2000. Phosphorylation

of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes

homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity.

Allosteric activation of ATP:citrate lyase by phosphorylated sugars. Biochemistry 39:1169-

1179.

Radenne, A., M. Akpa, C. Martel, S. Sawadogo, D. Mauvoisin, and C. Mounier. 2008. Hepatic

regulation of fatty acid synthase by insulin and T3: evidence for T3 genomic and

nongenomic actions. Am. J. Physiol. Endocrinol. Metab. 295:E884-E894.

Rollin, X., F. Médale, S. Gutieres, D. Blanc, and S. J. Kaushik. 2003. Short- and long-term

nutritional modulation of acetyl-CoA carboxylase activity in selected tissues of rainbow trout

(Oncorhynchus mykiss). Br. J. Nutr. 89:803-810.

Rozen, S., and H. J. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist

programmers. In: S. Krawetz and S. Misener (eds.) Bioinformatics Methods and Protocols:

Methods in Molecular Biology. p 365-386. Humana Press, Totowa, NJ.

Schwarz, J. M., P. Linfoot, D. Dare, and K. Aghajanian. 2003. Hepatic de novo lipogenesis in

normoinsulinemic and hyperinsulinemic subjects consuming high-fat, low-carbohydrate and

low-fat, high-carbohydrate isoenergetic diets. Am. J. Clin. Nutr. 77:43-50.



23

Seiliez, I., J. C. Gabillard, S. Skiba-Cassy, D. García-Serrana, J. Gutiérrez, S. Kaushik, S.

Panserat, and S. Tesseraud. 2008. An in vivo and in vitro assessment of TOR signaling

cascade in rainbow trout (Oncorhynchus mykiss). Am. J. Physiol. Regul. Integr. Comp.

Physiol. 295:R329-R335.

Sheridan, M. A., N. Y. S. Woo, and H. A. Bern. 1985. Changes in the rates of glycogenesis,

glycogenolysis, lipogenesis, and lipolysis in selected tissues of the coho salmon

(Oncorhynchus kisutch) associated with parr-smolt transformation. J. Exp. Zool. 236:35-44.

Skiba-Cassy, S., M. Lansard, S. Panserat, and F. Medale. 2009. Rainbow trout genetically

selected for greater muscle fat content display increased activation of liver TOR signaling

and lipogenic gene expression. Am. J. Physiol. Regul. Integr. Comp. Physiol. 297:1421-

1429.

Taniguchi, C. M., B. Emanuelli, and C. R. Kahn. 2006. Critical nodes in signalling pathways:

insights into insulin action. Nat. Rev. Mol. Cell. Biol. 7:85-96.

Walton, M. J., and C. B. Cowey. 1982. Aspects of intermediary metabolism in salmonid fish.

Comp. Biochem. Physiol. B-Biochem. Mol. Biol. 73:59-79.

Warman Iii, A. W., and N. R. Bottino. 1978. Lipogenic activity of catfish liver. Lack of response

to dietary changes and insulin administration. Comp. Biochem. Physiol. B-Biochem. Mol.

Biol. 59:153-161.

Witters, L. A., and B. E. Kemp. 1992. Insulin activation of acetyl-CoA carboxylase accompanied

by inhibition of the 5'-AMP-activated protein kinase. J. Biol. Chem. 267:2864-2867.



24

Figure legends

Figure 1. Effects of insulin infusion on liver Akt/PKB phosphorylation status in trout fed a high-

carbohydrate diet. Fish were implanted with pumps containing saline (control) or 2 insulin doses

(0.35 or 0.7 IU·kg–1·d-1) and then fed for 5 d. Gels were loaded with 20 µg total protein per lane.

Protein and phosphorylation levels were normalized to tissue β-tubulin levels and are indicated

as fold-change compared with the saline-treated group. Results are expressed as means ± SEM.

(n = 6).

Figure 2. Effects of insulin infusion on levels of ATP-citrate lyase (ACLY), acetyl-CoA

carboxylase (ACC), and fatty acid synthase (FAS) enzyme activity, protein levels, and mRNA

levels in livers of trout fed a high-carbohydrate diet. Gene expression was measured by realtime

PCR. Expression levels were normalized to elongation factor 1α (EF1α)-expressed transcripts

which did not change under the experimental conditions and are presented as fold-change against

the saline solution-treated group set at 1. Enzyme activity is expressed in mIU·mg-1 protein for

ACLY and µIU·mg-1 protein for ACC and FAS. Protein (20 µg per lane) and phosphorylation

levels were normalized to tissue β-tubulin levels and are indicated as fold-change compared to

the saline-treated group. Results are presented as means ± SEM. (n = 6).

Figure 3. Effects of insulin infusion on levels of glucose 6-phosphate dehydrogenase (G6PDH),

6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), and isocitrate

dehydrogenase (ICDH) enzyme activity and mRNA levels in livers of trout fed a high-

carbohydrate diet. Gene expression was measured by realtime PCR. Expression levels were

normalized to elongation factor 1α (EF1α)-expressed transcripts, which did not change under the



25

experimental conditions and are presented as fold-change against the saline solution-treated

group set at 1. Enzyme activity is expressed in mIU·mg-1 protein. Results are presented as means

± SEM (n = 6).



26

Table 1. Plasma glucose, tryglyceride, NEFA, and bovine insulin levels in rainbow trout fed a

high-carbohydrate diet and implanted with mini-osmotic pumps infusing saline alone (control)

or 2 insulin doses (0.35 or 0.7 IU·kg–1·d-1).

Parameter Saline Insulin 0.35 Insulin 0.7

Glucose (mM) 9.08 ± 0.42a 7.67 ± 0.42b 9.02 ± 0.44a

Tryglycerides (mM) 4.91 ± 0.56 6.36 ± 0.72 4.99 ± 0.40

NEFA (mEq·L-1) 0.126 ± 0.008a 0.107 ± 0.002b 0.097 ± 0.006b

Bovine insulin (ng·mL-1) no data 3.11 ± 0.34a 6.36 ± 0.65b



27

Table 2. Hepatic enzyme activity and mRNA levels of β-oxidation-related enzymes in rainbow

trout fed a high-carbohydrate diet and implanted with mini-osmotic pumps infusing saline alone

(control) or 2 insulin doses (0.35 or 0.7 IU·kg–1·d-1).

Parameter Saline Insulin 0.35 Insulin 0.7

HOAD

Enzyme activity 46.61 ±

5.14a

82.43 ± 15.43b

(P = 0.048)

86.79 ± 13.32b

(P = 0.021)

mRNA levels 1.00 ± 0.26 1.98 ± 0.30

(P > 0.10)

1.20 ± 0.08

(P > 0.10)

CPT-1

Enzyme activity 8.21 ± 1.18 9.56 ± 0.50

(P > 0.10)

11.20 ± 2.26

(P > 0.10)

mRNA levels CPT1A 1.00 ± 0.20a 0.57 ± 0.11a

(P = 0.051)

1.64 ± 0.29b

(P = 0.025)

mRNA levels CPT1B 1.00 ± 0.25a 0.26 ± 0.05b

(P = 0.017)

0.37 ± 0.07b

(P = 0.025)

HOAD, 3-hydroxyacyl-CoA dehydrogenase; CPT-1, carnitin palmitoyltransferase 1. Enzyme activity units

(mIU) are defined as nmol of substrate converted to product, per min, at assay temperature, expressed/mg

protein. Expression levels were normalized to elongation factor 1α (EF1α)-expressed transcripts which did

not change under the experimental conditions and are presented in arbitrary units.



28

Table 3. The P-value from one-way ANOVA of parameters measured in plasma and liver of high-carbohydrate-fed trout infused with saline solution alone or 2 insulin doses for 5 d.

Parameter P-value

Plasma

Glucose 0.044

Tryglycerides 0.738

NEFA 0.018

Bovine insulin 0.034

Liver mRNA

ACLY 0.010

FAS 0.030

ACC 0.248

G6PDH 0.039

6PGDH 0.007

ME 0.717

ICDH 0.046

HOAD 0.073

CPT1A 0.040

CPT1B 0.029

Liver proteins

Akt 0.941

ACLY 0.043

ACC 0.038

FAS 0.025

Liver activities

ACLY 0.136

FAS 0.042

ACC 0.015



29

G6PDH 0.005

6PGDH 0.038

ME 0.217

ICDH 0.195

CPT1 0.390

HOAD 0.049

Non-esterified fatty acids (NEFA), ATP-citrate lyase (ACLY), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), and isocitrate dehydrogenase (ICDH), 3-hydroxyacyl-CoA dehydrogenase (HOAD), carnitin palmitoyltransferase 1 (CPT-1), protein kinase B (Akt/PKB).



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Regulation of de novo hepatic lipogenesis by insulin infusion in rainbow trout fed a high-carbohydrate diet

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