Maternal Protein Restriction in Rats Alters Postnatal Growth and Brain Lipid Sensing in Female Offspring.

Perinatal nutrition is a key player in the susceptibility to developing metabolic diseases in adulthood, leading to the concept of "metabolic programming". The aim of this study was to assess the impact of maternal protein restriction during gestation and lactation on glucose homeostasis and eating behaviour in female offspring. Pregnant rats were fed a normal or protein-restricted (PR) diet and followed throughout gestation and lactation. Body weight, glucose homeostasis, and eating behaviour were evaluated in offspring, especially in females. Body weight gain was lower in PR dams during lactation only, despite different food and water intakes throughout gestation and lactation. Plasma concentration of leptin, adiponectin and triglycerides increased drastically before delivery in PR dams in relation to fat deposits. Although all pups had identical birth body weight, PR offspring body weight differed from control offspring around postnatal day 10 and remained lower until adulthood. Offspring glucose homeostasis was mildly impacted by maternal PR, although insulin secretion was reduced for PR rats at adulthood. Food intake, satiety response, and cerebral activation were examined after a lipid preload and demonstrated some differences between the two groups of rats. Maternal PR during gestation and lactation does induce extrauterine growth restriction, accompanied by alterations in maternal plasma leptin and adiponectin levels, which may be involved in programming the alterations in eating behaviour observed in females at adulthood.

Maternal Protein Restriction in Rats Alters the Expression of Genes Involved in Mitochondrial Metabolism and Epitranscriptomics in Fetal Hypothalamus.

Fetal brain development is closely dependent on maternal nutrition and metabolic status. Maternal protein restriction (PR) is known to be associated with alterations in the structure and function of the hypothalamus, leading to impaired control of energy homeostasis and food intake. The objective of this study was to identify the cellular and molecular systems underlying these effects during fetal development. We combined a global transcriptomic analysis on the fetal hypothalamus from a rat model of maternal PR with in vitro neurosphere culture and cellular analyses. Several genes encoding proteins from the mitochondrial respiratory chain complexes were overexpressed in the PR group and mitochondrial metabolic activity in the fetal hypothalamus was altered. The level of the N6-methyladenosine epitranscriptomic mark was reduced in the PR fetuses, and the expression of several genes involved in the writing/erasing/reading of this mark was indeed altered, as well as genes encoding several RNA-binding proteins. Additionally, we observed a higher number of neuronal-committed progenitors at embryonic day 17 (E17) in the PR fetuses. Together, these data strongly suggest a metabolic adaptation to the amino acid shortage, combined with the post-transcriptional control of protein expression, which might reflect alterations in the control of the timing of neuronal progenitor differentiation.

Maternal supplementation with citrulline or arginine during gestation impacts fetal amino acid availability in a model of intrauterine growth restriction (IUGR).

BACKGROUND: Supplementing maternal diet with citrulline or arginine during gestation was shown to enhance fetal growth in a model of IUGR induced by maternal dietary protein restriction in the rat. OBJECTIVE: The aims of this study were to determine in the same model whether maternal supplementation with citrulline or arginine would increase 1) citrulline and arginine concentration in fetal circulation; 2) the expression of placental amino acid transporters, and 3) the fetal availability of essential amino acids. METHODS: Pregnant rats (n = 8 per group) were fed either an isocaloric control (20% protein, NP) or a low protein (LP, 4% protein) diet, either alone or supplemented with 2 g/kg/d of l-citrulline (LP + CIT) or isonitrogenous Arginine (LP + ARG) in drinking water throughout gestation. Fetuses were extracted by C-section on the 21st day of gestation. The gene expression of system A (Slc38a1, Slc38a2, and Slc38a4) and L (Slc7a2, Slc7a5, Slc7a8) amino acid transporters was measured in placenta and amino acid concentrations determined in maternal and fetal plasma. RESULTS: Maternal LP diet decreased fetal (4.01 ± 0.03 vs. 5.45 ± 0.07 g, p < 0.0001) and placental weight (0.617 ± 0.01 vs. 0.392 ± 0.04 g, p < 0.001), by 26 and 36% respectively, compared with NP diet. Supplementation with either CIT or ARG increased fetal birth weight by ≈ 5 or 11%, respectively (4.21 ± 0.05 and 4.48 ± 0.05 g vs. 4.01 ± 0.03 g, p < 0.05). CIT supplementation produced a 5- and 2-fold increase in fetal plasma citrulline and arginine, respectively, whereas ARG supplementation only increased fetal arginine concentration. LP diet led to lower placental SNAT 4 mRNA, and higher LAT2 and SNAT1 expression, compared with NP. SNAT4, 4hFC, LAT2 mRNA were up-regulated in LP + CIT and LP + ARG group compared with the un-supplemented LP group. Higher level of LAT1 mRNA was also observed in the LP + CIT group than in the LP group (p < 0.01). SNAT2 expression was unchanged in response to CIT or ARG supplementation. Fetal amino acid concentrations were decreased by LP diet, and were not restored by CIT or ARG supplementation. CONCLUSIONS: The current findings confirm supplementation with citrulline or arginine enhances fetal growth in a rat model of IUGR. They further suggest that: 1) citrulline and arginine administered orally to the pregnant mother may reach fetal circulation; 2) citrulline effectively raises fetal arginine availability; and 3) although it failed to increase the concentrations of essential amino acids in fetal plasma, citrulline or arginine supplementation upregulates the gene expression of several placental amino acid transporters.

Perinatal Hypercholesterolemia Exacerbates Atherosclerosis Lesions in Offspring by Altering Metabolism of Trimethylamine-N-Oxide and Bile Acids.

OBJECTIVE: Experimental studies suggest that maternal hypercholesterolemia may be relevant for the early onset of cardiovascular disease in offspring. We investigated the effect of perinatal hypercholesterolemia on the atherosclerosis development in the offspring of apolipoprotein E-deficient mice and the underlying mechanism. APPROACH AND RESULTS: Atherosclerosis and related parameters were studied in adult male or female apolipoprotein E-deficient mice offspring from either normocholesterolemic or hypercholesterolemic mothers and normocholesterolemic fathers. Female born to hypercholesterolemic mothers had more aortic root lesions than female born to normocholesterolemic mothers. Lesions in whole aorta did not differ between groups. Higher trimethylamine-N-oxide levels and Fmo3 hepatic gene expression were higher in female born to hypercholesterolemic mothers offspring compared with female born to normocholesterolemic mothers and male. Trimethylamine-N-oxide levels were correlated with the size of atherosclerotic root lesions. Levels of hepatic cholesterol and gallbladder bile acid were greater in male born to hypercholesterolemic mothers compared with male born to normocholesterolemic mothers. At 18 weeks of age, female born to hypercholesterolemic mothers showed lower hepatic Scarb1 and Cyp7a1 but higher Nr1h4 gene expression compared with female born to normocholesterolemic mothers. Male born to hypercholesterolemic mothers showed an increase in Scarb1 and Ldlr gene expression compared with male born to normocholesterolemic mothers. At 25 weeks of age, female born to hypercholesterolemic mothers had lower Cyp7a1 gene expression compared with female born to normocholesterolemic mothers. DNA methylation of Fmo3, Scarb1, and Ldlr promoter regions was slightly modified and may explain the mRNA expression modulation. CONCLUSIONS: Our findings suggest that maternal hypercholesterolemia may exacerbate the development of atherosclerosis in female offspring by affecting metabolism of trimethylamine-N-oxide and bile acids. These data could be explained by epigenetic alterations.

Perinatal high methyl donor alters gene expression in IGF system in male offspring without altering DNA methylation.

AIM: To investigate the effect of a protein restriction and a supplementation with methyl donor nutrients during fetal and early postnatal life on the expression and epigenetic state of imprinted genes from the IGF system. MATERIALS & METHODS: Pregnant female rats were fed a protein-restricted diet supplemented or not with methyl donor. RESULTS: Gene expression of the Igf2, H19, Igf1, Igf2r and Plagl1 genes in the liver of male offspring at birth and weaning was strongly influenced by maternal diet. Whereas the methylation profiles of the Igf2, H19 and Igf2r genes were remarkably stable, DNA methylation of Plagl1 promoter was slightly modified. CONCLUSION: DNA methylation of most, but not all, imprinted gene regulatory regions was resistant to methyl group nutritional supply.

Maternal citrulline supplementation enhances placental function and fetal growth in a rat model of IUGR: involvement of insulin-like growth factor 2 and angiogenic factors.

OBJECTIVE: To determine the effects of maternal citrulline supplementation on fetal growth and placental efficiency in a rat model of intrauterine growth restriction (IUGR) induced by maternal protein restriction. METHODS: Pregnant Sprague-Dawley rats were randomly assigned to three groups: NP (receiving a control 20% protein diet), LP (a 4% protein diet), or LP-CIT (an LP diet along with L-citrulline, 2 g/kg/d in drinking water). On the 15th and 21st day of gestation (GD15 and GD21, respectively), dams underwent a C-section, by which fetuses and placentas were extracted. The expression of genes involved in placental growth and angiogenesis was studied by quantitative RT-PCR. RESULTS: Maternal citrulline supplementation increased fetal weight at GD21, and fetal weight/placental weight ratio, an index of placental efficiency, from mid gestation (p < 0.001). The expression of Igf2-P0, a placenta-specific variant of insulin-like growth factor 2 (Igf2) gene, and Vegf and Flt-1, involved in angiogenic pathways, was enhanced in the LP-CIT group (versus NP, p < 0.001, p < 0.01, and p < 0.05 for Igf2-P0, Vegf, and Flt-1, respectively). CONCLUSIONS: In a model of IUGR induced by protein deprivation, citrulline enhances fetal growth, placental efficiency, and the expression of genes involved in angiogenesis. The relevance of such effect in human pregnancies complicated by IUGR warrants further study.

[The new paradigm of the developmental origin of health and diseases (DOHaD)--Epigenetics and environment: evidence and missing links]. / Le nouveau paradigme de l'origine développementale de la santé et des maladies (DOHaD)--Épigénétique, environnement: preuves et chaînons manquants.

According to the new paradigm of the Developpemental Origins of Health and Disease (DOHaD), the environmental factors to which an individual is exposed throughout his life can leave an epigenetic footprint on the genome. A crucial period is the early development, where the epigenome is particularly sensitive to the effects of the environment, and during which the individual builds up his health capital that will enable him to respond more or less well to the vagaries of life. The research challenge is to decipher the modes of action and the epigenetic mechanisms put into play by environmental factors that lead to increased disease susceptibility or resilience. The challenge for health is to translate these scientific discoveries into action through, among others, the establishment of preventive recommendations to slow down the growing incidence of non communicable diseases.

[Epigenetics in transgenerational responses to environmental impacts: from facts and gaps]. / Épigénétique et réponses transgénérationnelles aux impacts de l'environnement--Des faits aux lacunes.

The existence of non-genetic and non-cultural mechanisms that transfer information on the memory of parental exposures to various environments, determining the reactivity of the following generations to their environments during their life, are of growing interest. Yet fundamental questions remain about the nature, the roles and relative importance of epigenetic marks and processes, non-coding RNAs, or other mechanisms, and their persistence over generations. A model incorporating the various transmission systems, their cross-talks and windows of susceptibility to the environment as a function of sex/gender of parent and offspring, has yet to be built.

[The sensory and nutritional experience of parents guide offsprings' feeding behavior]. / L'expérience sensorielle et nutritionnelle des parents et leur état métabolique orientent le comportement alimentaire de leur descendance.

Eating behavior is strongly regulated by intrinsic physiological factors and largely influenced by the individual and cultural environments. Excessive food intake and sedentary lifestyle are the main reasons for the global epidemic of obesity. The influence of family background on eating habits makes no doubt but the fact that the nutritional, metabolic and hormonal status of the parents before conception, and of the mother during gestation and lactation, may influence the child's future eating behavior is an innovative concept that opens the way for preventive policies. In the last decades, research on human cohorts and animal models have targeted biological mechanisms (neuroanatomical, epigenetic) that give some clues on how eating behavior can be formatted by early nutrition and related sensory experience.

A Common Genetic Variant in the Insulin Receptor Gene Is Associated with Eating Difficulties at 2 Years of Age in a Cohort of Preterm Infants.

BACKGROUND/AIMS: Children born preterm are more likely than full-term infants to develop eating difficulties that can affect their growth. Although this behavior is certainly influenced by their fetal and postnatal history, a large individual variability exists that results from a complex interaction between genetic and environmental factors. We performed an original pilot study to identify common genetic variants associated with eating difficulties at 2 years of age in the POLYNUCA cohort of preterm infants. METHODS: Eating behavior was assessed using a parental questionnaire in a cohort of 234 very preterm infants (including 38 pairs of twins). Eighty-two common single nucleotide polymorphisms (SNPs) were selected in a total of 40 candidate genes involved in the regulation of energy homeostasis and food intake. RESULTS: Eating behavior was strongly correlated in monozygotic (r = 0.92, p = 0.001) but not dizygotic twins (r = 0.27, p = 0.14), suggesting a strong heritability of this trait. One SNP (rs11671975) in the insulin receptor (INSR) gene was significantly associated with eating behavior. This effect was maintained after adjustment for birth weight Z score and maternal education level, two factors that are associated with eating difficulties at 2 years of age. CONCLUSION: The INSR gene is potentially associated with eating difficulties in preterm infants.

Protein content and methyl donors in maternal diet interact to influence the proliferation rate and cell fate of neural stem cells in rat hippocampus.

Maternal diet during pregnancy and early postnatal life influences the setting up of normal physiological functions in the offspring. Epigenetic mechanisms regulate cell differentiation during embryonic development and may mediate gene/environment interactions. We showed here that high methyl donors associated with normal protein content in maternal diet increased the in vitro proliferation rate of neural stem/progenitor cells isolated from rat E19 fetuses. Gene expression on whole hippocampi at weaning confirmed this effect as evidenced by the higher expression of the Nestin and Igf2 genes, suggesting a higher amount of undifferentiated precursor cells. Additionally, protein restriction reduced the expression of the insulin receptor gene, which is essential to the action of IGFII. Inhibition of DNA methylation in neural stem/progenitor cells in vitro increased the expression of the astrocyte-specific Gfap gene and decreased the expression of the neuron-specific Dcx gene, suggesting an impact on cell differentiation. Our data suggest a complex interaction between methyl donors and protein content in maternal diet that influence the expression of major growth factors and their receptors and therefore impact the proliferation and differentiation capacities of neural stem cells, either through external hormone signals or internal genomic regulation.

Excess of methyl donor in the perinatal period reduces postnatal leptin secretion in rat and interacts with the effect of protein content in diet.

Methionine, folic acid, betaine and choline interact in the one-carbon metabolism which provides methyl groups for methylation reactions. An optimal intake of these nutrients during pregnancy is required for successful completion of fetal development and evidence is growing that they could be involved in metabolic long-term programming. However, the biological pathways involved in the action of these nutrients are still poorly known. This study investigated the interaction between methyl donors and protein content in maternal diet during the preconceptual, pregnancy and lactation periods and the consequences on the rat offspring in the short and long term. Methyl donor supplementation reduced leptin secretion in offspring, whereas insulin levels were mostly affected by protein restriction. The joint effect of protein restriction and methyl donor excess strongly impaired postnatal growth in both gender and long term weight gain in male offspring only, without affecting food intake. In addition, rats born from protein restricted and methyl donor supplemented dams gained less weight when fed a hypercaloric diet. Methylation of the leptin gene promoter in adipose tissue was increased in methyl donor supplemented groups but not affected by protein restriction only. These results suggest that maternal methyl donor supplementation may influence energy homeostasis in a gender-dependent manner, without affecting food intake. Moreover, we showed that macronutrients and micronutrients in maternal diet interact to influence the programming of the offspring.

Effect of preterm birth and birth weight on eating behavior at 2 y of age.

BACKGROUND: Preterm infants frequently present eating difficulties in early childhood. Determinants of these difficulties are not known. OBJECTIVE: We assessed the influence of neonatal and maternal characteristics on eating behaviors at 2 y of age. DESIGN: The following 2 cohorts were compared: 234 preterm children born <33 wk of gestational age from the POLYmorphisme génétique, Nutrition et Comportement Alimentaire cohort and 245 term children from the Observation des Préférences ALImentaires du Nourrisson et de l'Enfant cohort. Eating behaviors were assessed by using the validated Children's Eating Difficulties Questionnaire, which assesses the following 2 dimensions: a low drive to eat and narrow food repertoire. Each dimension was graded from 2 to 10 with more severe difficulties reflected by a higher score. Children in the upper quintile were classified as having eating disorders. RESULTS: Compared with term children, preterm children had a worse drive-to-eat score (4.3 ± 1.6 compared with 3.6 ± 1.5, respectively; P = 0.001) and a marginally lower food-repertoire score (5.0 ± 1.5 compared with 4.8 ± 1.6, respectively; P = 0.05). In a multilevel logistic regression model, female sex [adjusted OR (aOR): 1.76; 95% CI: 1.08, 2.88; P = 0.025] and birth weight less than -1 z score (aOR: 2.88; 95% CI: 1.47, 5.67; P = 0.002) but not gestational age were associated with a worse drive to eat. A maternal level of education beyond high school was associated with lower risk of a poor food-repertoire score (aOR: 0.54; 95% CI: 0.32, 0.90; P = 0.02). CONCLUSIONS: Preterm children have more eating difficulties than term children do, but after adjustment for maternal and neonatal characteristics, gestational age is not associated with impaired eating behaviors at the age of 2 y. Female sex, a low maternal level of education, and less than -1 SD intrauterine growth are associated with eating difficulties at 2 y of age. This trial was registered at clinicaltrials.gov as NCT 00663572.

Nutritional programming affects hypothalamic organization and early response to leptin.

Nutritional programming, taking place in utero or early after birth, is closely linked with metabolic and appetite disorders in adulthood. Following the hypothesis that nutritional programming impacts hypothalamic neuronal organization, we report on discrepancies of multiple molecular and cellular early events that take place in the hypothalamus of rats submitted to intrauterine growth restriction (IUGR). Expression screening performed on hypothalami from IUGR rats at birth and at postnatal d 12 identified changes in gene expression of neurodevelopmental process (cell differentiation and cytoskeleton organization). Additionally, a slight reduction of agouti-related protein and a strong reduction of alpha-MSH-immunoreactive efferent fibers were demonstrated in the paraventricular nucleus of IUGR rats. Rapid catch-up growth of IUGR rats, 5 d after birth, had a positive effect on neurodevelopmental factors and on neuronal projections emanating from the arcuate nucleus. The molecular and cellular anomalies detected in IUGR rats can be related to the reduced and delayed plasma leptin surge from d 0-16 when compared with control and IUGR rats with catch-up growth. However, the ability of leptin to activate intracellular signaling in arcuate nucleus neurons was not reduced in IUGR rats. Other mechanism such as epigenetic regulation of the major appetite-regulating neuropeptides genes was analyzed in parallel with their mRNA expression during postnatal development. This study reveals the importance of an early catch-up growth that reduces abnormal organization of hypothalamic pathways involved in energy homeostasis, whereas protein restriction, maintained during postnatal development leads to an important immaturity of the hypothalamus.

Recovery of exfoliated cells from the gastrointestinal tract of premature infants: a new tool to perform "noninvasive biopsies?".

To gain insight into specific gene expression in the gastrointestinal (GI) tract of preterm infants, we adapted a method to isolate exfoliated epithelial cells. Gastric residual fluid aspirates (n = 89) or stool samples (n = 10) were collected from 96 neonates (gestational age, 24-36 wk). Cells were characterized by microscopic observation, cytokeratin-18 immunodetection, and expression of transcripts. The human origin of cellular DNA was confirmed by amplification of specific X and Y chromosome sequences. Isolation yielded 100-500 cells per sample for gastric aspirates (n = 8) and 10-20 cells for fecal samples (n = 5). Epithelial origin was confirmed by immunodetection of cytokeratin 18. Analyses of reverse transcribed products, using two independent methods, from 15 gastric fluid and two stool samples showed that 18S-rRNA and transcripts of beta-actin, glyceraldehyde-3-phosphate dehydrogenase (gapdh), and period1 were in quantities corresponding to at least 10 cells. On 59 aspirates, we found beta-actin transcripts (all but one), cytokeratin 18 (eight positive of eight samples), SLC26-A7-1 (13 positive of 19 samples), period2 (17 positive of 17 samples), and clock (25 positive of 26 samples). Exfoliated cells can be recovered from gastric aspirates and fecal samples and serve as a tool to investigate the impact of therapeutic and nutritional regimens on the maturation of GI functions.

Cotranscription and intergenic splicing of the PPARG and TSEN2 genes in cattle.

BACKGROUND: Intergenic splicing resulting in the combination of mRNAs sequences from distinct genes is a newly identified mechanism likely to contribute to protein diversity. Few cases have been described, most of them involving neighboring genes and thus suggesting a cotranscription event presumably due to transcriptional termination bypass. RESULTS: We identified bovine chimeric transcripts resulting from cotranscription and intergenic splicing of two neighboring genes, PPARG and TSEN2. These two genes encode the Peroxisome Proliferator Activated Receptors gamma1 and gamma2 and the tRNA Splicing Endonuclease 2 homolog and are situated in the same orientation about 50 kb apart on bovine chromosome 22q24. Their relative position is conserved in human and mouse. We identified two types of chimeric transcripts containing all but the last exon of the PPARG gene followed by all but the first exon of the TSEN2 gene. The two chimers differ by the presence/absence of an intermediate exon resulting from transcription of a LINE L2 sequence situated between the two genes. Both transcripts use canonical splice sites for all exons coming from both genes, as well as for the LINE L2 sequence. One of these transcripts harbors a premature STOP codon and the other encodes a putative chimeric protein combining most of the PPARgamma protein and the entire TSEN2 protein, but we could not establish the existence of this protein. CONCLUSION: By showing that both individual and chimeric transcripts are transcribed from PPARG and TSEN2, we demonstrated regulation of transcription termination. Further, the existence and functionality of a chimeric protein harboring active motifs that are a priori unrelated is hypothesized.

Characterization of the bovine PRKAG3 gene: structure, polymorphism, and alternative transcripts.

The bovine PRKAG3 gene encodes the AMPK gamma3 subunit, one isoform of the regulatory gamma subunit of the AMP-activated protein kinase (AMPK). The AMPK plays a major role in the regulation of energy metabolism and mutations affecting the genes encoding the gamma subunits have been shown to influence AMPK activity. The gamma3 subunit is involved in the regulation of AMPK activity in skeletal muscle and strongly influences glycogen metabolism. Glycogen content in muscle is correlated to meat quality in livestock because it influences postmortem maturation process and ultimate pH. Naturally occurring mutations in the porcine PRKAG3 gene highly affect meat quality by influencing glycogen content before slaughter. We present the characterization of the bovine PRKAG3 gene and a polymorphism analysis in three cattle breeds. Thirty-two SNPs were identified among which 13 are in the coding region, one is in the 3' UTR, and 18 are in the introns. Five of them change an amino acid in the PRKAG3 protein sequence. Allelic frequencies were determined in the three breeds considered, and mutant alleles affecting the coding sequence are found at a very low frequency. Alternative splicing sites were identified at two positions of the gene, introducing heterogeneity in the population of proteins translated from the gene.

Correlation between bovine calpastatin mRNA transcripts and protein isoforms.

Calpastatin is a specific calpain protease inhibitor: calpains are a family of calcium-activated neutral proteases, which have been implicated in various processes. Despite all the available data concerning calpastatin, little is known about how this gene is regulated, particularly in bovine. The existence of four types of transcripts differing at their 5' ends (Type I, II, III, and IV) has been demonstrated. Here, we show that the Type I, II, and III transcripts are ubiquitous while Type IV is testis-specific. In addition, a Northern blot analysis revealed that the Type III transcript may have three different 3' termini. Using specific anti-peptide anti-sera, a correspondence between a 145 and a 125 kDa isoforms, and Type I and/or II and III transcripts, respectively, has been established. Finally, we discuss the origin of a 70 kDa isoform, recognized by anti-sera directed against the N-terminal region.

Four promoters direct expression of the calpastatin gene.

Calpastatin is a specific endogenous protein inhibitor of the ubiquitous calcium dependent proteinases mu- and m-calpain. The calpain-calpastatin system is involved in various physiological and pathological processes. In the present study, we determined the bovine calpastatin gene structure and demonstrated that four promoters direct its expression. The gene harbours 35 exons spanning at least 130kb on genomic DNA. Its structure is similar to that of mouse, pig, and human gene. Transient transfection assays in both C2C12 and COS7 cell lines demonstrated that the putative promoter regions situated 5' to exon 1xa, 1xb, 1u, and 14t were functional. We also established that the region situated upstream exon 14t is subjected to a tissue specific regulation. The implication of numerous high-scoring cis acting transcriptional motifs which are present in these regions will need to be determined. The existence of four promoters suggests differential expression patterns which must have a physiological significance.

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle.

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.