Objective
We hypothesized that gestationally programmed nonalcoholic fatty liver disease in low-birthweight offspring is mediated through nutrient sensors nicotinamide adenine dinucleotide+–dependent histone deacetylase (SIRT1) and AMP-activated protein kinase (AMPK).
Study Design
Pregnant dams received ad libitum food or were 50% food restricted from pregnancy days 10-21 to produce control and low-birthweight newborn offspring, respectively. All pups were nursed by control dams and weaned to ad libitum feed. We determined hepatic SIRT1 and AMPK activities and protein expression of lipid targets in low-birthweight and control fetuses, newborns, and adult offspring (3 months).
Results
Low-birthweight fetuses demonstrated increased prenatal hepatic SIRT1 activity, although with increased lipogenesis. After birth, low-birthweight newborn offspring undergo postnatal suppression of hepatic SIRT1 and AMPK activities in conjunction with increased lipogenesis, decreased lipolysis, and increased fat stores.
Conclusion
These findings suggest that undernutrition stress in utero may program hepatic nutrient sensors to perceive normal postnatal nutrition as a state of nutrient excess with the induction of hepatic lipid storage.
The prevalence of adult and childhood nonalcoholic fatty liver disease (NAFLD) has increased markedly in conjunction with increasing prevalence of obesity. Although the incidence of NAFLD, which is associated with fat deposits within hepatocytes of the liver parenchyma, varies dependent on the diagnostic criteria (liver function enzymes, ultrasound scanning, or liver biopsy), it is estimated that 20% of adults and 10% of children (2-19 years) in the United States have NAFLD as diagnosed by liver biopsy.
NAFLD occurs as a result of dysregulated lipid homeostasis, of which increased de novo hepatic fatty acid lipogenesis and decreased fatty acid β-oxidation are the primary contributors. Lipogenesis is mediated by lipogenic transcription factor sterol regulatory element-binding protein (SREBP-1c) and its downstream lipogenic enzyme target fatty acid synthase (FAS). Fatty acid oxidation is facilitated by coactivator, peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and downstream transcription factor, hepatocyte nuclear factor (HNF-4α). Importantly, upstream factors that regulate both these pathways include the nutrient/metabolic sensors, nicotinamide adenine dinucleotide (NAD)+–dependent histone deacetylase (SIRT1) and AMP-activated protein kinase (AMPK).
SIRT1 is a histone deacetylase that responds to NAD/NADH levels ; AMPK is a heterotrimeric protein kinase that responds to AMP/ATP ratio. Notably, SIRT1 and AMPK can activate each other, and both of them modulate hepatic lipid homeostasis through their deacetylation/phosphorylation activities on similar downstream target genes, SREBP-1c and PGC-1α. SIRT1 deacetylation of SREBP-1c inhibits activity by decreasing its stability and its association with its lipogenic target gene promoters, whereas AMPK-mediated phosphorylation of SREBP-1 inhibits proteolytic cleavage transcriptional activation of SREBP-1. In contrast, SIRT1 deacetylation and AMPK phosphorylation of PGC-1α increases PGC-1α activity, thereby enhancing the expression of the metabolic genes that are involved in hepatic fatty acid β-oxidation. Thus, in response to low nutritional availability, increased SIRT1 and activated AMPK inhibit hepatic fat accumulation by suppressing SREBP-1-mediated lipogenesis and enhancing PGC-1α–mediated oxidation, respectively.
In obese human and animal models of diet-induced obesity that have NAFLD, hepatic SIRT1 and AMPK levels are reduced, which is consistent with increased energy status. Furthermore, in diet-induced obese mice, SREBP-1c acetylation levels are highly elevated, and the overexpression of SIRT1 decreases the acetylation levels with resultant down-regulation of lipogenesis.
It is now well-recognized that an important contributor to the high prevalence of obesity and metabolic syndrome is the “programming” effects of low birthweight (LBW). Both human and animal studies have demonstrated an association between LBW and NAFLD. Specifically, pediatric NAFLD, as confirmed by biopsy, has been shown in LBW Italian children. In animal studies, maternal food restriction during rat pregnancy results in LBW newborns, who have adult obesity that includes leptin/insulin resistance and NAFLD, when nursed normally. Despite relative undernutrition, LBW fetuses demonstrate excess lipid accumulation in embryonic liver at day 20 (e20) before the onset of offspring obesity, which suggests that NAFLD is programmed gestationally. However, the molecular mechanism that contributes to programmed NAFLD is unknown.
We hypothesized that gestationally programmed NAFLD in LBW offspring is mediated by nutrient sensors (eg, SIRT1/AMPK) that regulate hepatic lipogenesis. To address this hypothesis, we studied fetal (e20), newborn (p1), and adult (3 months) LBW and control offspring, and determined hepatic (1) SIRT1 activity and activated AMPK and (2) protein expression of common downstream lipid targets (PGC1-α, HNF4-α, SREBP-1c, FAS).
Materials and Methods
Maternal rat and offspring diet
The studies were approved by the Animal Research Committee of the Los Angeles BioMedical Research Institute at Harbor-University of California Los Angeles and were in accordance with the American Association for Accreditation of Laboratory Care and National Institutes of Health guidelines. We used our well-characterized rat model of maternal undernutrition. Briefly, first-time pregnant Sprague Dawley rats (Charles River Laboratories, Hollister, CA) were housed in a facility with constant temperature and humidity, and a controlled 12:12-hour light/dark cycle. At 10 days of gestation, rats were provided either an ad libitum diet (control; n = 18) of standard laboratory chow (Lab Diet 5001; Lab Diet, Brentwood, MO; protein, 23%; fat, 4.5%; metabolizable energy, 3030 kcal/kg) or a 50% food-restricted diet (n = 12) to produce LBW newborn offspring. Fifty percent diet was determined by quantification of normal intake in the ad libitum fed rats. The respective diets were given from 10 days of pregnancy to term. After delivery, litter size was standardized to 8 (4 male and 4 female), and all pups were cross-fostered to ad libitum fed control dams. At 21 days old, all offspring were weaned to ad libitum laboratory chow and maintained on this diet until 3 months of age. The 2 groups of offspring from ad libitum and food-restricted dams are designated as control and LBW, respectively.
Offspring study age and liver collection
At e20, dams (n = 6 per group) received an overdose of 4% isoflurane; the uterus was delivered intact operatively, and individual fetuses were removed. After birth at 1 day of age (p1), 1 male per litter (n = 4 from 4 litters per group) was decapitated. At 3 months of age, 1 male per litter (n = 6 per group) received an overdose of pentobarbital (200 mg/kg bodyweight). In all cases, liver was collected, snap frozen, and stored at −80°C for analysis. In addition, liver was fixed in 4% formaldehyde for lipid staining.
SIRT1 deacetylase assay
Liver nuclear extracts were prepared as previously described ; protein concentration was determined (bicinchoninic acid), and SIRT1 deacetylase activity was assayed with a deacetylase fluorometric kit (CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay Kit; MBL International Corporation, Woburn, MA). The final reaction mixture (50 μL) contained 50 mmol/L Tris-HCl (pH 8.8), 4 mmol/L MgCl 2 , 0.5 mmol/L dithiothreitol, 0.25 mAU/mL Lysyl Endopeptidase (Wako Chemicals USA Inc, Richmond, VA), 1 μmol/L trichostatin A, 200 μmol/L NAD, and 5 μL of nuclear extract. Immediately on the addition of fluorosubstrate peptide (20 μmol/L final concentration), the fluorescence intensity at 460 nm (excitation at 340 nm) was measured every 5 minutes over a 1-hour period period. All samples were analyzed in duplicate and normalized by protein concentration; the values are reported as relative fluorescence/micrograms of protein (AU).
Western blot
Protein was extracted from liver tissues (n = 4 from 4 litters per group per age) by homogenizing with RIPA buffer that contained cocktail proteinase inhibitors (Thermal Company, Rockford, IL). The lysate concentration was determined by bicinchoninic acid solution (Pierce, Rockford, IL). Equal amounts of protein (50 μg) were separated on a 4-12% Bis-Tris gel (Bio-Rad Laboratories, Hercules, CA). The separated proteins were transferred electrophoretically to nitrocellulose membrane. Western blot data were normalized to actin and presented as fold change. Anti-actin antibody was bought from Sigma (Sigma-Aldrich, St. Louis, MO); all other antibodies were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and diluted 1:1000. The band density was analyzed with Quantity One software (Bio-Rad Laboratories) for AMPK (63 kd), phosphorylated AMPK (pAMPK; 65 kd), HNF4α (53 kd), PGC-1α (90 kd), SREBP-1c (125 kd), and FAS (270 kd). For detection of pAMPK, NaF (50 mmol/L), which had been demonstrated to be an effective phosphoseryl and phosphothreonyl protein phosphatase inhibitor, was added to all buffers.
Hematoxylin-eosin and oil Red O staining of liver sections
Livers from 3-month-old LBW and control offspring were used for the current study; we have reported the results for e20 and p1 staining previously. After fixation of the livers with 10% formalin/phosphate-buffered saline solution, livers were sectioned (5μm) and stained with oil Red O (lipid) and hematoxylin and eosin (nuclei) for histologic examination.
Statistical analysis
Differences between control and LBW groups were compared by analysis of variance. Data are expressed as mean ± SE. Probability values of ≤ .05 were considered to be statistically significant.
Results
Offspring bodyweight
As previously reported, LBW fetuses were significantly growth restricted at e20 (2.29 ± 0.03 vs 2.64 ± 0.04 g; P < .001) and p1 (6.8 ± 0.2 vs 7.3 ± 0.1 g; P < .01) as compared with controls. When nursed by control dams, LBW offspring showed rapid catch-up growth and were markedly heavier at 3 months of age (498 ± 9 vs 473 ± 6 g; P < .01).
SIRT1 deacetylase activity and activated AMPK
Consistent with maternal/fetal undernutrition, LBW fetal liver at e20 demonstrated increased SIRT1 deacetylase activity compared with control liver. In response to maternal lactation and nursing, control p1 newborns demonstrated a marked increase in SIRT1 activity that persisted to 3 months. In contrast, LBW newborns exhibited markedly reduced SIRT1 activity at p1 and 3 months of age ( Figure 1 , A).
At e20, LBW fetuses had comparable pAMPK/AMPK ratio with control fetuses. Control pAMPK levels were stable from e20 through 3 months of age. However, LBW pAMPK/AMPK ratio was decreased significantly compared with control males at p1 and 3 months of age ( Figure 1 , B and C).
Downstream targets of SIRT1 and AMPK
Because SIRT1 deacetylase activity and AMPK phosphorylation directly influence downstream targets, we determined the protein expression of fatty acid oxidative (PGC-1α and HNF-4α) and lipogenic genes (SREBP-1c and FAS). LBW offspring showed no change in hepatic PGC-1α expression at e20; however, at p1 and 3 months of age, the expression was decreased significantly compared with controls ( Figure 2 , A and B). HNF-4α levels were significantly decreased in LBW offspring at all 3 ages compared with controls ( Figure 2 , A and C).
