Early life exposure to maternal insulin resistance has persistent effects on hepatic NAFLD in juvenile nonhuman primates

Abstract

The origins of nonalcoholic fatty liver disease (NAFLD) may lie in early intrauterine exposures. Here we examined the maternal response to chronic maternal high-fat (HF) diet and the impact of postweaning healthy diet on mechanisms for NAFLD development in juvenile nonhuman primate (NHP) offspring at 1 year of age. Pregnant females on HF diet were segregated as insulin resistant (IR; HF+IR) or insulin sensitive (IS; HF+IS) compared with control (CON)-fed mothers. HF+IR mothers have increased body mass, higher triglycerides, and increased placental cytokines. At weaning, offspring were placed on a CON or HF diet. Only offspring from HF+IR mothers had increased liver triglycerides and upregulated pathways for hepatic de novo lipid synthesis and inflammation that was irreversible upon switching to a healthy diet. These juvenile livers also showed a combination of classical and alternatively activated hepatic macrophages and natural killer T cells, in the absence of obesity or insulin resistance. Our findings suggest that maternal insulin resistance, including elevated triglycerides, insulin, and weight gain, initiates dysregulation of the juvenile hepatic immune system and development of de novo lipogenic pathways that persist in vitro and may be an irreversible "first hit" in the pathogenesis of NAFLD in NHP.

Figure 1

Study design and maternal HF diet classification. Females were placed on a CON or chronic HF diet beginning prior to pregnancy. Maternal metabolic phenotype was measured during the early third trimester. A: Based on GTTs during pregnancy, females were classified as HF and IR (HF+IR) if they had an insulin AUC greater than two SDs above CON females. The dashed line represents two SDs above the mean in the CON group. Females on the HF diet were IS (HF+IS) if they had an insulin AUC similar to CON females. B: Offspring were exposed to the maternal diet and maternal metabolic phenotype from conception to weaning. At weaning, around 7 months of age, offspring were placed on a postweaning CON (white bars) or HF (black bars) diet until the end of study at 1 year of age. Offspring diet treatment groups are indicated.

Figure 2

Effect of maternal and postweaning HF diet on juvenile offspring hepatic lipid accumulation and lipogenic capacity. A: Liver triglyceride concentrations measured in offspring from CON, HF+IS, and HF+IR mothers (horizontal axis labels) on the postweaning CON (white bars) or HF (black bars) diet. In panels A and C, when the main effect of maternal group was significant, comparisons between groups are indicated: *P < 0.05 vs. maternal CON and **P < 0.05 vs. maternal HF+IS. B: Histological analysis of livers stained with hematoxylin and eosin. C: Hepatic mRNA expression of lipid metabolism genes (n = 21, 9, 5, 5, 9, and 16 per group). The main effect of postweaning CON versus HF diet is shown as #P < 0.05. D: Primary hepatocytes were treated for 8 h with high glucose and lipogenic hormones (insulin and dexamethasone). Gene expression results are expressed as fold increase relative to basal treatment (n = 2 CON/CON in white bars, 3 HF+IR/CON in black bars). *P < 0.05 vs. CON/CON; #P < 0.10 vs. CON/CON.

Figure 3

Effect of maternal and postweaning HF diet on offspring hepatic inflammation and stress pathways. Hepatic mRNA expression measured in offspring from CON, HF+IS, and HF+IR mothers (horizontal axis labels) on the postweaning CON (white bars) or HF (black bars) diet (n = 21, 9, 5, 5, 9, and 16 per group). When the main effect of maternal group was significant, comparisons between groups are indicated: *P < 0.05 vs. maternal CON and **P < 0.05 vs. maternal HF+IS. The main effect of postweaning CON versus HF diet is shown as #P < 0.05.

Figure 4

Effect of maternal HF diet on hepatic macrophage and hepatocyte inflammatory activation. Hepatic macrophages and hepatocytes were isolated from juvenile livers and studied after 3 h of treatment with PALM and LPS compared with basal treatment (n = 2 CON/CON, 3 HF+IR/CON). A: Expression of canonical macrophage and activation markers in hepatic macrophages from CON/CON (white bars) and HF+IR/CON (dashed bars) offspring. Results shown are least square means and SEM for each offspring group. *P < 0.05 for main effect of offspring group; #P < 0.15. B: Expression of inflammatory genes in hepatic macrophages from CON/CON (white bars) and HF+IR (dashed bars) offspring following basal, LPS, and PALM treatment. C: Expression of inflammatory genes in hepatocytes from CON/CON (white bars) and HF+IR/CON (black bars) offspring following basal, LPS, and PALM treatment. In panels B and C, all results are expressed relative to hepatocyte basal CON/CON group. When the main effect of treatment was significant, comparisons between treatments are indicated: *P < 0.05 vs. BASAL. The main effect of maternal group (CON/CON versus HF+IR/CON) is shown as #P < 0.05.

Figure 5

Relationship between maternal outcomes on the HF diet with juvenile offspring hepatic triglycerides and gene expression. Correlations between (A) juvenile offspring liver triglycerides and maternal insulin AUC (log transformed) during GTT, pregnancy body weight, and plasma triglycerides and (B) offspring inflammation and lipogenic gene expression with maternal insulin AUC. Open circles represent HF+IS (n = 9) and closed circles represent HF+IR (n = 25) maternal–offspring pairs. C: Mechanisms for NAFLD in HF+IR offspring. HF+IR placentas have increased transplacental lipids, decreased uterine and fetal blood flow, and increased placental inflammation, which increases fetal cytokine exposure. We speculate that this combination of lipid, cytokine, and hypoxia exposure in utero produced by the HF+IR mother programs the offspring liver for increased NAFLD at 1 year of age.