（HP 戦略研究・若手大学院奨励研究報告）

Effect of maternal glucoraphanin intake on chronic inflammation in the livers
and hypothalamic tissues of high fructose-diet-fed rat offspring exposed tomaternal undernutrition.
Anishma Karmacharya＊
Graduate School of Health Sciences, Aomori University of Health and Welfare
1 Glucoraphanin, ○2 Fructose, ○3 Inflammation, ○4 AMPK, ○5 Maternal undernutrition
Key words ○Ⅰ．Background
The fetal and neonatal environment is associated with subsequent development of diseases in adulthood.
Maternal undernutrition during pregnancy and lactation can cause dyslipidemia, hyperglycemia which can affectdifferent organs and cause metabolic disorders such as chronic liver disease, Type 2 diabetes in offspring during
their adulthood (1). A high-fructose diet (HFD) increases glucose intolerance, insulin resistance, dyslipidemia,
and liver inflammation (2).
AMP-activated protein kinase (AMPK) plays a major role in regulating glucose and lipid metabolism invarious tissues and organs, including the liver and skeletal muscle.
Glucoraphanin (GR) derived from broccoli, is a water soluble, relatively inert precursor of sulforaphane; thereactive isothiocyanate which has shown to be effective in different metabolic disorders such obesity, insulin
resistance, and non-alcoholic fatty liver disease (NAFLD) (3). However, less is known about the effect of GR
intake during lactation on the insulin resistance and liver inflammation in the adult rat offspring programmed bymaternal protein restriction.
Ⅱ．Objectives
The aim of the present study was to explore the effect of maternal intake of GR-containing broccoli powder (BP)during lactation on inflammation and dysfunction of autophagy in livers and hypothalamic tissues of highfructose-diet-fed adult rat offspring from dams fed on normal- or low- protein diets during pregnancy andlactation.
Ⅲ．Materials and Methods
The Animal Research Committee, Aomori University of Health and Welfare, approved this study and all
experimental procedures were performed in accordance with the Institutional Guidelines for Animal
Experimentation (Permission number: 21008). Pregnant rats received diets containing 20% (Normal Protein;NP) or 8% (Low Protein; LP) casein. While 0 or 0.74% BP containing NP diets (NP/NP or NP/ NP+BP) inExperiment (Expt.) 1 and 0 or 0.74% BP containing LP diets (LP/LP or LP/LP+BP in Expt. 2 were provided
during lactation. At weaning, pups that received a diet of distilled water (W) or 10% fructose solution (Fr) weredivided into six groups: NP/NP/W, NP/NP/Fr, NP/NPBP/Fr in Expt. 1 and LP/LP/W, LP/LP/Fr, LP/ LPBP /Fr inExpt. 2. At week 13 after treatment, male offsprings were weighed, and blood samples were taken. The samples
that we analyzed have already collected in 2023. The plasma glucose (Glc) and insulin levels were measured.
Homeostatic model assessment of insulin resistance (HOMA-IR), a biomarker of insulin sensitivity, wasperformed according to the equation (4). Paraformaldehyde-fixed livers and hypothalamic tissues wereembedded in paraffin and cross-sections were prepared. Then, their sections were stained for macrophages by
incubating an anti-CD68 antibody, and the number of macrophages in the livers were counted. However, thenumber of microglia that serve as brain macrophages has not been determined, because the staining methods are＊

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still being established. To examine protein expression of AMPK and NFκB p65 in the western blot analysis oflivers was carried out. Because the western blot analysis for hypothalamic tissues are still being established, theprotein expression levels in the hypothalamic tissues have not been examined.
Ⅳ．Results and Discussion
1. Effect of BP intake during lactation on plasma insulin and HOMA-IR index
In Expt. 1, plasma Glc levels and HOMA-IR values were significantly higher in the NP/NP/Fr and NP/NPBP/Fr
than in the NP/NP/W. However, there was no significant difference between the NP/NP/Fr and NP/NPBP/Fr. No
significant differences in the plasma insulin levels were found among the three groups. In Expt. 2, plasma Glclevels in the LP/LP/Fr and LP/LPBP/Fr were increased significantly compared to the LP/LP/W. No significant
differences were observed between the LP/LP/Fr and LP/LPBP/Fr. Insulin levels in the LP/LP/Fr weresignificantly higher than those in the LP/LP/W. Conversely, the levels were significantly lower in theLP/LPBP/Fr than in the LP/LP/Fr. Similarly, the HOMA-IR values in the LP/LPBP/Fr were significantly lower
than in the LP/LP/Fr.
2. Effect of BP intake during lactation on macrophages infiltration
The number of macrophages in the NP/NP/Fr was significantly increased compared to the NP/NP/W; however,there was no significant difference between the NP/NP/Fr and NP/NPBP/Fr in Expt.1. In Expt. 2, themacrophage number in the LP/LP/Fr was significantly higher than that in the LP/LP/W. Conversely, the numberin the LP/LPBP/Fr was significantly lower than in the LP/LP/Fr, suggesting that BP intake during lactation may
suppress the macrophage infiltration in the liver of HFD-fed adult offspring programmed by maternal protein
restriction.
3. Effect of BP intake during BP on NFκB p65 protein expression
NFκB p65 protein expression levels were significantly higher in the NP/NP/Fr and NP/NPBP/Fr than theNP/NP/W, but not significantly different between the NP/NPBP/Fr and NP/NP/Fr. In Expt. 2, NFκB p65 protein
expression levels were significantly higher in the LP/LP/Fr than the LP/LP/W. Conversely, these levels weresignificantly lower in the LP/LPBP/Fr than the LP/LP/Fr.
V. Conclusion
We showed that maternal intake of BP ameliorated insulin resistance and liver inflammation via the upregulationof AMPK activation and/or the downregulation of NFκB activation of HFD-fed adult offspring programmed bymaternal protein restriction.
VI. References
1. Lillycrop KA et al. J Nutr. 2005; 135: 1382.
2. Castro M et al. Life Sci. 2015; 137: 1.
3. Zhang DM et al. Nutrients. 2017; 9: 335.
4. Okita K et al. Endocr J. 2013; 60: 283.
VII. Presentation・Karmacharya A, et al. Effect of maternal broccoli powder intake on autophagy flux in the livers of highfructose-fed male rat offspring from protein restricted mothers. 第 77 回日本栄養食糧学会大会, 2023 年 5 月,札幌市
・Karmacharya A, et al. Effect of maternal glucoraphanin intake on lipid and glucose metabolism in highfructose-fed male rat offspring exposed to maternal protein restriction. 2023 年度青森県保健医療福祉研究発
表会, 2023 年 12 月, 青森市