Partial replacement of glucose by galactose in the post-weaning diet improves parameters of hepatic health

 Replacing part of glucose with galactose in the post-weaning diet beneficially affects later life metabolic health in female mice

• Here, weanling female mice (C57BL/6JRccHsd) were fed a starch containing diet with glucose (32 en%) monosaccharide (GLU) or a diet supplemented with glucose/galactose (1:1 both 16 en%) GLU+GAL
• body weight, body composition, and food intake were determined weekly.
• Global hepatic mRNA expression, hepatic triglyceride (TG) and glycogen contents were determined by enzymatic assays.

The lactation period is followed by the period of weaning: a period when solid foods are gradually introduced in the diet

• The weaning period is characterized by a diversification of the diet, in particular in the carbohydrate fraction
• Due to the gradual reduction in milk intake, the relative intake from lactose decreases gradually during weaning, and, consequently, so does the intake of galactose.
• Galactose elimination from the blood is used as a (clinical) measure for hepatic function, although outcome is dependent on clearance by kidney cells and the magnitude of hepatic blood flow.

Animals and experimental setup

• All animal experimental procedures were approved by the national and local Animal Experimental Committees (AVD1040020171668) and executed in compliance with EU directive 2010/63/EU.
• Breeding pairs (C57BL/6JRccHsd mice) were purchased from Envigo and time-mated while on a standard rodent chow diet (AM-II, AB Diets, Woerden, The Netherlands)
• Litters were randomized, and standardized to six pups per nest, with two to four female pups each litter, at postnatal day (PN) one or two
• Female pups were weighed and their body composition (fat mass (FM) and lean mass (LM)) were determined with an EchoMRI 100 V
• BW, FM, LM, and food intake (FI) were determined weekly
• Whole blood was collected in MiniCollect serum tubes and stored at −80 °C

Indirect Calorimetry

• Continuous measurements of oxygen consumption, carbon dioxide production, hydrogen production, activity, water intake, and FI were executed as described in the Methods section.
• The first day of the measurements was considered as adaptation and the next 24 h (one 12 h light phase and one 12 h dark phase) were analyzed.

Serum measurements

• Commercial assays were used to measure serum concentrations of the following hormones and metabolites: insulin (Ultra-Sensitive Mouse Insulin ELISA Kit, Crystal Chem, Elk Grove Village, IL, USA), glucose (glucose GOD-PAP kit, Roche, Mannheim, Germany), leptin (Bio-Plex Pro Mouse diabetes assays, Bio-Rad laboratories, Veenendaal, The Netherlands), triglycerides (TG) (Liquicolour kit, Human), free fatty acids (NEFA-HR(s), Fujifilm Wako Chemicals Europe GMBH, Neuss, Germany)
• ALT
• Serum amyloid A 3 (SAA3) (Mouse SAA-3 ELISA, Merck chemicals B.V., Amsterdam, the Netherlands).
• Assays were performed according to the manufacturers' instructions; samples were tested in duplicate.

Hepatic measurements

• Hepatic glycogen content in the right lobe was determined with the Abcam Glycogen Assay kit (Abcam, Cambridge, UK)

Genome wide gene expression

• Two hundred ng RNA of each sample (n=12 per group) was assayed using mouse whole genome 8×60 K v. 2.2 microarrays (Agilent Technologies Inc.)
• Signals were extracted and normalized as described in [22]
• Analysis of pathway maps using all expressed genes with P<.05 was performed with MetaCore (version 6.37)
• Genes enriched in the top 10 pathway maps were gathered and presented as a heat map using GeneMaths XT 2.12 (Applied Maths, Sint-Martens-Latem, Belgium).

RT-qPCR

• data were normalized against β-2 microglobulin (B2m), Calnexin (Canx), and ribosomal protein S15 (Rps15).
• average gene expression per gene was set to 1.7 μg/mL.

Western blot

• Frozen liver tissue was grinded on liquid nitrogen, placed in ice-cold Tris-NaCL lysis buffer (50 mM, 150 mM) pH 7.4, with glycerol (10%), triton (1%) EDTA, trichostatin A (2 μM), nicotinamide, and phosphatase inhibitor-Mix I (Roche) and stored at−80 °C.
• Protein concentrations were determined in the supernatant with the DC protein assay (Bio-Rad).
• Samples were diluted to the same concentration, mixed with *** loading, heated to 95 °C for 10 min, and loaded on 4-12% Nupage Bis-Tris gels and ran for 40 min at 110 V and 60 min at 150 V, followed by transfer to a methanol-activated Immobilon-FL PVDF membrane (0.45 μm pore size, Merck). Protein transfer was verified with a Ponceau staining, and blots were rinsed with TBS+0.1% tween.

Statistics

• Graphpad Prism 5.04 was used for statistical analysis
• BW, FM, LM, and FI were analyzed using a two-way ANOVA
• For all other data, independent Student’s t tests were used to compare normally distributed data
• Normality was checked using a Saphiro-Wilk normality test
• When data was not normally distributed, data was log transformed and rechecked for normality

Body weight and food intake

• GLU+GAL mice had similar body weights as mice fed the glucose diet (GLU)
• Body composition analysis showed that fat mass, lean mass, and gonadal and mesenteric fat pad weights were similar in both groups
• Food intake was higher on the GLU diet, and cumulatively, mice on the diet consumed approximately 8.5% more food than those on GLU

Indirect Calorimetry

• To investigate metabolism and substrate utilization of animals on the GLU and GLU+GAL diets in more detail, animals were studied in an indirect calorimeter system.
• RER patterns showed no clear difference between GLU- and GAL-fed females
• Energy expenditure (EE) was not different during the 24-h period (Fig. 2E), nor during the light phase or the dark phase only (Table 1), nor was activity different (Fig 2F and Table 1).
• Water intake was, however, significantly higher in the animals on a galactose diet

Serum and hepatic parameters

• Despite higher overall food intake, serum insulin levels were significantly lower in the GLU+GAL animals compared to the control group (Fig. 3A).
• Serum glucose levels were not significantly different.
• Neither serum leptin nor serum FFA was significantly affected by the different monosaccharide composition of the diets, and liver mass was not affected.

Transcriptomics

• Overall, 1574 out of 29,287 expressed transcripts were significantly affected
• The majority of the (immune-related) regulated genes were lower expressed in the livers of GLU+GAL-fed vs. GLU-fed females.
• All other genes were expressed similarly in both dietary groups (Suppl. S2).

Protein expression

• To verify the effect on inflammation, acute phase protein SAA3, was studied in detail.
• The microarray data showed a significant downregulation of hepatic Saa3 mRNA expression in the livers of GLU+GAL-fed females (Fig. 6A), and this was confirmed by RT-qPCR and biochemical analysis (Fig 6B).
• Similarly, a non-significant downregulation was found in both the α- and the β-isoform of STAT1 protein.

Discussion

• The replacement resulted in slightly higher food intake (~8.5%) and substantially higher water intake (approximately twofold), without differences in body weight, body composition, or energy expenditure.
• In depth analysis of hepatic gene expression indicated a downregulation of inflammation-related pathways, and this downregulation was confirmed by lower serum SAA3 concentrations.
• Four weeks of feeding with a high glucose diet (60% w/w) was shown to increase hepatic TG levels in adult male mice.

Reference

10.1016/j.jnutbio.2019.108223