Lipid dysmetabolism in ceruloplasmin‐deficient mice revealed both in vivo and ex vivo by MRI, MRS and NMR analyses

Ceruloplasmin (Cp) is a ferroxidase that plays a role in cellular iron homeostasis and is mainly expressed in the liver and secreted into the blood. Cp is also produced by adipose tissue, which releases it as an adipokine. Although a dysfunctional interaction of iron with the metabolism of lipids has been associated with several metabolic diseases, the role of Cp in adipose tissue metabolism and in the interplay between hepatocytes and adipocytes has been poorly investigated. We previously found that Cp‐deficient (CpKO) mice become overweight and demonstrate adipose tissue accumulation together with liver steatosis during aging, suggestive of lipid dysmetabolism. In the present study, we investigated the lipid alterations which occur during aging in adipose tissue and liver of CpKO and wild‐type mice both in vivo and ex vivo. During aging of CpKO mice, we observed adipose tissue accumulation and liver lipid deposition, both of which are associated with macrophage infiltration. Liver lipid deposition was characterized by accumulation of triglycerides, fatty acids and ω‐3 fatty acids, as well as by a switch from unsaturated to saturated fatty acids, which is characteristic of lipid storage. Liver steatosis was preceded by iron deposition and macrophage infiltration, and this was observed to be already occurring in younger CpKO mice. The accumulation of ω‐3 fatty acids, which can only be acquired through diet, was associated with body weight increase in CpKO mice despite food intake being equal to that of wild‐type mice, thus underlining the alterations in lipid metabolism/catabolism in Cp‐deficient animals.


Supplementary Figure S2
Evaluation of liver size.The MRI analysis was performed using a 7-Tesla preclinical scanner as reported in Materials and Methods on both mice at 6 and 10 months of age.For liver volume quantification, manual segmentation was performed on each slice using the RARE-T2 sequence without fat suppression, and volume results from automatic summation of voxel volumes; images were analysed with MIPAV software.
At 10 months of age mice were euthanized by transcardial perfusion with saline and organs were collected.

Supplementary Figure S3
Glucose tolerance test (GTT) and insulin tolerance test (ITT).Tests were performed evaluating blood glucose level measured using a Contour Care glucometer (Ascensia Diabetes Care) as described in Materials and Methods in new groups of CpKO and WT mice at 6 and 10 months of age (groups "6 months bis" and "10 months bis").

Figure S3:
(A and C) Glucose and (B and D) insulin tolerance test (GTT and ITT respectively) performed on CpKO (purple) and WT (green) mice at 6 and 10 months of age (n= 6 mice/group, 3 males and 3 females).Data are shown as mean ± SEM of glucose concentration in the blood over 120 minutes from glucose or insulin administration.Statistical comparison of the two group of mice was done by Student's t test performed on the different time points and as comparison of the areas under the curve (AUC).For the value of each mouse AUC see Table S3.Food intake in light and dark cycles.Food intake in light and dark cycle was evaluated in selected group of mice at 6 and 10 months of age every 12 hours for a total of 3 days, as described in Materials and Methods.Mice were weighted to confirm CpKO overweight at 10 months of age.

Figure S4:
(A and C) Body weight evaluation of CpKO and WT mice at 6 and 10 months of age, respectively.(B and D) Evaluation of food intake in the 12 hours dark/light cycles assessed over 3 days and reported as grams of aet food at 6 and 10 months of age.Data are reported as mean ± SEM of the animal groups, each dot corresponds to one animal (n= 6 mice/group, 3 males and 3 females); statistical pvalues were evaluated by Student's t test comparing the two groups of animals at the same age and same phase of the cycle.For the value of each mouse see Table S3.
Food intake over 3 days in dark /light cycles (g)  S1 Supplementary Table S2 Fatty

Product code Lipid name Lipids classes
Lipid molecules from Merck/Sigma Aldrich used to build an "in-house" database for 1H-HR-NMR resonance Figure S1: Representative sagittal 2D-high-resolution rapid acquisition with relaxation enhancement T1 images acquired without fat suppression at abdominal (A) and renal (B) levels, used for in vivo quantification of the adipose tissue volume of all CpKO and WT experimental mice at 6 and 10 months of age.

Figure S2 :
Figure S2: (A) Representative manual segmentation of liver performed on slices of the RARE-T2 sequence and used for liver volume reconstruction.For the value of each mouse see TableS3.(B) Liver of CpKO and WT mice at 10 months of age.

Table S3
. (B) Liver of CpKO and WT mice at 10 months of age.