Development of insulin resistance preceded major changes in iron homeostasis in mice fed a high-fat diet

 Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) have been associated with dysregulation of iron metabolism

• The basis for this association is not completely understood.
• Here, we studied temporal associations between onset of insulin resistance and dysregulated iron homeostasis in a mouse model of T2DM.
• Male C57Bl/6 mice (aged 8 weeks) were fed a high-fat diet (HFD; 60% energy from fat) or a control diet for 4, 8, 12, 16, 20 and 24 weeks.- HFD-feeding induced weight gain, hepato-steatosis and IR in the mice.

Insulin resistance (IR) is the hallmark of type 2 diabetes mellitus (T2DM).

• Several studies have shown a strong association between high levels of serum ferritin (a marker of body iron stores) and increased risk of T2DM.
• These studies seem to suggest that increased body iron per se, and not inflammation, may mediate the link between increased seri-ferritin levels and TDM.
• The mechanisms that underlie such associations are complex and not completely understood.

Animals

• Male C57BL/6J mice were shifted from a "chow" diet (Diet no. #D131, Scientific Animal Food and Engineering, France) to a control diet (CD) (Research Diets, Inc., USA, #D12450J, with 10% of total energy derived from fat).
• At 8 weeks of age, mice were randomly allocated to receive either a high-fat diet (HFD) or a CD for 4, 8, 12, 16, 20 or 24 weeks.
• A subset of HFD-fed and CD-fed mice (n=6 in each group) were euthanized by cervical dislocation under deep inhalational anesthesia (using isoflurane).

Evaluation of insulin sensitivity in vivo

• Intraperitoneal glucose tolerance tests (ipGTT) and insulin tolerance tests were carried out to determine glucose tolerance and insulin sensitivity, as described earlier [38,39]. These were done at baseline and towards the end of the periods of feeding, 3 and 2 days prior to euthanasia, respectively.

Isolation of erythroid progenitor cells (Ter119+ cells) from bone marrow

• Single-cell suspensions of bone marrow were prepared as described previously [40].
• These were used for isolation of Ter119 + cells (erythridite precursors), by magnetically activated cell sorting (Octo-MACS) using anti-mouse Ter119 MicroBeads.

Quantitative real-time PCR (qPCR)

• RNA was isolated from the liver, adipose tissue and Ter119+ cells (isolated from bone marrow), using Tri-Reagent (Sigma, India), according to the manufacturer’s instructions.
• RNA isolates were subjected to DNase I treatment (Ambion DNA-free kit). RNA integrity was confirmed by denaturing agarose gel electrophoresis.

Western blotting

• Homogenized adipose tissue samples were homogenized in RIPA buffer and separated on 10% SDS-PAGE gels.
• Separated proteins were electro-transferred onto PVDF membrane (0.45 μm, Immobilon-P, Millipore, Merck, Germany) at 80 V for 2 h
• Bands were detected using a chemiluminescence substrate kit (SuperSignal West Dura, Thermo Fisher Scientific, USA)
• The intensities of the bands were quantified using ImageJ software and normalized to that of β-actin, which was used as a loading control

Tissue iron estimation

• Acid extracts were prepared and iron content was estimated by a colorimetric assay based on bathophenanthrolene-dye binding
• Atomic absorption spectrophotometry (AAS) was used to estimate iron content in adipose tissue samples
• Approximately 100 mg of eWAT was weighed and homogenized in 500 μL of RIPA buffer and then centrifuged at 14,000×g for 10 min.
• The supernatant obtained was used for estimation of iron

Visualization of tissue iron by Perls' perfusion staining

• In situ, mice under terminal anesthesia were perfused with 30 mL of phosphate-buffered saline containing heparin (5 U/mL), at a rate of 6 mL/min.

Biochemical analyses of serum samples

• Serum samples were used for estimation of the following analytes: insulin, hepcidin, adiponectin, erythropoietin, C-reactive protein, GDF-15, interleukin-6, iron, and triglyceride

Histological examination of liver

• Liver tissue was fixed in 10% buffered formalin, embedded in paraffin and four-micron thick sections were made for histological examination.
• Tissue sections were stained with hematoxylin and eosin (H&E) for light microscopy and additional sections stained with Prussian blue for visualization of iron deposits in the liver (Perls's staining).
• All slides were examined by a trained histopathologist. Grading of steatosis was done based on standard criteria

Statistical Analysis

• Statistical Package for Social Scientists (SPSS) version 16.11.0

High-fat feeding increased body weight and induced hepato-steatosis, glucose intolerance, insulin resistance, and hyperinsulinemia in mice

• HFD-fed mice progressively gained weight, while body weights in CD-fed were not significantly affected
• The content of triglycerides in the liver increased progressively, with increasing duration of feeding in mice fed HFD; this was not seen in those fed CD.
• There was no significant worsening of glucose tolerance.

HFD-feeding decreased hepatic hepcidin expression

• both type of diet and duration of feeding were found to individually affect hepatic Hamp1 expression.
• No significant interaction was found between the two, however, no significant interaction between the time of feeding and Hamp1 mRNA expression.

HFD-feeding decreased hepatic iron content and caused changes in expression of iron-related proteins in the liver

• Both the type of diet and duration of feeding had significant effects on Tfrc mRNA expression.
• Expression of mRNA for BMP6 and matriptase-2 was not affected by HFD.

HFD-feeding did not affect serum iron levels, markers of inflammation, or proteins involved in duodenal iron absorption.

• Serum ferritin levels increased with increasing durations of feeding, but this effect was not statistically significant (diet effect P=0.156) (Fig. 4A).
• Markers of inflammation (serum C-reactive protein [CRP], mRNA levels of Saa1, and IL-6 in the liver) were also not significantly affected in response to the HFD.

erythroid regulators of hepcidin expression in Ter119+ cells isolated from bone marrow was unaffected by HFD.

• Hemoglobin levels in HFD- and CD-fed mice were similar at the various time points studied, and serum erythropoietin levels were not significantly affected by the feeding.

Iron content in eWAT was higher in HFD-fed than in CD-fed mice.

• Protein levels of ferritin tended to increase and that of ferroportin tend to decrease over time in both types of diet fed mice
• Total iron content increased with duration of feeding
• A significant negative correlation was seen between the iron content in the liver and total iron content

Linear regression analyses

• IR in HFD-fed mice was associated positively with liver triglyceride levels, negatively with liver iron levels, and positively with eWAT iron content.
• Hepatic Hamp1 mRNA levels were found to be positively associated with serum ferritin levels in both groups of mice, and negatively associated with iron content in the CD-fed group.

Discussion C57Bl/6 mice fed a high-fat diet (HFD) develop many of the features of T2DM that are seen in humans

• Prior to this study, it was not clear how HFD feeding affected systemic iron homeostasis.
• The observed correlations of IR with hepatic steatosis and hepatic iron levels (Supplementary Table 3) are in agreement with previous literature in this area
• A decrease in intracellular iron levels, acting via the iron regulatory protein-iron response element (IRP-IRE) axis, has been shown to increase the stability of TfR1 mRNA
• Similarly, the IRP-RE axis would also be expected to decrease the translation of ferroportin, thus decreasing iron absorption.

Conclusions

• The onset of IR in HFD-fed mice preceded reductions in hepatic iron content and other alterations in iron-related parameters
• HFD did not affect serum iron levels, markers of inflammation, or expression of the erythroid regulators of hepcidin in the bone marrow
• Decreases in liver iron levels in response to HFD feeding were associated with a coincident increase in the iron content in adipose tissue
• Whether these increases play a role in the development of IR is unclear

Reference

10.1016/j.jnutbio.2020.108441