Iron elevation and adipose tissue remodeling in the epididymal depot of a mouse model of polygenic obesity

<div><p>Background</p><p>Iron dysregulation is a potential contributor to the pathology of obesity-related metabolic complications. KK/HIJ (KK) mice, a polygenic obese mouse model, have elevated serum iron levels. A subset of KK male mice display a bronzing of epididymal adipose tissue (eAT) associated with >100-fold (p<0.001) higher iron concentration.</p><p>Methods</p><p>To further phenotype and characterize the adipose tissue iron overload, 27 male KK mice were evaluated. 14 had bronzing eAT and 13 had normal appearing eAT. Fasting serum and tissues were collected for iron content, qPCR, histology and western blot.</p><p>Results</p><p>High iron levels were confirmed in bronzing eAT (High Iron group, HI) versus normal iron level (NI) in normal appearing eAT. Surprisingly, iron levels in subcutaneous and brown adipose depots were not different between the groups (p>0.05). The eAT histology revealed iron retention, macrophage clustering, tissue fibrosis, cell death as well as accumulation of HIF-2α in the high iron eAT. qPCR showed significantly decreased <i>Lep</i> (leptin) and <i>AdipoQ</i> (adiponectin), whereas <i>Tnfα</i> (tumor necrosis factor α), and <i>Slc40a1</i> (ferroportin) were up-regulated in HI (p<0.05). Elevated HIF-2α, oxidative stress and local insulin signaling loss was also observed.</p><p>Significance</p><p>Our data suggest that deposition of iron in adipose tissue is limited to the epididymal depot in male KK mice. A robust adipose tissue remodeling is concomitant with the high iron concentration, which causes local adipose tissue insulin resistance.</p></div

No systemic change despite local eAT remodeling and iron change.

<p>A) Serum iron levels between the NI and HI groups. B) Representative H&E staining for liver in NI and HI groups. C) Liver iron-related and inflammatory gene expression. <u><i>Abbreviations</i>:</u> <i>DcytB</i>, Duodenal cytochrome b; <i>Fpn</i>, ferroportin; <i>Hamp</i>, hepcidin; <i>Vegfa</i>, Vascular endothelial growth factor A; <i>Tnf</i>α, tumor necrosis factor; NI (open bar, n = 6) <i>v</i>.<i>s</i>. HI (filled bar, n = 6). *p< 0.05.</p

Iron deposition is associated with increased adipose tissue inflammation.

<p><b>A)</b> Representative adipose tissue F4/80 inmmunoflorescent staining from both NI (left) and HI (right) groups. Quantification for F4/80 antibody staining revealed macrophage clustering among the adipocytes in HI group. B) eAT gene expression with inflammatory gene markers. <u><i>Abbreviations</i>:</u> <i>Tnf</i>α, tumor necrosis factor; <i>Ccl2</i>, C-C Motif Chemokine Ligand 2; <i>Cxcl1</i>, C-X-C Motif Chemokine Ligand 1, <i>Il10</i>, interlukin 10; <i>Vegf</i>α, Vascular endothelial growth factor A. *p< 0.05.</p

The elevated iron deposition in the eAT is associated with increased HIF-2α accumulation.

<p><b>A)</b> eAT gene expression with iron-regulating gene markers. B)&C) adipose tissue iron-regulating protein levels and the quantifaction after normalizing with GAPDH. D) immunoflorescent staining for HIF-2α in NI and HI eAT. <u><i>Abbreviations</i>:</u> DMT1-IRE, Divalent metal transporter 1-Iron response element, <i>DcytB</i>, Duodenal cytochrome b; TfR1, <i>Fpn</i>, ferroportin; <i>Hamp</i>, hepcidin; Hif-2α, hypoxia inducible factor 2α. *p< 0.05.</p

Mouse tissue weights.

<p>Mouse tissue weights.</p

Iron deposition is tissue specific.

<p>A) Gross pictures of eAT from NI and HI groups. B) eAT iron was significantly elevated in HI group. C) Common iron deposition tissues such as liver, pancreas and heart had elevated iron levels in HI. However, iron deposition in duodenum or other adipose tissue depots (i.e. subcutaneous and brown adipose tissue) was not elevated in HI group. D) Serum testosterone and estradiol levels were not significantly different between NI and HI groups. <u><i>Abbreviations</i></u>: eAT, epididymal adipose tissue; SubQ, subcutaneous adipose tissue; BAT, brown adipose tissue; Pan, pancreas; Duo, duodenum. **p<0.01 NI (open bar) <i>vs</i> HI (filled bar).</p

A robust tissue remodeling in the HI mice epididymal fat pads.

<p><b>A)</b> Representitive adipose tissue histology from both NI (left) and HI (right) groups. H&E staining revealed robust adipose tissue cellularity changes in the HI group. A high number of non-adipocytes were observed residing between the adipocytes, Prussian blue staining also showed a robust iron overload in the eAT of HI group. Masson's trichrome stain revealed a significant amount of collagen fibers present among adipocytes in HI compared with NI. Caspase 3 staining suggested greater apoptosis in the HI group. B) Quantification for Prussian blue, Thricrome and caspase 3 staining. <u><i>Colors</i></u>: F4/80, brown; Trichrome, blue; Caspase 3, brown; Prussian blue, blue. Scale bars represent 100μm. **p<0.01 NI (open bar) <i>vs</i> HI (filled bar).</p

Local glucose homeostasis was impaired with increased adipose tissue iron accumulation.

<p>A) Insulin resistance index HOMA-IR, glucose tolerance test (GTT) and the area under the curve (AUC) in both NI and HI groups. B)Serum adiponectin and leptin in NI (n = 6) and HI (n = 4) mice. C) adipose tissiue gene expression with adipokine markers in NI and HI groups. D&E) adipose tissue insulin signaling protein levels and the quantification after normalizing with GAPDH. *p< 0.05.</p

Primers sequences for gene expression.

<p>Primers sequences for gene expression.</p

Nanotribological Characterization of Lubricants between Smooth Iron Surfaces

We performed the resonance shear measurement (RSM) for evaluating the nanorheological and tribological properties of model lubricants, hexadecane and poly­(α-olefin) (PAO), confined between iron surfaces. The twin-path surface forces apparatus (SFA) was used for determining the distance between the surfaces. The obtained resonance curves for the confined lubricants showed that the viscosity of the confined hexadecane and PAO increased due to liquid structuring when the surface separation (<i>D</i>) decreased to a value less than 24 and 20 nm, respectively. It was also determined that the iron surfaces were lubricated by the hexadecane when normal load (<i>L</i>) was less than 1.1 mN, while the confined hexadecane behaved almost solid-like and showed poor lubricity when <i>L</i> was greater than 1.1 mN. In contrast, PAO between the iron surfaces showed high lubricity even under the high load (<i>L</i> > 2 mN). The surface separation of hexadecane and PAO at a hard wall contact between the iron surfaces was determined to be 4.6 ± 0.5 and 5.0 ± 0.4 nm by applying the fringes of equal chromatic order (FECO) for half-transparent iron films deposited on mica surfaces as substrates. We also characterized hexadecane and PAO confined between mica surfaces for studying the effect of substrates on the confined lubricants