Hypoxia accentuates LOX expression via HIF-1, leading to enhanced collagen cross-linking.

<p>(A) Hypoxic exposure increases expression of HIF-1α in the nuclear extract of wild-type mouse hepatocytes. This change is abolished in hepatocytes from <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice. (B) LOX mRNA expression increases in wild-type hepatocytes exposed to hypoxia, but this increase in LOX is not observed in <i>Hif1a</i>^<i>-/-</i><i>hep</i> hepatocytes. (C) When culture media from wild-type hepatocytes is added to collagen, hypoxia causes increased collagen cross-linking and precipitation; this difference is not seen in <i>Hif1a</i>^<i>-/-</i><i>hep</i> hepatocytes. *, p<0.05; ††, p<0.001.</p

Expression of genes involved in fibrosis, hepatic lipid metabolism, and inflammation/apoptosis.

<p>Expression of genes involved in fibrosis, hepatic lipid metabolism, and inflammation/apoptosis.</p

Liver histology and collagen quantification.

<p>(A) Representative liver H&E (top) and Masson’s trichrome stains (bottom) from wild-type (left) and <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice. More fibrosis can be observed in the wild-type mice in the Masson’s trichrome stain. (B) Sirius red stain of collagen in wild-type (left) and <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice (right). (C) Collagen content of all samples by use of hydroxyproline assay. *, p<0.05.</p

Liver hypoxic profile.

<p>(A) Wild-type (left) and <i>Hif1a</i>^<i>-/-</i><i>hep</i> (right) liver tissue was similarly hypoxic after six months on an HTFD, as qualitatively assessed by Hypoxyprobe stain. (B) As expected, HIF-1α protein levels were reduced in <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice. Some HIF-1 activation in <i>Hif1a</i>^<i>-/-</i><i>hep</i> mouse liver tissue likely remains due to incomplete gene knockout, and preserved HIF-1α in non-hepatocyte cell types. †, p<0.005.</p

Metabolic characteristics of wild-type and <i>Hif1a</i>^<i>-/-</i><i>hep</i> mice.

<p>(A) Hepatocyte knockout of HIF-1α was confirmed by quantifying HIF-1α mRNA expression from the nuclear extract of isolated hepatocytes. (B) Body weight (top) and food intake (bottom) over experiment duration. (C) IPGTT (top) and ITT (bottom) at time of sacrifice. †, p<0.005.</p

Mouse Histologic Characteristics.

<p>Mouse Histologic Characteristics.</p

Expression of hypoxia inducible factor 1 alpha (HIF-1α, panel A) and a canonical HIF-1 regulated gene, Glut-1 (panel B) in different tissues of C57BL/6J mice with diet induced obesity (DIO) treated with HIF-1α antisense oligonucleotides (HIF-1α ASO), control ASO or observed untreated for 8 weeks.

<p>High fat diet was administered for 12 weeks prior to the ASO treatment and continued during the treatment. BAT, brown adipose tissue; EPI, epididymal adipose tissue; ING, inguinal adipose tissue; OM, omental adipose tissue. *, † and ‡ denote p<0.05, <0.01 and <0.001, respectively, for the difference with HIF-1α ASO treated mice.</p

Accumulation of glycogen during HIF-1α ASO treatment.

<p>(<b>A</b>) Representative images of liver sections in HIF-1α ASO treated (upper panel), control ASO treated (middle panel) and untreated mice (bottom panel). Periodic acid Schiff (PAS) staining, x 400 original magnification. HIF-1α ASO treated mice showed intense PAS positive staining, which was absent in other groups. Control ASO treated and untreated mice show macrovesicular steatosis, which was attenuated in HIF-1α ASO treated mice. (<b>B</b>) Glycogen levels in livers measured biochemically (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046562#s2" target="_blank">Methods</a>). * and † denote p<0.05 and <0.01, respectively, for the difference with HIF-1α ASO treated mice. (<b>C</b>) Western blots showing phosphorylation of glycogen synthase kinase (GSK) α in HIF-1α ASO treated, control ASO treated and untreated mice in representative samples. GAPDH, glyceraldehydes-3-phosphate dehydrogenase.</p

Basic Characteristics of DIO mice treated with Hif-1a ASO and control ASO compared to untreated animals.

*<p>, † and ‡ denote p<0.05, <0.01 and <0.001 respectively for the difference with mice treated with HIF-1α antisense oligonucleotides (ASO).</p

Fasting blood glucose (A) and serum insulin (B), leptin (C), adiponectin (D), fasting serum cholesterol (E) and triglycerides (F), liver cholesterol (G) and triglycerides (H) in DIO mice treated with HIF-1α ASO, control ASO or observed untreated.

<p>* and † denote p<0.05 and 0.01, respectively, for the difference with HIF-1α ASO treated mice.</p