Effects of Diet and Genotype on Adipose Tissue Size and Function

Although multiple adipose pads contribute differently to bodily pathophysiology (see Section 1.2.7), in this part of the study we measured only periovarian adipose tissue (referred to as visceral WAT) due to the demanding (of time) morphometric analyses required. Among chow-fed mice, visceral WAT weighed more in foz/foz and less in Tlr9-/- mice than WT (Fig. 4.3A). Atherogenic dietary feeding increased visceral WAT weight (~2-fold) in WT mice; a similar but limited trend was found in atherogenic diet- fed Tlr9-/- mice. In atherogenic diet-fed foz/foz mice, adiposity was the highest among

145 genotypes. However, as previously reported (232), visceral WAT as a percentage of body weight was less than in chow-fed WT mice (Fig. 4.3B).

Figure 4.3:Absolute and relative visceral WAT weight in WT, foz/foz and Tlr9-/- _mice.

(A) foz/foz mice had increased and Tlr9-/- _{decreased adiposity compared with WT, irrespective of}

diet. (B) However, adipose expansion as a proportion of body weight was limited in atherogenic

diet-fed foz/foz compared to chow-fed foz/foz or atherogenic diet-fed WT mice. Tlr9-/- _{mice also}

showed limited visceral adiposity when fed an atherogenic diet compared to WT counterparts.

Data are mean ± SEM (n=6-14/gp).

† P<0.05 vs. diet-matched WT (genotype effect); e.g., chow-fed foz/foz vs. WT

‡ P<0.05 vs. genotype-matched control (diet effect); e.g., atherogenic diet-fed vs. chow-fed WT

There was insufficient adipose in some groups (e.g., chow-fed Tlr9-/-) to perform both morphometry and the full range of molecular techniques. Priority was therefore given to use tissues for morphometric and mRNA expression assays. As a result, protein analyses in this Chapter are limited by logistics. In Figure 4.4, mRNA expression levels are shown for genes concerned with adipose function. Some of them are involved in adipogenesis (and adipocyte metabolism), whereas others contribute to whole body lipid metabolism.

As described in Section 1.5.4, adipsin is an adipokine with positive effects on metabolic regulation, such as improving pancreatic β cell function (294). In atherogenic diet-fed foz/foz mice, levels of visceral WAT adipsin mRNA were high than in atherogenic diet-fed WT mice, while adipsin mRNA was lower than WT in Tlr9-/- mice (Fig. 4.4A). Patatin-like phospholipase domain containing 2 (PNPLA2) (ATGL)

146 catalyzes the first step of triglyceride hydrolysis in adipocytes (397, 398). The expression profile for Pnpla2 mRNA was similar to adipsin (Fig. 4.4B). Perilipin 2, as explained in Section 1.4, is an important lipid droplet protein. Perilipin 2 mRNA levels were upregulated in atherogenic diet-fed foz/foz mice, and decreased in Tlr9-/- mice vs. WT counterparts (Fig. 4.4C). Caveolin 1 is a subunit in caveolae structures where insulin signalling molecules locate (see Section 1.4.4). Cav1 mRNA levels were significantly lower in atherogenic diet-fed foz/foz mice visceral WAT than WT mice, but there was no change in Tlr9-/- mice (Fig. 4.4D).

SCD1 forms double bonds in C16 and C18 units of LCFAs that transfers saturated FAs into unsaturated ones of the same chain length (399). There was no effect of genotype on mRNA expression of this gene (Fig. 4.4E). Peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) initiates FA β oxidation (400, 401). While there was a decrease in Acox1 mRNA expression in atherogenic diet-fed foz/foz mice visceral WAT compared to WT (Fig. 4.4F), there was no change in Tlr9-/- mice. A similar trend was found for fatty acid synthase (Fas) mRNA levels (Fig. 4.4G). As reviewed in Section 1.3, FOXO1 is a protein regulated by insulin action (196, 197). Foxo1 mRNA levels of visceral WAT were decreased in foz/foz vs. WT mice. In contrast, Tlr9 deletion was associated with an increase in Foxo1 visceral WAT mRNA levels compared to WT mice (Fig. 4.4H).

147

Figure 4.4: Visceral WAT mRNA expression levels for the genes indicated, which are

related to adipose function, in atherogenic diet-fed mice. Note: chow-fed mice not studied

here, as explained in the text. Data are mean ± SEM (n=8/gp). Values are expressed relative to

WT, which is set at 1.0.

† P<0.05 vs. WT control (genotype effect); e.g., atherogenic diet-fed foz/foz vs. WT

A detailed overview of adiponectin as an insulin-sensitizing, anti-inflammatory hormone produced by adipocytes was provided in Section 1.5. In atherogenic diet-fed WT mice, circulating adiponectin levels vs. chow-fed WT tended to fall, but this was not significant (Fig. 4.5A). Hypoadiponectemia was found in foz/foz mice irrespective of diet, but this decrease was more pronounced in atherogenic diet-fed animals. Atherogenic dietary intake did not alter circulating serum adiponectin levels in Tlr9-/- mice.

Visceral (periovarian) adipose tissue is an important source of adiponectin production (see Section 1.5.4). Comparison of WAT from atherogenic diet-fed foz/foz or Tlr9-/- with WT mice showed that tissue adiponectin mRNA levels were significantly less with foz/foz genotype, whereas Tlr9 deletion tended to increase (P = 0.07) adipose

148 adiponectin mRNA, compared to WT (Fig. 4.5B). Consequently, WAT adiponectin protein levels were less in foz/foz mice and higher in Tlr9-/- mice on atherogenic diet compared to WT mice (Fig. 4.5C).

Figure 4.5: foz/foz mice developed hypoadiponectemia, whereas Tlr9-/- _{mice expressed}

higher tissue adiponectin compared to WT counterparts. (A) Serum adiponectin levels

([HMW) fell markedly in foz/foz mice, particularly with atherogenic diet, but values were not

affected by atherogenic dietary feeding in Tlr9-/- _{and WT mice. (B) WAT adiponectin mRNA,}

and (C) protein expression (LMW) largely reflected serum adiponectin levels on atherogenic

diet, although an effect of atherogenic diet was also evident in WT mice. There was insufficient

tissue to assay adiponectin protein in WAT of chow-fed Tlr9-/- _mice.

Data are mean ± SEM (n=8/gp).

† P<0.05 vs. diet-matched WT (genotype effect); e.g., atherogenic diet-fed foz/foz vs. WT

‡ P<0.05 vs. genotype-matched control (diet effect); e.g., atherogenic diet-fed vs. chow-fed WT

4.4.3 Effects of Diet and Genotype on Adipose Morphometry, Including