Adipocyte Angptl8 deletion improves glucose and energy metabolism and obesity associated inflammation in mice

Figure 1. Construction and validation of adipose-specific conditional Angptl8 KO mice (A) Diagram of the Angptl8 gene with targeted ΔNeo allele, and the disrupted A8 allele. LoxP sites were placed upstream of exon 1 and downstream of exon 4. A vNEO cassette was inserted after exon 4. Mice with the targeted ΔNeo allele (Fl/Fl) were crossed with mice expressing Cre under the control of the adiponectin promoter to inactivate A8 in adipocytes (As-A8−/− mice). NDEL1 and NDEL2 primers used for genotyping are indicated. (B) The presence of the WT, Adipo-Cre transgene or the floxed A8 alleles was evaluated in DNA obtained from ear tissue biopsies using the primers as described in Methods. Specific amplification of a 686 bp DNA fragment corresponds to the floxed- A8 allele while a 569 bp DNA fragment corresponds to the WT A8 allele, the presence of a 600 bp DNA fragment denotes the Adipo-Cre transgene, as verified by genomic PCR. (C) Angptl8 mRNA expression in VAT, SAT, BAT and Liver tissue (n = 4) were measured in 11-week-old male Cre and AT-A8-KO mice, 3 weeks after TMX treatment. Values are means ± SEM. Multiple unpaired t-test for multiple comparison were performed with Holm-Šídák correction…

Figure 2. Physiological parameters of AT-A8-KO mice on HFHF diet (A) Experimental design depicting the timing of TMX treatment and ad libitum access to a control diet or high-fat and high-fructose diet (HFHF) for 23-week in Cre and AT-A8-KO mice. (B) Weekly measurement of body weight in mice from Cre-ND (n = 9), KO-ND (n = 10), Cre-HFHF (n = 10), and KO-HFHF (n = 10) groups. Multiple unpaired t-tests for multiple comparisons were conducted using the Holm-Šídák correction with GraphPad Prism 10. Cre-HFHF vs. KO-HFHF: ∗p ≤ 0.033. (C) Calculation of caloric intake from total food and water intake, measured weekly in Cre and KO mice fed ND or HFHF diet. (D) Measurement of total fat mass and (E) lean mass using magnetic resonance imaging (EchoMRI) in mice from all four groups after 20 weeks on either the ND or HFHF diet. (F) Fasted glycemia was measured in 6 h fasted mice. The following were measured in fed condition: (G) rectal temperature, (H) plasma triglycerides (TG), (I) liver TG, (J) plasma cholesterol and (K) ratio of plasma LDL cholesterol and HDL cholesterol, at the study’s end in Cre and KO mice fed ND or HFHF diet. Data are means ± SEM. Group comparisons were performed using two-way…

Figure 3. Increased energy expenditure in early dark cycle in HFHF diet fed AT-A8-KO mice (A) Male mice from Cre-ND (n = 5), KO-ND (n = 5), Cre-HFHF (n = 7–9), and KO-HFHF (n = 7–8) were housed individually in metabolic cages for three days at room temperature (22°C). After 24h of adaptation, both O2 consumption and (B) CO2 output (C) RER and (D) Locomotor activity were measured for 2 consecutive days and divided into Light D1, Dark D1, Light D2 and Dark D2 cycle. Values are means ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test were performed using GraphPad Prism 10. ∗p ≤ 0.05. (E) Energy expenditure was calculated and expressed as kcal/metabolic mass(g)/h. (F) Area under the curve were calculated from energy expenditure graphs and divided as follows: Light D1 (0-12h), Dark D1 (12-17h), Dark D1 (17-24h), Light D2 (0-12h), Dark D1 (12-17h) and Dark D2 (17-24h). Two-way ANOVA with Tukey’s multiple comparisons test were performed using GraphPad Prism 10. ∗p < 0.033.

Figure 4. Improved Glucose Tolerance and Insulin Sensitivity in AT-A8-KO Mice on HFHF Diet (A) Male mice from Cre-ND (n = 9), KO-ND (n = 10), Cre-HFHF (n = 10), and KO-HFHF (n = 10) groups, 20 weeks after ND or HFHF diet feeding, were subject to oral glucose tolerance test (OGTT) after a 6h fasting. Area under curve (AUC) during the OGTT was calculated. (B) Tail blood was collected at indicated times during the OGTT and analyzed for insulin by AlphaLISA and AUC for insulinemia was calculated. (C) Insulin tolerance test (ITT) was conducted after 23 weeks of ND or HFHF diet feeding in all four groups as indicated, following 6 h fasting. Area above curve (AAC) and (D) HOMA-IR were calculated in these mice. Values are means ± SEM. Groups were compared by two-way ANOVA test using GraphPad Prism 10. ∗p < 0.033, ∗∗p < 0.01, ∗∗∗p < 0.001. (E) ex vivo basal or insulin stimulated glucose uptake measurements were done using radioactive [3H]-2-deoxy-D-glucose in SAT mature adipocytes and (F) BAT tissues isolated from Cre (n = 3) and KO (n = 4) mice on HFHF diet. Data are means ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test was applied using GraphPad Prism 10. ∗p ≤ 0.033.

Figure 5. Adipose tissue mass, morphology and cell size (A) Tissue weights of fat pads (VAT, SAT, BAT) from Cre and KO mice on ND and HFHF diet for 23 weeks (n = 9–10/genotype). Values are means ± SEM. Groups were compared using two-way ANOVA test using GraphPad Prism 10. ∗p < 0.05, ∗∗∗p < 0.001. (B) Representative H&E staining of tissue sections (n = 4–6/genotype, original magnification ×20; scale bar = 200 μm). (C) Adipocyte size was quantified using ImageJ software (4 fields/tissue section) and presented as the frequency distribution of adipocyte cell/lipid droplet size, expressed as percentage.

Figure 6. AT-A8-KO Mice on HFHF diet Reduces Inflammatory Markers (A) MAC-2 immunostaining in VAT tissue section from Cre and KO mice on HFHF diet and (B) quantification of crown-like structures (CLS) (n = 6 mice, 4 fields/tissue section, original magnification ×20; scale bar = 400 μm). Values are means ± SEM and groups were compared using unpaired t-test. ∗∗p ≤ 0.002. (C) Plasma MCP-1, (D) Leptin and (E) Adiponectin levels in mice under fed state were quantified by ELISA or AlphaLISA (n = 8–10). Values are means ± SEM. Groups were compared using two-way ANOVA test using GraphPad Prism 10. ∗p ≤ 0.033, ∗∗p ≤ 0.002, ∗∗∗p < 0.001.