Figure 1.
Metabolic parameters in wt (black symbols) and Alms1GT/GT (white symbols) male mice.
The body weight (a), plasma glucose (b) and insulin levels (c) were evaluated at 4 week intervals. Dotted lines denote the age (6 weeks) of mice characterized in the present study. Data are expressed as mean values ±SEM, n = 15. *p<0.01 wt vs. Alms1GT/GT.
Figure 2.
Adipose tissue characterization in 6 week-old Alms1GT/GT mice.
(a) Weight of subcutaneous (SAT) and visceral (VAT) tissues of wt (black bars) and Alms1GT/GT (white bars) mice. *p<0.01 wt vs. Alms1GT/GT. (b) Average of 100 adipocyte diameters in SAT and VAT of 6 Alms1GT/GT (white circles) and 6 wt (black circles) expressed as mean values ±SD. *p<0.01 wt vs. mutant animals. (c) A representative H&E staining of adipose tissue depots in Alms1GT/GT and wt mice. Scale bar = 100 µm. (d) Leptin expression in SAT and VAT of 6 wt (black bars) and 6 Alms1GT/GT (white bars) mice. Data are normalized to Rn18s (18S) content and reported as arbitrary unit mean ratio ±SEM. *p<0.05 wt vs. Alms1GT/GT. (e) Circulating plasma leptin in 12 wt (black bars) and 12 Alms1GT/GT (white bars) mice. Data are expressed as mean values ±SEM. (f) mRNA expression of several enzymes involved in different lipogenic pathways in SAT and VAT of 6 wt (black bars) and 6 Alms1GT/GT (white bars) mice (Pck1: Phosphoenolpyruvate carboxykinase 1, Fasn: Fatty acid synthase, Dgat1: Diacylglycerol acyltransferase 1, Dgat2: Diacylglycerol acyltransferase 2, Lpl: Lipoprotein lipase). Each transcript was normalized to Rn18s content. Results are reported as arbitrary unit mean ratio ±SEM and are expressed as fold change with respect to wt, arbitrarily set as 1 for each transcript. *p<0.05 wt vs. Alms1GT/GT.
Figure 3.
Insulin signaling in adipose tissue and whole body glucose homeostasis of 6 week-old Alms1GT/GT mice.
(a) Representative immunoblot and quantification of VAT lysates probed with antibodies: pAKT (S473), AKT, and β-actin from 6 week old wild type (black bars) and Alms1GT/GT (white bars) mice collected 30 minutes following an intraperitoneal injection of saline (−) or insulin (+). Immunoblots were performed with three independent samples. *p<0.05 wt (−) vs. wt (+); Alms1GT/GT (−) vs. Alms1GT/GT (+). (b) HOMA-IR values of control (black bars) and mutant mice (white bars) at different ages (w = weeks) following a six hour fast (6W, n = 4–5/group;>20W, n = 3/group). Intraperitoneal glucose (c) and insulin (d) tolerance tests were performed at 6 weeks of age in wt (black circle) and Alms1GT/GT (white circle) mice. Glucose values are means ±SEM; n = 5–7 for each group. The areas under the curve (AUC) are arbitrary units shown as means ±SEM. *p<0.05 wt vs. Alms1GT/GT.
Figure 4.
GLUT4 content and subcellular distribution in adipose tissue depots of 6 week-old Alms1GT/GT mice before and after insulin stimulation.
(a) Representative western blot of total GLUT4 and (b) quantification (n = 6) normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content, in subcutaneous (SAT) and visceral (VAT) adipose tissues of wt (black bars) and Alms1GT/GT (white bars). (c) Representative western blot of GLUT4 distribution in the subcellular compartments (PM = plasma membrane; HD = high density microsome; LD = low density microsome) in basal conditions (bas) and after insulin stimulation (ins) in SAT pooled from 3 wt and Alms1GT/GT mice. (d) The plot represents the fold-increase (insulin/basal) in GLUT4 signal after insulin stimulation in every subcellular fraction from SAT of wt (black bars) and Alms1GT/GT (white bars) mice as mean values ±SEM of 3 western blot quantification. *p<0.05 wt vs. Alms1GT/GT.
Figure 5.
In vitro characterization of pre-adipocyte adipogenic potential and adipocyte insulin responsiveness from 6 week-old Alms1GT/GT mice.
(a) Representative pictures of in vitro adipogenic differentiation from wt and Alms1GT/GT SAT pre-adipocytes (32× magnification). Pre-adipocytes (left) were grown in adipogenic medium until fully differentiation (middle) and then stained with Oil Red-O (right). (b) Spectrophotometer ODs from Oil Red O staining of in vitro differentiated adipocytes (n = 3) are reported as mean values ±SEM. (c) mRNA expression of reported genes in pre-adipocytes (PA) and mature adipocytes cell cultures (AD) from wt (black bars) and Alms1GT/GT (white bars) mice was normalized to Rn18s content, reported as arbitrary unit mean ratio ±SEM and expressed as fold change with respect to wt AD, arbitrarily set as 1 for each transcript. *p<0.05 wt vs. Alms1GT/GT. (d) Insulin-induced 2DG-uptake of adipocyte cell cultures (n = 3) obtained from wt (black bars) and Alms1GT/GT (white bars) mice stimulated with different insulin concentration and normalized for total protein content. Data are reported as percent increase (%) over basal uptake (0 nM insulin) which was arbitrarily set as 100 for each group. (e) Representative western blot of GLUT4 distribution in the subcellular compartments (PM = plasma membrane; HD = high density microsome; LD = low density microsome) in basal conditions (bas) and after insulin stimulation (ins) in adipocyte cultures obtained from SAT of wt and Alms1GT/GT mice. (f) The plot represents the fold increase (insulin/basal) in GLUT4 signal after insulin stimulation in every subcellular fraction from adipocyte cultures (n = 3) of wt (black bars) and Alms1GT/GT (white bars) mice as mean values ±SEM. *p<0.05 wt vs. Alms1GT/GT.