ALMS1 interacts with α-actinin in mammalian cells.

<p>(A–D) Co-localization of α-actinin (A) and ALMS1-C (B) in MDCK cells. Both proteins are expressed within cytoplasmic dense bodies. Antibody overlay and orthogonal projection are shown in C & D, respectively. Scale bar = 5 µm. (E) Co-immunoprecipitation of ALMS1 and actinin from renal lysates of C57BL/6Ei mice. Lysates were incubated overnight with a C-terminal ALMS1 antibody (polyclonal rabbit) and the precipitated proteins were probed with anti-ACTN4 (polyclonal rabbit) antibody.</p

Y2H interaction domains of ALMS1 and α-actinin.

<p>(A) Direct interaction tests with truncated ALMS1 constructs reveals that both constructs were sufficient for the interaction with α-actinin. However the most C-terminal construct showed the strongest interaction. (B) Alignments of α-actinin 1 & 4 prey clone sequences from all three library screens. Potential ALMS1-interacting domains include the central rod and CaM-like domains. The number of clones identified with each unique actinin prey sequence is shown in parentheses. CH = calponin homology; SR = spectrin repeat; CaM = calmodulin-like; NM-nonmuscle; SM = smooth muscle.</p

GLUT4 Defects in Adipose Tissue Are Early Signs of Metabolic Alterations in <i>Alms1^GT/GT</i>, a Mouse Model for Obesity and Insulin Resistance

<div><p>Dysregulation of signaling pathways in adipose tissue leading to insulin resistance can contribute to the development of obesity-related metabolic disorders. Alström Syndrome, a recessive ciliopathy, caused by mutations in <i>ALMS1</i>, is characterized by progressive metabolic alterations such as childhood obesity, hyperinsulinemia, and type 2 diabetes. Here we investigated the role of <i>Alms1</i> disruption in AT expansion and insulin responsiveness in a murine model for Alström Syndrome. A gene trap insertion in <i>Alms1</i> on the insulin sensitive C57BL6/Ei genetic background leads to early hyperinsulinemia and a progressive increase in body weight. At 6 weeks of age, before the onset of the metabolic disease, the mutant mice had enlarged fat depots with hypertrophic adipocytes, but without signs of inflammation. Expression of lipogenic enzymes was increased. Pre-adipocytes isolated from mutant animals demonstrated normal adipogenic differentiation but gave rise to mature adipocytes with reduced insulin-stimulated glucose uptake. Assessment of whole body glucose homeostasis revealed glucose intolerance. Insulin stimulation resulted in proper AKT phosphorylation in adipose tissue. However, the total amount of glucose transporter 4 (SLC4A2) and its translocation to the plasma membrane were reduced in mutant adipose depots compared to wildtype littermates. Alterations in insulin stimulated trafficking of glucose transporter 4 are an early sign of metabolic dysfunction in Alström mutant mice, providing a possible explanation for the reduced glucose uptake and the compensatory hyperinsulinemia. The metabolic signaling deficits either reside downstream or are independent of AKT activation and suggest a role for ALMS1 in GLUT4 trafficking. Alström mutant mice represent an interesting model for the development of metabolic disease in which adipose tissue with a reduced glucose uptake can expand by de novo lipogenesis to an obese state.</p></div

Kinetics of transferrin recycling in ALMS and control fibroblasts.

<p>(A–L) ALMS and control fibroblasts were incubated with unlabelled transferrin for 30 minutes. TfR and nuclei are depicted in red and blue, respectively. Early and recycling endosomes were immunostained with Alexa Fluor 488-labelled EEA1 (A–F) and Rab11 (G–L), respectively. Scale bars = 25 µm. (M–N) Co-localization with pericentrin (PCTN;red) and TfR (green) shows overlay at the pericentrosome. (O–Q) Quantification of TfR and its colocalization with Rab11 at the pericentrosome. Mean fluorescence intensity per 2 µm diameter area (dashed circle) was measured by ImageJ/Fiji software and used as an estimate of the number of TfR positive endosomes. Results are from 40 patient and 47 control cells and error bars indicate ± SEM; Star indicates p<0.0001. Scale bars = 5 µm. (R) Cells were ‘pulsed’ with Tf-Alexa Fluor 647 for 30 min, followed by a ‘cold chase’ of unlabelled holo-transferrin for indicated times. Data was pooled from three fibroblast cell lines from ALMS and control subjects. The graph represents the mean +/− SEM of four independent experiments as a mean percentage of Tf internalization at each time point. The values (MFI) obtained at time 0 following the pulse were set at 100%. Star denotes p<0.0001.</p

F-actin staining in fibroblasts.

<p>(A,E) Staining with anti-ACTN4 (red) in control and patient fibroblasts show actinin staining along stress fibers and at focal adhesions. (B–C,E–F) F-actin stress fibers were visualized by staining with FITC-conjugated phalloidin at lower (20×: B–E) and higher (100×: C,F) magnifcations. Long densely packed fibers were observed in both samples, however, nonuniform and stunted filaments (white arrowhead) were noted in ALMS fibroblasts. Co-staining with anti-ACTN4 (red) and FITC-phalloidin (green) show actinin staining along the stress fibers (inset) and focal adhesions (white arrows) in both patient and control fibroblasts. Scale bars = 10 µm (a,c,d,e,g,h); 100 µm (b,f).</p

Yeast two hybrid analysis of ALMS1.

<p>(A) <i>Alms1</i>-C-terminal bait used for yeast two hybrid. (B) Bacterial induction of ALMS-C lumio fusion protein reveals a 55 kDa band of expected size. B = Benchmark fluorescent ladder (Invitrogen); P = pellet; S = supernatant (C) Immunoblot using anti-ALMS-C antibody shows specificity of the bacterially expressed protein to ALMS1, as indicated by blue coloration. (D) Y2H analysis reveals protein interactors with mouse ALMS1 (carboxy-terminal end).</p

In vitro characterization of pre-adipocyte adipogenic potential and adipocyte insulin responsiveness from 6 week-old Alms1^GT/GT mice.

<p>(a) Representative pictures of <i>in vitro</i> adipogenic differentiation from wt and <i>Alms1^GT/GT</i> 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 <i>in vitro</i> 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 <i>Alms1^GT/GT</i> (white bars) mice was normalized to <i>Rn18s</i> 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 <i>vs</i>. <i>Alms1^GT/GT</i>. (d) Insulin-induced 2DG-uptake of adipocyte cell cultures (n = 3) obtained from wt (black bars) and <i>Alms1^GT/GT</i> (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 <i>Alms1^GT/GT</i> 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 <i>Alms1^GT/GT</i> (white bars) mice as mean values ±SEM. *p<0.05 wt <i>vs</i>. <i>Alms1^GT/GT</i>.</p

Distribution of ALMS1 during cell division.

<p>(<b>A–C</b>) Spatial distribution of N-terminal ALMS1 and C-terminal ALMS1 during cytokinesis. ALMS-Ntr (A, green) is found within the centrioles at the spindle poles. ALMS1-C (B–C, green) redistributes to the acto-myosin contractile ring and to the cleavage furrow during late cytokinesis. Mitotic spindles are observed by acetylated α-tubulin (red) staining. Scale bars = 5 µm.</p

Metabolic parameters in wt (black symbols) and Alms1^GT/GT (white symbols) male mice.

<p>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 <i>vs</i>. <i>Alms1^GT/GT</i>.</p

GLUT4 content and subcellular distribution in adipose tissue depots of 6 week-old Alms1^GT/GT mice before and after insulin stimulation.

<p>(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 <i>Alms1^GT/GT</i> (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 <i>Alms1^GT/GT</i> 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 <i>Alms1^GT/GT</i> (white bars) mice as mean values ±SEM of 3 western blot quantification. *p<0.05 wt <i>vs</i>. <i>Alms1^GT/GT</i>.</p