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. 2011 Mar;60(3):925-35.
doi: 10.2337/db10-1334. Epub 2011 Jan 26.

Genetic defects in human pericentrin are associated with severe insulin resistance and diabetes

Isabel Huang-Doran  1 , Louise S Bicknell, Francis M Finucane, Nuno Rocha, Keith M Porter, Y C Loraine Tung, Ferenc Szekeres, Anna Krook, John J Nolan, Mark O'Driscoll, Michael Bober, Stephen O'Rahilly, Andrew P Jackson, Robert K Semple; Majewski Osteodysplastic Primordial Dwarfism Study Group
Collaborators, Affiliations

Genetic defects in human pericentrin are associated with severe insulin resistance and diabetes

Isabel Huang-Doran et al. Diabetes. 2011 Mar.

Abstract

Objective: Genetic defects in human pericentrin (PCNT), encoding the centrosomal protein pericentrin, cause a form of osteodysplastic primordial dwarfism that is sometimes reported to be associated with diabetes. We thus set out to determine the prevalence of diabetes and insulin resistance among patients with PCNT defects and examined the effects of pericentrin depletion on insulin action using 3T3-L1 adipocytes as a model system.

Research design and methods: A cross-sectional metabolic assessment of 21 patients with PCNT mutations was undertaken. Pericentrin expression in human tissues was profiled using quantitative real-time PCR. The effect of pericentrin knockdown on insulin action and adipogenesis in 3T3-L1 adipocytes was determined using Oil red O staining, gene-expression analysis, immunoblotting, and glucose uptake assays. Pericentrin expression and localization also was determined in skeletal muscle.

Results: Of 21 patients with genetic defects in PCNT, 18 had insulin resistance, which was severe in the majority of subjects. Ten subjects had confirmed diabetes (mean age of onset 15 years [range 5-28]), and 13 had metabolic dyslipidemia. All patients without insulin resistance were younger than 4 years old. Knockdown of pericentrin in adipocytes had no effect on proximal insulin signaling but produced a twofold impairment in insulin-stimulated glucose uptake, approximately commensurate with an associated defect in cell proliferation and adipogenesis. Pericentrin was highly expressed in human skeletal muscle, where it showed a perinuclear distribution.

Conclusions: Severe insulin resistance and premature diabetes are common features of PCNT deficiency but are not congenital. Partial failure of adipocyte differentiation may contribute to this, but pericentrin deficiency does not impair proximal insulin action in adipocytes.

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Figures

FIG. 1.
FIG. 1.
Stable knockdown of Pcnt in murine 3T3-L1 preadipocytes. A: Pcnt mRNA expression in shPcnt-infected preadipocytes (□, shPcnt1; ▨, shPcnt2) and luciferase knockdown control preadipocytes (■, shLuc) determined by quantitative real-time PCR. **P < 0.01 vs. shLuc (n = 4). B: Pericentrin expression in shPcnt and shLuc preadipocytes assessed by Western blotting. Calnexin expression was evaluated as a loading control. Representative example of three experiments. C: Expression and localization of pericentrin (green), γ-tubulin (red), α-tubulin (green, far right panels only), and nuclear material (blue, stained with DAPI) in shPcnt and shLuc preadipocytes, assessed by immunocytochemistry and confocal microscopy. Arrowheads indicate juxtanuclear pericentrin. Scale bars: 20 μm (far left and far right panels) and 5 μm (central panels). D: Pcnt mRNA expression during differentiation of wild-type 3T3-L1 cells, determined by quantitative real-time PCR. **P < 0.01; ***P < 0.005 vs. day 0 (n = 4). E: Pcnt mRNA expression during differentiation of shLuc (■) and shPcnt (□ and ▨) preadipocytes. #P < 0.005 vs. shLuc (n = 4). All data are means ± SE. AU, arbitrary units. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Pcnt knockdown in 3T3-L1 preadipocytes is associated with impaired adipocyte differentiation. A: Oil red O staining of triglycerides at day 10 of differentiation in Pcnt knockdown and control adipocytes. Representative of n = 9. B: Relative adipocyte gene expression in Pcnt knockdown (□ and ▨) and control (■) cells at day 10 after differentiation. *P < 0.05; **P < 0.01; #P < 0.005 vs. shLuc (n = 9). C: Proliferation of shPcnt (blue circles) and control (red squares) preadipocytes during the preconfluent growth phase (n = 4). Representative experiments are shown. Data are means ± SE of four wells. D: Relative preconfluent rate of proliferation in shPcnt (▨) and control (■) preadipocytes (n = 3). E: Cell proliferation during the preconfluent phase, confluency, and after hormonal induction of differentiation. Representative experiments are shown. Data are means ± SE of four wells. F: Relative rate of cell proliferation after induction of differentiation (n = 3). AU, arbitrary units. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 3.
FIG. 3.
Knockdown of Pcnt results in mildly impaired insulin-stimulated glucose uptake with normal proximal insulin signaling and GLUT4 localization in adipocytes. A: Insulin-stimulated phosphorylation of insulin receptor (INSR), AKT and extracellular signal–regulated kinase (ERK) 1/2 after a 5-min treatment of serum-starved mature adipocytes with 100 nmol/L insulin, assessed by Western blotting. Representative example of three independent experiments. B: Relative [3H]deoxyglucose uptake by Pcnt knockdown (□ and ▨) and control (■) adipocytes after serum starvation and treatment with 100 nmol/L insulin or PBS for 30 min. ***P < 0.005 vs. insulin treatment; aP < 0.005 vs. shLuc after PBS treatment; bP < 0.005 vs. shLuc after insulin treatment. Values are means ± SE (n = 9). C: Coimmunostaining of pericentrin (green) and endogenous GLUT4 (red) in shLuc (top panel) and shPcnt (bottom panel) mature adipocytes. Far right, magnified image. Blue = DAPI. Scale bar: 5 μm. AU, arbitrary units. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Pericentrin expression in human and murine tissues. Pcnt mRNA expression in a panel of human (A) and murine (B) tissues determined by quantitative real-time PCR. A: Data are means ± SE. Sample sizes are in parentheses. B: Data represent n = 1. C: Immunostaining of pericentrin (red) in human vastus lateralis sections. Nuclei stained with DAPI. Lower panel shows magnified images. Scale bars: 5 μm (top) and 1 μm (bottom). AU, arbitrary units; BAT, brown adipose tissue; Cortex, cerebral cortex; Hypoth, hypothalamus; mWAT, mesenteric white adipose tissue; scWAT, subcutaneous white adipose tissue; SkM, skeletal muscle; SmInt, Small intestine; WAT, white adipose tissue. (A high-quality digital representation of this figure is available in the online issue.)
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