Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity, Resistance to Hepatic Steatosis, and Defective Gluconeogenesis ▿

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice

Cell line panel screen and validation of BPTES as an on-target tool compound.

<p>(A) Sensitivity of a panel of NSCLC lines to inhibition of growth by 10 µM BPTES in a 72 hr proliferation assay. Growth rates (mu) plotted relative to DMSO control for each cell line (mu/max). (B,C) A427 parent cells or cells stably expressing an empty vector control (Con), wild-type GAC (GAC), or a BPTES-resistant GAC enzyme (GAC-BR) were treated with the indicated concentrations of BPTES (A) or the inactive BPTES analogue, AGX-4769 (C), in a 72 hr proliferation assay. Results are representative of three independent experiments with mean and standard deviation indicated. (D,E) Measurement of isotopomer labeled ¹³C(5)-Glu (D) or ¹³C(4)-Asp (E) from cells treated for 4 hr with BPTES or with inactive analogue AGX-4769. Results are the mean of three replicates with the standard deviation indicated. Calculated p-values from student t-test, ^*(3×10⁻⁸), ^**(10⁻⁹), ^#(10⁻⁷), ^##(2×10⁻⁶).</p

Metabolic profiling of epithelial and mesenchymal cells and effects of GLS inhibition.

<p>NCI-H358 epithelial (E) and mesenchymal (M) lines were treated with DMSO or 8 µM BPTES for 20 hrs. Cells were either unlabeled or U-¹³C-Glc or U-¹³C-Gln labels were included for the last 4 hrs of drug treatment and levels of central carbon metabolites were measured by LCMS. (A) Inhibition of GLS activity by BPTES. (B) Percentage of citrate pool that is either unlabeled or containing carbon derived from U-¹³C-Gln (DMSO treated cells). (C) TCA metabolite and glutathione pool sizes +/− BPTES. Data was collected in triplicate or quadruplicate from 3 independent experiments (for α-KG and citrate) and depicted as mean levels +/−SD relative to NCI-H358 DMSO treated cells; values were normalized for cell number; <i>P</i> =  *1×10⁻⁵; **4×10⁻⁶; ***9×10⁻⁸ (D) Citrate isotopomer percentages from ¹³C(6)-Glc labeled cells treated +/− BPTES. *<i>P</i> = 0.001, **<i>P</i> = 0.002 comparing % citrate m+2 in DMSO or BPTES treated E vs M cells, respectively. (E) Alterations in relative ratios of DHAP and 3PG levels in EvsM cells +/−BPTES. <i>P</i> values for comparison of metabolite levels +/−BPTES: *0.1, **0.57, ^#0.04, ^##0.07. Results representative of 2 independent experiments.</p

EMT drives GLS1 dependence in two independent NSCLC models.

<p>(<b>A</b>) A427 cells were transfected with non-targeting (NT) or β-catenin targeting siRNAs and treated with the indicated concentrations of BPTES (left panel). Growth rates were normalized to DMSO treated cells transfected with NT siRNAs. The right panel depicts the effects of the β-catenin siRNAs on growth of DMSO treated cells. <b>(B</b>) Immunoblot of A427 protein extracts collected from cells 72 hrs post transfection. (<b>C</b>) NCI-H358 cells were treated with 25ng/ml TGF-ß for 4 wks. Induction of EMT was confirmed by the loss of E-cadherin and gain of vimentin. EMT was accompanied by an increase in GAC relative to PC protein levels (results representative of 2 independent experiments). Actin levels indicated for protein normalization. (<b>D</b>) NCI-H358 parental and 6wk TGF-ß treated cells were treated in triplicate with BPTES for 72 hrs and cell growth assessed by CTG. Results are representative of 3 independent experiments and are plotted as average growth rates (+/−SD) compared to DMSO treated cells.</p

Oxygen consumption rates differentially altered upon drug perturbation in sensitive compared to insensitive cells.

<p>(A) NCI-H358 epithelial and mesenchymal lines or (B) NCI-H1703 (BPTES sensitive) and NCI-H1563 (BPTES insensitive) cells were treated with DMSO or 8 µM BPTES and oxygen consumption rate (OCR) was monitored over a 20 hr treatment. Data normalized to OCR at time zero (100%) and presented as mean values +/−SEM. <i>P</i> = 0.024 comparing changes in oxygen consumption with BPTES in the epithelial vs mesenchymal line; <i>P</i> = 0.00017 comparing NCI-H1703 and NCI-H1563 cells. (C) OCR following treatment with the indicated drugs to perturb mitochondrial respiration. Data normalized to OCR measurement prior to oligomycin treatment. (D) Addition of pyruvate to media restores the impaired FCCP response in the mesenchymal line. Average values +/−SEM indicated. Results representative of 2–3 independent experiments.</p

Association between EMT-related markers and BPTES sensitivity in NSCLC lines.

<p>(A) GeneGo analysis indicating four top-ranking mesenchymal signatures correlating with BPTES sensitivity. P-values are calculated within the GeneGo software using the hypergeometric distribution. (B) Anti-correlation between high E-cadherin low vimentin RNA expression levels and sensitivity to BPTES. (C) Mu/mumax values depicted following 72 hr treatment of NSCLC lines with 10 µM BPTES. Equal protein amounts of tumor cell line extracts were electrophoresed, with the group of more sensitive lines on the left and least sensitive on the right, and immunoblotted for the indicated proteins. Histone H3 was used as a loading control. (D) Quantitation of average GAC protein levels relative to PC protein expression (+/−SEM) in BPTES sensitive (n = 7), intermediate (n = 7), and insensitive (n = 7) lines. <i>P</i> = 0.03 comparing GAC/PC protein levels in sensitive compared to insensitive lines; <i>P</i> = 0.06 comparing sensitive to intermediate groups (unpaired 2-tail t-test).</p