Distinct Effects of Carrageenan and High-Fat Consumption on the Mechanisms of Insulin Resistance in Nonobese and Obese Models of Type 2 Diabetes

Exposure to low concentration of the common food additive carrageenan (10 mg/L) for only six days led to glucose intolerance and insulin resistance in the C57BL/6J mouse. Longer exposure produced fasting hyperglycemia but with no increase in weight, in contrast to the HFD. Glucose intolerance was attributable to carrageenan-induced inflammation and to increased expression of GRB10. Both HFD and carrageenan increased p(Ser32)-IκBα and p(Ser307)-IRS1, and the increases were greater following the combined exposure. The effects of carrageenan were inhibited by the combination of the free radical inhibitor Tempol and BCL10 siRNA, which had no impact on the HFD-mediated increase. In contrast, the PKC inhibitor sotrastaurin blocked the HFD-induced increases, without an effect on the carrageenan-mediated effects. HFD had no impact on the expression of GRB10. Both carrageenan and high fat increased hepatic infiltration by F4/80-positive macrophages. Serum galectin-3 and galectin-3 binding to the insulin receptor increased by carrageenan and by HFD. Tyrosine phosphorylation of the insulin receptor declined following either exposure and was further reduced by their combination. Carrageenan reduced the activity of the enzyme N-acetylgalactosamine-4-sulfatase (ARSB; arylsulfatase B), which was unchanged following HFD. Dietary exposure to both high fat and carrageenan can impair insulin signaling through both similar and distinct mechanisms

Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF.

Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events

Exposure to Common Food Additive Carrageenan Alone Leads to Fasting Hyperglycemia and in Combination with High Fat Diet Exacerbates Glucose Intolerance and Hyperlipidemia without Effect on Weight

Aims. Major aims were to determine whether exposure to the commonly used food additive carrageenan could induce fasting hyperglycemia and could increase the effects of a high fat diet on glucose intolerance and dyslipidemia. Methods. C57BL/6J mice were exposed to either carrageenan, high fat diet, or the combination of high fat diet and carrageenan, or untreated, for one year. Effects on fasting blood glucose, glucose tolerance, lipid parameters, weight, glycogen stores, and inflammation were compared. Results. Exposure to carrageenan led to glucose intolerance by six days and produced elevated fasting blood glucose by 23 weeks. Effects of carrageenan on glucose tolerance were more severe than from high fat alone. Carrageenan in combination with high fat produced earlier onset of fasting hyperglycemia and higher glucose levels in glucose tolerance tests and exacerbated dyslipidemia. In contrast to high fat, carrageenan did not lead to weight gain. In hyperinsulinemic, euglycemic clamp studies, the carrageenan-exposed mice had higher early glucose levels and lower glucose infusion rate and longer interval to achieve the steady-state. Conclusions. Carrageenan in the Western diet may contribute to the development of diabetes and the effects of high fat consumption. Carrageenan may be useful as a nonobese model of diabetes in the mouse

Phospho-p38 inhibitor blocks increases in GPNMB and nuclear MITF.

<p><b>(A)</b> When HepG2 cells were treated with the phospho-p38 MAPK inhibitor SB20350, the ARSB siRNA- induced increase in GPNMB declined from 26.4 ± 1.7 ng/mg protein to 1.4 ± 0.2 ng/mg protein (p<0.001, n = 3). Baseline GPNMB declined >80% (from 5.1 ± 0.2 ng/mg protein to 1.0 ± 0.04 ng/mg protein) following exposure to SB20350. Genistein also significantly reduced GPNMB, to 21.7 ± 0.4 ng/mg protein (p<0.001, n = 3), but the PI3K/AKT inhibitor (LY294002) had no effect. <b>(B)</b> The increase in nuclear MITF induced by ARSB siRNA was inhibited by SB20350, declining from 1.25 ± 0.02 ng/mg protein to 0.084 ± 0.015 ng/mg protein (p<0.001; n = 3). Genistein also significantly inhibited the ARSB siRNA-induced increase (to 1.04 ± 0.07 ng/mg protein; p<0.001; n = 3), but LY294002 had no effect. <b>(C)</b> The phospho-p38 MAPK to total p38 ratio was determined by fast-activated cell-based ELISA. The ratio more than doubled following ARSB silencing (from 0.21 ± 0.02 to 0.43 ± 0.02), and declined significantly following exposure to SB20350 (to 0.015 ± 0.002 with control siRNA and to 0.032 ± 0.003 with ARSB siRNA), demonstrating the effectiveness of the inhibitor. [ARSB = arylsulfatase B; GPNMB = glycoprotein (transmembrane) NMB; MITF = microphthalmia-associated transcription factor; si = siRNA]</p

Decline in SHP2 activity follows decline in ARSB in HepG2 cells and in ARSB-null mice.

<p><b>(A)</b> The SHP2 activity declined ~36% from (2.49 ± 0.15 nmol/mg protein/0.5 h to 1.59 nmol/mg protein/0.5h) following ARSB silencing (p<0.001; n = 6), and increased to 3.70 ± 0.16 nmol.mg protein/0.5h when ARSB was overexpressed in the HepG2 cells (p<0.001; n = 3). <b>(B)</b> SHP2 activity was markedly reduced in the ARSB-null mice (declining from 1.99 ± 0.12 to 1.33 ± 0.10 nmol/mg protein/0.5h, p = 0.001 (F); and 2.09 ± 0.13 to 1.28 ± 0.06 nmol/mg protein/h, p = 0.0006 (M), unpaired t-test, two-tailed; n = 3 per group). <b>(C)</b> The impact of different SHP2 DNA constructs on the SHP2 activity was tested. Constitutively active(CA)- and wild-type(WT)-SHP2 DNA increased the SHP2 activity (p<0.001, p<0.05), and dominant negative (DN)-SHP2 DNA inhibited the SHP2 activity (p<0.001). ARSB siRNA, the SHP2 inhibitor PHSP1, and PHSP1 in combination with ARSB siRNA or with ARSB OE reduced the activity (p<0.001; n = 3), in comparison to the controls. SHP2 activity in the CA was ~50 times greater than in the DN (5.04 ± 0.10 nmol/mg protein/0.5h vs. 0.10 ± 0.01 nmol/mg protein/0.5h). [AF = ARSB-null female; AM = ARSB-null male; ARSB = arylsulfatase B; CA = constitutively active; CF = control female; CM = control male; DN = dominant negative; OE = overexpression;si = siRNA; WT = wild-type; * for decrease; # for increase]</p

Increase in SHP2 that co-immunoprecipitates with C4S following ARSB knockdown.

<p><b>(A)</b> Western blot of SHP2 co-immunoprecipitated with C4S shows increased band density following ARSB silencing compared to control silencing (n = 2). The C4S content increased following ARSB silencing, with similar protein. <b>(B)</b> Densitometry showed increased intensity of the immunoprecipitated bands following ARSB silencing (p = 0.007, unpaired t-test, two-tailed; n = 2). <b>(C)</b> HepG2 cells were treated with PHSP1, a chemical SHP2 inhibitor and with DN, CA, or WT SHP2 DNA vectors. These exposures had no effect on the ARSB activity. (p>0.05, one-way ANOVA with Tukey-Kramer post-test; n = 3). <b>(D)</b> Treatment with PHSP1 or with DN. CA, or WT SHP2 DNA vectors did not modify the chondroitin 4-sulfate in the HepG2 cells. [ARSB = arylsulfatase B; CA = constitutively active; con si = control siRNA; DN = dominant negative; Inh = inhibitor; N.D. = no difference; OE = overexpression; si = siRNA; Vcon = vector control; WT = wild-type]</p

Decline in SHP2 leads to increases in phospho-p38 to total p38 ratio, nuclear MITF, GPNMB promoter activity, and GPNMB in HepG2 cells.

<p><b>(A)</b> The ratio of phospho-p38 to total p38 increased to 0.43 ± 0.02 following ARSB siRNA and to 0.38 ± 0.02 following PHSP1 treatment from a control level of 0.21 ± 0.02 (p<0.001; n = 5). DN-SHP2 DNA also increased the ratio (to 0.39 ± 0.03). In contrast, ARSB overexpression (p<0.01) and CA-SHP2 DNA construct (p<0.001) decreased the ratio to 0.10 ± 0.01 and 0.02 ± 0.002, respectively.<b>(B)</b> Nuclear MITF increased following ARSB silencing (1.25 ± 0.23 ng/mg protein) and treatment with the DN-SHP2 DNA vector (to 1.37 ± 0.08 ng/mg protein) and the SHP2 inhibitor PHSP1 (1.20 ± 0.10 ng/mg protein) from a control level of 0.36 ± 0.001 ng/mg protein(p<0.001; n = 5), in contrast to the declines following ARSB overexpression or treatment with the CA-SHP2 or WT-SHP2 DNA vectors. PHSP1 treatment of cells in which ARSB was overexpressed reversed the decline in nuclear MITF (p<0.001; n = 5). <b>(C)</b> GPNMB promoter activity was increased by PHSP1 to ~4.4 times the control level (p<0.001; n = 5) and by ARSB silencing (to ~4.0 times control), as previously noted (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153463#pone.0153463.g002" target="_blank">Fig 2C</a></b>). The combination of ARSB siRNA and PHSP1 had no greater effect. <b>(D)</b> GPNMB protein was increased by ARSB silencing, PHSP1, and DN SHP2 and reduced by ARSB overexpression, CA SHP2, and WT SHP2. Increase was greatest following CASHP2, increasing to 30.9 ± 1.7 ng/mg protein from control level of 5.13 ± 0.13 ng/mg protein. [ARSB = arylsulfatase B; CA = constitutively active; DN = dominant negative; GPNMB = glycoprotein (transmembrane) NMB; Inh = inhibitor; MITF = microphthalmia-associated transcription factor; OE = overexpression; RLU = relative luciferase units; si = siRNA; Vcon = vector control; WT = wild-type]</p

Schematic illustration of signaling pathway from ↓ARSB → ↑chondroitin 4-sulfate → ↑SHP2 bound → ↓SHP2 activity → ↑phospho-p38 → ↑MITF → ↑GPNMB.

<p><b>(A)</b> When ARSB activity is normal, the 4-sulfate group at the non-reducing end of the C4S chain is removed from C4S and the binding of SHP2 to C4S is not increased. Hence, SHP2 can remove tyrosine phosphate from p38. <b>(B)</b> When ARSB is reduced and chondroitin 4-sulfation is increased, SHP2 binding to C4S is increased and SHP2 activity is reduced. There is inhibition of removal of tyrosine phosphate from p38, leading to increased phosphorylation of p38 and of MITF, leading to activation of the GPNMB promoter. [ARSB = arylsulfatase B; C4S = chondroitin 4-sulfate; GPNMB = glycoprotein (transmembrane) NMB; MITF = microphthalmia-associated transcription factor; P = phosphate; S = sulfate]</p