Hematopoietic cell-specific deletion of toll-like receptor 4 ameliorates hepatic and adipose tissue insulin resistance in high-fat-fed mice - PubMed (original) (raw)

Hematopoietic cell-specific deletion of toll-like receptor 4 ameliorates hepatic and adipose tissue insulin resistance in high-fat-fed mice

Maziyar Saberi et al. Cell Metab. 2009 Nov.

Abstract

Chronic low-grade inflammation, particularly in adipose tissue, is an important modulator of obesity-induced insulin resistance. The Toll-like receptor 4 (Tlr4) is a key initiator of inflammatory responses in macrophages. We performed bone marrow transplantation (BMT) of Tlr4lps-del or control C57Bl/10J donor cells into irradiated wild-type C57Bl6 recipient mice to generate hematopoietic cell-specific Tlr4 deletion mutant (BMT-Tlr4(-/-)) and control (BMT-WT) mice. After 16 weeks of a high-fat diet (HFD), BMT-WT mice developed obesity, hyperinsulinemia, glucose intolerance, and insulin resistance. In contrast, BMT-Tlr4(-/-) mice became obese but did not develop fasting hyperinsulinemia and had improved hepatic and adipose insulin sensitivity during euglycemic clamp studies, compared to HFD BMT-WT controls. HFD BMT-Tlr4(-/-) mice also showed markedly reduced adipose tissue inflammatory markers and macrophage content. In summary, our results indicate that Tlr4 signaling in hematopoietic-derived cells is important for the development of hepatic and adipose tissue insulin resistance in obese mice.

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Figures

Figure 1

Figure 1. Hematopoietic homeostasis is maintained in mice following bone marrow transplantation of Tlr4 deleted hematopoietic cells

Panel A shows real-time PCR analysis for the wt Tlr4 genomic locus in peripheral blood mononuclear cells from transplanted mice, demonstrating near complete reconstitution of white blood cells in bone marrow transplant recipient mice with Tlr4 deletion mutant hematopoietic cells (BMT-Tlr4-/-) (n=8). Panels B, and C, show hematogram analyses demonstrating normal blood cell lineage distributions including monocytes, lymphocytes and neutrophils, in mice BMT-Tlr4-/-), as compared with control mice transplanted with wild type Tlr4 (BMT-wt) cells. High fat diet fed mice had slightly altered lineage distributions with slightly increased neutrophil counts and decreased lymphocyte counts, but this was similar for both the BMT-wt and BMT-Tlr4-/- mice. Panel D, E, and F show weight gain of mice given high fat diet (HFD) or normal chow diet (NCD) over 12 weeks following BMT, with no statistical difference in weight gain or food intake/day between BMT-Tlr4-/-and BMT-wt mice. Panel G and H shows cross section and coronal section views using 3D magnetic resonance imaging (MRI) of mice and software based analyses of tissue volumes to determine the total weight of abdominal adipose tissues and subcutaneous fat tissues demonstrating similar distribution of adipose tissue in the BMT-wt and BMT-Tlr4-/- mice. Red arrows signify visceral fat, and yellow dots signify hepatic tissue. Panel I shows weight gain results of a control experiment where the global Tlr4 knockout mice and wild type mice were placed on HFD. No weight gain was seen in the global Tlr4 knockout mice on a HFD.

Figure 2

Figure 2. Insulin tolerance tests in BMT-Tlr4-/- mice on HFD show normal insulin sensitivity

The insulin tolerance test (ITT) performed on bone marrow transplanted mice reveals a significantly increased insulin sensitivity in BMT-Tlr4-/-mice fed a high fat diet (HFD) compared to BMT-wt mice also fed HFD, Panel A. The glucose tolerance test (GTT) reveals less efficient glucose clearing over time in obese mice compared to normal weight control mice for either BMT group, Panel B. * and ** signifies statistical significance between BMT-Tlr4-/- and BMT-wt glucose levels for p<0.05 and p<0.01, respectively, using t-test. Panels C and D reveal the partial correction of insulin levels in obese BMT-Tlr4-/- mice compared to BMT-wt mice. Area-under-curve analysis of insulin data from Panel C shows a statistical difference between BMT-Tlr4-/- and BMT-wt mice both fed HFD.

Figure 3

Figure 3. The hyperinsulinemic euglycemic clamp test shows obese BMT-Tlr4-/- mice have normal insulin sensitivity of hepatocytes and adipocytes

Panel A shows glucose infusion rates during clamp test of mice with the BMT-Tlr4-/- mice showing partial correction of insulin function as measured by partially restored glucose infusion rates (n=8). Panel B shows the hepatic glucose production (HGP) both at basal levels and during the clamp test. On HFD, HGP in the BMT-Tlr4-/- mice was not different from HGP on chow diet, whereas, HFD led to an increase in HFP in the BMT-wt group. The percent suppression of HGP is shown in Panel C. Panel D demonstrates the percentage suppression of free fatty acid secretion from adipose tissue, with obese BMT-Tlr4-/- mice showing normalized adipocyte insulin sensitivity. Panel E shows the insulin stimulated glucose disposal rate (IS-GDR) on NCD or HFD for the two genotypes. * signifies statistical significance between mice groups p<0.05, using t-test, n>8. Panel F shows Western blot analyses of adipose and hepatic tissue for makers of insulin signaling. Analysis demonstrates the up regulated Phosphorylated Akt in the adipose and liver of Tlr4-/- BMT mice compared to the wt BMT, all mice fed the high fat diet, suggesting increased insulin sensitivity. Data shows representative wells with Tubulin as a loading control. Panel G and H show lipid analyses of Liver and muscle cell lysates from BMT mice. The triacylglycerol (TAG) and diacylglycerol (DAG) levels were determined via lipid extraction by organic solvents and separation via thin layer chromoatography. Consistent with the in vivo BMT data, where increase insulin sensitivity was seen in the Tlr4-/- mice, liver lysates from these mice showed reduced TAG and DAG. At least 8 mice were analyzed per group and the measurements were averaged from triplicate wells. Statistical analyses were performed on the samples using student's T-test; blue bar with asterix indicates p value less than 0.1.

Figure 4

Figure 4. Insulin sensitivity in hepatocytes of obese mice correlates with reduced inflammatory signaling of Tlr4-/- hepatic macrophages

CD11b liver macrophages (predominantly Kupffer cells) play an important role in regulating inflammation in the liver. Panels A and B show the increase in liver weight of HFD fed mice compared to NCD mice, and the near complete reconstitution of CD11b positive cells of the Tlr4-/- genotype in the liver of obese mice following bone marrow transplantation. Homogenized liver tissue from bone marrow transplanted mice was enriched for the macrophage specific marker CD11b and real time PCR performed to assay for wild type Tlr4 genomic DNA content. Panel C shows the fold reductions of macrophage specific and non-specific pro-inflammatory cytokines, chemoattractants, and signaling molecules of BMT-Tlr4-/- mice compared to BMT-wt mice fed a HFD. Panels D and E confirm levels of TNF-α and the chemoattractant RANTES via ELISA.

Figure 5

Figure 5. Reduced inflammatory cell recruitment to adipose tissue in BMT-Tlr4-/- mice on HFD

Panel A shows decreased expression of common proinflammatory cytokines in adipose tissue of BMT-Tlr4-/- mice compared to BMT-wt mice fed a HFD. Panels B-E show histological analysis of adipose tissue stained with MAC2 antibody for macrophage detection. Significantly increased macrophage infiltration between adipocytes is detectable and visually quantitated as crown-like-structures with significant reductions in BMT-Tlr4-/- mice compared to BMT-wt mice fed HFD (Panel F). * and ** signifies statistical significance between BMT-Tlr4-/- and BMT-wt levels for p<0.05 and p<0.01, respectively, using student's t-test.

Figure 6

Figure 6. Lentiviral vector mediated knockdown of Tlr4 in a hematopoietic stem cell gene therapy setting maintains insulin sensitivity in HFD mice

Lentiviral vector driving expression of small interfering RNA (siRNA) targeted against Tlr4 in bone marrow transplanted mice of transduced and sorted hematopoietic stem cells, yields partial but significant knockdown of Tlr4 expression in peripheral blood mononuclear cells of transplanted mice as determined by real time PCR, (n=3), Panel A. Panel B shows average weights of mice fed either a high fat diet or a normal chow diet. Insulin tolerance test (ITT) was performed at 8 weeks posttransplant with LV-siTlr4 and control vector transduced bone marrow mice receiving normal chow (Panel C) or high fat diet (Panel D).

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