Energy content analysis of dietary treatments.

<p>CO: corn oil, 8.14 kcal/ml, 100%kcal from fat. EP: Ensure-plus, 1.48 kcal/ml, 28.5%kcal from fat.</p

Body weight and food intake of gKD mice on HFD.

<p>Male gKD mice and wild type littermate raised no normal chow diet were switched to HFD at 4 months of age, and (A) body weight and (B) food intake were monitored for 8 weeks. No statistical significant difference was observed. N = 17–18.</p

Gut-selective DGAT1 knockdown and its effects on TG and gut hormones.

<p>(A) Design of the transgenic construct used to generate DGAT1 gKD mice. A transgenic vector was constructed that contains a 12.4 kb <i>Villin1</i> promoter, a sequence encoding EGFP, followed by miR155-embedded <i>Dgat1</i> shRNA and a poly-A tail. Sequence of the miR155-embedded <i>Dgat1</i> shRNA is shown, with the sequences homologous to <i>Dgat1</i> underlined. (B) DGAT1 mRNA expression in selected tissues from two DGAT1-shRNA transgenic lines and wild type control littermates. N = 3–5. Data are normalized against Rplp0 mRNA. ND: not determined. (C, D) 4-month-old female gKD mice and wild type littermates were fasted overnight, then p.o. dosed with corn oil at 10 ml/kg. Plasma TG was measured at 0, 1, 2, and 4 h after oil challenge, and net area-under-the-curve (AUC) compared to time 0 was calculated. Overnight fasted gKD mice and wild type littermates were p.o. dosed with a mixed meal containing (E–G) corn oil:Ensure-plus = 1∶1 (52.6% lipid load, 4.81 kcal/ml) or (H–J) corn oil:Ensure-plus = 3∶17 (19.4% lipid load) at 10 ml/kg. Plasma levels of active GLP-1, PYY, and GIP were measured at 2 h after meal challenge. N = 10–14. *P<0.05, **P<0.01 vs. wild type.</p

Postprandial triglyceridemia, gut hormones, and gastric emptying in <i>Dgat1</i>+/− and <i>Dgat1</i>−/− mice.

<p>4- to 5-month-old <i>Dgat1</i>+/−, <i>Dgat1</i>−/−, and wild type mice maintained on normal chow diet were fasted overnight, then p.o. dosed with (A–F) corn oil (100% lipid load, 8.14 kcal/ml) or (G–L) a mixed meal containing corn oil:Ensure-plus = 3∶17 v/v (19.4% lipid load, 2.48 kcal/ml) at 10 ml/kg body weight. At 2 h after meal challenge, plasma levels of (A) TG, (B, H) active GLP-1, (C, I) total GLP-1, (D, J) PYY, (E, K) GIP, and (F, L) stomach weight were measured. (G) In a separate cohort, plasma TG at 0, 1, 2, and 4h after 19.4% lipid load was determined. N = 8–12. *P<0.05, **P<0.01, ***P<0.001 vs. wild type. ^#P<0.05, ^##P<0.01, ^###P<0.001 <i>Dgat1</i>−/− vs. <i>Dgat1</i>+/−.</p

Effects of DGAT1 inhibition on postprandial GLP-1.

<p>3-month-old lean C57BL/6N mice were p.o. dosed with DGAT1i at 3 mg/kg body weight and fasted overnight. The next morning, mice were p.o. dosed with mixed meals containing 2.6 (water:Ensure-plus = 1:1), 5.2, 28.9, 68.4, or 100% (corn oil:Ensure-plus = 0∶1, 1∶3, 2∶1, 1∶0 v/v) lipid load at 10 ml/kg body weight (0.74, 1.48, 3.15, 5.92, or 8.14 kcal/ml). At 1, 2, or 3h after meal challenge, plasma levels of (A–C) active GLP-1, (D–F) total GLP-1, and (H–J) blood glucose were measured. (G) 3-month-old lean C57BL/6N mice were p.o. dosed with 3 mg/kg DGAT1i or vehicle and fasted overnight. 18h after dosing, jejunal mucosa was harvested, and Gcg mRNA expression was measured by realtime PCR. Data are normalized against β-actin mRNA. N = 8. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 between groups.</p

Biochemical characterization of DGAT1 heterozygous and homozygous knockout mice.

<p>(A) DGAT1 mRNA expression determined by quantitative RT-PCR in jejunal mucosa, white adipose tissue, and liver from <i>Dgat1</i>+/−, <i>Dgat1</i>−/−, and age-matched C57BL/6N wild type mice. N = 3. DGAT1 mRNA and were normalized against β-actin mRNA. All data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. wild type. ^##P<0.01, ^###P<0.001 <i>Dgat1</i>−/− vs. <i>Dgat1</i>+/−. (B) DGAT enzymatic activity in intestinal mucosa of <i>Dgat1</i>−/− and wild type mice measured by TG synthesis in the presence of various concentrations of DGAT1i. (C) Binding of intestinal microsomes of wild type, <i>Dgat1</i>+/−, and <i>Dgat1</i>−/− mice to ³H-labeled DGAT1i. Microsome preps pooled from 2–5 mice were used in the assay. (D) Scatchard analysis of ³H-labeled DGAT1i binding in intestinal microsomes of wild type and <i>Dgat1</i>+/− mice.</p

Whole-genome sequencing of L638^R mutants.

<p>Heat map summary of all non-synonymous mutations identified by Illumina-based whole-genome sequencing (100X genome coverage) of L638^R mutants in MRSE CLB26329 (A) or MRSA COL (B). Red, non-synonymous mutation; grey, no change versus parental genome sequence; yellow, non-synonymous mutations in genes other than <i>mnaA</i>. Genome position, base pair change, and resulting amino acid residue substitution are highlighted. Note: with only one exception (<i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>), no additional non-synonymous mutations besides the indicated <i>mnaA</i> mutation were identified in each of the drug resistant strains examined.</p

Genetic inactivation of <i>mnaA</i> in methicillin-resistant <i>Staphylococci</i> restores β-lactam susceptibility.

<p>Genetic inactivation of <i>mnaA</i> in methicillin-resistant <i>Staphylococci</i> restores β-lactam susceptibility.</p

MRSA and MRSE MnaA LOF mutants are highly susceptible to imipenem in a murine thigh infection.

<p>Immune-suppressed CD-1 mice (5 per group) were challenged intramuscularly with the parental MRSA COL strain, MRSA <i>Δcap5P</i>, or MRSA <i>Δcap5P mnaA</i>_<i>Sa</i> LOF mutants (A) or with the parental MRSE strain versus <i>mnaA</i>_<i>Se</i>, <i>tarO</i>_<i>Se</i> and <i>tarA</i>_<i>Se</i> LOF mutants (B) and treated three times daily (TID) with imipenem (IPM). Thighs were harvested at 24hrs, homogenized and plated to determine CFU per thigh. (A) Restored efficacy of IPM (10 mg kg^-1) against MRSA <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i>. Following IPM treatment, bacterial burden amongst mice infected with <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>P12L</i>, <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>Y194</i>*, and <i>Δcap5P mnaA</i>_<i>Sa</i>^<i>D281Y</i> is reduced approximately 2–3 log at 24 hrs versus those infected with MRSA COL or <i>Δcap5P</i> controls. * p<0.01 versus parent at 24 hr; $ p<0.05 versus respective 24 hr vehicle. (B) Restored efficacy of IPM (2.5 mg kg^-1) against MRSE <i>mnaA</i>, <i>tarO</i>, and <i>tarA</i> LOF mutants. Reduction in bacterial burden of mice infected with the <i>mnaA</i>_<i>Se</i>^<i>G171D</i> is comparable to those infected with <i>tarO</i>_<i>Se</i>^<i>G84</i>* or <i>tarA</i>_<i>Se</i>^<i>G129R</i> mutants, yielding an approximate 3 log reduction in 24 hr IPM treatment versus the wild-type control. Note, as MRSE CLB26329 is more susceptible to IPM than MRSA COL, its dose was reduced to 4-fold versus the MRSA efficacy study (A).</p

MnaA loss of function mutants in MRSA and MRSE fail to produce WTA.

<p>WTA extraction and SDS PAGE analysis from L638^R MRSE CLB26329 (A) and MRSA COL (B) mutants. Note, wild-type MRSA WTA polymers appear as a ladder of discretely sized bands whereas a more diffuse staining of MRSE WTA polymer is observed. WTA material was normalized to cell biomass prior to loading. Wild-type copies of <i>cap5P</i>, <i>mnaA</i>_<i>Sa</i>, and <i>mnaA</i>_<i>Se</i>, as well as the empty vector introduced into these strains for complementation studies are indicated. The <i>tarO</i> and <i>tarA</i> deletion mutants serve as a control for complete impairment of WTA polymer production.</p