Diet Induced Obesity Alters Intestinal Cytoplasmic Lipid Droplet Morphology and Proteome in the Postprandial Response to Dietary Fat

Dietary fat absorption by the small intestine is an efficient, multistep process that regulates the uptake and delivery of essential nutrients and energy. Fatty acids taken up by enterocytes, the absorptive cells of the small intestine, are resynthesized into triacylglycerol (TAG) and either secreted in chylomicrons or temporarily stored in cytoplasmic lipid droplets (CLDs). Proteins that associate with CLDs are thought to regulate the dynamics of TAG storage and mobilization. It is currently unclear what effect diet induced obesity (DIO) has on the balance between dietary fat storage and secretion. Specifically, there is limited knowledge of how DIO affects the level and diversity of proteins that associate with CLDs and regulate CLD dynamics. In the current study, we characterize CLDs from lean and DIO mice through histological and proteomic analyses. We demonstrate that DIO mice have larger intestinal CLDs compared to lean mice in response to dietary fat. Additionally, we identified 375 proteins in the CLD fraction isolated from enterocytes of lean and DIO mice. We identified a subgroup of lipid related proteins that are either increased or unique to the DIO CLD proteome. These proteins are involved in steroid synthesis, TAG synthesis, and lipolysis. This analysis expands our knowledge of the effect of DIO on the process of dietary fat absorption in the small intestine (D’Aquila, 2016)

Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

<div><p>Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.</p></div

A sub group of proteins are associated with known lipid related functions.

<p>Thirty seven proteins associated with known lipid metabolism pathways were identified, of which twenty three proteins have been previously identified in other CLD proteomic analyses. Relative levels of the proteins were determined by LFQ and the average is reported (n = 4 mice).</p><p>A sub group of proteins are associated with known lipid related functions.</p

Isolated CLD fraction is enriched with CLD marker, Plin3, and has a high TAG to protein ratio.

<p>Enterocytes were isolated from the jejunum section of mouse small intestine, two hours after a 200 μl olive oil bolus. (A) Cells were lysed and fractionated using sucrose gradient ultracentrifugation. After freezing, the sample was cut into 7 fractions. (B) TAG/ protein ratio of isolated fractions. (C) Immunoblot analysis of the fractions with known markers of CLDs (Plin3), cytosol (Gapdh), and membranes (Cnx). (D) Negative staining transmission electron micrograph of fraction 1. Scale bar, 0.5 μm.</p

Lipid accumulates in CLDs in enterocytes in response to a dietary fat challenge.

<p>Representative transmission electron micrograph of a mouse enterocyte from the jejunum section of the small intestine, two hours after an 200 μl olive oil bolus. Neutral lipids, stained with osmium tetroxide, accumulate in CLDs indicated by white asterisks. Golgi apparatus (white arrows) contain smaller chylomicron sized particles. White areas between enterocytes contain secreted chylomicrons (black cross). Scale bar, 1 μm.</p

Immunofluoresence and immunoelectron imaging of Acsl5 demonstrates localization on or around CLDs.

<p>Representative confocal immunofluorescence images (n = 4 mice) and immunoelectron micrographs (n = 2 mice) of enterocytes two hours after a 200 μl olive oil bolus. Frozen sections were immunostained for Acsl5 (A). Lipids were stained with Bodipy (orange), nuclei stained with Dapi (blue), and the signals were merged. Bars = 5 μm (B). A 3D volume view was generated from Z-series images with the dimensions of 21.21 x 21.21 x 3.25 μm and indicates Acsl5 localizes to the area on or around the CLD (C). An immunoelectron micrograph of an enterocyte containing CLDs two hours post a 200 μl olive oil bolus with areas of interest highlighted by colored arrows. Scale bar = 1 μm. Colored arrows correspond with colored boxes of images E-H (D). CLDs are labeled with nanogold conjugated anti-Acsl5 antibody as indicated by the white arrows. Bars = 100 nm (E-G). Additionally, Acsl5 gold labeling was also observed in mitochondria. Bars = 100 nm (H).</p

Lipid related proteins identified cluster based on predicted physical and functional interactions.

<p>A STRING analysis was conducted to map predicted interactions of lipid related proteins. The resulting network shows several clusters, including proteins responsible for lipoprotein synthesis and modifiers of fatty acids.</p

Immunofluorescence imaging of Plin3 and ApoA-IV demonstrates localization on or around CLDs.

<p>Representative confocal immunofluorescence images of enterocytes two hours after a 200 μl olive oil bolus (n = 4 mice). Frozen sections were immunostained for Plin3 in green (A) and ApoA-IV in red (B). Lipids were stained with Bodipy (orange) and nuclei stained with Dapi (blue) and the signals were merged. Bars = 5 μm(C). The signals from Plin3 and ApoA-IV have a Pearson’s correlation of 0.59 and a Mander’s overlap of 0.96 indicating a high degree of colocalization. A capped white line denotes a region of interest and the intensity profile along the line of the signals was generated (D) and indicates the signals of Plin3 and ApoA-IV overlap and flank the CLD. A 3D volume view was generated from Z-series images (E) with the dimensions of 15.91 x 15.91 x 7.5 μm was generated and indicates Plin3 and ApoA-IV localizes to the area on or around the CLD.</p

The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

<div><p>Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.</p></div

PF-04991532 improves glucose metabolism in rats.

<p>PF-04991532 increased the rate of glucose infusion in order to maintain hyperglycemia in Goto-Kakizaki rats (n = 6/group) (<b>A</b>) which can be attributed to the increased glucose disposal and decreased glucose production (n = 6/group) during steady state (<b>B</b>). PF-04991532 decreased plasma glucose in Goto-Kakizaki rats over 28 days of dosing (n = 6–8/group) [P<0.05 for 30, 60, and 100 mg/kg compared to vehicle] (<b>C</b>) which was accompanied by an increase in plasma triglycerides at the highest dose (n = 6–8/group) [P<0.05 for 100 mg/kg] (<b>D</b>). These plasma changes were not associated with any changes in liver triglycerides compared to vehicle treated animals (<b>E</b>). **P<0.01 One-way ANOVA and Tukey’s Multiple Comparison Test were used for A,C,D, & E. Student’s t-test was used for B.</p