Kimberly K. Buhman

Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth by Relocalizing from the ER to Lipid Droplets

Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.

Deficiency of acyl CoA:cholesterol acyltransferase 2 prevents atherosclerosis in apolipoprotein E-deficient mice

Deficiency of acyl CoA:cholesterol acyltransferase 2 (ACAT2) in mice results in a reduction in cholesterol ester synthesis in the small intestine and liver, which in turn limits intestinal cholesterol absorption, hepatic cholesterol gallstone formation, and the accumulation of cholesterol esters in the plasma lipoproteins. Here we examined the contribution of ACAT2-derived cholesterol esters to atherosclerosis by crossing ACAT2-deficient (ACAT2–/–) mice with apolipoprotein (apo) E-deficient (ApoE–/–) mice, an atherosclerosis-susceptible strain that has impaired apoE-mediated clearance of apoB-containing lipoproteins. ACAT2–/– ApoE–/– mice and ACAT2+/+ ApoE–/– (control) mice had similar elevations of plasma apoB and total plasma lipids; however, the lipid cores of the apoB-containing lipoproteins in ACAT2–/– ApoE–/– mice contained primarily triglycerides rather than cholesterol esters. At 30 wk of age, only the control mice had significant atherosclerosis, which was nearly absent in ACAT2–/– ApoE–/– mice. ACAT2 deficiency in the apoE-deficient background also led to a compensatory increase in the activity of lecithin/cholesterol acyltransferase, the major plasma cholesterol esterification enzyme, which increased high-density lipoprotein cholesterol esters. Our results demonstrate the crucial role of ACAT2-derived cholesterol esters in the development of atherosclerosis in mice and suggest that triglyceride-rich apoB-containing lipoproteins are not as atherogenic as those containing cholesterol esters. Our results also support the rationale of pharmacological inhibition of ACAT2 as a therapy for atherosclerosis.

1α,25-Dihydroxyvitamin D hydroxylase in adipocytes ☆

High vitamin D intake is associated with reduced insulin resistance. Expression of extra-renal 1alpha,25-dihydroxyvitamin D hydroxylase (1alpha-hydroxylase) has been reported in several tissues and contributes to local synthesis of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D) from the substrate 25-hydroxyvitamin D (25OHD). Expression and dietary regulation of 1alpha-hydroxylase in tissues associated with energy metabolism, including adipose tissue, has not been assessed. Male Wistar rats were fed a high calcium (1.5%) and high vitamin D (10,000IU/kg) or a low calcium (0.25%), low vitamin D (400IU/kg) with either a high fat (40% energy) or high sucrose (66% energy) dietary background for 14 weeks. Expression of 1alpha-hydroxylase, assessed by real time PCR, was detected in adipose tissue and did not differ with dietary level of calcium and vitamin D. 1alpha-Hydroxylase mRNA was also detected in 3T3-L1 preadipocytes and 25OHD treatment at 10nM levels induced 1,25(OH)(2)D responsive gene, CYP24, and this response was reduced in the presence of the p450 inhibitor, ketoconazole. In addition, (3)H 25OHD was converted to (3)H 1,25(OH)(2)D in intact 3T3-L1 preadipocytes. Cumulatively, these results demonstrate that 1alpha-hydroxylase is expressed in adipose tissue and is functional in cultured adipocytes. Thus, the capacity for local production may play a role in regulating adipocyte growth and metabolism.

ACAT2 deficiency limits cholesterol absorption in the cholesterol‐fed mouse: Impact on hepatic cholesterol homeostasis

Acyl CoA:cholesterol acyltransferase (ACAT) 2 is the major cholesterol‐esterifying enzyme in mouse enterocytes and hepatocytes. Male ACAT2+/+ and ACAT2−/ − mice were fed chow containing added cholesterol (0%‐0.500% w/w) for 24 days. Over this range, fractional cholesterol absorption in the ACAT2+/+ mice fell from 41.4% ± 6.6% to 21.0% ± 5.2%, and in their ACAT2−/− counterparts it fell from 35.1% ± 4.5% to 7.9% ± 0.8%. The mass of dietary cholesterol absorbed (mg/d per 100 g body weight) increased from 1.2 ± 0.2 to 14.7 ± 4.4 in the ACAT2+/+ mice and from 1.0 ± 0.2 to 5.5 ± 0.6 in those without ACAT2. In the ACAT2+/+ mice, hepatic cholesterol concentrations increased as a function of intake despite compensatory changes in cholesterol and bile acid synthesis and in the expression of adenosine triphosphate–binding cassette transporter G5 (ABCG5) and ABC transporter G8 (ABCG8). In contrast, in ACAT2−/− mice in which the amount of cholesterol absorbed at the highest intake was only 37% of that in the ACAT2+/+ mice, suppression of synthesis was a sufficient adaptive response; there was no change in bile acid synthesis, ABCG5/G8 expression, or hepatic cholesterol concentration. The expression of adenosine triphosphate–binding cassette transporter A1 (ABCA1) in the jejunum was markedly elevated in the ACAT2−/− mice, irrespective of dietary cholesterol level. In conclusion, although ACAT2 deficiency limits cholesterol absorption, the extent to which it impacts hepatic cholesterol homeostasis depends on cholesterol intake. Loss of ACAT2 activity may result in unesterified cholesterol being absorbed via an ABCA1‐mediated basolateral efflux pathway. (HEPATOLOGY 2004;40:1088–1097.)

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.

Intestine-specific expression of acyl CoA:diacylglycerol acyltransferase 1 reverses resistance to diet-induced hepatic steatosis and obesity in Dgat1-/- mice.

Mice deficient in acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triacylglycerol (TG) biosynthesis, are resistant to high-fat (HF) diet-induced hepatic steatosis and obesity. DGAT1-deficient (Dgat1−/−) mice have no defect in quantitative absorption of dietary fat; however, they have abnormally high levels of TG stored in the cytoplasm of enterocytes, and they have a reduced postprandial triglyceridemic response. We generated mice expressing DGAT1 only in the intestine (Dgat1IntONLY) to determine whether this phenotype contributes to resistance to HF diet-induced hepatic steatosis and obesity in Dgat1−/− mice. Despite lacking DGAT1 in liver and adipose tissue, we found that Dgat1IntONLY mice are not resistant to HF diet-induced hepatic steatosis or obesity. The results presented demonstrate that intestinal DGAT1 stimulates dietary fat secretion out of enterocytes and that altering this cellular function alters the fate of dietary fat in specific tissues.

High ACAT1 expression in estrogen receptor negative basal-like breast cancer cells is associated with LDL-induced proliferation

The specific role of dietary fat in breast cancer progression is unclear, although a low-fat diet was associated with decreased recurrence of estrogen receptor alpha negative (ER−) breast cancer. ER− basal-like MDA-MB-231 and MDA-MB-436 breast cancer cell lines contained a greater number of cytoplasmic lipid droplets compared to luminal ER+ MCF-7 cells. Therefore, we studied lipid storage functions in these cells. Both triacylglycerol and cholesteryl ester (CE) concentrations were higher in the ER− cells, but the ability to synthesize CE distinguished the two types of breast cancer cells. Higher baseline, oleic acid- and LDL-stimulated CE concentrations were found in ER− compared to ER+ cells. The differences corresponded to greater mRNA and protein levels of acyl-CoA:cholesterol acyltransferase 1 (ACAT1), higher ACAT activity, higher caveolin-1 protein levels, greater LDL uptake, and lower de novo cholesterol synthesis in ER− cells. Human LDL stimulated proliferation of ER− MDA-MB-231 cells, but had little effect on proliferation of ER+ MCF-7 cells. The functional significance of these findings was demonstrated by the observation that the ACAT inhibitor CP-113,818 reduced proliferation of breast cancer cells, and specifically reduced LDL-induced proliferation of ER− cells. Taken together, our studies show that a greater ability to take up, store and utilize exogenous cholesterol confers a proliferative advantage to basal-like ER− breast cancer cells. Differences in lipid uptake and storage capability may at least partially explain the differential effect of a low-fat diet on human breast cancer recurrence.

Differential association of adipophilin and TIP47 proteins with cytoplasmic lipid droplets in mouse enterocytes during dietary fat absorption.

Recently, we found that enterocytes dynamically store triglycerides (TGs) in cytoplasmic lipid droplets (CLDs) during dietary fat absorption. A dynamic pool of TG in the form of CLDs which expands and depletes relative to time post dietary fat challenge is present in the absorptive cells of the small intestine, enterocytes. To identify cellular factors which may play a role in the regulation of this dynamic process we investigated the expression and localization of a lipid droplet associated protein family, PAT proteins, in enterocytes of mice chronically and acutely challenged by dietary fat. We found that adipophilin and Tip47 are the only PAT genes present in mouse intestinal mucosa and both genes are present at higher levels after high-fat challenges. We found TIP47 protein present in the intestine from chow and high-fat challenged mice; however, adipophilin protein was only present after high-fat challenges. In addition, TIP47 protein level was higher after an acute than a chronic high-fat challenge whereas adipophilin protein level was higher after a chronic than an acute high-fat challenge. We co-imaged TG in CLDs using CARS microscopy and TIP47 or adipophilin using immunocytochemistry in isolated enterocytes from mice challenged chronically and acutely by high levels of dietary fat. TIP47, but not adipophilin, coats CLDs in enterocytes after an acute high-fat challenge suggesting that TIP47 plays a role in the synthesis of CLDs from newly synthesized TG at the beginning of the process of dietary fat absorption in enterocytes. Adipophilin, on the other hand, coats CLDs only in enterocytes of chronic high-fat fed mice suggesting that adipophilin may play a role in the stabilization of TG stored in CLDs in longer term. These results suggest distinct roles for TIP47 and adipophilin in dietary fat absorption.

Assessing Cholesterol Storage in Live Cells and C. elegans by Stimulated Raman Scattering Imaging of Phenyl-Diyne Cholesterol

We report a cholesterol imaging method using rationally synthesized phenyl-diyne cholesterol (PhDY-Chol) and stimulated Raman scattering (SRS) microscope. The phenyl-diyne group is biologically inert and provides a Raman scattering cross section that is 88 times larger than the endogenous C = O stretching mode. SRS microscopy offers an imaging speed that is faster than spontaneous Raman microscopy by three orders of magnitude, and a detection sensitivity of 31 μM PhDY-Chol (~1,800 molecules in the excitation volume). Inside living CHO cells, PhDY-Chol mimics the behavior of cholesterol, including membrane incorporation and esterification. In a cellular model of Niemann-Pick type C disease, PhDY-Chol reflects the lysosomal accumulation of cholesterol, and shows relocation to lipid droplets after HPβCD treatment. In live C. elegans, PhDY-Chol mimics cholesterol uptake by intestinal cells and reflects cholesterol storage. Together, our work demonstrates an enabling platform for study of cholesterol storage and trafficking in living cells and vital organisms.

Dietary Intervention with Vitamin D, Calcium and Whey Protein Reduced Fat Mass and Increased Lean Mass in Rats

The aim of the current study was to determine the effects and the mechanisms of inclusion of dietary whey protein, high calcium, and high vitamin D intake with either a high-sucrose or high-fat base diets on body composition of rodents. Male Wistar rats were assigned to either no whey protein, suboptimal calcium (0.25%), and vitamin D (400 IU/kg) diet (LD), or a diet containing whey protein, high calcium (1.5%), and vitamin D (10 000 IU/kg) diet (HD), and either high-fat (40% of energy) or high-sucrose (60%) base diets for 13 weeks. Liver tissue homogenates were used to determine [(14)C]glucose and [(14)C]palmitate oxidation. mRNA expression of enzymes related to energy metabolism in liver, adipose, and muscle, as well as regulators of muscle mass and insulin receptor was assessed. The results demonstrated that there was reduced accumulation of body fat mass (P = .01) and greater lean mass (P = .03) for the HD- compared to LD-fed group regardless of the background diet. There were no consistent differences between the LD and HD groups across background diets in substrate oxidation and mRNA expression for enzymes measured that regulate energy metabolism, myostatin, or muscle vascular endothelial growth factor. However, there was an increase in insulin receptor mRNA expression in muscle in the HD compared to the LD groups. In conclusion, elevated whey protein, calcium, and vitamin D intake resulted in reduced accumulation of body fat mass and increased lean mass, with a commensurate increase in insulin receptor expression, regardless of the level of calories from fat or sucrose.