Lipid Hydrolysis

The regulation of lipid hydrolysis and synthesis of LDs is strongly associated with the differential regulation of LD proteins, perilipin, Cide/FSP27, and Serpin (Greenberg et al., 2011).

From: Advances in Pharmacology , 2013

ADIPOSE TISSUE | Structure and Function of White Adipose Tissue

R.G. Vernon , D.J. Flint , in Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

Regulation of Adipose Tissue Metabolism

Both lipid synthesis and hydrolysis are under complex hormonal control. Hormones regulate the amounts of key enzymes and other proteins involved, as well as their activities. In addition, the 'signal transduction' systems (a series of reactions transmitting hormone-induced signals to targets in the cell), through which hormones achieve their effects, are also subject to endocrine control themselves, and changes in the ability of adipocytes to transmit such signals are an important part of the adaptations to some physiological states (e.g., lactation).

Regulation of fatty acid synthesis depends on the precursor. For glucose, control begins at the point of entry into the cell where its transport is dependent on a specific carrier protein (transporter); the major glucose transporter of adipocytes is called 'glut 4.' Insulin stimulates glucose transport both by promoting recruitment of glut 4 into the plasma membrane and by increasing its activity. Within the cell, glucose is initially phosphorylated and then metabolized by a long series of reactions, some in the cytosol, some in the mitochondria, to produce acetyl coenzyme A (CoA) in the cytosol. Several enzymes, in particular phosphofructokinase and pyruvate dehydrogenase, have key roles in controling this flux. Insulin, for example, activates pyruvate dehydrogenase. For acetate, the control is much simpler as its initial reaction results in the production of acetyl CoA. The conversion of acetyl CoA to fatty acid is catalyzed by two enzymes, acetyl CoA carboxylase and fatty acid synthetase. The former is thought to be the most important enzyme controling flux. Both the amount of acetyl CoA carboxylase and its activation status (it is an enzyme that exists in active and inactive forms in the cell) change markedly with physiological, nutritional, and pathological condition. The amount and activity, for example, are decreased by fasting, high-fat diets, diabetes, and lactation. Insulin increases both the amount and activity of the enzyme. These effects of insulin are antagonized by growth hormone. Catecholamines and glucagon also cause inactivation of the enzyme and hence a fall in the rate of fatty acid synthesis.

Insulin increases the synthesis and secretion of lipoprotein lipase; this effect is accentuated by glucocorticoids. Gastric inhibitory polypeptide also increases lipoprotein lipase activity; this effect is likely to be important for promoting fat deposition in animals eating high-fat diets as such diets stimulate secretion of this hormone. Thus, insulin and certain gut hormones increase fat synthesis by increasing the supply of fatty acids for esterification. Insulin also promotes glycerol 3-phosphate formation, in part at least, by increasing glucose uptake by adipocytes. The rate of fatty acid esterification itself may not be stimulated directly by hormones but varies directly with fatty acid availability. Curiously, adipocytes secrete adipsin and two related proteins, which interact in the presence of chylomicrons, to produce acylation-stimulating protein, which then acts on adipocytes to stimulate esterification and glucose uptake.

The enzyme controling lipolysis, hormone-sensitive lipase, exists in active and inactive states in the fat cell. Glucagon and adrenaline (epinephrine), and also noradrenaline (norepinephrine) (which is released from nerve endings of the sympathetic nervous system within the tissue itself), interact with specific receptor proteins in the plasma membrane (Figure 5). This causes activation of a key enzyme, adenylate cyclase, which synthesizes cyclic adenosine monophosphate (cAMP). Increased concentrations of cAMP both activate hormone-sensitive lipase and promote its movement from the cytosol to the surface of the lipid droplet, resulting in increased lipolysis. This stimulatory mechanism is attenuated by several inhibitory systems. Adenosine and prostaglandin E2, which are both produced within adipose tissue, interact with their own receptors, leading to inhibition of adenylate cyclase. Curiously, adrenaline and noradrenaline can both activate and inhibit adenylate cyclase. They activate adenylate cyclase by interacting with β-adrenergic receptors and inhibit by interacting with α2-adrenergic receptors. The effect of adrenaline and noradrenaline on lipolysis will thus depend in part on the relative number of β- and α2-adrenergic receptors in the adipocytes. There is considerable site- and gender-specific variation in the ratio of α2- to β-adrenergic receptor number of adipocytes in some species. For example, in women, intraabdominal adipocytes have a ratio of about 1:1, whereas subcutaneous femoral and gluteal adipocytes have a ratio of about 10:1 α2-:β-adrenergic receptors. This ratio is thought to be responsible for the very poor lipolytic response to catecholamines of these subcutaneous adipocytes in women and hence the relatively large size of these cells compared with adipocytes elsewhere in the body. In addition to the above, insulin activates the enzyme, cAMP-phosphodiesterase, which catalyzes the degradation of cAMP and so reduces its concentration. The rate of lipolysis then will depend on the concentration of a whole range of hormones, locally produced factors, and neurohumoral transmitters (substances, such as noradrenaline, which are released by nerve endings in tissues). In addition, the ability of the 'signal transduction' system to transmit signals varies with age and with physiological state. For example, during lactation, when fat is often mobilized to support milk production, the system can become more responsive to agents that promote lipolysis. Thyroid hormones, glucocorticoids, sex steroids, and growth hormone all act on one or more components of the signal transduction system, altering its ability to respond to stimulatory and/;or inhibitory agents.

Figure 5. Control of triacylglycerol hydrolysis (lipolysis) by the catecholamines (adrenaline and noradrenaline) and insulin. AMP, adenosine monophosphate; ↑, ↓, activity/concentration increased or decreased by stimulus, respectively.

Adipose tissue metabolism is thus under complex control. In general, insulin promotes fat synthesis and inhibits lipolysis, whereas catecholamines and glucagon inhibit synthesis and promote lipolysis. In addition, steroid hormones, thyroid hormones, and growth hormone act to modulate the effects of insulin and catecholamines, in part at least, by modifying the ability of the signal transduction systems to transmit signals.

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Liposome-based carrier systems and devices used for pulmonary drug delivery

Iftikhar Khan , ... Waqar Ahmed , in Biomaterials and Medical Tribology, 2013

9.4.3 Freeze drying

Freeze drying, also called lyophilization, is employed to minimize the rate of lipid hydrolysis during storage ( Nounou et al. 2005). However, freeze drying itself may cause a damage to the liposome structures because it involves two stressful stages, namely freezing at which ice crystals can pierce the liposomes and drying at which vacuum is applied to sublime the ice. This stressful operation might result in aggregation or fusion of liposomes and subsequent leakage of the originally entrapped material. The damaging effect of freeze drying can be minimized by the addition of cryoprotectants (e.g. carbohydrates) prior to freezing the liposomes (Nounou et al. 2005, Stark et al. 2010). Thus, cryoprotectants like lactose, trehalose, sucrose and other sugars are necessary in freeze drying (lyophilization) to guard the liposomes against aggregation, fusion and leakage of the originally entrapped material. The phase transition alterations that can possibly happen to liposomes following freeze drying are measured by using differential scanning calorimetry (DSC) (Elhissi A. M. et al. 2006b, Vemuri and Rhodes 1995). The freeze-dried product can be reconstituted by rehydration to get liposomes prior to administration. However, it is important to bear in mind that freeze drying is time-consuming and expensive.

Liposome preparations did not show any physical change before or after freeze drying. However, it increased stability and decreased reconstitution time ((Elhissi A. M. A. and Taylor 2005, Lee et al. 2007).

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Metabolic Analysis Using Stable Isotopes

Sergey Tumanov , ... Jurre J. Kamphorst , in Methods in Enzymology, 2015

3.2 LC-MS Analysis

We routinely analyze negatively charged free (i.e., underivatized) fatty acids generated by lipid hydrolysis with high resolution orbitrap mass spectrometry ( Kamphorst, Fan, Lu, White, & Rabinowitz, 2011) and envision that TOF, and perhaps other types of mass spectrometers may also be used successfully for this type of analysis. The ESI used to introduce the fatty acids into the mass spectrometer does not cause fragmentation. Therefore, only the intact molecular ion and its isotopologs are analyzed (Fig. 3). Our current setup is a Q Exactive orbitrap mass spectrometer coupled to a Dionex UltiMate 3000 LC system (Thermo Scientific). The LC settings we use are 2   μL of sample is injected onto a 1.9   μm particle 100   ×   2.1   mm id Thermo Hypersil GOLD column (Thermo Scientific) which is heated to 60   °C. A binary gradient solvent system of (A) water/acetonitrile (40:60, v/v) with 10   mM ammonium formate and (B) acetonitrile/isopropanol (10:90, v/v) with 10   mM ammonium formate is used. Sample analysis is performed using a linear gradient from 30% to 60% B over 6   min followed by raising gradient to 100% B within next 0.5   min and holding at this composition for another 1.5   min. Thereafter, the column is equilibrated with the initial conditions for another 2   min. The flow rate we use is 0.3   mL/min. Fatty acids are analyzed in negative ionization mode. The electrospray and mass spec settings are as follows: spray voltage 3.5   kV, capillary temperature 300   °C, sheath gas flow rate 25 (arbitrary units), and auxiliary gas flow rate 15 (arbitrary units). The mass spec resolution is set to 70,000, automatic gain control is set at 1   ×   106 with a maximum injection time of 100   ms and the scan range is 240–400 m/z for MS mode. Xcalibur 2.2 and MAVEN are used for data processing (Melamud, Vastag, & Rabinowitz, 2010).

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Protein Kinase C: Relaying Signals from Lipid Hydrolysis to Protein Phosphorylation

Alexandra C. Newton , in Handbook of Cell Signaling, 2003

Introduction

Protein kinase C (PKC) has been in the spotlight since the discovery a quarter of a century ago that, through its activation by diacylglycerol, it relays signals from lipid hydrolysis to protein phosphorylation [ 1]. The subsequent discovery that PKCs are the target of phorbol esters resulted in an avalanche of reports on the effects on cell function of phorbol esters, nonhydrolyzable analogs of the endogenous ligand, diacylglycerol [2–4]. Despite the enduring stage presence of PKC and tremendous advances in understanding the enzymology and regulation of this key protein, an understanding of the function of PKC in biology is still the subject of intense pursuit. Its uncontrolled signaling wreaks havoc in the cell, as epitomized by the potent tumor-promoting properties of phorbol esters. In fact, the pluripotent effects of phorbol esters, compounded with the existence of multiple isozymes of PKC, has made it difficult to uncover the precise cellular function of this key enzyme [5]. Studies with knockout mice have underscored the problem, with knockouts of most isozymes having only subtle phenotypic effects [6]. This chapter summarizes our current understanding of the molecular mechanisms of how protein kinase C transduces information from lipid mediators to protein phosphorylation.

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Volume 2

Alexandra C. Newton , in Handbook of Cell Signaling (Second Edition), 2010

Introduction

Protein kinase C (PKC) has been in the spotlight since the discovery over a quarter of a century ago that, through its activation by diacylglycerol, it relays signals from lipid hydrolysis to protein phosphorylation [1]. The subsequent discovery that PKCs are targets of phorbol esters, non-hydrolyzable functional analogs of the endogenous ligand, diacylglycerol, resulted in an avalanche of reports on the effects of phorbol esters on cell function [2–6]. Biophysical, biochemical, cellular, and physiological studies over the past two decades, complemented by recent structural and cellular imaging advances, have unveiled many of the secrets of this center-stage signal transducer [7–9]. Its uncontrolled signaling wreaks havoc in the cell, as epitomized by the potent tumor-promoting properties of phorbol esters, poising it as a therapeutic target in diseases such as cancer. This chapter summarizes our current understanding of the molecular mechanisms of how protein kinase C transduces information from lipid mediators to protein phosphorylation.

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SPOILAGE PROBLEMS | Problems Caused by Bacteria

D.A. Bautista , in Encyclopedia of Food Microbiology (Second Edition), 2014

Spoilage of Other Dairy Products

The microflora of whole milk tends to be present in the cream component. Because cream is the main ingredient of butter, microbial spoilage can be a problem. Microorganisms associated with the lipid hydrolysis of triglycerides to free fatty acids can produce increased acidity, rancidity, and soapiness in butter. Causative agents include Pseudomonas, Micrococcus, and Serratia spp. Surface taint or putridity results from the growth of S. putrefaciens.

Bacterial contamination of cheese usually is the result of manufacturing with milk that has a high microbial content (≥1000   cfu   ml−1). The undesirable growth of lactic acid bacteria, such as Leuconostoc spp. and L. lactis, can cause an undesirable pink discoloration near the surface of cheese. Bacillus, Leuconostoc, and Pseudomonas spp. can attack proteins and produce carbon dioxide. Production of large amounts of gas may result in the formation of undesirable holes in curd during cheese manufacturing. These bacteria are responsible for bitter flavor and slime in soft and hard cheeses (e.g., Brie and Parmesan, respectively).

In cottage cheese, Pseudomonas spp., namely P. fragi, can alter the flavors leaving a putrid, rancid, bitter, or fruity taste. Another problem is the growth of Flavobacterium spp., which can alter the color of cottage cheese. Escherichia coli in high enough numbers (100   000   cfu   g−1) can result in an unclean or barny taste, especially when cottage cheese is left at room temperature (22   °C).

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Primary Mixed Dyslipidemias

Rafael Carmena , in Encyclopedia of Endocrine Diseases (Second Edition), 2019

HL Deficiency

HL is a key liver enzyme involved in catalyzing the hydrolysis of triglycerides and phospholipids in remnant lipoproteins and HDL particles, which thus plays an important role in the conversion of IDL to LDL. HL lipid hydrolysis in remnant particles contributes to their hepatic uptake via an Apo E-mediated process. In addition, HL is involved in remodeling remnant particles, HDL, and LDL, as well as in the production of small, dense LDL (Carmena et al., 2004; Kobayashi et al., 2015).

HL deficiency is a rare autosomal-recessive disease resulting in mixed hyperlipidemia, characterized by cholesterol and triglyceride elevations caused by the accumulation of lipoprotein remnants, and this may be accompanied by normal or elevated levels of HDL-C (Hegele et al., 1993). Different mutations in the HL gene (LIPC) have been shown to give rise to loss of circulating HL activity, causing an increase in plasma remnants and triglyceride-rich HDL, which produces an increased CHD risk (Chatterjee and Sparks, 2011). The phenotype is similar to that found in FDBL, with elevated levels of total cholesterol and triglycerides, premature arcus cornea, palmar and tubero-eruptive xanthomas, and premature CVD (Semenkovich et al., 2016).

The diagnosis requires the demonstration of HL deficiency with in vitro assays of HL activity in postheparin plasma samples or DNA analysis to identify a mutation. As indicated in the treatment of FDBL, statin therapy is recommended to reduce remnant lipoproteins and CVD risk in this disease.

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Formulating Creams, Gels, Lotions, and Suspensions

Vitthal S. Kulkarni Ph.D. , Charles Shaw Ph.D. , in Essential Chemistry for Formulators of Semisolid and Liquid Dosages, 2016

4.4 Formulating with Liposomes

Liposomes have an aqueous solution core surrounded by a hydrophobic membrane, in the form of a lipid bilayer. During the formulation process, hydrophilic materials can be dissolved in the aqueous core and hydrophobic materials made to associate with the bilayer. Consequently, liposomes can be used in formulations as "carriers" for both hydrophilic and hydrophobic ingredients. The cell membranes in human skin are also made up of lipid bilayer structures. The lipid bilayers of the liposomes in formulated products can "fuse" with the bilayers in the cell membrane, thereby delivering the liposome contents to a site of action. In this way, liposomes can be used as a vehicle for the administration of nutrients and pharmaceutical drugs.

The presence of surfactants in a formulation will compromise the integrity of liposomes. As a result, stability becomes a major concern when attempting to formulate liposomes in emulsion systems. For topical or transdermal routes of administration, liposomes can be safely formulated in gel-type dosages. Gels made from carbomer, cellulose derivatives, and hyaluronic acids are most suitable for formulating with liposomes. Entrapping the liposomes in a thickened matrix helps to prolong their shelf life by reducing the possibility of liposome–liposome collisions. For injectable liposome formulations, the liposomes can be freeze-dried and supplied with a separate suitable carrier medium (such as saline- or dextrose-injection solutions). The liposomes are resuspended by mixing with the carrier medium immediately prior to injecting into the body.

Some of the critical factors that affect the stability of liposomes include [4]:

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pH of the formulation: A finished-formulation pH of 6.5 is ideal, because, at this pH, the rate of lipid hydrolysis is lowest.

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Storage temperature: Liposomes are very susceptible to temperatures that promote oxidation and leakage of the entrapped cargo. Therefore, storage at 2-8   °C is ideal. Additionally, it is critical not to subject loaded liposomes to freeze and thaw conditions as it is known that the loaded cargo is likely to leak after freeze–thaw stress.

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Container-closure system: The selection of the container-closure system used for storing liposome formulations is crucial. Liposomes are not compatible with certain plastic materials. For injectable liposome suspensions, testing compatibility with the elastomeric stoppers to be used with the injection vials is essential. Using glass ampoules rather than stoppered injection vials is often safer. As lipids are susceptible to photooxidation, protecting them from light during storage is highly recommended.

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Infusion tubing: For infusible liposome formulations, establishing compatibility of the liposome suspensions with intravenous tubing is critical, because this tubing is made of synthetic plastic materials. The product label needs to specify the parts/types of tubing that can be used during drug administration.

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Enzymes, Industrial (overview)

B.C. Saha , ... R.J. Bothast , in Encyclopedia of Microbiology (Third Edition), 2009

Enzymes in the Modification of Fats and Oils

Lipases (EC 3.1.1.3, glycerol ester hydrolases) are a ubiquitous class of enzymes which catalyze hydrolysis, esterification (synthesis), and transesterification (group exchange of esters). These enzymes are used for diverse purposes such as fat hydrolysis, flavor development in dairy products, ester synthesis, transesterification of fats and oils, production of chiral organic compounds, washing and cleaning products, and treatment of domestic and industrial products. Lipolytic reactions occur at the lipid–water interface. There are two broad types of lipases based on their positional specificity. Nonspecific lipase releases fatty acids from all three positions of the glycerol moiety and is used to hydrolyze fats and oils completely to free fatty acids and glycerol. These are produced by Candida sp., Staphylococcus sp., and Geotrichum sp. The other type of the enzyme is 1,3-specific lipase which releases fatty acids from 1,3 positions and preferentially free fatty acids and di- and monoglycerides as the reaction products. This type of lipase is produced by Aspergillus, Mucor, Rhizopus, and Pseudomonas sp.

Conventional fat hydrolysis by high-temperature steam splitting (250–260   °C at 50   bar) consumes large amounts of energy and creates environmental concerns. Lipases can be used to hydrolyze fats and oils with excellent yields, but the high cost of the enzymes, exacerbated by enzyme stability problems, currently makes the process uneconomical. Similarly, lipase-catalyzed transesterification of plant glycerides to make alkyl esters (plus glycerol byproduct) for use as biodiesel and replace base-catalyzed processes would be a huge market for enzyme biotechnology; the need to overcome severe cost restraints to become economically competitive is an ongoing research effort. In the meantime, higher value and lower bulk products such as edible and nonedible fats and oils with specialized, improved, or new properties are produced by the action of regio-specific lipases. For example, the 1,3-regio-specific lipase from Rhizomucor miehei is used on the industrial scale to replace palmitic acid moities of palm oil with stearic acid yielding stearic–palmitic–stearic triacylglycerol, which is a cocoa butter substitute. Similarly, enrichment of oils with highly unsaturated fatty acids can be made by lipase-mediated transesterification reactions to produce nutraceuticals. Lipases are also used to assist in the extraction of fats and oils and for the development of cheese flavor. Owing to their stability in organic solvents, lack of cofactor requirement, and range of substrate selectivities, lipases are the most utilized enzymes in mediating reactions in organic synthesis with hundreds of research papers on the subject. There is an increasing adaptation of the versatile microbial lipases in the large-scale synthesis of fine chemicals, in various organic synthesis processes including the preparation of chiral compounds in enantiomerically pure form.

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Solid-State Fermentation for the Production of Lipases for Environmental and Biodiesel Applications

Erika C.G. Aguieiras , ... Denise M.G. Freire , in Current Developments in Biotechnology and Bioengineering, 2018

2.5 Applications of Lipases Produced by Solid-State Fermentation

Although numerous lipases have been produced by SSF, only a few reports describe the application of these biocatalysts.

The most-studied applications for SSF lipases are the synthesis of flavor and fragrance esters [48,69–72], the synthesis of biodiesel [50,54,57,59,68,73–76] , and the lipid hydrolysis of wastewater with high oil and grease content [77–84]. SSF lipases were also used to remove oil from cotton fabric [85], enhance the aroma of black tea [86], enantioselective reaction [67], lipid interesterification [87], selective oil hydrolysis [65], and oil and fat hydrolysis [56,88].

Considering the wide variety of lipase applications and the few areas covered with SSF lipases, there is still a large area remaining to be explored. The next section deals with the application of SSF lipases for biodiesel purposes in more detail.

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