John M. Dietschy

Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1−/− mouse

Niemann-Pick type C disease is largely attributable to an inactivating mutation of NPC1 protein, which normally aids movement of unesterified cholesterol (C) from the endosomal/lysosomal (E/L) compartment to the cytosolic compartment of cells throughout the body. This defect results in activation of macrophages in many tissues, progressive liver disease, and neurodegeneration. In the npc1−/− mouse, a model of this disease, the whole-animal C pool expands from 2,082 to 4,925 mg/kg body weight (bw) and the hepatic C pool increases from 132 to 1,485 mg/kg bw between birth and 49 days of age. A single dose of 2-hydroxypropyl-β-cyclodextrin (CYCLO) administered at 7 days of age immediately caused this sequestered C to flow from the lysosomes to the cytosolic pool in many organs, resulting in a marked increase in cholesteryl esters, suppression of C but not fatty acid synthesis, down-regulation of genes controlled by sterol regulatory element 2, and up-regulation of many liver X receptor target genes. There was also decreased expression of proinflammatory proteins in the liver and brain. In the liver, where the rate of C sequestration equaled 79 mg·d−1·kg−1, treatment with CYCLO within 24 h increased C movement out of the E/L compartment from near 0 to 233 mg·d−1·kg−1. By 49 days of age, this single injection of CYCLO resulted in a reduction in whole-body C burden of >900 mg/kg, marked improvement in liver function tests, much less neurodegeneration, and, ultimately, significant prolongation of life. These findings suggest that CYCLO acutely reverses the lysosomal transport defect seen in NPC disease.

Unesterified Cholesterol Accumulation in Late Endosomes/Lysosomes Causes Neurodegeneration and Is Prevented by Driving Cholesterol Export from This Compartment

While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase, which hydrolyzes C esters, and NPC1 (Niemann-Pick type C1). In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1−/− mouse, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a t½ of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of sterol regulatory-binding protein 2 (SREBP2) target genes, and activation of liver X receptor-controlled genes. These findings indicated that HP-β-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED50 for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted >7 d. Continuous infusion of HP-β-CD into the ventricular system of npc1−/− animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-β-CD into the CNS.

Derivation of the equations that describe the effects of unstirred water layers on the kinetic parameters of active transport processes in the intestine

Abstract Unidirectional flux of solutes into the intestinal mucosal cells is determined by the rate of movement of these molecules across both an unstirred water layer and the microvillus membrane of the epithelial cell. Therefore, an equation is derived in this paper that describes the velocity of active transport as a function of the characteristics of both the transport carrier in the membrane and the resistance of the overlying unstirred water layer. Using this equation a series of curves are presented that depict the effect on the kinetics of active transport of varying the thickness (d) or surface area (Sw) of the unstirred water layer, the free diffusion coefficient (D) of the solute, the distribution of active transport sites along the villus ( ƒ n ), the maximal transport velocity (Jmd) and the true Michaelis constant (Km). These theoretical curves illustrate the serious limitations inherent in interpretation of previously published data dealing with active transport processes in the intestine.

Weekly Cyclodextrin Administration Normalizes Cholesterol Metabolism in Nearly Every Organ of the Niemann-Pick Type C1 Mouse and Markedly Prolongs Life

Niemann-Pick type C1 (NPC1) disease arises from a mutation inactivating NPC1 protein that normally moves unesterified cholesterol from the late endosomal/lysosomal complex of cells to the cytosolic compartment for processing. As a result, cholesterol accumulates in every tissue of the body causing liver, lung, and CNS disease. Treatment of the murine model of this disease, the npc1−/− mouse, s.c. with β-cyclodextrin (4000 mg/kg) one time each week normalized cellular cholesterol metabolism in the liver and most other organs. At the same time, the hepatic dysfunction seen in the untreated npc1−/− mouse was prevented. The severity of cerebellar neurodegeneration also was ameliorated, although not entirely prevented, and the median lifespan of the animals was doubled. However, in contrast to these other organs, lung showed progressive macrophage infiltration with development of lipoid pneumonitis. These studies demonstrated that weekly cyclodextrin administration overcomes the lysosomal transport defect associated with the NPC1 mutation, nearly normalizes hepatic and whole animal cholesterol pools, and prevents the development of liver disease. Furthermore, this treatment slows cerebellar neurodegeneration but has little or no effect on the development of progressive pulmonary disease.

Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters

Niemann-Pick C1-like 1 (NPC1L1) facilitates the uptake of sterols into the enterocyte and is the target of the novel cholesterol absorption inhibitor, ezetimibe. These studies used the Golden Syrian hamster as a model to delineate the changes in the relative mRNA expression of NPC1L1 and other proteins that regulate sterol homeostasis in the enterocyte during and following cessation of ezetimibe treatment and also to address the clinically important question of whether the marked inhibition of cholesterol absorption alters biliary lipid composition. In hamsters fed a low-cholesterol, low-fat basal diet, the abundance of mRNA for NPC1L1 in the small intestine far exceeded that in other regions of the gastrointestinal tract, liver, and gallbladder. In the first study, female hamsters were fed the basal diet containing ezetimibe at doses up to 2.0 mg.day(-1).kg body wt(-1). At this dose, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased by 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition. The ezetimibe-induced changes in intestinal cholesterol handling were reversed when treatment was withdrawn. In a second study, male hamsters were given a diet enriched in cholesterol and safflower oil without or with ezetimibe. The lipid-rich diet raised the absolute and relative cholesterol levels in bile more than fourfold. This increase was largely prevented by ezetimibe. These data are consistent with the recent finding that ezetimibe treatment significantly reduced biliary cholesterol saturation in patients with gallstones.

Dual modulation of hepatic and intestinal acyl-CoA: cholesterol acyltransferase activity by (de-)phosphorylation and substrate supply in vitro

Acyl‐CoA: cholesterol acyltransferase (ACAT) activity in microsomes from rat liver and rat intestinal epithelial cells was increased by incubation of the microsomes with the 100 000 × g supernatant fraction in the presence of ATP/MgCl2 and NaF. The measured activity was further increased by including cholesterol‐rich liposomes in the preincubation. The ACAT activity in rat liver microsomes could be inhibited by preincubation in the presence of 100 000 × g supernatant and MgCl2 and microsomes preactivated by ATP/MgCl2 could also be inhibited in this way. The results suggest that ACAT activity in vitro is modulated by substrate supply and also reversibly by an ATP‐dependent process which may be protein phosphorylation.

Rates of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vivo

The absolute rate of cholesterol acquisition from de novo synthesis and from receptor-dependent and receptor-independent low-density lipoprotein (LDL) uptake was determined in the adrenal glands of the rat, hamster and rabbit under in vivo conditions. The rate of incorporation of [3H]water into cholesterol in the adrenal gland was much higher in the hamster (1727 nmol/h per g) and rabbit (853 nmol/h per g) than in the rat (71 nmol/h per g). Assuming that 23 atoms of 3H are incorporated into the cholesterol molecule during its biosynthesis, the absolute rates of cholesterol synthesis were then calculated to equal 59, 29 and 2.4 micrograms/h per g of adrenal gland in the hamster, rabbit and rat, respectively. Rates of LDL-cholesterol uptake were measured using a primed continuous infusion of [14C]sucrose-labeled homologous LDL (total LDL transport) and methylated human LDL (receptor-independent LDL transport). The rate of total LDL-cholesterol uptake in the adrenal gland was much higher in the rabbit (227 micrograms/h per g) than in the rat (18 micrograms/h per g) or hamster (6 micrograms/h per g). In all three species LDL uptake was mediated largely (greater than 93%) by receptor-dependent mechanisms. In terms of total cholesterol acquisition, the hamster adrenal gland derived 10-times more cholesterol from de novo synthesis than from LDL uptake, whereas the converse was true in the rabbit. Rates of de novo synthesis and LDL-cholesterol uptake were both low in the rat adrenal gland, which is known to derive cholesterol mainly from circulating high-density lipoproteins. Thus, the adrenal gland acquires cholesterol for hormone synthesis from at least three different sources and the quantitative importance of these sources varies markedly in different animal species, including man.

Relative rates of sterol synthesis in the liver and various extrahepatic tissues of normal and cholesterol-fed rabbits: Relationship to plasma lipoprotein and tissue cholesterol levels

The relative rates of sterol synthesis in the liver and ten extrahepatic tissues of normal and cholesterol-fed rabbits were determined by measuring the rates of incorporation of [1-14C]octanoate into digitonin-precipitable sterols by tissue slices. In normal rabbits the rate of sterol synthesis in the liver was very low compared to that in several extrahepatic tissues, particularly the small intestine. The rate of synthesis in the small intestine showed marked regional variation, with the highest rate occurring in the section proximal to the entry of the common bile duct and the lowest rate in the mid-sections of the intestine. The regional differences in intestinal sterol synthesis correlated inversely with the cholesteryl ester content of the tissue. Rabbits fed the cholesterol diet developed marked hypercholesterolemia, with much of the additional cholesterol appearing in the VLDL and LDL fractions. The cholesteryl ester content of the liver, small intestine and various other extrahepatic tissues increased significantly. Coincident with these changes was a marked suppression of sterol synthesis, not only in the liver, but also in the small intestine, adrenal gland, kidney, lung, spleen and ovary. Thus, the rabbit, like the guinea pig, normally exhibits a very low rate of hepatic sterol synthesis compared to that found in other species such as the rat, squirrel monkey and baboon and, furthermore, manifests feedback inhibition of both hepatic and extrahepatic sterol synthesis when dietary cholesterol intake is increased. This general suppression of synthesis correlates with an accumulation of cholesteryl ester in the tissues which, in turn, presumably is related to the uptake of lipoprotein cholesterol from the hypercholesterolemic plasma that develops under such dietary conditions.

Experimental Demonstration of the Effect of the Unstirred Water Layer on the Kinetic Constants of the Membrane Transport of D-Glucose in Rabbit Jejunum

SummaryThe rate of active transport of a probe molecule into the intestinal mucosal cells is determined by the rate of movement of the solute molecule across two barriers, the unstirred water layer and the microvillus membrane of the epithelial cell. Previously a theoretical equation has been derived which describedJd, the velocity of unidirectional flux, as a function of the characteristics of the transport carrier in the membrane and of the resistance of the overlying unstirred water layer (UWL). The predictions of these equations have been tested experimentally by studying the effect of the rate of stirring of the bulk phase on thein vitro uptake ofd-glucose by rabbit jejunum. These studies demonstrated that, first, alterations in the UWL have a profound effect on the magnitude of the apparent affinity constant, xKm*, of the active transport process. Second, at bulk phase concentrations in excess of theKm the passive component of the experimentally determined flux rate becomes of such magnitude as to introduce significant error into the estimate of both the maximal transport rate,Jdm, and the trueKm. Third, as a result of the UWL, the use of double-reciprocal plots to determineJdm andKm leads to the overestimation of these constants. Finally, failure to account for the UWL leads to important quantitative errors describing a number of the characteristics of the transport process: these include an underestimation of the Q10 and the effect of sodium ion on the active transport of glucose in the jejunum. The results confirm that the kinetic characteristics of the uptake of an actively transported molecule are a complex function of the resistance of both the UWL and the mucosal cell membrane, and this transport process can be adequately described by a newly-derived equation. It is apparent that there are serious limitations in the interpretation of much of the previously published data dealing with active transport processes in the intestine, since these studies failed to account for the effect of the UWL.

Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED50 values for this reversal varied from ∼40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2−/− and npc1−/− mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.