NPC1L1 inhibition disturbs lipid trafficking and induces large lipid droplet formation in intestinal absorptive epithelial cells

ABSTRACTEzetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1) protein, which mediates intracellular cholesterol trafficking from the brush border membrane to the endoplasmic reticulum, where chylomicron assembly takes place in enterocytes or in the intestinal absorptive epithelial cells. Cholesterol is a minor lipid component of chylomicrons; however, whether or not a shortage of cholesterol attenuates chylomicron assembly is unknown. The aim of this study was to examine the effect of NPC1L1 inhibition on trans-epithelial lipid transport, and chylomicron assembly and secretion in enterocytes. Caco-2 cells, an absorptive epithelial model, grown onto culture inserts were given lipid micelles from the apical side, and chylomicron-like triacylglycerol-rich lipoprotein secreted basolaterally were analyzed after a 24-h incubation period in the presence of ezetimibe up to 50 μM. The secretion of lipoprotein and apolipoprotein B48 were reduced by adding ezetimibe (30%, p<0.01 and 34%, p<0.05, respectively). Additionally, ezetimibe accelerated intracellular apoB protein degradation by approximately 2.8-fold and activated sterol regulatory element binding protein 2 by approximately 1.5-fold: These are indicators whether the cells are sensing cellular cholesterol shortage. Thus, ezetimibe appeared to limit cellular cholesterol mobilization required for lipoprotein assembly. In such conditions, large lipid droplet formation in Caco-2 cells and the enterocytes in mice were induced, implying that unprocessed triglyceride was sheltered in these compartments. Although ezetimibe did not reduce the post-prandial lipid surge appreciably in triolein-infused mice, the results of the present study indicated that NPC1L1-mediated supply chylomicron with cholesterol may participate in a novel regulatory mechanism for the efficient chylomicron assembly and secretion.

Brown adipose tissue: development, metabolism and beyond

Obesity represents a major risk factor for the development of several of our most common medical conditions, including Type 2 diabetes, dyslipidaemia, non-alcoholic fatty liver, cardiovascular disease and even some cancers. Although increased fat mass is the main feature of obesity, not all fat depots are created equal. Adipocytes found in white adipose tissue contain a single large lipid droplet and play well-known roles in energy storage. By contrast, brown adipose tissue is specialized for thermogenic energy expenditure. Owing to its significant capacity to dissipate energy and regulate triacylglycerol (triglyceride) and glucose metabolism, and its demonstrated presence in adult humans, brown fat could be a potential target for the treatment of obesity and metabolic syndrome. Undoubtedly, fundamental knowledge about the formation of brown fat and regulation of its activity is imperatively needed to make such therapeutics possible. In the present review, we integrate the recent advancements on the regulation of brown fat formation and activity by developmental and hormonal signals in relation to its metabolic function.

Major thermogenic defect associated with insulin resistance in brown adipose tissue of obese diabetic SHR/N-cp rats

The effects of norepinephrine and insulin on thermogenesis were investigated in adipocytes isolated from brown adipose tissue (BAT) of obese non-diabetic LA/N-cp rats (obese LA), obese diabetic SHR/N-cp rats (obese SHR), and their corresponding lean controls. The maximal calorigenic response (Vmax) and the sensitivity [50% effective concentration (EC50)] to norepinephrine (1 microM) were markedly reduced in brown adipocytes from obese SHR rats compared with their lean controls (3- to 4-fold decrease in the Vmax and 50% increase in the EC50 value). In the same cells, there was a similar decrease in the respiratory response to dibutyryl adenosine 3',5'-cyclic monophosphate, indicating the presence of a major postreceptor defect. Remarkably, total BAT cytochrome oxidase activity (an index of cellular mitochondrial content) was also diminished three to four times in obese SHR rats, suggesting that a reduced BAT mitochondrial content is responsible for the decreased thermogenesis. Ultrastructural studies revealed that the cytoplasm of brown adipocytes from obese SHR rats contained a large lipid droplet, numerous tiny droplets, and few atypical mitochondria with loosely packed cristae. Adipocytes from obese SHR rats were also characterized by a significant resistance to the antithermogenic effect of insulin but not to that of the nonmetabolizable adenosine analogue N6-phenylisopropyl adenosine. In contrast, all the above biochemical parameters were normal in obese LA rats. These results demonstrate that the marked insulin resistance in BAT of obese SHR rats is associated with a decreased responsiveness and sensitivity to norepinephrine, indicating the presence of receptor and postreceptor defects. It is suggested that insulin resistance and/or diabetes in SHR/N-cp rats lead to a decreased mitochondriogenesis in BAT, which results in a reduced thermogenic capacity, thereby contributing to the development of obesity.

Ultrastructure of basidiospores of the mycorrhizal fungus Pisolithus tinctorius

Basidia of Pisolithus tinctorius develop from terminal hyphal cells. The distal portion of the basidium enlarges, meiosis occurs, and basidiospore initials develop on short, stout, sterigmata, Young basidia and basidiospore initials are embedded in a fibrillar matrix. As spore initials develop, the basidium becomes highly vacuolate as the contents of the basidium move into the spore initials. A single nucleus typically enters each spore initial. A spore initial is eventually delimited from the basidium near the distal end of the sterigma. Wall material is subsequently deposited in this region sealing off the young spore from the basidium. The basidium then collapses leaving the short sterigma attached to the spore. Young basidiospores are initially highly vacuolate but a large lipid droplet eventually develops and displaces the other cellular components to the periphery of the spore. The oldest spores examined possessed a four-layered wall, the outer layer of which bears the surface spines. Many aberrant spores possessing more or less typical surface spines but lacking cellular components were routinely observed in this study side by side with normal spores.

Fine structure of the retinal pigment epithelium and photoreceptor cells of an Australian marsupial Setonix brachyurus

The morphology of the retinal pigment epithelium and photoreceptor cells has been studied in the quokka (Setonix brachyurus), an Australian marsupial, by light and electron microscopy.The pigment epithelium is formed by a single layer of cuboidal cells which are separated from the choriocapillaris by multilayered Bruch's membrane. Each epithelial cell is rich in organelles and inclusions, including smooth endoplasmic reticulum, mitochondria, Golgi complexes, phagosomes, and pigment granules. The outer border of the epithelial cells is highly infolded while the inner surface displays numerous processes which surround both rod and cone photoreceptor outer segments.Three photoreceptor types are seen, single rods, single cones, and twin cones. The rod photoreceptors outnumber the cones about 50 to 1 and are smaller and more electron-dense than the cones. The cones possess a large lipid droplet within their inner segments. Twin cones are seen only occasionally. They are formed by two cones lying in close apposition, with each member being morphologically quite similar to the other and to the single cone.Photoreceptor synapses in both rods and cones appear to be formed by superficial and invaginated contacts with bipolar and horizontal cells.

STRUCTURE-FUNCTION RELATIONSHIPS IN THE ADIPOSE CELL

A method is described for preparing isolated rat adipose cells for electron microscopy. The ultrastructure of such cells and their production of 14CO2 from U-glucose-14C were studied simultaneously in the presence of insulin or epinephrine. Each adipose cell consists of a large lipid droplet surrounded by a thin rim of cytoplasm. In addition to typical subcellular organelles, a variety of small lipid droplets and an extensive system of membranes characterize the cell's cytoplasm. A fenestrated envelope surrounds the large, central lipid droplet. Similar envelopes surround cytoplasmic lipid droplets occurring individually or as aggregates of very small, amorphous droplets. Groups of individual droplets of smaller size also occur without envelopes. The system of membranes consists of invaginations of the cell membrane, vesicles possibly of pinocytic origin, simple and vesiculated vacuoles, vesicles deeper in the cytoplasm, flattened and vesicular smooth surfaced endoplasmic reticulum, and Golgi complexes. Neither insulin nor epinephrine produced detectable ultrastructural alterations even when cells were incubated under optimal conditions for the stimulation of 14CO2 evolution. Structural responses of the isolated adipose cell to hormones, if such occur, must, therefore, be dynamic rather than qualitative in nature; the extensive system of smooth surfaced membranes is suggestive of compartmentalized transport and metabolism.