Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.     

Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice

Combined lipase deficiency (cld) is a recessive mutation which causes a severe deficiency of lipoprotein lipase and hepatic lipase activities and lethal hypertriacylglycerolemia within 3 days in newborn mice. The effect of this genetic defect on lipoprotein lipase was studied in primary cultures of brown adipocytes derived from tissue of newborn mice. Cells cultured from cld/cld mice replicated, accumulated triacylglycerol, and differentiated into adipocytes at normal rates. Lipoprotein lipase activity in unaffected cells was detectable on Day 0 of confluence and increased to 1.3 units/mg DNA by Day 6, while that in cld/cld cells was less than 4% of that in unaffected cells on Days 4-6. Unaffected cells released 1.2% of their lipase activity in 30 min in the absence of heparin, and 11% in 10 min in the presence of heparin, whereas cld/cld cells released no lipase activity. cld/cld cells contained 2-3 times as much lipoprotein lipase protein as unaffected cells, and released no lipase protein to the medium. Immunofluorescent lipoprotein lipase was not detectable in unaffected adipocytes unless lipase secretion was blocked with monesin, causing retention of the lipase in Golgi. cld/cld adipocytes, in contrast, contained immunofluorescent lipoprotein lipase distributed in a diffuse reticular pattern, indicating retention of lipase in endoplasmic reticulum. Lipoprotein lipase immunoprecipitated from cells incubated 1-3 h with [35S]methionine was digested with or without endoglycosidase H (endo H) or F, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoprotein lipase in unaffected cells (Mr = 56,000-58,000) consisted of three glycosylated forms, of which the most prevalent was endo H-resistant, the next was totally endo H-sensitive, and the least was partially endo H-sensitive. In contrast, lipoprotein lipase in cld/cld cells (Mr = 56,000) consisted of a single, totally endo H-sensitive form. Lipoprotein lipase in both groups of cells contained two oligosaccharide chains. Chromatography studies with heparin-Sepharose indicated that at least some of the lipoprotein lipase in cld/cld cells was dimerized. The findings demonstrate that brown adipocytes cultured from cld/cld mice synthesize lipoprotein lipase with two high mannose oligosaccharide chains, but it is inactive and retained in endoplasmic reticulum. Whether the cld mutation affects primarily processing of oligosaccharide chains of lipoprotein lipase in endoplasmic reticulum, transport of the lipase from the reticulum, or some other process, is to be resolved.     

Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion.

Combined lipase deficiency (cld) is a recessively inherited disorder in mice associated with a deficiency of LPL and hepatic lipase (HL) activity. LPL is synthesized in cld tissues but is retained in the endoplasmic reticulum (ER), whereas mouse HL (mHL) is secreted but inactive. In this study we investigated the effect of cld on the secretion of human HL (hHL) protein mass and activity. Differentiated liver cell lines were derived from cld mice and their normal heterozygous (het) littermates by transformation of hepatocytes with SV40 large T antigen. After transient transfection with lipase expression constructs, secretion of hLPL activity from cld cells was only 12% of that from het cells. In contrast, the rate of secretion of hHL activity and protein mass per unit of expressed hHL mRNA was identical for the two cell lines. An intermediate effect was observed for mHL, with a 46% reduction in secretion of activity from cld cells. The ER glucosidase inhibitor, castanospermine, decreased secretion of both hLPL and hHL from het cells by approximately 70%, but by only approximately 45% from cld cells. This is consistent with data suggesting that cld may result from a reduced concentration of the ER chaperone calnexin. In conclusion, our results demonstrate a differential effect of cld on hLPL, mHL, and hHL secretion, suggesting differential requirements for activation and exit of the enzymes from the ER.     

Human monocytes in culture synthesize and secrete lipoprotein lipase

Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III₂; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.     

Hepatic lipase in the rat ovary. Ovaries cannot synthesize hepatic lipase but accumulate it from the circulation

Hepatic lipase is proposed to have a role in steroidogenesis through its involvement in the metabolism of high density lipoproteins. We examined the activity, synthesis, distribution, and uptake of this enzyme and assessed the content of its mRNA in luteinized ovaries. We found that during peak steroidogenesis, ovaries of pregnant mare's serum gonadotropin-human chorionic gonadotropin-treated immature rats contained heparin-releasable hepatic lipase-like activity which was neutralized in a dose-dependent manner by purified antibodies to hepatic lipase isolated from post-heparin perfusates of rat livers. Quantitative immunoelectron microscopy revealed that ovarian hepatic lipase occurred along endothelial cells and was 3-fold more abundant in blood vessels of corpora lutea than those of stroma. However, hepatic lipase was not synthesized by the ovary since radiolabeled enzyme was not immunoisolated from the medium of dispersed luteinized granulosa cells incubated with [35S]methionine whereas it was present in the medium of control cells (hepatocytes). Similarly, hepatic lipase mRNA was detectable in liver but not ovaries or kidneys by Northern or slot blot analyses or by the polymerase chain reaction. Finally, 125I-labeled hepatic lipase injected into tail veins was quickly cleared from the systemic circulation, accumulating in liver, ovaries, kidneys, and spleen. Subsequent heparin injection caused rapid reappearance of radioactivity in the bloodstream and a marked decline of radiolabel in liver and ovaries but a modest decrease of that in kidneys and none in spleen. Exogenous 125I-bovine serum albumin also accumulated in all four organs but was not displaced from liver or ovaries by subsequent administration of heparin. Taken together, these data suggest that steroidogenically active ovaries possess but do not synthesize hepatic lipase. Instead, hepatic lipase originating elsewhere, presumably in the liver, is accumulated from the circulation at heparin-sensitive sites in ovarian blood vessels.     

Lipoprotein lipase mRNA in neonatal and adult mouse tissues: comparison of normal and combined lipase deficiency (cld) mice assessed by in situ hybridization

Combined lipase deficiency (cld) is a genetic abnormality in mice resulting in the production of enzymatically inactive lipoprotein lipase (LPL). After suckling, these mice have markedly elevated levels of circulating triglyceride. An alteration of LPL gene expression in cld mice may affect the amount and/or the distribution of LPL mRNA in different cell types. Therefore, we performed in situ hybridization for LPL mRNA in tissues from normal and cld pups and adult mice using an antisense 35S-labeled cRNA probe. LPL mRNA had the same pattern of distribution in both cld and normal newborn mice; the probe hybridized strongly to pyramidal neurons of the hippocampus, heart myocytes, and hepatocytes. Despite the lack of noticeable fat stores, LPL mRNA was found in the dermal layer of the skin of cld mice and normal littermates. In adult mice, the cRNA probe for LPL hybridized to the hippocampus, to the heart, and to localized areas of the kidney. We conclude that despite great variation in plasma triglyceride levels, LPL gene is similarly expressed in animals with or without LPL activity.     

Removal of chylomicron remnants in transgenic mice overexpressing normal and membrane-anchored hepatic lipase

The LDL receptor and the LDL receptor-related protein (LRP) mediate the removal of chylomicron remnants. The LRP pathway involves sequestration of particles in the space of Disse. It has been proposed that either alone or in combination with other factors, such as apolipoprotein E and proteoglycans, hepatic lipase (HL) may contribute to the sequestration of chylomicron remnants. To test this hypothesis, we generated two lines of transgenic mice producing rat HL as a native or as a membrane-anchored form. These animals express HL at levels similar to normal rat. Chylomicron remnants were perfused in a single nonrecirculating pass into the livers of the rat HL transgenic, HL-deficient, and wild-type (WT) mice for 20 min, and the rate of chylomicron remnant removal was measured. Chylomicron remnants were removed at a rate of approximately 50% per pass in WT mice. It was slightly increased in both transgenic mice and reduced in HL-deficient mice compared with the WT mice. Confocal microscopy of liver sections showed that a modest amount of HL colocalized with chylomicron remnant clusters in the transgenic mice, suggesting that HL is a component of the LRP-proteoglycan clusters. These data suggest that HL helps to direct cholesterol to the tissues in which it is localized by a nonenzymatic mechanism.     

Intestinal absorption of dietary cholesteryl ester is decreased but retinyl ester absorption is normal in carboxyl ester lipase knockout mice

Carboxyl ester lipase (CEL; EC 3.1.1.13) hydrolyzes cholesteryl esters and retinyl esters in vitro. In vivo, pancreatic CEL is thought to liberate cholesterol and retinol from their esters prior to absorption in the intestine. CEL is also a major lipase in the breast milk of many mammals, including humans and mice, and is thought to participate in the processing of triglycerides to provide energy for growth and development while the pancreas of the neonate matures. Other suggested roles for CEL include the direct facilitation of the intestinal absorption of free cholesterol and the modification of plasma lipoproteins. Mice with different CEL genotypes [wild type (WT), knockout (CELKO), heterozygote] were generated to study the functions of CEL in a physiological system. Mice grew and developed normally, independent of the CEL genotype of the pup or nursing mother. Consistent with this was the normal absorption of triglyceride in CELKO mice. The absorption of free cholesterol was also not significantly different between CELKO (87 +/- 26%, mean +/- SD) and WT littermates (76 +/- 10%). Compared to WT mice, however, CELKO mice absorbed only about 50% of the cholesterol provided as cholesteryl ester (CE). There was no evidence for the direct intestinal uptake of CE or for intestinal bacterial enzymes that hydrolyze it, suggesting that another enzyme besides CEL can hydrolyze dietary CE in mice. Surprisingly, CELKO and WT mice absorbed similar amounts of retinol provided as retinyl ester (RE). RE hydrolysis, however, was required for absorption, implying that CEL was not the responsible enzyme. The changes in plasma lipid and lipoprotein levels to diets with increasing lipid content were similar in mice of all three CEL genotypes. Overall, the data indicate that in the mouse, other enzymes besides CEL participate in the hydrolysis of dietary cholesteryl esters, retinyl esters, and triglycerides.     

Appraisal of hepatic lipase and lipoprotein lipase activities in mice

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.     

Elevated oxidative stress and sensorimotor deficits but normal cognition in mice that cannot synthesize ascorbic acid

Oxidative stress is implicated in the cognitive deterioration associated with normal aging as well as neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. We investigated the effect of ascorbic acid (vitamin C) on oxidative stress, cognition, and motor abilities in mice null for gulono-γ-lactone oxidase (Gulo). Gulo−/− mice are unable to synthesize ascorbic acid and depend on dietary ascorbic acid for survival. Gulo−/− mice were given supplements that provided them either with ascorbic acid levels equal to- or slightly higher than wild-type mice (Gulo-sufficient), or lower than physiological levels (Gulo-low) that were just enough to prevent scurvy. Ascorbic acid is a major anti-oxidant in mice and any reduction in ascorbic acid level is therefore likely to result in increased oxidative stress. Ascorbic acid levels in the brain and liver were higher in Gulo-sufficient mice than in Gulo-low mice. F₄-neuroprostanes were elevated in cortex and cerebellum in Gulo-low mice and in the cortex of Gulo-sufficient mice. All Gulo−/− mice were cognitively normal but had a strength and agility deficit that was worse in Gulo-low mice. This suggests that low levels of ascorbic acid and elevated oxidative stress as measured by F₄-neuroprostanes alone are insufficient to impair memory in the knockouts but may be responsible for the exacerbated motor deficits in Gulo-low mice, and ascorbic acid may have a vital role in maintaining motor abilities.     

Hormone-sensitive lipase is localized at synapses and is necessary for normal memory functioning in mice

Hormone-sensitive lipase (HSL) is mainly present in adipose tissue where it hydrolyzes diacylglycerol. Although expression of HSL has also been reported in the brain, its presence in different cellular compartments is uncertain, and its role in regulating brain lipid metabolism remains hitherto unexplored. We hypothesized that HSL might play a role in regulating the availability of bioactive lipids necessary for neuronal function and therefore investigated whether dampening HSL activity could lead to brain dysfunction. In mice, we found HSL protein and enzymatic activity throughout the brain, localized within neurons and enriched in synapses. HSL-null mice were then analyzed using a battery of behavioral tests. Relative to wild-type littermates, HSL-null mice showed impaired short-term and long-term memory, yet preserved exploratory behaviors. Molecular analysis of the cortex and hippocampus showed increased expression of genes involved in glucose utilization in the hippocampus, but not cortex, of HSL-null mice compared with controls. Furthermore, lipidomics analyses indicated an impact of HSL deletion on the profile of bioactive lipids, including a decrease in endocannabinoids and eicosanoids that are known to modulate neuronal activity, cerebral blood flow, and inflammation processes. Accordingly, mild increases in the expression of proinflammatory cytokines in HSL mice compared with littermates were suggestive of low-grade inflammation. We conclude that HSL has a homeostatic role in maintaining pools of lipids required for normal brain function. It remains to be tested, however, whether the recruitment of HSL for the synthesis of these lipids occurs during increased neuronal activity or whether HSL participates in neuroinflammatory responses.     

Synthesis of hepatic lipase in liver and extrahepatic tissues

Immunoprecipitations of hepatic lipase from pulse-labeled rat liver have demonstrated that hepatic lipase is synthesized in two distinct molecular weight forms, HL-I (Mr = 51,000) and HL-II (Mr = 53,000). Both forms are immunologically related to purified hepatic lipase, but not to lipoprotein lipase. HL-I and HL-II are also kinetically related and represent different stages of intracellular processing. Glycosidase experiments suggest that HL-I is the high mannose microsomal form of the mature, sialylated HL-II enzyme. Hepatic lipase activity was detected in liver and adrenal gland but was absent in brain, heart, kidney, testes, small intestine, lung, and spleen. The adrenal and liver lipase activities were inhibited in a similar dose-dependent manner by hepatic lipase antiserum. Immunoblot analysis of partially purified adrenal lipase showed an immunoreactive band co-migrating with HL-II at 53,000 daltons which was absent in a control blot treated with preimmune serum. Adrenal lipase and authentic hepatic lipase yielded similar peptide maps, confirming the presence of the lipase in adrenal gland. However, incorporation of L-[35S]methionine into immunoprecipitable hepatic lipase was not detected in this tissue. In addition, Northern blot analysis showed the presence of hepatic lipase mRNA in liver but not adrenal gland. The presence of hepatic lipase in adrenal gland in the absence of detectable synthesis or messenger suggests that hepatic lipase originates in liver and is transported to this extrahepatic site.     

Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice

Both LPL and HL are synthesized in parenchymal cells, are secreted, and bind to endothelial cells. To learn where endothelial lipase (EL) is synthesized in adult animals, the localization of EL in mouse and rat liver was studied by immunohistochemical analysis. Furthermore, to test whether EL could play a role in atherogenesis, the expression of EL in the aorta and liver of apolipoprotein E knockout (EKO) mice was determined. EL in both mouse and rat liver was colocalized with vascular endothelial cells but not with hepatocytes. In contrast, HL was present in both hepatocytes and endothelial cells. By in situ hybridization, EL mRNA was present only in endothelial cells in liver sections. EL was also present at low levels in aorta of normal mice. We fed EKO mice and wild-type mice a variety of diets and determined EL expression in liver and aorta. EKO mice showed significant expression of EL in aorta. EL expression was lower in the liver of EKO mice than in normal mice. Cholesterol feeding decreased EL in liver of both types of mice. In the aorta, EL was higher in EKO than in wild-type mice, and cholesterol feeding had no effect. Together, these data suggest that EL may be upregulated at the site of atherosclerotic lesions and thus could supply lipids to the area.     

Endocytosis of hepatic lipase and lipoprotein lipase into rat liver hepatocytes in vivo is mediated by the low density lipoprotein receptor-related protein

In isolated cell studies, the internalization and degradation of hepatic lipase (HL) has been linked to its binding to the low density lipoprotein receptor-related protein (LRP). We have utilized the receptor-associated protein (RAP), a universal inhibitor of high affinity ligand binding to LRP, to evaluate the participation of LRP in the endocytosis of HL and lipoprotein lipase (LPL). We isolated a total endosome fraction from rat livers after a 30-min infusion of recombinant RAP, administered as a glutathione S-transferase conjugate (GST-RAP). GST-RAP infusion had no effect on the concentration of HL in liver homogenates, but its concentration in blood plasma increased progressively by 20%, and enrichment over homogenate of HL in endosomes was reduced by 50% as compared with infusion of GST alone. The concentrations of LPL in liver and plasma were 1.4 and 0.5%, respectively, those of HL, but endosomal enrichment of the two enzymes was similar ( approximately 10-fold). GST-RAP infusion had no effect on the concentration of LPL in liver but increased its concentration in blood plasma by 250% and reduced its endosomal enrichment by 95% or greater. GST-RAP infusion also reduced endosomal enrichment of LRP by 40%, but enrichment of several other endocytic receptors was unaffected. Endosomal enrichment of several membrane trafficking proteins associated with the endocytic pathway in hepatocytes was unaffected by GST-RAP with the exception of early endosome endosome antigen 1, which was reduced by 85%. We conclude that HL is partially and LPL almost exclusively taken up into rat hepatocytes after binding to the endocytic receptor LRP.     

Diet-induced obesity and hepatic steatosis in L-Fabp / mice is abrogated with SF, but not PUFA, feeding and attenuated after cholesterol supplementation

Liver fatty acid (FA)-binding protein (L-Fabp), a cytoplasmic protein expressed in liver and small intestine, regulates FA trafficking in vitro and plays an important role in diet-induced obesity. We observed that L-Fabp(-/-) mice are protected against Western diet-induced obesity and hepatic steatosis. These findings are in conflict, however, with another report of exaggerated obesity and increased hepatic steatosis in female L-Fabp(-/-) mice fed a cholesterol-supplemented diet. To resolve this apparent paradox, we fed female L-Fabp(-/-) mice two different cholesterol-supplemented low-fat diets and discovered (on both diets) lower body weight in L-Fabp(-/-) mice than in congenic wild-type C57BL/6J controls and similar or reduced hepatic triglyceride content. We extended these comparisons to mice fed low-cholesterol, high-fat diets. Female L-Fabp(-/-) mice fed a high-saturated fat (SF) diet were dramatically protected against obesity and hepatic steatosis, whereas weight gain and hepatic lipid content were indistinguishable between mice fed a high-polyunsaturated FA (PUFA) diet and control mice. These findings demonstrate that L-Fabp functions as a metabolic sensor with a distinct hierarchy of FA sensitivity. We further conclude that cholesterol supplementation does not induce an obesity phenotype in L-Fabp(-/-) mice, nor does it play a significant role in the protection against Western diet-induced obesity in this background.     

Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein

Adipose differentiation-related protein (ADFP; also known as ADRP or adipophilin), is a lipid droplet (LD) protein found in most cells and tissues. ADFP expression is strongly induced in cells with increased lipid load. We have inactivated the Adfp gene in mice to better understand its role in lipid accumulation. The Adfp-deficient mice have unaltered adipose differentiation or lipolysis in vitro or in vivo. Importantly, they display a 60% reduction in hepatic triglyceride (TG) and are resistant to diet-induced fatty liver. To determine the mechanism for the reduced hepatic TG content, we measured hepatic lipogenesis, very-low-density lipoprotein (VLDL) secretion, and lipid uptake and utilization, all of which parameters were shown to be similar between mutant and wild-type mice. The finding of similar VLDL output in the presence of a reduction in total TG in the Adfp-deficient liver is explained by the retention of TG in the microsomes where VLDL is assembled. Given that lipid droplets are thought to form from the outer leaflet of the microsomal membrane, the reduction of TG in the cytosol with concomitant accumulation of TG in the microsome of Adfp-/- cells suggests that ADFP may facilitate the formation of new LDs. In the absence of ADFP, impairment of LD formation is associated with the accumulation of microsomal TG but a reduction in TG in other subcellular compartments.     

Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice

The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBbeta, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728-1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBalpha, display a conspicuous impairment in organismal growth. Akt1(-/-) mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBbeta null mice, Akt1/PKBalpha-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 and Akt2 genes with respect to organismal growth and insulin-regulated glucose metabolism.     

ApoA-II maintains HDL levels in part by inhibition of hepatic lipase. Studies In apoA-II and hepatic lipase double knockout mice.

High density lipoprotein (HDL) cholesterol levels are inversely related to the risk of developing coronary heart disease. Apolipoprotein (apo) A-II is the second most abundant HDL apolipoprotein and apoA-II knockout mice show a 70% reduction in HDL cholesterol levels. There is also evidence, using human apoA-II transgenic mice, that apoA-II can prevent hepatic lipase-mediated HDL triglyceride hydrolysis and reduction in HDL size. These observations suggest the hypothesis that apoA-II maintains HDL levels, at least in part, by inhibiting hepatic lipase. To evaluate this, apoA-II knockout mice were crossbred with hepatic lipase knockout mice. Compared to apoA-II-deficient mice, in double knockout mice there were increased HDL cholesterol levels (57% in males and 60% in females), increased HDL size, and decreased HDL cholesteryl ester fractional catabolic rate. In vitro incubation studies of plasma from apoA-II knockout mice, which contains largely apoA-I HDL particles, showed active lipolysis of HDL triglyceride, whereas similar studies of plasma from apoA-I knockout mice, which contains largely apoA-II particles, did not. In summary, these results strongly suggest that apoA-II is a physiological inhibitor of hepatic lipase and that this is at least part of the mechanism whereby apoA-II maintains HDL cholesterol levels.     

Abdominal obesity in BTBR male mice is associated with peripheral but not hepatic insulin resistance

Insulin resistance is a common feature of obesity. BTBR mice have more fat mass than most other inbred mouse strains. On a chow diet, BTBR mice have elevated insulin levels relative to the C57BL/6J (B6) strain. Male F1 progeny of a B6 x BTBR cross are insulin resistant. Previously, we reported insulin resistance in isolated muscle and in isolated adipocytes in this strain. Whereas the muscle insulin resistance was observed only in male F1 mice, adipocyte insulin resistance was also present in male BTBR mice. We examined in vivo mechanisms of insulin resistance with the hyperinsulinemic euglycemic clamp technique. At 10 wk of age, BTBR and F1 mice had a >30% reduction in whole body glucose disposal primarily due to insulin resistance in heart, soleus muscle, and adipose tissue. The increased adipose tissue mass and decreased muscle mass in BTBR and F1 mice were negatively and positively correlated with whole body glucose disposal, respectively. Genes involved in focal adhesion, actin cytoskeleton, and inflammation were more highly expressed in BTBR and F1 than in B6 adipose tissue. The BTBR and F1 mice have higher levels of testosterone, which may be related to the pathological changes in adipose tissue that lead to systemic insulin resistance. Despite profound peripheral insulin resistance, BTBR and F1 mice retained hepatic insulin sensitivity. These studies reveal a genetic difference in body composition that correlates with large differences in peripheral insulin sensitivity.