The role of leptin and adiponectin as mediators in the relationship between adiposity and hand and knee osteoarthritis.

OBJECTIVES: To investigate associations of leptin and adiponectin levels with knee and hand osteoarthritis, and explore whether these mediate the association between adiposity and osteoarthritis. METHODS: This is a cross-sectional analysis of baseline data from the population-based Netherlands Epidemiology of Obesity study. Adiposity was assessed with body mass index (BMI) and percentage total body fat (%TBF). Osteoarthritis, defined as hand or knee osteoarthritis, was determined using American College of Rheumatology criteria. Fasting serum adipokine levels were measured using immunoassays. Associations between adiposity and osteoarthritis were examined with logistic regression, adjusted for age, sex, ethnicity and education, and additionally for leptin and adiponectin as potential mediators. RESULTS: In 6408 participants (56% women, median age 56 years), prevalence of osteoarthritis was 22% (10% isolated knee and 8% isolated hand osteoarthritis). Leptin levels were positively associated with osteoarthritis, while adiponectin levels were not. Leptin partially mediated the association of adiposity with osteoarthritis (OR 1.40 (95%CI 1.30; 1.52) attenuated to 1.38 (1.24; 1.54) per 5 units BMI and OR 1.25 (1.17; 1.35) to 1.20 (1.10; 1.32) per 5 units %TBF, representing 4% and 17% mediation, respectively). Larger proportion mediation by leptin was found in knee (13%/27%) than in hand osteoarthritis (9%/18%). Sex-stratified analyses generally showed stronger associations between adiposity, leptin and osteoarthritis in women than in men. CONCLUSIONS: Serum leptin levels were associated with osteoarthritis, and partially mediated the association between adiposity and osteoarthritis, while adiponectin levels were not associated with osteoarthritis. These findings provide evidence for systemic effects of adipose tissue in osteoarthritis.

Genetic variation in the obesity gene FTO is not associated with decreased fat oxidation: the NEO study.

BACKGROUND: The fat mass and obesity-associated (FTO) gene harbors the strongest common genetic variant associated with obesity. Recently, rs1421085-T to -C substitution mapped in FTO was shown to induce a developmental shift of human adipocytes from an energy-combusting beige to an energy-storing white phenotype in vitro. As browning of adipocytes selectively enhances fat oxidation (FatOx), we hypothesized that rs1421085-C in FTO is associated with deceased FatOx compared with carbohydrate oxidation (CarbOx) and an increased respiratory quotient (RQ). METHODS: In the Netherlands Epidemiology of Obesity study, a population-based cohort study of middle-aged individuals (45-65 years), anthropometry and genotyping was performed (n=5744), in addition to indirect calorimetry (n=1246). With linear regression analyses, we examined associations of rs1421085 genotype with FatOx, CarbOx and RQ. RESULTS: In the total study population, 36.7% carried the rs1421085-TT genotype, 47.6% rs1421085-CT and 15.7% rs1421085-CC. Mean (s.d.) age was 56 (6) years, mean (s.d.), body mass index (BMI) was 26.3 (4.4) kg m-2 and 56% of the total population were women. Measures of adiposity (difference, 95% confidence interval) were higher in CC carriers compared with that in rs1421085-TT carriers: BMI +0.56 (0.15, 0.98) kg m-2, waist circumference +1.25 (0.02, 2.49) cm and total body fat mass +1.21 (0.28, 2.14) kg. However, no differences in mean FatOx (+2.5 (-2.4, 7.4) mg min-1), CarbOx (-6.1 (-17.4, 5.2) mg min-1) or RQ (-0.01 (-0.02, 0.01)) were observed between the two genotypes. CONCLUSIONS: We observed no evidence for associations of rs1421085 in FTO with FatOx and RQ. This indicates that the rs1421085-C allele in FTO induces obesity likely via other pathways than via reduced FatOx.

How the COVID-19 pandemic highlights the necessity of animal research.

Recently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible, there has been a push by the non-scientific community and politicians for a rapid transition to animal-free innovations. Although there are benefits for both animal welfare and researchers, advances on alternative methods have not progressed enough to be able to replace animal research in the foreseeable future. This trend has led first and foremost to a substantial increase in the administrative burden and hurdles required to make timely advances in research and treatments for human and animal diseases. The current COVID-19 pandemic clearly highlights how much we actually rely on animal research. COVID-19 affects several organs and systems, and the various animal-free alternatives currently available do not come close to this complexity. In this Essay, we therefore argue that the use of animals is essential for the advancement of human and veterinary health.

Prevalence and heritability of the metabolic syndrome and its individual components in a Dutch isolate: the Erasmus Rucphen Family study.

BACKGROUND: Metabolic syndrome (MetS) is defined by a combination of abnormalities that are all individual risk factors for the development of type 2 diabetes and/or cardiovascular disease. The aetiology of MetS includes both an environmental and genetic component. We studied the prevalence and heritability of MetS and its individual components Dutch genetic isolate. METHODS: The Erasmus Rucphen Family study (ERF) consists of some 3000 genealogically documented individuals from a Dutch genetic isolate. Data on waist circumference (WC), blood pressure (BP), high density lipoprotein cholesterol (HDL-C), triglycerides (TG) and fasting plasma glucose values (FPG) are available. MetS was defined according to the International Diabetes Federation (IDF) (2003) and National Cholesterol Education program Adult Panel III (NCEP ATPIII) criteria. Variance component analysis was applied to extended family data to test for evidence of heritability. RESULTS: The prevalence of MetS in the ERF cohort ranged from 23-37% depending on MetS definition and gender considered. Low HDL-C and high WC are the main contributors to MetS. The heritability of MetS corrected for sibship effect was 10.6% (p = 0.01) according to IDF and 13.2% (p = 0.07) according to NCEP ATPIII criteria. In addition, the heritability of individual components of MetS were analysed and found to range from 21.9-42.9%. The highest heritability was found for HDL-C (42.9%, p<0.0001) and WC (37.8%, p<0.0001). In addition, WC, systolic BP, HDL-C and TG showed low to moderate genetic correlation (RhoG) between genders, whereas FPG and diastolic BP showed absolute genetic correlation between genders. CONCLUSION: Although the prevalence of MetS was high, the heritability of MetS in the ERF population was found to be moderate. The high heritability of the individual components of MetS indicates that the genetic dissection of MetS should be approached from its individual components.

In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway.

To study the role of apoC1 in lipoprotein metabolism, we have generated transgenic mice expressing the human APOC1 gene. On a sucrose-rich diet, male transgenic mice with high APOC1 expression in the liver showed elevated levels of serum cholesterol and triglyceride compared with control mice (5.7+/-0.7 and 3.3+/-2.1 vs. 2.7+/-0.1 and 0.4+/-0.1 mmol/liter, respectively). These elevated levels were mainly confined to the VLDL fraction. Female APOC1 transgenic mice showed less pronounced elevated serum lipid levels. In vivo VLDL turnover studies revealed that, in hyperlipidemic APOC1 transgenic mice, VLDL particles are cleared less efficiently from the circulation as compared with control mice. No differences were observed in the hepatic production and extrahepatic lipolysis of VLDL-triglyceride. Also, VLDL isolated from control and APOC1 transgenic mice were found to be equally good substrates for bovine lipoprotein lipase in vitro. These data indicate that the hyperlipidemia in APOC1 transgenic mice results primarily from impaired hepatic VLDL particle clearance, rather than a defect in the hydrolysis of VLDL-triglyceride. To investigate which hepatic receptor is involved in the apoC1-mediated inhibition of VLDL clearance, APOC1 transgenic mice were bred with an LDL receptor-deficient (LDLR(-/-)) background. In addition, control, LDLR(-/-), and LDLR(-/-)/APOC1 mice were transfected with adenovirus carrying the gene for the receptor-associated protein (Ad-RAP). Both serum cholesterol and triglyceride levels were strongly elevated in LDLR(-/-)/APOC1 mice compared with LDLR(-/-) mice (52+/-19 and 36+/-19 vs. 8.4+/-0.9 and 0.5+/-0.2 mmol/liter, respectively), indicating that apoC1 inhibits the alternative VLDL clearance pathway via the remnant receptor. Transfection of LDLR(-/-) mice with Ad-RAP strongly increased serum cholesterol and triglyceride levels, but to a lesser extent than those found in LDLR(-/-)/APOC1 mice (39+/-8 and 17+/-8 vs. 52+/-19 and 36+/-19 mmol/liter, respectively). However, in LDLR(-/-)/APOC1 mice the transfection with Ad-RAP did not further increase serum cholesterol and triglyceride levels (52+/-19 and 36+/-19 vs. 60+/-10 and 38+/-7 mmol/liter, respectively). From these studies we conclude that, in the absence of the LDLR, apoC1 inhibits the hepatic uptake of VLDL via a RAP-sensitive pathway.

Genomic and cDNA cloning of a novel mouse lipoxygenase gene.

A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA sequences of a series of overlapping RT-PCR products. This novel mouse lipoxygenase gene was found to contain an open reading frame encoding 662 amino acids. The cDNA sequence is 68% identical to the mouse leukocyte type 12-lipoxygenase and 65% identical to the mouse platelet type 12-lipoxygenase cDNA. Nucleotide identities to the human 15-lipoxygenase cDNA and the human platelet type 12-lipoxygenase cDNA are 70% and 67%, respectively. The deduced amino acid sequence is 60% identical to the mouse platelet and leukocyte type 12-lipoxygenase sequences and the human 15- and 12-lipoxygenase sequences. This novel mouse lipoxygenase gene was found to be transcribed predominantly in epidermal tissue and named Aloxe.

Mice expressing only the mutant APOE3Leiden gene show impaired VLDL secretion.

Apolipoprotein E (apoE)-deficient mice develop hepatic steatosis and show impaired very low density lipoprotein (VLDL)-triglyceride (TG) secretion. These effects are normalized on the introduction of the human APOE3 gene. To assess whether this apoE effect is isoform specific, we studied hepatic lipid metabolism in mice expressing either APOE3 or the mutant APOE3Leiden on apoe-/- or apoe+/- backgrounds. The transgenes were expressed mainly in periportal hepatocytes, as revealed by in situ hybridization. Mice expressing APOE3Leiden, on the apoe-/- and apoe+/- backgrounds, had fatty livers, which were absent in APOE3/apoe-/- mice. APOE3Leiden/apoe-/- mice showed a strongly reduced VLDL-TG secretion compared with APOE3/apoe-/- mice (48+/-14 versus 82+/-10 micromol/kg per hour, respectively). The presence of a single mouse apoe allele increased VLDL-TG secretion in APOE3Leiden/apoe+/- mice (121+/-43 micromol/kg per hour) compared with APOE3Leiden/apoe-/- mice. These results show that APOE3Leiden does not prevent development of a fatty liver and does not normalize VLDL-TG secretion in mice with an apoE-deficient background. The presence of a single mouse apoe allele is sufficient to normalize the APOE3Leiden-associated reduction of VLDL-TG secretion but does not prevent steatosis. We conclude that apoE-mediated stimulation of VLDL secretion is isoform specific.

Protection from obesity in mice lacking the VLDL receptor.

It has previously been reported that mice lacking the VLDL receptor (VLDLR-/-) exhibit normal plasma lipid levels and a modest decrease in adipose tissue mass. In the present study, the effect of VLDLR deficiency on profound weight gain was studied in mice. Obesity was induced either by feeding of a high-fat, high-calorie (HFC) diet or by crossbreeding mice onto the genetically obese ob/ob background. After 17 weeks of HFC feeding, VLDLR-/- mice remained lean, whereas their wild-type littermates (VLDLR+/+) became obese. Similarly, the weight gain of ob/ob mice was less profound in the absence of the VLDLR. Moreover, VLDLR deficiency led to increased plasma triglycerides after HFC feeding. The protection from obesity in VLDLR-/- mice involved decreased peripheral uptake of fatty acids, because VLDLR-/- mice exhibited a significant reduction in whole-body free fatty acid uptake, with no clear differences in food intake and fat absorption. These observations were supported by a strong decrease in average adipocyte size in VLDLR-/- mice of both obesity models, implying reduced adipocyte triglyceride storage in the absence of the VLDLR. These results suggest that the VLDLR plays a role in the delivery of VLDL-derived fatty acids into adipose tissue.

Essential role for the (hepatic) LDL receptor in macrophage apolipoprotein E-induced reduction in serum cholesterol levels and atherosclerosis.

Apolipoprotein E (apoE) is a high affinity ligand for several receptor systems in the liver, including the low-density lipoprotein (LDL) receptor, and non-LDL receptor sites, like the LDL receptor-related protein (LRP), the putative remnant receptor and/or proteoglycans. Although the liver is the major source of apoE synthesis, apoE is also produced by a wide variety of other cell types, including macrophages. In the present study, the role of the LDL receptor in the removal of lipoprotein remnants, enriched with macrophage-derived apoE from the circulation, was determined using the technique of bone marrow transplantation (BMT). Reconstitution of macrophage apoE production in apoE-deficient mice resulted in a serum apoE concentration of only 2% of the concentration in wild-type C57Bl/6 mice. This low level of apoE nevertheless reduced VLDL and LDL cholesterol 12-fold (P<0.001) and fourfold (P<0.001), respectively, thereby reducing serum cholesterol levels and the susceptibility to atherosclerosis. In contrast, reconstitution of macrophage apoE synthesis in mice lacking both apoE and the LDL receptor induced only a twofold (P<0.001) reduction in VLDL cholesterol and had no significant effect on atherosclerotic lesion development, although serum apoE levels were 93% of the concentration in normal C57Bl/6 mice. In conclusion, a functional (hepatic) LDL receptor is essential for the efficient removal of macrophage apoE-enriched lipoprotein remnants from the circulation and thus for normalization of serum cholesterol levels and protection against atherosclerotic lesion development in apoE-deficient mice.

Scavenger receptor deficiency leads to more complex atherosclerotic lesions in APOE3Leiden transgenic mice.

Apolipoprotein (apo) E3Leiden is a dysfunctional apo E variant associated with familial dysbetalipoproteinemia in humans. Transgenic mice carrying the APOE3Leiden gene develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. An early step in atherosclerosis is foam cell formation, which is thought to result from the unrestricted uptake of modified lipoproteins by macrophages. To investigate the role of the macrophage scavenger receptor type I and II (MSR-A) in this process, APOE3Leiden transgenic mice were crossed onto a MSR-A deficient background and the development of atherosclerosis was examined. In view of recent results with apo E deficient mice (Suzuki H et al., A role for the macrophage scavenger receptors in atherosclerosis. Nature 1997; 386(6622):292-296), absence of the MSR-A in APOE3Leiden mice was expected to lead to a reduction of atherosclerosis. In our study we compared APOE3Leiden/MSR-A deficient mice (E3L MSR-A -/-) to APOE3Leiden/MSR-A wild-type mice (E3L MSR-A +/+). These animals were fed an atherogenic diet for 10 weeks. Quantification of the lesion area showed no significant difference between E3L MSR-A -/- and E3L MSR-A +/+ mice although there was a trend towards the development of larger lesions in the E3L MSR-A -/- mice. All lesions were typed according to their cellular composition. In both male and female E3L MSR-A -/- mice, significantly more severe lesions developed as compared to E3L MSR-A +/+ mice. These results indicate that the effect of MSR-A deficiency on atherogenesis may depend on the presence or absence of apo E.

Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages.

Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated. A 180-kb yeast artificial chromosome (YAC) containing the human MSR gene (MSR1) with 60- and 40-kb flanking sequence at the 5' and 3' end, respectively, was obtained by reducing the size of a 1050-kb YAC by homologous recombination. This 180-kb YAC was microinjected into mouse oocytes. In the resulting transgenic mice, high levels of mRNA for both type I and type II human MSR1 were detected in peritoneal macrophages and trace levels in other organs, known to contain macrophage-derived cells. Using an antibody against the human MSR, the Kupffer cells in the liver were shown to contain the MSR protein. In vivo clearance of acetyl-LDL was not changed in the MSR1-transgenic mice. However, in vitro studies using peritoneal macrophages from the transgenic mice showed a two-fold increased degradation of acetyl-LDL and cholesterolester accumulation concomitant with a four-fold increase in foam cell formation, as compared to wild-type macrophages. Thus, macrophage specific overexpression of the MSR may lead to increased foam cell formation, which is one of the initial and crucial steps in atherogenesis.

Use of transgenic mice in lipoprotein metabolism and atherosclerosis research.

In APOE*3-Leiden transgenic mice the atherosclerotic lesion size is correlated with plasma cholesterol. In these mice the plasma lipid levels are positively correlated with the relative amount of APOE 3-Leiden protein on the VLDL particle. The plasma cholesterol levels are influenced by diet, age and gender, mainly due to an effect of these factors on VLDL production rate. Excess of APOC1 protein does inhibit the hepatic clearance of VLDL remnant particles, whereas excess of apoE leads to a hampered extra-hepatic lipolysis of VLDL triglyceride.

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation.

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.

Transgenic mouse models to study the role of the macrophage scavenger receptor class A in atherosclerosis.

Several in vivo studies have been performed on the role of the macrophage scavenger receptor class A (SR-A) in atherosclerosis using SR-A knockout mice. The results indicate both an antiatherogenic and a proatherogenic role of SR-A, depending on the nature of the animal model serving as the athero-susceptible background. To study the role of SR-A in a different model, we generated a transgenic mouse model with high level expression of the human SR-A gene using a 180 Kb yeast artificial chromosome (MSR1 transgenic mice). These mice show increased expression of SR-A according to the natural expression pattern. The MSR1 transgenic mice were crossed onto a low-density lipoprotein receptor deficient background and were fed a high fat diet for 10 weeks. After this period, the size of the atherosclerotic lesions in the proximal aorta was measured. Surprisingly, atherosclerosis was significantly reduced in the MSR1 transgenic mice. In a second study, the effect of SR-A was examined in APOE-3 Leiden mice providing a different athero-susceptible background. To exclude nonmacrophage effects, bone marrow was transplanted from MSR1 mice and wild-type littermates to APOE-3 Leiden transgenic mice. After 8 weeks on a high fat diet, atherosclerosis in the mice that had received MSR1 bone marrow was reduced compared with mice that had received wild-type bone marrow. This difference reached statistical significance when individual cholesterol exposure of the mice was taken into account. Both experiments indicated an antiatherogenic role of the SR-A. This observation cannot be explained easily by SR-A function in foam cell formation because in MSR1 macrophages in vitro foam cell formation is increased. Alternatively, however, SR-A may affect the activation of macrophages. Hence the response to lipopolysaccharide was measured in MSR1-transgenic macrophages. These macrophages showed a reduction in their activation in response to lipopolysaccharide, as measured by nitric oxide production. These data show that an elevated level of SR-A expression reduces atherosclerosis, potentially by modifying the response of macrophages to activation signals in the plaque.

Mapping of human H chain V region genes (VH4) using deletional analysis and pulsed field gel electrophoresis.

We have used molecular genetic mapping techniques to establish the order and approximate chromosomal locations of VH4 elements on both chromosomes 14 from a single patient. A total of 10 BglII restriction fragments carrying VH4 elements was characterized. The genomic order of VH4-carrying restriction fragments was determined by analysis of the pattern of loss of hybridizing bands from the genomes of a panel of monoclonal lymphoblastoid cell lines which had well characterized rearrangements of the Ig locus on each chromosome. Some individual elements were identified using sequence-specific oligonucleotide probes. Physical dimensions were estimated by the assignment of these ordered elements to large (50-350-kb) restriction fragments using two-dimensional pulse field gel electrophoresis. Six such fragments spanning approximately 890 kb were physically linked and ordered. The chromosomes differed with respect to the complement of VH4 elements, although no evidence was found of major differences in organization. The establishment of a panel of well characterized deletion lines facilitates the rapid mapping of defined restriction fragments carrying VH elements.

Macrophage scavenger receptor class A: A multifunctional receptor in atherosclerosis.

In atherogenesis, elevated plasma levels of low density lipoprotein (LDL) lead to the chronic presence of LDL in the arterial wall. There, LDL is modified (eg, oxidized), and these modified lipoproteins activate endothelial cells, which attract circulating monocytes. These monocytes enter the vessel wall, differentiate into macrophages, and subject the modified lipoproteins to endocytosis through scavenger receptor pathways. This unrestricted uptake, which is not limited by intracellular cholesterol levels, eventually leads to the formation of lipid-filled foam cells, the initial step in atherosclerosis. Macrophage scavenger receptor class A (SRA) is thought to be one of the main receptors involved in foam cell formation, mediating the influx of lipids into the macrophages. In addition to this role in modified lipoprotein uptake by macrophages, the SRA has been shown to be important in the inflammatory response in host defense, cellular activation, adhesion, and cell-cell interaction. Given the importance of these processes in atherogenesis, these latter functions may prove to make the SRA a multifunctional player in the atherosclerotic process.

Dissection of the complex role of apolipoprotein E in lipoprotein metabolism and atherosclerosis using mouse models.

Transgenic and knockout mice have been instrumental in delineating the role of apolipoprotein (apo) E in lipoprotein metabolism and atherosclerosis. The severe hypercholesterolemia and premature atherosclerosis of the apoE knockout mouse have been the starting point from which various physiologic processes have been identified in which apoE plays a critical role. These processes include 1) very low density lipoprotein (VLDL) triglyceride production; 2) lipoprotein lipase mediated triglyceride lipolysis; 3) VLDL remnant clearance and intracellular processing; and 4) the efflux of cellular cholesterol. In this review we will discuss the recent insight in the role of apoE in these processes, which has been obtained using a variety of in vivo and in vitro approaches to modify apoE expression and function.

Polymorphism of the human Ig VH4 gene family.

In this study, we analyze the human VH4 gene family and find it to exhibit a level of polymorphism similar to that of the much larger VH3 family. A cloned VH4 probe detected an average of 10 hybridizing BgIII restriction fragments in genomic DNA derived from 75 unrelated individuals and a total of 15 distinct bands. Of these 15 restriction fragments, 12 were polymorphic, as demonstrated by band absence in some individuals. Oligonucleotide probes specific to CDR1 and CDR2 sequences of known VH4 genes detected limited numbers of bands and revealed sequence polymorphisms that correlated with several of the RFLP detected by the cloned probe. The prevalence of the individual polymorphic restriction fragments was highly variable, ranging from 1% to 97%, with a mean prevalence of 51%. These values resemble those previously observed among VH3 elements. Analysis of linkage disequilibrium suggests that most VH4 gene segments are in genetic equilibrium. These results indicate that the VH4 loci, like those of VH3, are dominated by relatively few, perhaps two to four, alleles/locus and further suggest that the haplotype organization of the human VH locus is very complex.