Overexpression of Slc22a18 facilitates fat accumulation in mice.

We previously reported that solute carrier family 22 member 18 (Slc22a18) regulates lipid accumulation in 3T3-L1 adipocytes. Here, we provide additional evidence derived from experiments with adenoviral vector expression and genetic manipulation of mice. In primary cultured rat hepatocytes, adenoviral overexpression of mouse Slc22a18 increased triglyceride accumulation and triglyceride synthetic activity, which was decreased in an adenoviral knockdown experiment. Adenoviral overexpression of mouse Slc22a18 in vivo caused massive fatty liver in mice, even under normal dietary conditions. Conversely, adenoviral knockdown of mouse Slc22a18 reduced hepatic lipid accumulation induced by a high-glucose and high-sucrose diet. We created Slc22a18 knockout mice, which grew normally and showed no obvious spontaneous phenotypes. However, compared with control littermates, the knockout mice exhibited decreased hepatic triglyceride content under refeeding conditions, significantly reduced epididymal fat mass, and tended to have lower liver weight in conjunction with leptin deficiency. Finally, we created transgenic mice overexpressing rat Slc22a18 in an adipose-specific manner, which had increased body weight and epididymal fat mass primarily because of increased adipocyte cell volume. In these transgenic mice, a positive correlation was observed between adiposity and the expression levels of the rat Slc22a18 transgene. Taken together, these results indicate that Slc22a18 has positive effects on lipid accumulation in vivo.

Current Diagnosis and Management of Primary Chylomicronemia.

Primary chylomicronemia (PCM) is a rare and intractable disease characterized by marked accumulation of chylomicrons in plasma. The levels of plasma triglycerides (TGs) typically range from 1,000 - 15,000 mg/dL or higher.PCM is caused by defects in the lipoprotein lipase (LPL) pathway due to genetic mutations, autoantibodies, or unidentified causes. The monogenic type is typically inherited as an autosomal recessive trait with loss-of-function mutations in LPL pathway genes (LPL, LMF1, GPIHBP1, APOC2, and APOA5). Secondary/environmental factors (diabetes, alcohol intake, pregnancy, etc.) often exacerbate hypertriglyceridemia (HTG). The signs, symptoms, and complications of chylomicronemia include eruptive xanthomas, lipemia retinalis, hepatosplenomegaly, and acute pancreatitis with onset as early as in infancy. Acute pancreatitis can be fatal and recurrent episodes of abdominal pain may lead to dietary fat intolerance and failure to thrive.The main goal of treatment is to prevent acute pancreatitis by reducing plasma TG levels to at least less than 500-1,000 mg/dL. However, current TG-lowering medications are generally ineffective for PCM. The only other treatment options are modulation of secondary/environmental factors. Most patients need strict dietary fat restriction, which is often difficult to maintain and likely affects their quality of life.Timely diagnosis is critical for the best prognosis with currently available management, but PCM is often misdiagnosed and undertreated. The aim of this review is firstly to summarize the pathogenesis, signs, symptoms, diagnosis, and management of PCM, and secondly to propose simple diagnostic criteria that can be readily translated into general clinical practice to improve the diagnostic rate of PCM. In fact, these criteria are currently used to define eligibility to receive social support from the Japanese government for PCM as a rare and intractable disease.Nevertheless, further research to unravel the molecular pathogenesis and develop effective therapeutic modalities is warranted. Nationwide registry research on PCM is currently ongoing in Japan with the aim of better understanding the disease burden as well as the unmet needs of this life-threatening disease with poor therapeutic options.

Integrative genomic analysis of blood pressure and related phenotypes in rats.

Despite remarkable progress made in human genome-wide association studies, there remains a substantial gap between statistical evidence for genetic associations and functional comprehension of the underlying mechanisms governing these associations. As a means of bridging this gap, we performed genomic analysis of blood pressure (BP) and related phenotypes in spontaneously hypertensive rats (SHR) and their substrain, stroke-prone SHR (SHRSP), both of which are unique genetic models of severe hypertension and cardiovascular complications. By integrating whole-genome sequencing, transcriptome profiling, genome-wide linkage scans (maximum n=1415), fine congenic mapping (maximum n=8704), pharmacological intervention and comparative analysis with transcriptome-wide association study (TWAS) datasets, we searched causal genes and causal pathways for the tested traits. The overall results validated the polygenic architecture of elevated BP compared with a non-hypertensive control strain, Wistar Kyoto rats (WKY); e.g. inter-strain BP differences between SHRSP and WKY could be largely explained by an aggregate of BP changes in seven SHRSP-derived consomic strains. We identified 26 potential target genes, including rat homologs of human TWAS loci, for the tested traits. In this study, we re-discovered 18 genes that had previously been determined to contribute to hypertension or cardiovascular phenotypes. Notably, five of these genes belong to the kallikrein-kinin/renin-angiotensin systems (KKS/RAS), in which the most prominent differential expression between hypertensive and non-hypertensive alleles could be detected in rat Klk1 paralogs. In combination with a pharmacological intervention, we provide in vivo experimental evidence supporting the presence of key disease pathways, such as KKS/RAS, in a rat polygenic hypertension model.

Critical Role of SREBP-1c Large-VLDL Pathway in Environment-Induced Hypertriglyceridemia of Apo AV Deficiency.

Objective- APOA5 variants are strongly associated with hypertriglyceridemia, as well as increased risks of cardiovascular disease and acute pancreatitis. Hypertriglyceridemia in apo AV dysfunction often aggravates by environmental factors such as high-carbohydrate diets or aging. To date, the molecular mechanisms by which these environmental factors induce hypertriglyceridemia are poorly defined, leaving the high-risk hypertriglyceridemia condition undertreated. Previously, we reported that LXR (liver X receptor)-SREBP (sterol regulatory element-binding protein)-1c pathway regulates large-VLDL (very low-density lipoprotein) production induced by LXR agonist. However, the pathophysiological relevance of the finding remains unknown. Approach and Results- Here, we reconstitute the environment-induced hypertriglyceridemia phenotype of human APOA5 deficiency in Apoa5-/- mice and delineate the role of SREBP-1c in vivo by generating Apoa5-/- ;Srebp-1c-/- mice. The Apoa5-/- mice, which showed moderate hypertriglyceridemia on a chow diet, developed severe hypertriglyceridemia on high-carbohydrate feeding or aging as seen in patients with human apo AV deficiency. These responses were nearly completely abolished in the Apoa5-/- ;Srebp-1c-/- mice. Further mechanistic studies revealed that in response to these environmental factors, SREBP-1c was activated to increase triglyceride synthesis and to permit the incorporation of triglyceride into abnormally large-VLDL particles, which require apo AV for efficient clearance. Conclusions- Severe hypertriglyceridemia develops only when genetic factors (apo AV deficiency) and environmental effects (SREBP-1c activation) coexist. We demonstrate that the regulated production of large-sized VLDL particles via SREBP-1c determines plasma triglyceride levels in apo AV deficiency. Our findings explain the long-standing enigma of the late-onset hypertriglyceridemia phenotype of apo AV deficiency and suggest a new approach to treat hypertriglyceridemia by targeting genes that mediate environmental effects.

KLF15 Enables Rapid Switching between Lipogenesis and Gluconeogenesis during Fasting.

Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link between glucose and lipid metabolism and have therapeutic implications for the treatment of hyperlipidemia.

[Genetic basis of primary hypertriglyceridemia].

Rare monogenic and severe hypertriglyceridemia usually results from genetic abnormalities that lead to severely reduced activity of lipoprotein lipase (LPL), a crucial player in the hydrolysis of triglyceride(TG)-rich lipoproteins. These include mutations in the genes for LPL, APOC2, GPIHBP1, LMF1 and APOA5. On the other hand, recent results from genome-wide association studies(GWAS) or resequencing studies have revealed that common polygenic and mild hypertriglyceridemia results from accumulation of both common as well as rare risk variants under the influence of classical environmental conditions. Interestingly, some primary hyperlipoproteinemiae appear to share a common genetic basis, providing a recent interesting and unique hypothesis. Genetic analysis also could provide novel candidates for therapeutic targets and fundamental basis of establishment of more rational genetic risk scoring for hypertriglyceridemia.

A novel link between Slc22a18 and fat accumulation revealed by a mutation in the spontaneously hypertensive rat.

Two different strains of the spontaneously hypertensive rat (SHR) exist, either with or without a Cd36 mutation. In the F2 population derived from a cross between these two SHR strains, the mutant Cd36 allele was tightly linked to differences in metabolic phenotypes but not to those in fat pad weight. This suggested the existence of another crucial mutation related to adiposity. Linkage analysis of this F2 population showed a significant linkage between the rat chromosome 1 region (D1Rat240-D1Wox28) and fat pad weight. By integrating both positional and expression information, we identified a donor splice site mutation in the gene for solute carrier family 22 member 18 (Slc22a18) in SHR with reduced fat pad weight. This mutation was located at the linkage peak with a maximum logarithm of odds score of 7.7 and caused skipping of the whole exon 9 that results in a complete loss of a whole membrane-spanning region of the rat Slc22a18 protein. Slc22a18 mRNA was abundantly expressed in isolated adipocytes and in a differentiation-dependent manner in 3T3-L1 cells. Knockdown of the Slc22a18 mRNA via infection of adenoviral vectors markedly inhibited both triglyceride accumulation and adipocyte differentiation in 3T3-L1 cells. By contrast, overexpression of the Slc22a18 mRNA had the opposite effects. These results reveal a novel link between Slc22a18 and fat accumulation and suggest that this gene could be a new therapeutic target in obesity.

Apolipoprotein C-II deficiency with no rare variant in the APOC2 gene.

AIM: Familial apolipoprotein C-II (apoC-II) deficiency is a rare autosomal recessive disorder with marked hypertriglyceridemia resulting from impaired activation of lipoprotein lipase. In most cases of apoC-II deficiency, causative mutations have been found in the protein-coding region of APOC2; however, several atypical cases of apoC-II deficiency were reported to have markedly reduced, but detectable levels of plasma apoC-II protein (hereafter referred to as hypoapoC-II), which resulted from decreased promoter activity or improper splicing of apoC-II mRNA due to homozygous mutations in APOC2. Here we aim to dissect the molecular bases of a new case of hypoapoC-II. METHODS: We performed detailed biochemical/genetic analyses of our new case of hypoapoC-II, manifesting severe hypertriglyceridemia (plasma triglycerides, 3235 mg·dL(-1)) with markedly reduced levels of plasma apoC-II (0.6 mg·dL(-1)). RESULTS: We took advantage of a monocyte/macrophage culture system to prove that transcription of apoC-II mRNA was decreased in the patient's cells, which is compatible with the reported features of hypoapoC-II. Concomitantly, transcriptional activity of the minigene reporter construct of the patient's APOC2 gene was decreased; however, no rare variant was detected in the patient's APOC2 gene. Fifty single nucleotide variants were detected in the patient's APOC2, but all were common variants (allele frequencies ＞35%) that are supposedly not causative. CONCLUSIONS: A case of apoC-II deficiency was found that is phenotypically identical to hypoapoC-II but with no causative mutations in APOC2, implying that other genes regulate apoC-II levels. The clinical entity of hypoapoC-II is discussed.

Background to discuss guidelines for control of plasma HDL-cholesterol in Japan.

A decrease in high density lipoprotein-cholesterol (HDL-C) is a strong risk factor for atherosclerotic disorders in Japan, probably more important than an increase in low density lipoprotein-cholesterol (LDL-C). While there are rational grounds for the argument that elevation of HDL-C leads to decreased risk, there has as yet been no direct evidence of such an effect. If elevation of HDL-C decreases the risk, this effect is expected throughout the normal range of HDL-C or perhaps even higher than that. Simulation based on epidemiological data indicated that it may eventually reduce the incidence of ischemic heart disease by 60-70% in Japan. In the risk management guideline, "low" HDL-C is presently defined as 40 mg/dL or below. While there is no evidence that strongly urges a change in this definition, the results of epidemiological studies support "The higher the HDL-C level, the lower the risk,"even in the "normal range". Elevation of the HDL-C level may reduce the risk, probably at least up to 70 mg/dL; however, there are no supportive data for this effect still being obtained over 80 mg/dL. Patients with homozygous CETP deficiency should be followed-up while controlling other risk factors, so as not to dismiss the possibility of a risk increase with an extremely elevated HDL-C level.

Management of type IIb dyslipidemia.

Although the Japan Atherosclerosis Society guideline for the diagnosis and prevention of atherosclerosis cardiovascular diseases for the Japanese population provides targets for low-density lipoprotein (LDL) cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol to prevent cardiovascular disease in patients with dyslipidemia, there is no guideline specifically targeting the treatment of type IIb dyslipidemia, which is one of the most common types of dyslipidemia, along with type IIa and type IV dyslipidemia. Type IIb dyslipidemia is important because it sometimes accompanies atherogenic lipid profiles, such as small, dense LDL, remnants, low HDL cholesterolemia. It is also associated with type 2 diabetes mellitus, metabolic syndrome, and chronic kidney disease (CKD), and most patients with familial combined hyperlipidemia (FCHL) show this phenotype; therefore, it is assumed that patients with type IIb dyslipidemia have a high risk for cardiovascular disease. Thus, the management of type IIb dyslipidemia is very important for the prevention of cardiovascular disease, so we have attempted to provide a guideline for the management of type IIb dyslipidemia.

Diagnosis and management of type I and type V hyperlipoproteinemia.

Both type I and type V hyperlipoproteinemia are characterized by severe hypertriglyceridemia due to an increase in chylomicrons. Type I hyperlipoproteinemia is caused by a decisive abnormality of the lipoprotein lipase (LPL)- apolipoprotein C-II system, whereas the cause of type V hyperlipoproteinemia is more complicated and more closely related to acquired environmental factors. Since the relationship of hypertriglyceridemia with atherosclerosis is not as clear as that of hypercholesterolemia, and since type I and V hyperlipoproteinemia are relatively rare, few guidelines for their diagnosis and treatment have been established; however, type I and V hyperlipoproteinemia are clinically important as underlying disorders of acute pancreatitis, and appropriate management is necessary to prevent or treat such complications. Against such a background, here we propose guidelines primarily concerning the diagnosis and management of type I and V hyperlipoproteinemia in Japanese.

[Metabolic syndrome].

Metabolic syndrome is a clinical condition characterized by the clustering of multiple cardiovascular risk factors and predisposing people to type 2 diabetes and its macrovascular complications. Although various diagnostic criteria have been proposed so far, none of those criteria have successfully provided great positive predictive value for cardiovascular diseases, indicating that its diagnosis is of limited clinical importance for macrovascular risk stratification in diabetic patients. Nevertheless, metabolic syndrome remains to be an important clinical entity for overcoming the diabetic macrovascular complications, because it provides basis not only for the incidence of type 2 diabetes but also for rational and comprehensive management of multiple cardiovascular risks in diabetic patients.

Polyunsaturated fatty acids selectively suppress sterol regulatory element-binding protein-1 through proteolytic processing and autoloop regulatory circuit.

Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBP-binding element on the promoter ("autoloop regulatory circuit"), although liver X receptor, an activator for Srebf1c transcription, is not involved in this regulation by PUFA. The mechanisms for PUFA suppression of SREBP-1 confirm that the autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.

Protein kinase Cbeta mediates hepatic induction of sterol-regulatory element binding protein-1c by insulin.

Sterol-regulatory element binding protein-1c (SREBP-1c) is a transcription factor that controls lipogenesis in the liver. Hepatic SREBP-1c is nutritionally regulated, and its sustained activation causes hepatic steatosis and insulin resistance. Although regulation of SREBP-1c is known to occur at the transcriptional level, the precise mechanism by which insulin signaling activates SREBP-1c promoter remains to be elucidated. Here we show that protein kinase C beta (PKCbeta) is a key mediator of insulin-mediated activation of hepatic SREBP-1c and its target lipogenic genes. Activation of SREBP-1c in the liver of refed mice was suppressed by either adenoviral RNAi-mediated knockdown or dietary administration of a specific inhibitor of protein kinase C beta. The effect of PKCbeta inhibition was cancelled in insulin depletion by streptozotocin (STZ) treatment of mice. Promoter analysis indicated that PKCbeta activates SREBP-1c promoter through replacement of Sp3 by Sp1 for binding to the GC box in the sterol regulatory element (SRE) complex, a key cis-element of SREBP-1c promoter. Knockdown of Sp proteins demonstrated that Sp3 and Sp1 play reciprocally negative and positive roles in nutritional regulation of SREBP-1c, respectively. This new understanding of PKCbeta involvement in nutritional regulation of SREBP-1c activation provides a new aspect of PKCbeta inhibition as a potential therapeutic target for diabetic complications.

Hormone-sensitive lipase deficiency suppresses insulin secretion from pancreatic islets of Lep ob/ob mice.

It has long been a matter of debate whether the hormone-sensitive lipase (HSL)-mediated lipolysis in pancreatic beta-cells can affect insulin secretion through the alteration of lipotoxicity. We generated mice lacking both leptin and HSL Lep(ob/ob)/HSL(-/-) and explored the role of HSL in pancreatic beta-cells in the setting of obesity. Lep(ob/ob)/HSL(-/-) developed elevated blood glucose levels and reduced plasma insulin levels compared with Lep(ob/ob)/HSL(+/+) in a fed state, while the deficiency of HSL did not affect glucose homeostasis in Lep(+/+) background. The deficiency of HSL exacerbated the accumulation of triglycerides in Lep(ob/ob) islets, leading to reduced glucose-stimulated insulin secretion. The deficiency of HSL also diminished the islet mass in Lep(ob/ob) mice due to decreased cell proliferation. In conclusion, HSL affects insulin secretary capacity especially in the setting of obesity.

[Monogenic disorders of human obesity].

Although the recent pandemic of obesity is apparently driven by environmental factors, the influence of genetic factors has also increasingly been recognized. Monogenic inheritance of obesity has been documented in several human disorders and some of their etiological basis has been elucidated at the molecular level. The frequency of these monogenic diseases is generally very rare, but their elucidation gives an important insight into our understanding of the molecular basis of the more common form of human obesity. In fact, the identification of rare mutations in the genes for ligands and receptors of the leptin-melanocortin pathway established that the pathway is physiologically essential for normal homeostasis of food intake and energy expenditure.