Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

An epididymis-specific secretory protein Clpsl2 critically regulates sperm motility, acrosomal integrity, and male fertility.

The epididymis performs an important role in the maturation of spermatozoa including their acquisition of progressive motility and fertilizing ability. However, the molecular mechanisms that govern these maturational events are still poorly defined. Here we report that Clpsl2, a novel colipase homology, is exclusively expressed in the caupt epididymis and conserved in mammalian. Clpsl2 was secreted into the lumen and covered the acrosome region and principal piece of spermatozoa tail. And during epididymal transit, the binding rate between Clpsl2 protein and the spermatozoa gradually decreased. Though Clpsl2 had the highest identifies with pancreatic colipase (Clps), Clpsl2 lacked those conserved amino acids in pancreatic Clps that interacting with lipase, correspondingly, the recombinant Clpsl2 protein did not possess the Clps function such as promoting the hydrolysis of lipase to its substrate glycerine trioleate. However, sequence analysis showed that Clpsl2 has the potency to bind with lipid. Knockdown expression of Clpsl2 by lentivirus-mediated RNAi in vivo caused an attenuation of spermatozoa motility, a suppressed acrosomal reaction, a decrease of cauda spermatozoa number, and subfertility. This study identified a novel and conserved molecule, Clpsl2, was specifically expressed in epididymis and involved in the regulation of spermatozoa motility, acrosomal integrity, and male fertility.

Lid dynamics of porcine pancreatic lipase in non-aqueous solvents.

BACKGROUND: Understanding the dynamics of enzymes in organic solvents has wider implications on their industrial applications. Pancreatic lipases, which show activity in their lid open-state, demonstrate enhanced activity in organic solvents at higher temperatures. However, the lid dynamics of pancreatic lipases in non-aqueous environment is yet to be clearly understood. METHODS: Dynamics of porcine pancreatic lipase (PPL) in open and closed conformations was followed in ethanol, toluene, and octanol using molecular simulation methods. In silico double mutant D250V and E254L of PPL (PPLmut-Cl) was created and its lid opening dynamics in water and in octanol was analyzed. RESULTS: PPL showed increase in solvent accessible surface area and decrease in packing density as the polarity of the surrounded solvent decreased. Breaking the interactions between D250-Y115, and D250-E254 in PPLmut-Cl directed the lid to attain open-state conformation. Major energy barriers during the lid movement in water and in octanol were identified. Also, the trajectories of lid movement were found to be different in these solvents. CONCLUSIONS: Only the double mutant at higher temperature showed lid opening movement suggesting the essential role of the three residues in holding the lid in closed conformation. The lid opening dynamics was faster in octanol than water suggesting that non-polar solvents favor open conformation of the lid. GENERAL SIGNIFICANCE: This study identifies important interactions between the lid and the residues in domain 1 which possibly keeps the lid in closed conformation. Also, it explains the rearrangements of residue-residue interactions during lid opening movement in water and in octanol.

Lid closure dynamics of porcine pancreatic lipase in aqueous solution.

BACKGROUND: Pancreatic lipases hydrolyze fatty acids in dietary pathway. The activity of porcine pancreatic lipase (PPL) is controlled by lid domain along with a coenzyme, colipase. The active open-state conformation of the protein could be induced by detergents or bile salts which would be further stabilized by binding of colipase. In the absence of these interactions, the lid preferably attains a closed conformation in water. METHODS: Molecular dynamic simulation was used to monitor the lid movement of PPL in open and closed conformations in water. Free energy surface was constructed from the simulation. Energy barriers and major structural changes during lid opening were evaluated. RESULTS: The lid closure of PPL in water from its open conformation might be initiated by columbic interactions which initially move the lid away from domain 1. This is followed by major dihedral changes on the lid residues which alter the trajectory of motion. The lid then swirls back towards domain 1 to attain closed conformation. This is accompanied with conformational changes around ß5- and ß9-loops as well. However, PPL in closed conformation shows only the domain movements and the lid remains in its closed conformation. CONCLUSIONS: PPL in closed conformation is stable in water and the open conformation is driven towards closed state. The lid follows a swirling trajectory during the closure. GENERAL SIGNIFICANCE: Conformational state of the lid regulates the activity and substrate specificity of PPL. Hence, it is essential to understand the lid dynamics and the role of specific amino acid residues involved.

Rapid analysis of colipase gene variants by multicapillary electrophoresis.

Despite of the fact that the Human Genome Project was completed more than a decade ago, identification of the genetic background of polygenic diseases is still challenging. Several somewhat different approaches are available to investigate inheritable factors of complex phenotypes, all require, however efficient, high-throughput techniques for SNP genotyping. In this paper, we report a robust and reliable multiplex PCR-RFLP for genotype and haplotype analysis of six SNPs (rs41270082, rs3748051, rs142027015, rs3748048, rs73404011, and rs72925892) of the colipase (CLPS) gene. A multicapillary (12 capillaries) electrophoresis unit was used for high throughput and sensitive analysis of the digestion fragments. A Microsoft Excel-based spreadsheet was designed for the flexible visualization and evaluation of the electrophoretic separations, which is readily adaptable for any kind of electrophoresis application. Haplotype analysis of the two loci localized in close proximity of each other was carried out by molecular method, extended haplotypes including all five SNPs in the 5' upstream region were calculated. The techniques were applied in a case-control association study of type 2 diabetes mellitus. Although, single marker analysis did not reveal any significant association, it was observed that the rare GGCCG haplotype of the five 5' upstream region SNPs was about three times more frequent among patients compared to healthy control population. Our results demonstrated the applicability of multicapillary CGE in large-scale, high-throughput SNP analysis, and suggested that the CLPS gene polymorphisms might be considered as genetic risk factor for type 2 diabetes mellitus.

Differential regulation of pancreatic digestive enzymes during chronic high-fat diet-induced obesity in C57BL/6J mice.

Exocrine pancreatic digestive enzymes are essential for the digestion of dietary components and are regulated by them. Chronic excess dietary high fat (HF) consumption is a contributing factor of diet-induced obesity (DIO) and associated chronic diseases and requires adaptation by the pancreas. The aim of the present study was to investigate the effects of chronic HF diet feeding on exocrine pancreatic digestive enzyme transcript levels in DIO C57BL/6J mice. C57BL/6J mice were fed diets containing either 10 or 45% energy (E%) derived from fat for 12 weeks (n 10 mice per diet group). Pancreatic tissue and blood samples were collected at 0, 4 and 12 weeks. The expression of a panel of exocrine pancreatic digestive enzymes was analysed using quantitative RT-PCR and Western blot analysis. The HF (45 E%) diet-fed C57BL/6J mice developed obesity, hyperleptinaemia, hyperglycaemia and hyperinsulinaemia. The transcript levels of pancreatic lipase (PL), pancreatic lipase-related protein 2 (PLRP2) and pancreatic phospholipase A2 (PLA2) were initially elevated; however, they were down-regulated to basal control levels at week 12. The transcript levels of colipase were significantly affected by diet and time. The protein levels of PL and PLRP2 responded to HF diet feeding. The transcript levels of amylase and proteases were not significantly affected by diet and time. The transcript levels of specific lipases in hyperinsulinaemic, hyperleptinaemic and hyperglycaemic DIO C57BL/6J mice are down-regulated. However, these mice compensate for this by the post-transcriptional regulation of the levels of proteins that respond to dietary fat. This suggests a complex regulatory mechanism involved in the modulation of fat digestion.

Identification of amino acids in human colipase that mediate adsorption to lipid emulsions and mixed micelles.

The adsorption of colipase is essential for pancreatic triglyceride lipase activity and efficient dietary fat digestion. Yet, little is known about which specific amino acids in the hydrophobic surface of colipase influence adsorption. In this study, we systematically substituted alanine or tryptophan at residues implicated in adsorption of colipase to an interface. We expressed, purified recombinant colipase mutants and characterized the ability of each alanine mutant to restore activity to lipase in the presence of bile salts. The functions of L16A, Y55A, I79A and F84A colipase were most impaired with activities ranging from 20 to 60% of wild-type colipase. We next characterized the fluorescence properties of the tryptophan mutants in the absence and presence of bile-salt-oleic acid mixed micelles. We performed steady-state emission spectra to determine peak shift and I330/I350 ratio and acrylamide quenching curves to characterize the environment of the residues. The analysis supports a model of adsorption that includes residues Leu 34 and Leu 36 on the 2nd loop, Tyr 55 and Tyr 59 on the 3rd loop and Ile 75 and Ile 79 on the 4th loop. The analysis confirms that Phe 84 is not part of the adsorption surface and likely stabilizes the conformation of colipase. Contrary to the predictions of computer modeling, the results provide strong support for an essential role of Tyr 55 in colipase adsorption to mixed micelles. The results indicate that the adsorption of colipase to mixed micelles is mediated by specific residues residing in a defined surface of colipase.

The Arg92Cys colipase polymorphism impairs function and secretion by increasing protein misfolding.

Colipase is essential for efficient fat digestion. An arginine-to-cysteine polymorphism at position 92 of colipase (Arg92Cys) associates with an increased risk for developing type-2 diabetes through an undefined mechanism. To test our hypothesis that the extra cysteine increases colipase misfolding, thereby altering its intracellular trafficking and function, we expressed Cys92 colipase in HEK293T cells. Less Cys92 colipase is secreted and more is retained intracellularly in an insoluble form compared with Arg92 colipase. Nonreducing gel electrophoresis suggests the folding of secreted Cys92 colipase differs from Arg92 colipase. Cys92 colipase misfolding does not trigger the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress. The ability of secreted Cys92 colipase to stimulate pancreatic triglyceride lipase (PTL) is reduced with all substrates tested, particularly long-chain triglycerides. The reaction of Cys92 colipase with triolein and Intralipid has a much longer lag time, reflecting decreased ability to anchor PTL on those substrates. Our data predicts that humans with the Arg92Cys substitution will secrete less functional colipase into the duodenum and have less efficient fat digestion. Whether inefficient fat digestion or another property of colipase contributes to the risk for developing diabetes remains to be clarified.

Kinetic properties of mouse pancreatic lipase-related protein-2 suggest the mouse may not model human fat digestion.

Genetically engineered mice have been employed to understand the role of lipases in dietary fat digestion with the expectation that the results can be extrapolated to humans. However, little is known about the properties of mouse pancreatic triglyceride lipase (mPTL) and pancreatic lipase-related protein-2 (mPLRP2). In this study, both lipases were expressed in Pichia Pastoris GS115, purified to near homogeneity, and their properties were characterized. Mouse PTL displayed the kinetics typical of PTL from other species. Like mPTL, mPLRP2 exhibited strong activity against various triglycerides. In contrast to mPTL, mPLRP2 was not inhibited by increasing bile salt concentration. Colipase stimulated mPLRP2 activity 2- to 4-fold. Additionally, mPTL absolutely required colipase for absorption to a lipid interface, whereas mPLRP2 absorbed fully without colipase. mPLRP2 had full activity in the presence of BSA, whereas BSA completely inhibited mPTL unless colipase was present. All of these properties of mPLRP2 differ from the properties of human PLRP2 (hPLRP2). Furthermore, mPLRP2 appears capable of compensating for mPTL deficiency. These findings suggest that the molecular mechanisms of dietary fat digestion may be different in humans and mice. Thus, extrapolation of dietary fat digestion in mice to humans should be done with care.

The mPlrp2 and mClps genes are involved in the hydrolysis of retinyl esters in the mouse liver.

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5'-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.

Enterostatin deficiency increases serum cholesterol but does not influence growth and food intake in mice.

A pentapeptide released from procolipase, enterostatin, selectively attenuates dietary fat intake when administered peripherally or centrally. Enterostatin may act through the afferent vagus nerve and in the hypothalamus and amygdala, primarily in the central nucleus of the amygdala. To investigate the physiological role of endogenous enterostatin, we created an enterostatin-deficient, colipase-sufficient (Ent(-/-)) mouse. Ent(-/-) mice are viable, normally active, and fertile. They exhibit normal growth on low-fat and high-fat diets. Furthermore, Ent(-/-) mice develop diet-induced obesity, as do Ent(+/+) mice, and have normal responses to a two-macronutrient choice diet and to a switch from a high-fat to a low-fat diet. Levels of total serum (P = 0.004) and non-HDL (P

Procolipase gene: no association with early-onset obesity or fat intake.

BACKGROUND: Several lines of evidence in volvement of procolipase (CLPS) or its derivative enterostatin in dietary fat absorption, regulation of fat intake, and body weight in rodents. We explored the relationship between genetic variation in CLPS, early-onset obesity and fat intake in humans. METHODS: We screened the CLPS in 93 extremely obese children and adolescents and 96 underweight young adults for sequence variations and genotyped single nucleotide polymorphisms (SNPs) in extremely obese children and adolescents, healthy normal-and underweight young adults and obesity trios. Case-control and family-based association analyses were performed. RESULTS: Five sequence variations were identified: two non-synonymous SNPs: rs2766597 (Leu8Pro), rs41270082 (Arg109Cys); one SNP in the 5'UTR: rs3748050; one intronic SNP: rs3748051; and one infrequent novel non-synonymous variant: Arg55His. For rs2766597, rs3748050, and rs3748051 we obtained no evidence for an association with obesity in the case-control comparison. For rs41270082 there was a trend for association which could not be substantiated in the family-based association analysis. Additionally, we found no association in subgroup analyses pertaining to the extremely obese children and adolescents in the lowest and highest quartile of the percentage of energy consumed as fat. CONCLUSIONS: We found no evidence for an association of CLPS SNPs rs2766597, rs41270082, rs3748050, and rs3748051 with obesity or percentage of dietary fat intake.

Genetic variability of procolipase associates with altered insulin secretion in non-diabetic Caucasians.

AIMS: Procolipase (CLPS) is secreted from the exocrine pancreas into the gastrointestinal tract and becomes proteolytically cleaved into colipase and the pentapeptide enterostatin. While colipase is an indispensable cofactor for pancreatic lipase, enterostatin acts as a hormone that inhibits insulin secretion and confers satiety signals to the brain, thereby restricting further food intake in animal models. As both high fat diet and obesity contribute to insulin resistance, we investigated whether genetic variability of CLPS associates with metabolic traits in non-diabetic humans at diabetes risk. METHODS: Tagging single nucleotide polymorphisms (SNPs) in the human CLPS locus on chr6p21.1 were selected using HapMap data. 498 humans, phenotyped for different glucose and lipid metabolic traits, were genotyped by bidirectional sequencing and multivariate linear regression analyses were undertaken. RESULTS: 2 tagging SNPs (rs3748050 in the Kozak sequence: A/G and rs3748051 in intron 1: A/G), covering 100% of CLPS variability including 8 kb of its promoter, were genotyped for association analyses. The minor alleles of both tagging SNPs associated significantly with a reduced insulin secretion (-20.2%, both SNPs) in various estimation models derived from the oral glucose tolerance test (OGTT; rs3748050/51: 30 min C-peptide levels: p=0.001/0.01, insulinogenic index: p=0.02/0.02, AUC C-peptide/AUC glucose: p=0.01/0.01) after adjustment for relevant covariates. No significant associations with fasting total cholesterol (c), HDL-c, LDL-c, triglycerides and free fatty acids were found (all p > 0.11). CONCLUSIONS: CLPS genetic variability associates with insulin secretory function in non-diabetic humans and may represent a novel candidate gene for development of type 2 diabetes.

The environmental light influences the circulatory levels of retinoic acid and associates with hepatic lipid metabolism.

Environmental light is involved in the regulation of photochemical reaction in mouse retina. It remains unclear whether light-mediated increase in all-trans retinoic acid (ATRA) synthesis in retina will result in altering the circulatory levels of ATRA and regulating downstream gene expression and physiological function. Here we showed circulatory levels of ATRA decreased in mice under constant darkness and elevated by light exposure. Fat gene pancreatic lipase-related protein 2 (mPlrp2) and its partner procolipase (mClps), but not hepatic lipase (mHl), activated in livers for responding to lack of light illuminating. Light-triggered alterations in circulatory ATRA levels regulated ecto-5'-nucleotidase gene expression by retinoic acid receptor retinoic acid receptor-alpha and modulated 5'-AMP levels in blood and were associated with mPlrp2 and mClps expression in the livers. Mice deficient in adenosine receptors displayed mPlrp2 and mClps expression in livers under 12-h light, 12-h dark cycles. Caffeine blocked adenosine receptors and induced hepatic mPlrp2 and mClps expression in wild-type mice. Mice activated in hepatic mPlrp2 and mClps expression lowered hepatic and serum lipid levels and markedly elevated circulatory levels of all-trans retinol. Our results suggest environmental light influence hepatic lipid homeostasis by light-modulated retinoic acid signaling associated with mPlrp2 and mClps gene expression in livers.

Production of recombinant porcine colipase secreted by Pichia pastoris and its application to improve dietary fat digestion and growth of postweaning piglets.

Recombinant porcine pancreatic colipase (pCoL) was produced in the methylotrophic yeast Pichia pastoris. A synthetic yeast secretion cassette was constructed with the constitutive promoter of the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene and the yeast alpha-mating factor signal peptide. The pCoL cDNA corresponding in the coding sequence, excluding a 16-amino acid segment of the native signal sequence, was cloned into the pGAPZalphaB vector and integrated into the genome of P. pastoris. Yeast transformants were cultured and bioactive pCoL protein was detected in the supernatant at a high-level of 126.8 mg/L after 3 days of culture. The transformed yeast containing the highest recombinant colipase level (pCoL yeast) and native yeast GS 115 not containing pCoL (non-pCoL yeast, as a control group) were separately cultured and the supernatants were adsorbed by dried skim milk. In an animal trial, two concentrations of colipase activity (0 vs. 5,000 U/kg in the diet) were blended with the pig corn-soybean basal diet and fed to weaned piglets for 4 weeks. The pCoL-administrated test group gained significantly more weight than piglets in the control group when measured at Day 15 (11.84 +/- 0.70 vs. 10.59 +/- 0.39 kg, P < 0.05), Day 22 (15.84 +/- 0.95 vs. 14.32 +/- 0.59 kg, P < 0.01), and Day 28 (20.19 +/- 1.47 vs. 18.54 +/- 0.92 kg, P < 0.01) after weaning. The blood triglyceride (TG) concentrations were significantly increased in the experimental test group that received recombinant colipase on the 28th day of postweaning when compared with that of the control group (32.50 vs. 16.37 mg/dL; P < 0.0001). These experimental data suggest that the use of recombinant porcine colipase as a dietary supplement provides an alternative approach for improving fat digestion and enhancing growth in postweaning piglets.

Consequences of metabolic challenges on hypothalamic colipase and PLRP2 mRNA in rats.

The hypothalamus is the main appetite-regulating center in the brain receiving peripheral signals regarding the metabolic status of the body. Pancreatic procolipase has recently been identified in rat hypothalamus. Procolipase is known mainly for its actions in the intestine where it is cleaved to colipase, an enzyme required for the maintenance of pancreatic lipase activity, and enterostatin, a peptide involved in appetite regulation through the gut-brain axis. Colipase is able to increase the activity of pancreatic lipase-related protein-2 (PLRP2), a lipase also expressed in extra-pancreatic tissues. This study was performed to elucidate if PLRP2, in addition to colipase, is expressed in the hypothalamus and if the mRNAs of colipase and PLRP2 respond to metabolic challenges such as fasting, high-fat feeding or feeding sugar solutions. RNA from rat hypothalamus was extracted and subjected to RT-PCR. For quantitative mRNA analysis of hypothalamic tissue from the different metabolic situations real-time RT-PCR was used. We found PLRP2 and colipase mRNA to be expressed in the hypothalamus. An overnight fast resulted in down-regulated colipase (3-fold) and PLRP2 (7-fold) mRNA compared to freely fed rats. Conversely, high-fat feeding resulted in up-regulated colipase and PLRP2 mRNA (1.3-fold and 1.8-fold, respectively) compared to standard chow-fed rats. A similar up-regulation in mRNA expression was observed after offering sugar solutions. In conclusion, PLRP2 mRNA is expressed in the rat hypothalamus and both procolipase and PLRP-2 mRNA are down-regulated during fasting and up-regulated during conditions of metabolic excess, suggesting an involvement in signaling energy availability.

A polymorphism in the gene encoding procolipase produces a colipase, Arg92Cys, with decreased function against long-chain triglycerides.

Type 2 diabetes mellitus is a multifactorial and polygenic disorder with increasing prevalence. Recently, a polymorphism in the gene encoding procolipase, a cysteine for arginine substitution at position 92, was associated with type 2 diabetes in two human populations. Because procolipase plays a critical role in dietary fat metabolism, polymorphisms that affect the function of procolipase could influence the development of type 2 diabetes. We hypothesized that the Arg92Cys polymorphism has functional consequences. To test our hypothesis, we expressed recombinant cysteine 92 (Cys92) procolipase in a yeast expression system and compared the function and stability of purified Cys92 with that of the more common arginine 92 (Arg92) procolipase. Cys92 fully restored the activity of bile-salt inhibited lipase with short- and medium-chain triglycerides but only had 50% of Arg92 function with long-chain triglycerides. After storage at 4 degrees C, Cys92 lost the ability to restore pancreatic triglyceride lipase activity with medium- and long-chain triglycerides. The loss of function correlated with the inability of Cys92 to anchor lipase on an emulsion surface and oxidation of the cysteine. No detectable degradation or intramolecular disulfide formation occurred in Cys92 after storage. Our findings demonstrate that the Arg92Cys polymorphism decreases the function of Cys92 colipase. This change may contribute to the development of type 2 diabetes.

Candidate gene association study of type 2 diabetes in a nested case-control study of the EPIC-Potsdam cohort - role of fat assimilation.

To search for common variants etiological for type 2 diabetes, we screened 15 genes involved in fat assimilation for sequence variants. Approximately 55 kb in promoter and coding regions, and intron/splice sites were sequenced by cycle sequencing. In the set of 15 genes, 71 single nucleotide polymorphisms (SNPs) were detected. 33 SNPs were presumed to be functionally significant and were genotyped in 192 incident type 2 diabetes subjects and 384 matched controls from the European Prospective Investigation into Cancer and Nutrition-Potsdam cohort. A total of 27 SNPs out of 15 genes showed no statistical association with type 2 diabetes in our study. Six SNPs demonstrated nominal association with type 2 diabetes, with the most significant marker (FABP6 Thr79Met) having an adjusted odds ratio of 0.45 (95% CI 0.22-0.92) in homozygous Met allele carriers. Evidence for an association with disease status was also found for a novel Arg109Cys (g.2129C > T) variant of colipase, 5'UTR (rs2084202) and Met71Val (rs8192506) variants of diazepam-binding inhibitor, Arg298His (rs13283456) of PTGES2, and a novel promoter variant (g.-1324G > A) of SLC27A5. The results presented here provide preliminary evidence for the association of common variants in genes involved in fat assimilation with the genetic susceptibility of type 2 diabetes. However, they definitely need further verification.

Constant darkness is a mammalian biological signal.

Environmental light is a potent modulator of mammalian circadian rhythm and expression of clock genes. Constant darkness (DD) is regarded as a "free-running" circadian state. In nature, hibernating mammals encounter constant darkness (DD) seasonally. Circadian expression of enzymes involved in fat catabolism, procolipase (CLP) and pancreatic-lipase-related protein 2 (PLRP2), were identified in many peripheral organs of mice during DD but not during regular light/dark (LD) cycles. Circulating 5'-adenosine monophosphate (5'-AMP) was associated with DD-activated gene expression. Synthetic 5'-AMP, when injected into LD mice, activated procolipase expression in their peripheral organs and the animals become severely hypothermic, both key features of hibernating mammals. These findings identified a circadian-regulated metabolic cycle in mammals that may be associated with hypometabolic behaviors such as hibernation and torpor.