Patent Application: US-201515500845-A

Abstract:
the disclosure provides methods of preventing or treating metabolic syndrome in a subject by administering an effective amount of an inhibitor of laminin α4 expression , laminin α4 activity , or both .

Description:
in the experiments disclosed herein , mice with a null mutation of the laminin α4 gene ( lama4 −/− ) were used to examine a potential role for α4 chain laminins in adipose tissue expansion and function . weight gain , adipose function and adipose structure were examined in lama4 −/− mice and compared to control animals . the lama4 −/− mice were found to be resistant to age - related and diet - induced obesity , and exhibited a depot - specific change in adipose structure , volume and function . differentiated adipocytes are surrounded by a thin bm layer . ecm in general , and bm specifically , have been shown to regulate cell behavior in a number of tissues and organs [ 3 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ]. however , there is little knowledge of its role in adipocyte behavior . laminins are assembled at the cell surface as one component of the basement membrane . cell receptors maintain the laminins at the cell surface , mediate basement membrane assembly and regulate intracellular signals . laminin - cell interactions are primarily dependent on the c - terminus domain of the specific laminin a chain . the laminins interact with integrins , dystroglycan , or sulfated glycolipids on the cell surface . integrins are heterodimeric cell surface receptors consisting of α and β subunits ( not to be confused with the α and β chains of laminin ). the α4 chain of laminin has been shown to bind to integrins α6β1 and α7β1 in a number of cells . in endothelial cells , laminin α4 signals through the β1 integrin . altering interactions between laminin α4 and integrins , dystroglycan , or sulfated glycolipids provides an indirect approach to modulate signaling and enhance beiging or adipose metabolic function . in the studies disclosed herein , mice completely lacking a specific bm protein , laminin α4 , were used to investigate its role in adipose tissue . using these mice , a profound influence of laminin α4 on the expansion and function of adipose tissue was found . lama4 −/− mice were found to gain weight at a much slower rate than control mice . this occurred during normal aging and was more pronounced on a high fat diet . in fact , lama4 −/− mice did not exhibit any differences in weight whether on normal chow or a high fat diet . differences from wild type animals did not result from reduced food intake , and a reduction in adipose was observed both grossly and via ct scans in the mice deficient in laminin α4 . while these results clearly showed that lama4 −/− mice have reduced adiposity , it was initially not clear if this resulted in normal or impaired adipose function . under both normal and high fat diets , livers in the lama4 −/− mice exhibited little steatosis , suggesting that the decreased body fat did not represent a form of lipodystrophy . serum profiles of the lama4 −/− mice on normal chow did not indicate severe metabolic defects , as there were no significant changes in the levels of glucose , free fatty acids , triglycerides or cholesterol . leptin levels were lower in the knockout mice , which was expected because levels are proportional to adipose tissue mass . a high - fat diet resulted in elevated levels of insulin and leptin in control and knockout animals compared to standard diet . the same parameters were also elevated in the lama4 −/− mice fed the high - fat diet . laminin α4 is present around cells in the kidney , vasculature and muscle and has been shown to regulate different cell behaviors [ 22 - 31 ]. in order to further evaluate the specific role of laminin α4 , adipocytes from these tissues were isolated and their metabolic function was analyzed . surprisingly , decreased laminin α4 was found to lead to impaired adipocyte lipogenesis . insulin - stimulated lipogenesis resulted in increased lipogenesis in adipocytes from both lama4 −/− and control animals indicating that insulin responsiveness appears to be intact , but basal lipogenesis levels were lower in lama4 −/− mice . interestingly , this difference was depot - specific . adipocytes isolated from epididymal adipose exhibited impaired lipogenesis compared to controls while the subcutaneous adipose depot had similar lipogenesis to control . this depot - specific impairment is further supported by the fact that epididymal adipose depots in lama4 −/− mice are significantly smaller in volume than control animals at about 3 . 5 months . this difference was observed prior to any measurable differences in total weight . while the total amount of epididymal adipose in lama4 −/− mice was lower than controls , the cells from lama4 −/− mice were larger than cells from control mice . the subcutaneous depots were similar in both total volume and cell size in comparisons between lama4 −/− and control mice . overall , these results indicate an impaired function in adipose tissue from mice lacking laminin α4 and this impairment is manifested primarily in the epididymal depot . the experimental studies disclosed herein showed a profound role for laminin α4 on adipose expansion and function . the mechanism underlying this influence , however , has been unclear . there is very little knowledge of the role of the ecm on adipocyte behavior . cell culture and tissue engineering studies have shown that ecm substrata influence adipocyte behavior . in 2d culture “ laminin ” was found to be more potent than type iv collagen , fibronectin or type i collagen at promoting differentiation of preadipocytes [ 32 ]. this study did not identify which specific laminin isoform was used , but it is likely that this commercial laminin was purified from a mouse soft tissue tumor that has been shown to contain laminin 111 ( α1 : β1 : γ1 ). studies with laminin α4 are less common , in part because it only recently became available commercially . preadipocytes have been shown to produce ln - 411 ( α4 : β1 : γ1 ) during induction to an adipocyte phenotype [ 14 ] and ecm mixtures isolated from adipose tissue that are rich in laminin α4 promote greater adipogenesis than ecm from other tissues [ 33 , 34 ]. α4 chain laminin could directly regulate adipocyte function through modulation of cell receptor signaling . laminins containing the α4 chain could also influence adipocyte behavior based on indirect effects on the local cell microenvironment . the bm structure appeared thicker in immunohistochemical stains and previous studies have shown that the vascular bm in lama4 −/− mice is altered structurally [ 15 ]. this structural change could reflect altered mechanical properties that may influence adipocyte function [ 35 ]; shoham , 2012 , mechanotransduction in adipocytes }. in addition , ln - 411 , which contains the α4 chain , has a chondroitin sulfate chain [ 36 , 37 ] which may function to sequester growth factors in the vicinity of a cell . this contributes to the regulation of growth factor availability and signaling near cell surface receptors [ 38 ]. alterations in the laminin composition could alter the ability of the bm to bind growth factors and control the local concentration of regulatory factors . as supported by the following examples , the disclosure establishes that lama4 −/− mice exhibited reduced weight gain in response to both age and high - fat diet . the mice had adipose tissue mass and altered function in a depot - specific manner . in particular , epididymal adipose tissue exhibited decreased mass and altered lipogenesis in lama4 −/− mice , but no differences were observed in the subcutaneous depot . the results indicate that : ( 1 ) impaired lipogenesis leads to diminished fat mass in lama4 −/− mice ; ( 2 ) alterations in lipogenesis are adipose tissue depot - specific ; and ( 3 ) specific ecm components dramatically influence adipose tissue function . the working examples that follow include example 1 , which provides the materials and methods for the experiments conducted in the ensuing examples , which collectively provide experimental results supporting the claimed subject matter . example 2 shows the adipose composition of mice genetically deficient in laminin α4 ; example 3 establishes that lama4 −/− mice are resistant to obesity induced by age or diet ; examples 4 , 5 , and 6 provide results characterizing lama4 −/− mice in terms of food consumption , serum profile ( insulin , leptin , igf - 1 , glucose , triglyceride , free fatty acids , and cholesterol ), and adipose tissue structure , respectively ; and example 7 establishes adipose tissue function in lama4 −/− and control wild - type mice . the generation of laminin α4 null mice ( lama4 ) was previously described [ 15 ], and that description is incorporated herein by reference . the mice were backcrossed to c57 bl / 6 mice ( charles river ) for more than 10 generations . mice were fed a standard diet , or a high - fat diet containing 45 kcal % fat ( d12451 , research diets ), beginning at 4 weeks of age . the animals were fed ad libitum and their food was weighed weekly . the mice were given a food refill up to 500 g after each weighing . the amount of food consumed was divided by the number of animals in a cage as an estimate of intake . all animal procedures were approved by the iacuc at karolinska institutet or the university of chicago . the animals were housed either in mixed cages ( two lama4 −/− and two wild - type control animals ) or in cages with only lama4 −/− mice or wild - type animals , in order to rule out the possibility that the weight differences observed were due to differences in dominance behavior . no differences were observed due to housing conditions . for immunostaining in mouse tissues , animals at 4 months of age were sacrificed and tissue harvested . samples were placed in tissuetek ® ( sakura ) in plastic molds and frozen in isopentane cooled to its freezing point . cryosections of 8 - 12 mm in thickness were made at − 38 ° c . the sections were allowed to dry for 1 hour at room temperature and then fixed in acetone for 10 minutes before staining , except for antibody to laminin α4 , where the sections were additionally treated for 5 minutes in boiling 1m urea and washed in distilled water . the antibodies used were anti - nidogen / entactin ( mab 1946 , chemicon ), anti - collagen type iv ( polyclonal # ab756p , chemicon ), anti - perlecan ( clone hk - 102 , seikagaku corp ), anti - laminin α1 ( clone 198 ( 35 )), anti - laminin α2 ( clone 4h8 - 2 ), anti - laminin α4 ( polyclonal s8 ( 36 )), and anti - laminin α5 ( serum 405 ). secondary antibodies were fitc - or cy3 - conjugated and purchased from jackson immunoresearch laboratories , inc . tissue sections were examined with a leica mdrb microscope ( leica ) and pictures were taken with a hamamatsu digital camera with openlab ( improvision ) software . digital images were further processed with photoshop 5 . 0 ( adobe ). computerized tomography in 12 - month - old mice was performed 5 mm proximal to the crista iliaca ( iliac crest ), identified by a longitudinal prescan , with the stratec pct xct research m , software version 5 . 4b ( norland medical systems ) operating at a resolution of 70 μm [ 16 ] [ 17 ]. dxa was used to determine body composition measurements and for measurements of body fat in the 12 - month - old mice . dxa measurements were performed with the norland pdxa sabre and the sabre research software ( version 3 . 9 . 2 ), as previously described [ 18 ]. that description is incorporated herein by reference . litter matched , ( n = 10 ) lama4 −/− and control mice at 10 months of age were used for serum analysis . the animals were fasted for two hours prior to sampling . blood was taken from the tail vein and allowed to coagulate for 20 - 30 minutes at room temperature before centrifugation and separation of serum . serum leptin and insulin levels were measured by radioimmunoassay ( chrystal chem . inc .). free fatty acids ( ffa ) were measured by an enzymatic colorimetric method ( acs - acod ; wako chemicals usa , inc .). insulin - like growth factor - i ( igf - i ) levels were measured by double antibody binding igf binding protein - blocked radioimmunoassay [ 19 ]. serum glucose was determined with the glucose oxidase method [ 20 ]. triglycerides and cholesterol were assayed using commercially available kits and following the manufacturers &# 39 ; instructions ( catalog no . 450032 , triglycerides / gb , roche diagnostics gmbh , respective catalog no . 2016630 , cholesterol , chod - pap , boehringer mannheim gmbh ). animals used for serum and dxa analyses were sacrificed and the livers harvested for histopathological evaluation ( 10 lama4 −/− and 10 lama +/− mice on both diets ). for histological staining , the tissue samples were fixed in 10 % neutral buffered formalin , paraffin - embedded and stained according to standard protocols . tissue sections were examined with a leica mdrb microscope ( leica ) and pictures were taken with a hamamatsu digital camera with openlab ( improvision ) software . digital images were further processed with photoshop 5 . 0 ( adobe ). lama4 +/+ and lama4 mice were fed a standard diet . at 3 months of age , mice were sacrificed . epididymal and subcutaneous fat depots were harvested , and the masses were assessed . mass of adipose tissues from each depot was normalized to the total individual animal weight the depot was harvested from using equation ( 1 ). the normalized % fat pad mass takes into account variation introduced from individual total animal weights . a portion of each fat pad type was then placed in formaldehyde and paraffin embedded . samples were sectioned and stained with hematoxylin and eosin . five images were taken with an axiovert 200 inverted microscope using a 5 × objective ( 1 . 3 μm / pixel ) ( carl zeiss microimaging , inc ., thornwood , n . y .) for each fat pad . the images were used to manually measure the diameters of individual adipocytes using axiovision ( carl zeiss microimaging ). the fat pads were weighed prior to functional analysis with a lipogenesis assay . the assay was performed as described previously [ 21 ], and incorporated herein by reference . briefly , adipocytes were isolated from the harvested fat pads by collagenase digestion and centrifugation . isolated adipocytes were incubated with radioactive glucose in krebs - ringer bicarbonate containing 10 nm insulin and 1 % ( w / v ) bsa . the lipid fraction was extracted and radioactivity in the triglyceride fraction was measured . student &# 39 ; s t test was used for all statistical analysis ( two - tailed ), p & lt ; 0 . 05 was considered significant . immunofluorescence staining was first performed to compare bm composition surrounding adipocytes in lama4 −/− and lama4 +/+ mice . staining was performed for known adipose bm proteins , including the α1 , α2 , α4 and α5 chains of laminin , type iv collagen , nidogen and perlecan . in control mice , the α2 and α4 chains of laminin were present in the bm surrounding mature adipocytes ( fig1 ). laminin α5 was not observed in mouse adipocyte bm . type iv collagen , perlecan and nidogen were present in the murine pericellular adipocyte bm . when examining lama4 −/− bm , the only difference in composition was the complete absence of laminin α4 . all other bm proteins present in the adipose bm of control mice were also observed in the pericellular bm of lama4 −/− mice . the adipocyte bm appeared somewhat thicker in the lama4 −/− mice . lama4 −/− mice are resistant to age - and diet - induced obesity the birth weight of laminin α4 deficient mice was about 10 % lower than control littermates , as previously reported [ 15 ]. at the time of weaning ( 6 weeks ), no difference in weight was observed between lama4 −/− or control animals ( lama4 −/− 20 . 6 ± 0 . 32 g , n = 8 ; lama +/− 22 . 3 ± 0 . 44 g , n = 10 ). animal weights were monitored first on a standard diet . a significant difference in weight was observed by 4 months of age ( fig2 a ; lama4 −/− 27 . 3 ± 0 . 48 g , n = 8 ; lama +/− 30 . 1 ± 0 . 76 g , n = 10 ; p = 0 . 003 ). lama4 −/− weighed less than control animals from this time point and increased steadily over time . to further investigate weight gain in these animals , mice were supplied a high - fat diet starting at four weeks of age . lama4 −/− animals gained weight at a much lower rate and differences were observed from control animal weights within one month on this diet ( lama4 −/− 21 . 7 ± 0 . 57 g , n = 8 ; lama +/+ 23 . 4 ± 0 . 47 g , n = 8 ; p = 0 . 05 , fig2 c ). this difference continued to increase over time with lama4 −/− exhibiting slow weight gain even on the high - fat diet . in fact , at 12 months of age , there was no difference in the average weight of lama4 −/− mice whether they were on standard or high - fat diets ( standard diet 34 . 3 ± 1 . 09 versus high - fat diet 35 . 5 ± 2 . 12 , p = 0 . 6 ). control animals rapidly gained weight on the high fat diet resulting in dramatic differences from lama4 −/− at 3 . 25 months ( lama4 −/− 25 . 4 ± 0 . 89 g , n = 8 ; lama +/+ 30 . 7 ± 1 . 27 . g , n = 8 ; p = 0 . 01 ). at sacrifice there was a visible difference in the epididymal fat pad volume in lama4 −/− mice compared to controls ( fig3 ). the amount of body fat in the mice was quantified using dual x - ray absorptiometry ( dxa ). drastically reduced adipose volumes were observed in lama4 −/− mice compared to controls ( standard diet lama4 −/− 50 . 62 ± 7 . 29 %, n = 6 ; lama +/+ 100 . 0 ± 5 . 80 %, n = 4 ; p & lt ; 0 . 05 ; high fat diet lama4 −/− 71 . 63 ± 9 . 40 %, n = 6 ; lama 125 . 24 ± 7 . 67 %, n = 8 ; p & lt ; 0 . 05 ; fig4 a , b ). an abdominal ct scan of lama4 −/− mice at 12 months further illustrated the fact that the adipose volumes were reduced . as shown in fig3 c , at this time point the amounts of both subcutaneous and intra abdominal adipose appeared lower than in littermate controls . there was no difference in the weights of livers from lama4 −/− and controls on standard diet , but the livers were smaller in lama4 −/− animals on high - fat diet ( fig5 ). the livers of control mice on standard diet showed mild liver steatosis after 12 months , while this was not seen in lama4 −/− mice ( fig6 a , c ) suggesting that the decreased body fat in lamaα4 −/− mice does not represent a form of lipodystrophy . on a high - fat diet , the control mice had moderate to severe steatosis ( fig6 b ), while lama4 −/− mice only had very mild steatosis ( fig6 d ). this decreased steatosis could help explain the smaller mass of livers of lama4 −/− mice relative to controls on the high - fat diet . in order to evaluate whether the observed differences in weight and body composition resulted from lower food consumption , food intake was quantified weekly . there was no difference in the total amount of food consumed between lama4 −/− and control animals ( p & gt ; 0 . 3 at any time - point , fig1 d ). these results suggest that the weight differences observed did not result from hypophagia in the lama4 −/− mice . the serum profiles of mice at 10 months of age on normal and high - fat diet were determined . serum samples were taken from 10 - month - old males and heterozygote littermates ( n = 5 - 10 in each group , housed 2 lama4 −/− and 2 littermate lama4 +/− in each cage ) after two hours of fasting . the levels of leptin , glucose , insulin , insulin - like growth factor - 1 ( igf - 1 ), free fatty acids ( ffa ), triglycerides ( tg ) and cholesterol were measured . the results are shown in table 1 , where *, p & lt ; 0 . 05 ; ** p ≦ 0 . 01 ; ***, p ≦ 0 . 001 . as revealed by the data in table 1 , serum levels of insulin , leptin , and igf - 1 were decreased in lama4 −/− mice , while serum levels of glucose , triglyceride , and free fatty acids were unchanged . interestingly , lama4 −/− animals had reduced serum cholesterol compared to controls , even on a high - fat diet . lama4 −/− and age matched control animals 13 to 15 week on a standard diet were used to further examine adipose structure and function . this time range was selected because it is prior to any statistically significant weight differences between lama4 −/− and control animals , allowing for the examination of adipose function without confounding results due to obesity . at the time of sacrifice there were no differences in total animal mass between lama4 −/− and control animals ( lama4 −/− 25 . 93 ± 0 . 77 g , n = 4 ; lama +/+ 26 . 60 ± 2 . 35 g , n = 4 ; p = 0 . 80 ). epididymal and subcutaneous adipose tissue were harvested from the mice . the normalized percentage mass of lama4 −/− mice &# 39 ; s epididymal adipose was significantly less than control mice ( lama4 −/− 0 . 80 ± 0 . 09 % , n = 4 ; lama +/+ 1 . 32 ± 0 . 14 %, n = 4 ; p = 0 . 022 ) ( fig7 ). the mass of lama4 −/− mice &# 39 ; s epididymal adipose was less than control mice ( lama4 −/− 0 . 21 ± 0 . 03 g , n = 4 ; lama +/+ 0 . 36 ± 0 . 07 g , n = 4 ; p = 0 . 10 ). interestingly , no differences in mass were observed between subcutaneous adipose mass ( lama4 −/− 0 . 18 ± 0 . 04 g , n = 4 ; lama +/+ 0 . 21 ± 0 . 06 g , n = 4 ; p = 0 . 69 ). these results are at a much earlier time point ( about 3 . 5 months ) than the dxa results shown above ( 12 months ) when all adipose depots appeared reduced in volume . these results indicate that prior to any phenotypic observations in total animal weight gain , epididymal volume was reduced in lama4 −/− mice . histomorphometrix analysis was used to further analyze adipose tissue in the different depots . mean adipocyte size determined from histological stains was greater in epididymal adipose tissue than in the subcutaneous adipose depots in both types of mice . in both subcutaneous and epididymal depots the mean adipose size was greater in the lama4 −/− mice ( fig8 ). cell density ( number of cells per area ) was lower in both epididymal and subcutaneous adipose depots for lama4 −/− mice relative to controls ( fig9 ). the reduced adiposity may be an indication of altered metabolic function . adipose function was examined by quantifying basal and insulin - 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( 2012 ) static mechanical stretching accelerates lipid production in 3t3 - l1 adipocytes by activating the mek signaling pathway . american journal of physiology - cell physiology 302 : c429 - c441 . 36 . sasaki t , mann k , timpl r ( 2001 ) modification of the laminin alpha 4 chain by chondroitin sulfate attachment to its n - terminal domain . febs letters 505 : 173 - 178 . 37 . kortesmaa j , doi m , patarroyo m , tryggvason k ( 2002 ) chondroitin sulphate modification in the alpha 4 chain of human recombinant laminin - 8 ( alpha 4 beta 1 gamma 1 ). matrix biology 21 : 483 - 486 . 38 . park p w , reizes o , bernfield m ( 2000 ) cell surface heparan sulfate proteoglycans : selective regulators of ligand - receptor encounters . journal of biological chemistry 275 : 29923 - 29926 . each of the references cited herein is hereby incorporated by reference in its entirety , or in relevant part , as would be apparent from the context of the incorporation . the disclosed subject matter has been described with reference to various specific embodiments and techniques . it should be understood , however , that many variations and modifications may be made while remaining within the spirit and scope of the disclosed subject matter .