A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression

Abstract

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor γ. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function. The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor γ. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function. Obesity is a major risk factor for insulin resistance and associated metabolic aberrations. Although visceral fat accumulation is of pathophysiologic importance (1Björntorp P. “Portal” adipose tissue as a generator of risk factors for cardiovascular disease and diabetes..Arteriosclerosis. 1990; 10: 493-496Crossref PubMed Google Scholar), associations with insulin resistance have been found also for increased total and subcutaneous fat mass (2Heilbronn L. Smith S.R. Ravussin E. Failure of fat cell proliferation, mitochondrial function and fat oxidation results in ectopic fat storage, insulin resistance and type II diabetes mellitus..Int. J. Obes. Relat. Metab. Disord. 2004; 28: 12-21Crossref PubMed Scopus (320) Google Scholar). Thus, enlargement of subcutaneous adipocytes showing both resistance to insulin-mediated glucose uptake (3Czech M.P. Cellular basis of insulin insensitivity in large rat adipocytes..J. Clin. Invest. 1976; 57: 1523-1532Crossref PubMed Scopus (85) Google Scholar) and increased rate of lipolysis (4Large V. Arner P. Regulation of lipolysis in humans. Pathophysiological modulation in obesity, diabetes, and hyperlipidaemia..Diabetes Metab. 1998; 24: 409-418PubMed Google Scholar) may also play an important role in insulin resistance development. It has been suggested that the reduced triglyceride storage capacity of enlarged fat cells is followed by ectopic fat storage in liver, skeletal muscle, and pancreas, resulting in insulin resistance and other features of the metabolic syndrome (2Heilbronn L. Smith S.R. Ravussin E. Failure of fat cell proliferation, mitochondrial function and fat oxidation results in ectopic fat storage, insulin resistance and type II diabetes mellitus..Int. J. Obes. Relat. Metab. Disord. 2004; 28: 12-21Crossref PubMed Scopus (320) Google Scholar, 5Bays H. Mandarino L. DeFronzo R.A. Role of the adipocyte, free fatty acids and ectopic fat in the pathogenesis of type 2 diabetes mellitus: peroxisome proliferator-activated receptor agonists provide a rational therapeutic approach..J. Clin. Endocrinol. Metab. 2004; 89: 463-478Crossref PubMed Scopus (516) Google Scholar). This overflow hypothesis is supported by findings from studies in patients lacking subcutaneous fat, lipodystrophy (6Hegele R.A. Phenomics, lipodystrophy and the metabolic syndrome..Trends Cardiovasc. Med. 2004; 14: 133-137Crossref PubMed Scopus (31) Google Scholar), a condition characterized by often severe insulin resistance due to excess accumulation of nonadipose tissue fat. In mice, the gene responsible for this syndrome, Lpin1, was recently isolated through positional cloning and found to encode an 891 amino acid protein, lipin, with a putative nuclear localization but hitherto unknown function (7Peterfy M. Phan J. Reue K. Lipodystrophy in the fld mouse results from a mutation in a new gene encoding a nuclear protein, lipin..Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (469) Google Scholar). Lipin mRNA expression was induced during 3T3-L1 preadipocyte differentiation and was found predominately in adipose tissue, skeletal muscle, and testis. Moreover, two mutant alleles of the Lpin1 gene, fld1 and fld2, were associated with a markedly reduced adipose tissue mass, consistent with an important role for lipin in adipose tissue development and function (7Peterfy M. Phan J. Reue K. Lipodystrophy in the fld mouse results from a mutation in a new gene encoding a nuclear protein, lipin..Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (469) Google Scholar). Altogether, these findings suggest that Lpin1 is a candidate gene in the pathogenesis of various adipose-related conditions, including obesity, insulin resistance, and type 2 diabetes mellitus. In screening for mutations in the human gene ortholog, LPIN1 (gene ID: 23175), no sequence variants exclusive for lipodystrophy were found (8Cao H. Hegele R.A. Identification of single-nucleotide polymorphisms in the human LPIN1 gene..J. Hum. Genet. 2002; 47: 370-372Crossref PubMed Scopus (20) Google Scholar). However, in a recent study, findings on both gene variation and adipose mRNA levels implicate LPIN1 in human glucose metabolism (9Suviolahti E. Reue K. Cantor R.M. Phan J. Gentil M. Naukkarinen J. Soro-Paavonen A. Oksanen L. Kaprio J. Rissanen A. et al.Cross-species analyses implicate Lipin 1 involvement in human glucose metabolism..Hum. Mol. Genet. 2006; 15: 377-386Crossref PubMed Scopus (86) Google Scholar). Here, we further investigate the adipose tissue expression of the human LPIN1 gene in relation to clinical variables and fat cell function. Four separate cohorts including obese and lean women were used. All were healthy and free of medication and ate a standard Swedish diet. They were all living in the Stockholm area and were at least second generation Scandinavian. None were completely sedentary or involved in athletic performances and none had undergone a slimming effort or experienced a change in body weight >1 kg within 6 months of the study, according to self-report. The study was explained in detail to each subject and her informed consent was obtained. The study was approved by the hospital's committee on ethics. Cohort 1 was composed of 80 obese (body mass index [BMI] 31–53 kg/m2) and 16 lean (BMI 20–24 kg/m2) women. At ∼07.30 AM, after an overnight fast, a venous blood sample was obtained for analyses of plasma levels of glucose, insulin, triglycerides, cholesterol, and HDL cholesterol, which were performed by the hospital's accredited chemistry laboratory. Insulin sensitivity was calculated from the homeostasis model assessment algorithm (HOMA) based on plasma glucose and plasma insulin (p-insulin × p-glucose/22.5). Systolic and diastolic blood pressures were measured in the supine position after 15 min of rest. Thereafter, an adipose sample (1–4 g) was obtained by needle biopsy from the abdominal subcutaneous area under local anesthesia, as previously described (10Kolaczynski J.W. Morales L.M. Moore Jr., J.H. Considine R.V. Pietrzkowski Z. Noto P.F. Colberg J. Caro J.F. A new technique for biopsy of human abdominal fat under local anaesthesia with Lidocaine..Int. J. Obes. Relat. Metab. Disord. 1994; 18: 161-166PubMed Google Scholar). This tissue was frozen in liquid nitrogen, kept frozen at −70°C, and used for subsequent mRNA analysis (see below). In 70 of the obese subjects, there was adipose tissue available also for fat cell glucose transport studies (see below). Cohort 2 consisted of 18 obese (BMI 38–53 kg/m2) women undergoing weight reduction surgery at the Department of Surgery, Karolinska University Hospital, Huddinge. From these women, perioperative fat biopsies from subcutaneous and omental adipose tissue (1–2 g) were taken for subsequent mRNA analysis using a laparoscopic technique. mRNA analyses were performed as described below. In 13 of these women, there was a second subcutaneous fat biopsy taken 2–4 years after surgery (cohort 3). All subjects were weight stable after the weight reduction, and their BMI had decreased by an average of approximately 15 kg/m2. Cohort 4 consisted of 11 subjects from which adipose tissue biopsies were taken for preadipocyte differentiation, see below. Isolated fat cells were prepared and isolated as described previously (11Rodbell M. Metabolism of isolated fat cells..J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar). In brief, adipocytes were separated from stroma cells by treatment in a shaking bath at 37°C for 60 min with 0.5 mg/l collagenase in 5 ml Krebs-Ringer phosphate (KRP) buffer (pH 7.4) with 40 g/l of purified BSA. Adipocyte suspensions were then rinsed three times in collagenase-free buffer using nylon filters. Fat cell sizes were measured by direct microscopy, and the mean adipocyte diameter was calculated from measures of 100 cells. The total lipid weight of the incubated fat cells was determined after organic extraction. The number of fat cells incubated was determined by dividing total lipid weight by the fat cell weight. We used an indirect method, which is described in detail elsewhere (12Arner P. Engfeldt P. Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women..Am. J. Physiol. 1987; 253: 193-201PubMed Google Scholar). In brief, isolated fat cells were incubated at a concentration of 2% (v/v) in KRP buffer (pH 7.4) containing albumin (20 mg/ml), (3-3H]glucose (5 × 106 dpm/ml), unlabeled glucose (1 μmol/l), and human insulin in different concentrations (10−15–10−6 M). The incubations were conducted for 2 h at 37°C with air as the gas phase. Incubations were stopped by rapidly chilling the incubation vials to 4°C and adding H2SO4. Thereafter, the incorporation of radiolabeled glucose into adipocyte lipids was determined. This incorporation into lipids (i.e., lipogenesis) reflects glucose transport, because at micromolar glucose concentrations, glucose transport is the rate-limiting step for lipogenesis in human fat cells, as discussed previously (12Arner P. Engfeldt P. Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women..Am. J. Physiol. 1987; 253: 193-201PubMed Google Scholar). Glucose transport, basal and maximal insulin-stimulated minus basal, was expressed as the amount of glucose incorporated per fat cell number (nmol/2 h/107 cells), as described previously (12Arner P. Engfeldt P. Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women..Am. J. Physiol. 1987; 253: 193-201PubMed Google Scholar). The half-maximum effective concentration (EC50) of insulin was calculated by log-logit transformation of concentration-response curves and converted to the negative logarithm of mol/l (pD2), which corresponds to insulin receptor sensitivity (12Arner P. Engfeldt P. Fasting-mediated alteration studies in insulin action on lipolysis and lipogenesis in obese women..Am. J. Physiol. 1987; 253: 193-201PubMed Google Scholar). From 11 subjects, 2 males and 9 females (cohort 4), adipose tissue (9 abdominal subcutaneous, 2 hip) biopsies were taken, from which preadipocytes were isolated and differentiated into adipocytes as described previously (13van Harmelen V. Dicker A. Ryden M. Hauner H. Lonnqvist F. Naslund E. Arner P. Increased lipolysis and decreased leptin production by human omental as compared with subcutaneous preadipocytes..Diabetes. 2002; 51: 2029-2036Crossref PubMed Scopus (157) Google Scholar). The mean BMI and age in this subject group were 28.4 ± 6.1 kg/m2 and 42 ± 11 years, respectively. The differentiation process was enhanced by adding rosiglitazone (10 μM) during the first 6 days of the differentiation process. The cells reached full differentiation after 12–14 days. At days 4, 8, and 12, cells were lysed for the isolation of total RNA or for the measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity for assessment of differentiation capacity, which was performed as described previously (14van Harmelen V. Skurk T. Rohrig K. Lee Y.M. Halbleib M. Aprath-Husmann I. Hauner H. Effect of BMI and age on adipose tissue cellularity and differentiation capacity in women..Int. J. Obes. Relat. Metab. Disord. 2003; 27: 889-895Crossref PubMed Scopus (204) Google Scholar). Total RNA was extracted from 300 mg of adipose tissue or adipocytes using the RNeasy mini kit (Qiagen; Hilden, Germany). The RNA concentration and purity were assessed spectrophotometrically. One microgram of total RNA from each sample was reverse transcribed to cDNA using the Omniscript RT kit (Qiagen) and random hexamer primers (Invitrogen; Tåstrup, Denmark). To minimize methodological errors resulting from variation in cDNA synthesis, cDNA synthesis was performed simultaneously for all subjects included in the first, second, third, and fourth cohorts, respectively, using exactly the same mix of primers and the same RT kit. The Agilent 2100 Bioanalyzer (Agilent Technologies; Kista, Sweden) was used to confirm the integrity of the RNA. In a final volume of 25 μl, 5 ng of cDNA was mixed with 2× SYBR green PCR master mix (Bio-Rad Laboratories, Inc.; Hercules, CA) and primers (Invitrogen). The primer pairs were selected to yield a single amplicon based on dissociation curves and analysis by agarose gel electrophoresis. The primers used were 5′-GAAAGGACAGGGCAGAAGAAC-3′ (sense) and 5′-TGCTCAATGGGCTGGACTC-3′ (antisense) for lipin (NM_145693); 5′-ACCCAGAAAGCGATTCCTTCAC-3′ (sense) and 5′-CCACGG-AGCTGATCCCAAAG-3′ (antisense) for peroxisome proliferator-activated receptor γ transcript variant 2 (PPARγ2) (NM_015869); 5′-CATTCCTTGGTTCATCGTG-3′ (sense) and 5′-ATAGCCTCCGCAACATAC-3′ (antisense) for GLUT4 (NM_001042); 5′-GGTCTCGAACTCCTGGCCTA-3′ (sense) and 5′-TGAGATATCGACTGGGCATGGT-3′ (antisense) for adiponectin (NM_004797); and 5′-TGACTCAACACGGGAAACC-3′ (sense) and 5′-TCGCTCCACCAACTAAGAAC-3′ (antisense) for 18S rRNA. Quantitative real-time PCR was performed in an iCycler IQ™ (Bio-Rad Laboratories, Inc.). The mRNA levels were determined by a comparative Ct method (ABI Prism 7700, Applied Biosystems; Foster City, CA). The subject with the highest Ct value was used as a reference; all other Ct values for the target gene and reference gene, respectively, were subtracted from this Ct value. The Ct values were then normalized to rRNA for 18S. All PCR reactions were run in duplicate. Parameter distributions were normalized when necessary by 10logarithm transformation mean ± The or and single and analyses were used for A value of or was The analyses were performed using Lipin mRNA levels were measured during preadipocyte differentiation and compared with mRNA Lipin expression and mRNA expression were at 4 and increased Lipin mRNA after 8, showed a at in with GLUT4 mRNA on the other were at 4, but increased from to The of GLUT4 mRNA levels during differentiation was to the of which was used as an index of the differentiation capacity of the cells 1 Lipin mRNA adipose expression in subcutaneous adipocytes adipose tissue was measured using from of 18 women of in the lipin as well as adiponectin rRNA was approximately as in the fat cells as in the tissue, ± ± and 100 ± ± The expression of lipin was further compared between subcutaneous and omental adipose tissue from 18 obese women (cohort adipose tissue difference in lipin mRNA levels was found 100 ± omental 100 ± the omental adiponectin mRNA levels ± were than in the subcutaneous ± The of on LPIN1 gene expression was in subcutaneous adipose tissue from 80 obese and 16 lean women (cohort in the lipin rRNA was markedly downregulated in obese subjects, ± 100 ± The values for adiponectin rRNA were ± and 100 ± A expression was found in 13 obese women (cohort undergoing by weight reduction, an increased of lipin, ± 100 ± as well as adiponectin ± 100 ± was found The obese women of 1 were according to the or of the metabolic syndrome, as by the of the of the on and treatment of blood in J. Med. Scholar). to this women the of the metabolic syndrome, In the of the metabolic syndrome on the subcutaneous adipose tissue mRNA levels of lipin and adiponectin is In women with the metabolic syndrome, the lipin ± as well as the adiponectin ± rRNA were reduced as compared with women that the metabolic syndrome ± and 100 ± In 70 of the obese subjects of there was tissue available to investigate the of lipin mRNA with human fat cell adipocyte as assessed by basal and maximal minus basal insulin-stimulated glucose transport as well as for insulin-mediated glucose transport, was to adipose tissue lipin mRNA in 4 between both basal and maximal insulin-stimulated minus basal glucose transport were found that were of and insulin sensitivity measured as A but was also between lipin mRNA expression and for insulin-stimulated glucose transport receptor However, this when for The subcutaneous adipose tissue adiponectin mRNA levels were also with both basal and maximal minus basal glucose transport and for insulin-mediated glucose transport the after for BMI or A major regulator of glucose uptake in fat cells is glucose transporter 4, We that LPIN1 gene expression also to levels of GLUT4 mRNA in the 80 obese subjects of A between lipin and GLUT4 mRNA levels was found that was of and 5 A but with GLUT4 was found also for adiponectin mRNA studies have that the LPIN1 gene is an important regulator of fat cell function and a candidate gene for insulin resistance and type 2 diabetes mellitus. Here, we show that lipin mRNA was in both human subcutaneous and omental adipose tissue and showed a of expression in both depots. We also found that lipin mRNA levels in human adipose tissue were downregulated in and that weight reduction in obese subjects was to an of lipin mRNA Moreover, lipin mRNA levels were reduced in women with the metabolic syndrome. in with findings from that lipin mRNA and or have a but glucose and insulin resistance (7Peterfy M. Phan J. Reue K. Lipodystrophy in the fld mouse results from a mutation in a new gene encoding a nuclear protein, lipin..Nat. Genet. 2001; 27: 121-124Crossref PubMed Scopus (469) Google Scholar), in has been to a of the adipose tissue mass in these The results also in with two recent human studies in which reduced levels of human adipose tissue lipin mRNA were in subjects with insulin resistance (9Suviolahti E. Reue K. Cantor R.M. Phan J. Gentil M. Naukkarinen J. Soro-Paavonen A. Oksanen L. Kaprio J. Rissanen A. et al.Cross-species analyses implicate Lipin 1 involvement in human glucose metabolism..Hum. Mol. Genet. 2006; 15: 377-386Crossref PubMed Scopus (86) Google Scholar) and lipodystrophy L. F. E. J. K. and K. tissue lipin expression levels patients with and J. Obes. of 2006; Scholar). no for human lipin is available at the we mRNA levels to the expression of a with measures of lipin adipose tissue levels of adiponectin mRNA were and the results in with studies that adiponectin levels reduced in and insulin resistance, increased after weight reduction T. T. K. K. K. and adiponectin in insulin resistance, diabetes, and the metabolic Clin. Invest. 2006; PubMed Scopus Google Scholar, A. A. J. M. A. The of surgery on and other cardiovascular risk factors in severe and severe 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), and expressed at levels in omental as compared with subcutaneous adipose tissue P.F. expression of adiponectin mRNA in visceral adipose tissue in lean and obese Endocrinol. 2004; PubMed Scopus Google Scholar). A of insulin resistance is reduced insulin-mediated glucose uptake in and adipose tissue, a process by GLUT4 A. Glucose transporter and PubMed Scopus Google Scholar). In skeletal muscle, this is due to of GLUT4 to the plasma GLUT4 expression M. Insulin in human skeletal the in II PubMed Scopus Google Scholar). In insulin resistance in adipocytes is associated with reduced expression of GLUT4 R.A. of type 2 metabolic and for diabetes Scholar, Glucose and insulin for insulin resistance and diabetes J. Med. PubMed Scopus Google Scholar). Moreover, an reduction of GLUT4 show a markedly insulin-stimulated glucose uptake in adipocytes by insulin resistance in both and liver, which is in with the overflow and results in ectopic fat storage E. K. E. T. of the GLUT4 gene insulin action in and 2001; PubMed Scopus Google Scholar). Here, we found that lipin mRNA levels in adipose tissue were to both basal and insulin-stimulated adipocyte glucose transport as well as GLUT4 mRNA levels in a large of obese human The relation between lipin mRNA and insulin receptor sensitivity on the other a study has that insulin may lipin activity by a target of the I. Jr., of lipin by the target of 2002; PubMed Scopus Google Scholar). This is to and in the and has been to adipocyte glucose transport F. A. A. A. A. of the target of insulin to and glucose transport in 3T3-L1 and human PubMed Scopus Google Scholar). lipin is a regulator of adipocyte glucose transport to In and mouse has been found that lipin is a factor in the gene of adipocyte differentiation, to of including and J. M. Reue K. Lipin expression peroxisome proliferator-activated is for in and in Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In human we also show that lipin mRNA is expressed at an early of the differentiation process and that is further induced or in parallel with that lipin may also have a role in human In LPIN1 gene expression is induced at an early in human preadipocyte differentiation in parallel with Moreover, adipose tissue LPIN1 expression levels reduced in and the metabolic syndrome and associated with both basal and insulin-stimulated glucose transport in human fat cells as well as adipose GLUT4 Altogether, these suggest a role for the LPIN1 gene in human and fat cell function. This study was supported by from the Swedish and the Swedish body mass index glycerol-3-phosphate dehydrogenase homeostasis model assessment algorithm peroxisome proliferator-activated receptor γ transcript variant 2