Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α.

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are short endogenous RNAs that regulate multiple biological processes including adipogenesis and fat metabolism. We sought to identify miRNAs that correlate with BMI and to elucidate their upstream regulation and downstream targets. METHODS: Microarray-based expression profiling of 233 miRNAs was performed on subcutaneous abdominal adipose tissue biopsies from 29 non-diabetic Pima Indian participants. Correlation of the expression levels of eight miRNAs with BMI was assessed by quantitative reverse transcription (QRT) PCR in adipose samples from 80 non-diabetic Pima Indians with a BMI of 21.6-54.0 kg/m(2). The upstream regulation of one of these miRNAs, miR-221, was tested by treating cultured human pre-adipocytes with leptin, TNF-α and insulin. Predicted targets of miR-221 were validated using QRT-PCR, immunoblots and luciferase assays. The downstream effects of miR-221 overexpression were assayed by proteomic analysis. RESULTS: Expression levels of miR-221 were positively correlated with BMI (particularly in women) and fasting insulin concentrations, while the levels of miR-193a-3p and miR-193b-5p were negatively correlated with BMI; other miRNAs did not show significant associations in the 80 samples. miR-221 was downregulated by leptin and TNF-α treatment in cultured human pre-adipocytes. Conversely, miR-221 overexpression upregulated several proteins involved in fat metabolism, mimicking peroxisome proliferator-activated receptor (PPAR) activation. Furthermore, miR-221 directly downregulated the adiponectin receptor 1 (ADIPOR1) and the transcription factor v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1). Adiponectin signalling is known to promote insulin sensitivity, and ETS1 is crucial for angiogenesis. CONCLUSIONS/INTERPRETATION: Our data suggest that miR-221 may contribute to the development of the insulin resistance that typically accompanies obesity, by affecting PPAR signalling pathways and by directly downregulating ADIPOR1 and ETS1.

HLA-DRB1 reduces the risk of type 2 diabetes mellitus by increased insulin secretion.

AIMS/HYPOTHESIS: We sought to identify the physiological implications of genetic variation at the HLA-DRB1 region in full-heritage Pima Indians in Arizona. METHODS: Single-nucleotide polymorphisms from the HLA region on chromosome 6p were tested for association with skeletal muscle mRNA expression of HLA-DRB1 and HLA-DRA, and with type 2 diabetes mellitus and prediabetic traits. RESULTS: The A allele at rs9268852, which tags HLA-DRB1 02(1602), was associated both with higher HLA-DRB1 mRNA expression (n = 133, p = 4.27 × 10(-14)) and decreased risk of type 2 diabetes (n = 3,265, OR 0.723, p = 0.002). Among persons with normal glucose tolerance (n = 266) this allele was associated with a higher mean acute insulin response during an intravenous glucose tolerance test (p = 0.005), higher mean 30 min insulin concentration during an oral glucose tolerance test (p = 0.017) and higher body fat percentage (p = 0.010). The polymorphism was not associated with HLA-DRA mRNA expression or insulin sensitivity. CONCLUSIONS/INTERPRETATION: HLA-DRB1*02 is protective for type 2 diabetes, probably by enhancing self tolerance, thereby protecting against the autoimmune-mediated reduction of insulin secretion.

Human primary myoblast cell cultures from non-diabetic insulin resistant subjects retain defects in insulin action.

Insulin resistance is a predictor of the development of noninsulin-dependent diabetes mellitus (NIDDM) in humans. It is unclear whether insulin resistance is a primary defect leading to NIDDM or the result of hyperinsulinemia and hyperglycemia. To determine if insulin resistance is the result of extrinsic factors such as hyperinsulinemia primary skeletal muscle cell cultures were established from muscle biopsies from Pima Indians with differing in vivo insulin sensitivities. These cell cultures expressed a variety of muscle-specific phenotypes including the proteins alpha-actinin and myosin, muscle-specific creatine kinase activity, and RNA encoding GLUT4, MYF5, MYOD1, and MYOGENIN. Labeled glucose was used to measure the insulin-stimulated conversion of glucose to glycogen in these cultures. The in vivo rates of insulin-stimulated glycogen production (insulin resistance) were correlated with in vitro measures of glycogen production (P = 0.007, r = 0.58). This defect in insulin action is stable in a uniform culture environment and is retained over time. The retention of insulin resistance in myoblast derived cell cultures is consistent with the expression of an underlying biochemical defect in insulin resistant skeletal muscle.

Evidence for linkage between a region on chromosome 1p and the acute insulin response in Pima Indians.

A low acute insulin response (AIR) is a predictor of non-insulin-dependent diabetes mellitus (NIDDM) in insulin-resistant Pima Indians. We have initiated a search for regions of the genome linked with the AIR using sib-pair linkage analysis as a first step in identifying genes that are determinants of this phenotype. Eighteen short tandem-repeat polymorphisms from chromosome 1 were genotyped in over 900 Pima Indians and tested for linkage with NIDDM and in a subset of Pima Indians for linkage with AIR. The anonymous DNA marker D1S198 on chromosome 1p was linked with AIR (P = 0.000056) in 175 sib pairs from 60 families, all with normal glucose tolerance, but no linkage was observed between D1S198 and NIDDM (P = 0.44, 996 sib pairs). Additional markers genotyped on chromosome 1 did not show linkage with AIR or NIDDM. This study indicates that a locus on chromosome 1p may be a determinant of the phenotypic variation seen in the AIR.

Tyrosine kinase activity of skeletal muscle during insulin infusion in humans.

Insulin receptor tyrosine kinase is stimulated by insulin in vivo, and this provides a mechanism by which the signal from insulin is transmitted into target cells. This study examined the time course of the in vivo activation of the kinase. Five nondiabetic Pima Indians had a euglycemic clamp at an insulin dose of 600 mU/min.m2, resulting in plasma insulin concentrations of about 15 nM by 30 min. Percutaneous muscle biopsies were taken from the vastus lateralis before and at regular intervals during insulin infusion, and the in vivo and in vitro tyrosine kinase activities were measured. There was a rapid in vivo activation of the kinase, detectable at less than 10 min and reaching a maximum within 30 min of insulin infusion. The time course of in vivo kinase activity, plasma insulin concentrations, and insulin-mediated glucose disposal rates displayed parallel patterns, indicating close interrelationships among these variables. The insulin concentration at which the kinase activity was maximal was about 10 nM both in vivo and in vitro. In vitro, however, this maximum increased with the degree of the kinase activation in vivo, indicating that the kinase potential in vitro is dependent on previous insulin exposure in vivo. We conclude that in vivo activation of the insulin receptor tyrosine kinase in human skeletal muscle is a rapid process, related to insulin action on glucose disposal, and that circulating insulin primes inactive insulin receptor molecules for subsequent tyrosine kinase activation by a mechanism that remains to be elucidated.

Insulin-sensitive tyrosine kinase activity changes in parallel with plasma insulin and glucose concentrations in humans.

Insulin receptor tyrosine kinase is an important step in insulin action. We examined the relationship between diet-induced changes in glucose metabolism and changes in skeletal muscle insulin-sensitive tyrosine kinase activity in 12 nondiabetic subjects. Subjects were fed a traditional, high carbohydrate Pima Indian diet and a modern, high fat western diet for 2 weeks in a randomized cross-over design. At the end of each dietary period, glucose tolerance was assessed, insulin sensitivity (SI) was estimated by Bergman's minimal model method, and insulin receptor concentration and tyrosine kinase activity were determined on lectin-purified extracts from quadriceps femoris muscle. Compared to the traditional diet, the modern diet was associated with a deterioration of glucose tolerance and an increase in glucose-induced plasma insulin levels. As expected, SI changes were associated with opposite changes in plasma insulin levels. However, the changes in maximal tyrosine kinase activity were negatively correlated with changes in SI (r = -0.69; P less than 0.01) and positively correlated with changes in plasma glucose (r = 0.70; P less than 0.01) and insulin response to glucose (r = 0.57; P less than 0.025). These results suggest that the site of diet-induced changes in insulin action is beyond the insulin-sensitive tyrosine kinase. The results further suggest that the kinase activity is modulated by prevailing plasma insulin levels.

Molecular characterization of the human PEA15 gene on 1q21-q22 and association with type 2 diabetes mellitus in Pima Indians.

The PEA15 gene encoding a protein kinase C substrate is widely expressed, and its overexpression may contribute to impairment of glucose uptake. PEA15 is located within a region on human 1q linked with type 2 diabetes in both Pima Indians and Caucasians. To assess the potential contribution of genetic alterations within this locus to disease susceptibility in the Pimas, we have investigated its genomic sequences. The PEA15 locus is composed of four exons spanning approximately 10.2kb of genomic DNA, flanked upstream by an potentially expressed Alu element, downstream by the H326 gene, and is located within 250kb of KCNJ9. We also sequenced over 2kb of the promoter region and identified various motifs analogous to known transcription factor binding sites. By analysis of 22 Pimas, including 13 diabetic subjects, we detected four single nucleotide polymorphisms (SNPs) in the non-coding regions of PEA15, including three frequent variants that were in allelic disequilibrium, and one variant found only in a single Pima. The three SNPs were not associated with type 2 diabetes mellitus in 50 affected and 50 control Pimas (p=0.12-0.17), and we conclude that mutations in this gene probably do not contribute significantly to disease susceptibility in this Native American tribe. However, knowledge of the genomic structure of PEA15 provides the basis for similar systematic examinations of this candidate locus in relation to type 2 diabetes and other metabolic disorders in other populations.

Linkage between stature and a region on chromosome 20 and analysis of a candidate gene, bone morphogenetic protein 2.

Sib-pair linkage analysis of the quantitative trait, structure, in over 500 Pima Indians indicates that a genetic determinant of governing stature is located on chromosome 20. Analysis of 10 short tandem repeat polymorphisms localized this linkage to a 3.2cM region that includes D20S98 and D20S66. Using all possible sib-pair combinations, linkage was detected to both stature (P = 0.0001) and to leg length (P = 0.001), but not to sitting height. Single-strand conformational polymorphism analysis of exon 3 of the bone morphogenetic protein 2 (BMP2) gene, a candidate gene in this region, in genomic DNA of 20 of the tallest and 20 of the shortest individuals did not show any consistent differences associated with leg length or height. Sequence analysis of the region encoding the mature protein revealed a single nucleotide substitution, a T to G transversion, not detected by single-strand conformational polymorphism (SSCP) analysis. This transversion results in a conservative amino acid substitution of glycine for valine at codon 80 of BMP2. The frequency of this allele was 0.23 in the sample. No significant differences in height were noted in persons carrying either allele. This indicates that this structural alteration is the mature BMP2 protein does not contribute to the differences in stature observed in the Pima Indians, nor is this structural change in the mature protein likely to be responsible for the linkage observed with stature on chromosome 20.

A physical map at 1p31 encompassing the acute insulin response locus and the leptin receptor.

Recently, we reported genetic linkage in Pima Indians between the acute insulin response to an intravenous glucose challenge and the short tandem repeat marker D1S198, indicative of a genetic element in this region that controls the phenotypic variation in the first phase of insulin secretion. As a first step to isolating the gene responsible for the acute insulin response, we have constructed a yeast artificial chromosome (YAC) contig map that spans the DNA microsatellites D1S438 through D1S464. The contig comprises 34 YACs on which we have mapped 44 ends of the genomic DNA inserts from the 34 YACs, 13 short tandem repeats, eight expressed sequence tags, and six genes. In addition, we have used this contig to construct a physical map encompassing approximately 9 Mb of DNA in this region.

Insulin-inducible changes in insulin receptor mRNA splice variants.

Alternative splicing of insulin receptor pre-mRNA has been shown to be regulated in a tissue-specific and developmental manner. We investigated whether the receptor ligand might regulate the relative distribution of alternatively spliced mRNA in insulin-sensitive cells and found that changes in the relative expression of the two alternatively spliced insulin receptor RNA isotypes expressed in hepatocytes are regulated by insulin. In addition, we observed significant differences (p < or = 0.001) in insulin receptor isotype expression in subjects who were hyperinsulinemic and insulin-resistant versus subjects who were insulin-sensitive. These results support a role for insulin in the regulation of the relative expression of alternatively spliced mRNA expressed in insulin-responsive cells and tissues.

Variant screening of PRKAB2, a type 2 diabetes mellitus susceptibility candidate gene on 1q in Pima Indians.

The AMP-activated protein kinase (AMPK) is a key enzyme involved in the regulation of lipid and glucose metabolism. There are multiple isoforms of the three subunits of this enzymatic complex, each encoded by a different gene in humans. We have investigated the PRKAB2 gene encoding the beta2 subunit, which is located on chromosome 1q within a region linked with type 2 diabetes mellitus (T2DM) in the Pima Indians and four different Caucasian populations. The gene consists of eight exons spanning about 15 kb, and we detected nine variants in the introns and 3' UTR, including eight informative single nucleotide polymorphisms (SNPs) and one rare 4 bp insertion/deletion. In an analysis of representative markers in selected Pima Indians including 149 diabetic cases (onset age < 25 years) and 150 controls (at least 45 years old, with normal glucose tolerance), we found no evidence for association of this locus with T2DM. We conclude that variants in PRKAB2 are unlikely to contribute to the disease susceptibility in Pima Indians.

Insulin-sensitive tyrosine kinase: relationship with in vivo insulin action in humans.

To investigate the relationship of insulin receptor kinase with insulin resistance in humans, we studied insulin-sensitive tyrosine kinase activity in muscle biopsies taken from 20 Pima Indians [14 nondiabetics, 6 with non-insulin-dependent mellitus (NIDDM)] during euglycemic clamps, at insulin concentrations of approximately 68 microU/ml (low dose) and approximately 1,170 microU/ml (high dose). In the nondiabetics, the low dose, insulin-induced kinase activation in vivo was 1.5-fold the activity in the fasting state (P less than 0.05), whereas in the diabetics, the kinase activity actually decreased by 40% relative to fasting (P less than 0.05). The difference in delta-kinase in vivo was significant (P less than 0.01) between the two groups. Similarly, the kinase activation in vitro in response to 1 nM insulin was lower in diabetic subjects compared with nondiabetics (P less than 0.01). These data indicate that, in NIDDM, both in vitro and in vivo insulin-stimulated tyrosine kinase activity is impaired. Among nondiabetics, the kinase sensitivity to insulin, calculated as the ratio of the kinase activity at 1 nM insulin in vitro to the kinase activity at 100 nM insulin, was positively correlated with plasma insulin concentrations 2 h after an oral glucose load (r = 0.69, P less than 0.01). Thus, in nondiabetic subjects with insulin resistance, insulin activation of the kinase is not reduced, but the kinase sensitivity to insulin increases with increasing plasma insulin levels. Therefore, the site of insulin resistance in nondiabetic subjects is distal to the insulin receptor kinase. Furthermore, it is possible that circulating insulin, by increasing the kinase sensitivity to insulin, is a determinant of the receptor kinase activity.

The Gln223Arg polymorphism of the leptin receptor in Pima Indians: influence on energy expenditure, physical activity and lipid metabolism.

Leptin regulates body weight by its receptor-mediated anorectic, thermogenic and antisteatotic effects. Recently, lower leptin binding to the soluble form of the leptin receptor (LEPR) was shown in carriers of the Arg223-encoding allele of the Gln223Arg polymorphism of the LEPR. To investigate whether this variant influences energy metabolism and adiposity in Pima Indians, we genotyped non-diabetic Pima Indians in whom we had measured body composition and 24 h energy expenditure (24 h EE), physical activity level (PAL) and 24 h respiratory quotient (24 h RQ) in a respiratory chamber (n=268) and who had undergone percutaneous fat biopsies from the periumbilical region (n=184). Genotype was not associated with percent body fat (P>0.39), but was associated with 24 h EE, PAL and mean subcutaneous abdominal adipocyte size (SAAS all P<0.05). Homozygotes for the Arg223-encoding allele had lower 24 h EE (P=0.04) and PAL (P=0.007), but larger SAAS (P=0.01) than Gln homozygotes. These findings are consistent with a role of the Gln223Arg polymorphism in reducing peripheral and central leptin binding to the LEPR in humans. However, these effects do not seem to have a major impact on adiposity in this population.