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Keywords:

  • adiponectin;
  • c-Cbl—associated protein;
  • 11β-hydroxysteroid dehydrogenase type 1;
  • sterol regulatory element binding protein;
  • type 2 diabetes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Objective: Adipose tissue secretes several molecules that may participate in metabolic cross-talk to other insulin-sensitive tissues. Thus, adipose tissue is a key endocrine organ that regulates insulin sensitivity in other peripheral insulin target tissues. We have studied the expression and acute insulin regulation of novel genes expressed in adipose tissue that are implicated in the control of whole body insulin sensitivity.

Research Methods and Procedures: Expression of adiponectin, c-Cbl—associated protein (CAP), 11-β hydroxysteroid dehydrogenase type 1 (11β-HSD-1), and sterol regulatory element binding protein (SREBP)-1c was determined in subcutaneous adipose tissue from type 2 diabetic and age- and BMI-matched healthy men by real-time polymerase chain reaction analysis.

Results: Expression of adiponectin, CAP, 11β-HSD-1, and SREBP-1c was similar between healthy and type 2 diabetic subjects. Insulin infusion for 3 hours did not affect expression of CAP, 11β-HSD-1, or adiponectin mRNA in either group. However, insulin infusion increased SREBP-1c expression by 80% in healthy, but not in type 2 diabetic, subjects.

Discussion: Our results provide evidence that insulin action on SREBP-1c is dysregulated in adipose tissue from type 2 diabetic subjects. Impaired insulin regulation on gene expression of select targets in adipose tissue may contribute to the pathogenesis of type 2 diabetes.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Evidence is emerging that there is a coordinated dysregulation of genes along metabolic pathways important for insulin action in type 2 diabetes (1). We have determined the expression and acute insulin regulation of several genes that have been identified to contribute to the regulation of insulin action. We have focused on adipose tissue because it is a key endocrine organ that regulates insulin sensitivity in other peripheral insulin target tissues (2).

The importance of functional adipose tissue in normal metabolic homeostasis is highlighted by reports of severe whole body insulin resistance in individuals suffering from lipodystrophy (3). Conversely, excess adipose tissue, especially in the visceral compartment, also leads to whole body insulin resistance and predisposes to type 2 diabetes (4). In obese insulin-resistant and type 2 diabetic patients, plasma concentrations of adiponectin, an adipose-specific—secreted protein, are decreased (5, 6, 7). Interestingly, improvement in insulin sensitivity during troglitazone treatment is associated with a correlative increase in plasma and adipocyte adiponectin concentrations (8, 9). Even more striking is the finding that adiponectin treatment improves insulin sensitivity in lipodystrophic and obese mice (10). Collectively, these data provide evidence that adiponectin is an important regulator of whole body insulin sensitivity.

c-Cbl—associated protein (CAP)1 is integral in a novel pathway that has been proposed to mediate insulin signaling to glucose transport (11). CAP recruits the proto-oncogene product c-Cbl to the insulin receptor. On phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched membrane fraction by forming a ternary complex with flotillin. Overexpression of mutant CAP that is unable to complex with either flotillin or Cbl completely blocks insulin-stimulated glucose transport and glucose transport protein-4 (GLUT4) translocation in 3T3-L1 adipocytes (12). Thus, the CAP-Cbl pathway provides an alternative insulin-signaling pathway that is parallel to the insulin receptor substrate-phosphatidylinositol (PI) 3-kinase pathway.

The enzyme 11β-hydroxysteroid dehydrogenase type-1 (11β-HSD-1) catalyzes the interconversion of cortisone to active cortisol. Overexpression of 11β-HSD-1, specifically in adipose tissue, leads to visceral obesity and insulin resistance (13). Conversely, 11β-HSD-1 null mice have improved glucose tolerance and lipid profile (14), and treatment of spontaneously hyperglycemic KKAy mice with a pharmacological inhibitor of 11β-HSD-1 decreases blood glucose and serum insulin concentrations (15). Sterol regulatory element binding protein-1c (SREBP-1c) is a transcription factor that regulates the expression of numerous genes involved in glucose and lipid metabolism (16, 17, 18). Collectively, these data suggest important roles for 11β-HSD-1 and SREBP-1c in the regulation of whole body insulin sensitivity.

An important facet of insulin action is the regulation of expression of key genes in target tissues (19). Insulin increases the mRNA concentrations of p85α regulatory subunit of phosphatidylinositol 3-kinase, hexokinase II, GLUT4, and SREBP-1c, and these responses are blunted in skeletal muscle and adipose tissue from type 2 diabetic subjects (1). Thus, alterations in the transcriptional control of gene expression may contribute to the pathogenesis of type 2 diabetes. This study was undertaken to examine whether the expression and acute insulin regulation of adiponectin, CAP, 11β-HSD-1, and SREBP-1c are altered in subcutaneous adipose tissue from type 2 diabetic subjects.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Subjects

The study protocol was reviewed and approved by the institutional ethical committee of the Karolinska Institute, and informed consent was received from all subjects before participation. Subjects with a normal resting electrocardiogram; a normal blood count; and normal kidney-, liver-, and thyroid functions were studied. Non-diabetic subjects with impaired glucose tolerance, determined by 2-hour plasma glucose concentration ≥7.8 mM after a standard 75-g oral glucose tolerance test (20); smokers, and subjects using anti-hypertensive medication (β-blocking agent, angiotensin converting enzyme-inhibitors, Ca2+-inhibitors or diuretics) were excluded. Diabetic subjects were treated with diet (n = 1), sulfonylurea (n = 4), metformin (n = 1), a combination of sulfonylurea and metformin (n = 1), or insulin only (n = 1). The mean duration of diabetes was 4 years (range, 2 to 8 years). All subjects were instructed to avoid strenuous exercise for 72 hours before the study. Subjects reported to the laboratory after an overnight fast, and in the case of diabetic patients, before administration of any anti-diabetic medication.

Euglycemic Hyperinsulinemic Clamp and Adipose Tissue Biopsy

Whole body insulin-stimulated glucose disposal was determined using the euglycemic hyperinsulinemic (insulin infusion, 40 mU/m2 per minute for 180 minutes) clamp technique (21, 22). Glucose infusion rate required to maintain euglycemia during the last hour (120 to 180 minutes) of the clamp was used as a measure of whole body insulin sensitivity. Subcutaneous adipose tissue biopsies were taken by needle aspiration (23) under local anesthesia (mepivacain chloride, 5 mg/mL) in the basal state and after 180 minutes of insulin infusion.

Real-Time Polymerase Chain Reaction Analysis of Gene Expression

Total RNA was isolated from adipose tissue samples using the RNAeasy kit (Qiagen, Hilden, Germany). The mRNA concentrations of target genes were measured by real-time polymerase chain reaction (PCR) (TaqMan; Applied Biosystems, Foster City, CA) using the standard curve method (user bulletin 2; ABI PRISM 7700 Sequence Detection System). The cDNA was prepared from total RNA samples using the TaqMan reverse transcription reagent. All samples were analyzed in triplicate, with primers and probe, TaqMan Universal PCR Master Mix, and cDNA. The ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) was used for analysis. Results were normalized to the endogenous β-microglobulin mRNA concentration. The sequences of primers and probe were designed using Primer Express software (Perkin Elmer, Boston, MA) and are shown in Table 1. In each case, the amplicon included a sequence to an exon—intron border in the genome. To verify the lack of contamination by genomic DNA, each sample was run in parallel under identical conditions but without the reverse transcription reagent. The control samples indicated that there was no genomic contamination in the total RNA preparation (data not shown).

Table 1. . Sequences of primers and probes
 SenseProbeAntisense
AdiponectinGGC CGT GAT GGC AGA GATCTG GTG AGA AGG GTG AGA AAG GAG ATC CAGAT GTC TCC CTT AGG ACC AAT AAG AC
CAPTCG CAG TGC CAC TGC AAAGC AGC GAA GAG TCA CCC CCG ATTG ATA GCT AAA TAA ATC TTG TGA GGT TTG
11β-HSD-1GCG AGG TCA AAA GAA ACT CTA CAGATGG TAT CCC ACT GCC TGG AGC TTG GCAA TGT AGT GTG CTG AGG CTG C
SREBP-1cGGG CGG GCG CAG ATCGG AGC CAT GGA TTG CAC TTT CGAGGT TGT TGA TAA GCT GAA GCA TGT

Biochemical and Anthropometric Analysis

Plasma glucose concentration was determined using the glucose oxidase method (Beckman Instruments, Fullerton, CA), plasma-free insulin and C-peptide concentrations were determined with commercial radioimmunoassays (Pharmacia, Uppsala, Sweden), and hemoglobin A1c was determined using an immunologic method. Maximal oxygen uptake (Vo2max) was determined on a bicycle ergometer on a separate occasion. Vo2max was measured continuously using a breath-by-breath data collection technique (Erich Jaeger, Hoechberg, Germany). Regional analysis of lean body mass, body fat, and bone mineral content was performed by DXA (Lunar, Madison, WI).

Statistical Analysis

Data are presented as mean ± SE. Student's paired and unpaired t tests were used in the analysis of paired and unpaired data, respectively. p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Subject Characteristics

Type 2 diabetic and non-diabetic (control) men were matched for age, BMI, percentage of body fat, and physical fitness (Table 2). Concentrations of serum total and high-density lipoprotein-cholesterol and serum triglycerides were comparable between non-diabetic and type 2 diabetic subjects. The glucose infusion rate needed to maintain euglycemia was 50% lower in type 2 diabetic subjects (p < 0.001, Table 2).

Table 2. . Subject characteristics
 Non-diabeticType 2 diabetic
  • Results are mean ± SEM (non-diabetic n = 9, type 2 diabetic n = 8)

  • *

    p < 0.01;

  • p < 0.05;

  • p < 0.001.

  • Glucose infusion rate was calculated during 120 to 180 minutes of the euglycemic hyperinsulinemic clamp.

Age (years)57 ± 259 ± 1
BMI (kg/m2)26.9 ± 0.627.9 ± 1.1
Waist (cm)97 ± 2101 ± 3
Hip (cm)100 ± 1101 ± 2
Body fat (%)23.6 ± 1.525.9 ± 2.2
Vo2max (mL/kg/min−1)31.7 ± 1.527.6 ± 3.0
Glucose infusion rate (μmol/kg/min)31.4 ± 1.415.8 ± 2.6
Plasma glucose (mM)5.8 ± 0.19.5 ± 0.6*
Insulin (mU/L)6 ± 114 ± 2
C-peptide (nM)0.67 ± 0.051.04 ± 0.11*
Hemoglobin Alc (%)4.7 ± 0.15.9 ± 0.4
Cholesterol (mM)5.5 ± 0.34.6 ± 0.3
High-density lipoprotein cholesterol (mM)1.21 ± 0.061.15 ± 0.08
Triglyceride (mM)1.7 ± 0.31.5 ± 0.3
Diabetes duration (years) 4 ± 1

Gene Expression in Adipose Tissue

The expression of the reference gene β-microglobulin was similar in control and type 2 diabetic subjects and was not modified by insulin infusion for 180 minutes in either group (data not shown). Thus, the expression of other target genes was normalized to β-microglobulin levels. The expression of adiponectin, CAP, 11β-HSD-1, and SREBP-1c was similar between control and type 2 diabetic subjects in the basal state (Table 3). Insulin infusion for 180 minutes did not modify the expression of adiponectin, CAP, or 11β-HSD-1 mRNA (Table 3). However, insulin infusion increased the expression of SREBP-1c mRNA 80% in non-diabetic subjects (p < 0.05) but not in type 2 diabetic subjects (Figure 1).

Table 3. . Adiponectin, CAP, and 11β-HSD-1 mRNA in adipose tissue before (basal) and after 180 minutes of insulin infusion
mRNANon-diabeticType 2 diabetic
 Basal180 minutesBasal180 minutes
  1. Results are mean ± SEM arbitrary units (non-diabetic n = 9, type 2 diabetic n = 8).

Adiponectin0.35 ± 0.050.77 ± 0.250.83 ± 0.360.67 ± 0.25
CAP1.89 ± 0.532.29 ± 0.553.15 ± 1.062.25 ± 0.61
11β-HSD-10.92 ± 0.240.98 ± 0.241.74 ± 0.451.21 ± 0.23
image

Figure 1. Effect of physiological hyperinsulinemia on SREBP-1c mRNA concentration in subcutaneous adipose tissue from type 2 diabetic (▴) and non-diabetic (▵) subjects. Solid lines show mean values.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Adipose tissue plays a major role in the regulation of insulin sensitivity. Both excess adipose tissue mass and lack of adipose tissue are associated with impaired glucose metabolism (4). For example, when adipogenesis is impaired by expression of dominant negative A-ZIP/F (24), pre-adipocyte marker Pref-1 (25), or induction of a combined deficiency of IRS-1 and IRS-3 (26), ensuing phenotype of lipodystrophy with impaired glucose tolerance and decreased insulin sensitivity arises. Conversely, in A-ZIP/F mice, these metabolic abnormalities can be corrected by implantation of adipose tissue (27) or by therapy with the adipose-derived proteins, adiponectin and leptin (10). Thus, functional adipose tissue is needed for normal insulin sensitivity, highlighting the importance of adipose-derived factors in mediating metabolic cross-talk with other insulin-sensitive tissues.

Adiponectin is a novel, adipose-specific protein abundantly present in the circulation that improves insulin sensitivity in animal models (10). In skeletal muscle, adenosine monophosphate-activated protein kinase activation by adiponectin results in increased glucose uptake and lipid oxidation (28, 29). However, direct metabolic effects of adiponectin in human tissues are still unknown. Plasma adiponectin concentrations are decreased in obese and type 2 diabetic subjects (6, 7). Here we report that adipose tissue expression of adiponectin mRNA is similar between type 2 diabetic patients and age- and BMI-matched normal glucose tolerant subjects. Reduced subcutaneous adipose tissue adiponectin mRNA levels have been observed in non-diabetic obese compared with non-diabetic lean subjects (30). Thus, obesity, but not diabetes per se, may lead to reduced adipose tissue adiponectin expression.

CAP (11) is integral in a novel pathway that mediates insulin signaling to glucose transport. Overexpression of mutant CAP blocks insulin-stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes (12), highlighting the importance of this pathway in insulin action. Moreover, CAP is the first insulin-signaling molecule that is up-regulated by peroxisome proliferator-activated receptor γ (PPARγ) agonists and thus may play a role in insulin sensitization induced by these regimens (31). In this report, CAP mRNA expression in subcutaneous adipose tissue was similar between type 2 diabetic and control subjects. Therefore, changes in CAP expression in subcutaneous adipose tissue are not likely to contribute to type 2 diabetes.

Obese subjects are likely to have impaired hepatic 11β-HSD-1 activity, because they excrete a greater proportion of glucocorticoid as metabolites of cortisone rather than cortisol (32). Furthermore, they convert less cortisone to cortisol after oral administration. In contrast, 11β-HSD-1 activity in subcutaneous adipose tissue is increased. Because overexpression of 11β-HSD-1 in adipose tissue leads to insulin resistance and visceral obesity in rodents (13), enhanced activity of 11β-HSD-1 in adipose tissue may contribute to reduced whole body insulin action. In agreement, an 11β-HSD-1 deficiency is associated with an improved lipid profile and hepatic insulin sensitization in experimental animals (14). Furthermore, treatment of obese diabetic KKAy mice with a small molecule, nonsteroidal, isoform-selective inhibitor of 11β-HSD-1 lowered hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA, blood glucose, and serum insulin concentrations compared with vehicle-treated mice, whereas hepatic 11β-HSD-1 mRNA, liver function marker enzyme expression (aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatases), daily food intake, and body weight were not altered by the treatment (15). In contrast, insulin-sensitizing therapy with PPARγ antidiabetic agents down-regulates 11β-HSD-1 expression in adipose tissue (33). Here we report that expression of subcutaneous adipose tissue 11β-HSD-1 was similar between type 2 diabetic and normal glucose tolerant subjects, despite a marked difference in insulin sensitivity between the groups. Whereas a cautionary note can be raised in the interpretation of gene expression data with respect to protein expression and enzymatic activity, our findings suggest that obesity per se and not insulin sensitivity may be the major regulator of 11β-HSD-1 expression in adipose tissue.

The SREBP family of transcription factors regulates several genes involved in glucose use and lipogenesis. Adipose tissue—specific overexpression of the nuclear form of SREBP-1a (nSREBP-1a) leads to enlarged, fully differentiated white and brown adipocytes that have increased fatty acid synthesis and enhanced fatty acid secretion (34). nSREBP-1a transgenic mice develop fatty livers as a consequence of increased production and release of fatty acids from adipocytes, but not diabetes. In contrast, adipose tissue—specific overexpression of the alternative SREBP-1 isoform, nSREBP1-c, inhibits adipocyte differentiation (35). nSREBP-1c mice exhibit many of the features of congenital generalized lipodystrophy, including fatty livers from birth, elevated plasma triglyceride levels later in life, marked insulin resistance, and type 2 diabetes. Thus, SREBP isoforms have a distinct role in regulating adipocyte fat metabolism. In most cells, SREBP-1c is predominant. SREBP-1c expression in adipose tissue was similar between type 2 diabetic and non-diabetic subjects. Insulin induced a 1.8-fold increase in the expression of SREBP-1c mRNA in adipose tissue from healthy subjects, whereas type 2 diabetic subjects were insulin resistant for this target. This defect in dynamic regulation of SREBP-1c expression by insulin is in agreement with a previous study, where a defect in insulin-mediated increase in gene expression of not only SREBP-1c, but also hexokinase II, GLUT4, and p85α regulatory subunit of phosphatidyl inositol 3-kinase, was observed (1). Collectively, these data support the hypothesis that insulin resistance in type 2 diabetes involves defects in the regulation of gene expression. Because SREBP-1c is a major transcriptional regulator of lipogenic genes, such as fatty acid synthetase (16), lipoprotein lipase (36), and PPARγ (37), dysregulation of SREBP-1c expression upon insulin stimulation is likely to contribute to impaired fatty acid metabolism in the postprandial state. This may be a contributing factor to impaired postprandial lipidemia in type 2 diabetes (38), with more fatty acids available for deposition in muscle and liver, which would then lead to impaired insulin action in these tissues.

In conclusion, subcutaneous adipose tissue mRNA expression of adiponectin, CAP, 11β-HSD-1, and SREBP-1c is similar between type 2 diabetic and healthy subjects. Insulin increases SREBP-1c mRNA in adipose tissue from healthy subjects, but this response is impaired in type 2 diabetic subjects. Thus, defects in insulin regulation of gene expression of select targets are likely to contribute to the pathogenesis of type 2 diabetes. SREBP-1c is one target that is dysregulated in adipose tissue from type 2 diabetic subjects.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This study was supported by grants from the Swedish Medical Research Council, the Swedish Diabetes Association, the Foundation for Scientific Studies of Diabetology, and Novo-Nordisk Foundation. H.A.K. was supported by fellowships from the Emil Aaltonen Foundation, Finnish Academy of Science (Grant 52841), Finnish Diabetes Research Foundation, Finnish Medical Foundation, Helsingin Sanomat Centennial Foundation, and the governmental subsidy for research of Helsinki University Central Hospital (“EVO”).

Footnotes
  • 1

    Nonstandard abbreviations: CAP, c-Cbl—associated protein; GLUT4, glucose transport protein 4; IRS, insulin receptor substrate; 11β-HSD-1, 11β-hydroxysteroid dehydrogenase type-1; SREBP, sterol regulatory element binding protein; Vo2max, maximal oxygen uptake; PPARγ, peroxisome proliferator-activated receptor γ nSREBP-1a, nuclear form of SREBP-1a.

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  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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