Genotype and Tissue-Specific Effects on Alternative Splicing of the Transcription Factor 7-Like 2 Gene in Humans

Context: Noncoding single-nucleotide polymorphisms (SNPs) within the TCF7L2 gene are confirmed risk factors for type 2 diabetes, but the mechanism by which they increase risk is unknown. Objective: We hypothesized that associated SNPs alter TCF7L2 splicing and that splice forms have altered biological roles. Design: Splice forms and 5′ and 3′ untranslated regions were characterized in sc adipose, muscle, liver, HepG2 cells, pancreas, and islet. Isoform-specific transcript levels were quantified in sc adipose. Alternative splice forms were characterized in HepG2 liver cells under glucose and insulin conditions and in SGBS cells with differentiation. Major isoforms were characterized by transfection. Setting: The study was conducted at an ambulatory general clinical research center. Patients: Patients included 78 healthy, nondiabetic study subjects characterized for insulin sensitivity and secretion. Results: We identified 32 alternatively spliced transcripts and multiple-length 3′ untranslated region transcripts in adipose, muscle, islet, and pancreas. Alternative exons 3a, 12, 13, and 13a were observed in all tissues, whereas exon 13b was islet specific. Transcripts retaining exons 13 and 13a but not total TCF7L2 transcripts were significantly correlated with both obesity measures (P < 0.01) and rs7903146 genotype (P < 0.026) in sc adipose. Insulin (5–10 nm) suppressed all TCF7L2 isoforms in SGBS cells but suppressed exon 13a-containing isoforms most significantly (P < 0.001). The isoform distribution differed throughout SGBS cell differentiation. Isoforms with predicted early stop codons yielded stable proteins of the predicted size, bound β-catenin, and targeted correctly to the nucleus. Conclusions: Intronic TCF7L2 variants may regulate alternative transcript isoforms, which in turn may have distinct physiologic roles.

Maturation of Luteinizing Hormone (Gonadotropin-Releasing Hormone) Secretion across Puberty: Evidence for Altered Regulation in Obese Peripubertal Girls

Context: Peripubertal obesity (body mass index-for-age ≥ 95%) in girls is associated with hyperandrogenemia. LH likely contributes to this relationship, but overnight LH secretion in obese girls is poorly characterized. Objective: The aim of the study was to evaluate LH pulse characteristics in obese girls throughout pubertal maturation. Design: We conducted a cross-sectional analysis. Setting: The study was performed in a general clinical research center. Participants: Eight nonobese and five obese Tanner 1–2 girls participated, as well as 32 nonobese and 12 obese Tanner 3–5 girls. Intervention: Blood samples were collected every 10 min overnight (from 1900 to 0700 h). Main Outcome Measures: LH pulse frequency, amplitude, and mean LH were measured in three 4-h time blocks (block 1, 1900–2300 h; block 2, 2300–0300 h; and block 3, 0300–0700 h). Results: Tanner stage 1–2 nonobese girls demonstrated nocturnal increases of LH frequency (P < 0.01, block 1 vs. 2) and mean LH (P < 0.05, block 1 vs. 2 and 3). Obese Tanner 1–2 girls had lower 12-h LH frequency and LH amplitude (P < 0.05 for both), with no overnight changes of LH pulse parameters. Compared to normal, LH frequency was elevated in Tanner 3–5 obese girls (P < 0.01 in all blocks), whereas LH amplitude was low (P < 0.05 in all blocks). Overnight increases of LH amplitude were observed in nonobese Tanner 3–5 girls (P < 0.0001), but not in obese Tanner 3–5 girls. Conclusions: Obesity in prepubertal and early pubertal girls is associated with reduced LH secretion and reduced nocturnal changes of LH. In later pubertal girls, obesity is linked with reduced LH amplitude, but elevated LH frequency; the latter may reflect effects of hyperandrogenemia.

Effects of Continuous Versus Intermittent Exercise, Obesity, and Gender on Growth Hormone Secretion

Context: Obesity attenuates spontaneous GH secretion and the GH response to exercise. Obese individuals often have low fitness levels, limiting their ability to complete a typical 30-min bout of continuous exercise. An alternative regimen in obese subjects may be shorter bouts of exercise interspersed throughout the day. Objective: The objective of the study was to examine whether intermittent and continuous exercise interventions evoke similar patterns of 24-h GH secretion and whether responses are attenuated in obese subjects or affected by gender. Design: This was a repeated-measures design in which each subject served as their own control. Setting: This study was conducted at the University of Virginia General Clinical Research Center. Subjects: Subjects were healthy nonobese (n = 15) and obese (n = 14) young adults. Interventions: Subjects were studied over 24 h at the General Clinical Research Center on three occasions: control, one 30-min bout of exercise, and three 10-min bouts of exercise. Main Outcome Measures: Twenty-four hour GH secretion was measured. Results: Compared with unstimulated 24-h GH secretion, both intermittent and continuous exercise, at constant exercise intensity, resulted in severalfold elevation of 24-h integrated serum GH concentrations in young adults. Basal and pulsatile modes of GH secretion were attenuated both at rest and during exercise in obese subjects. Conclusions: The present data suggest that continuous and intermittent exercise training should be comparably effective in increasing 24-h GH secretion.

Evidence for Acyl-Ghrelin Modulation of Growth Hormone Release in the Fed State

Context: The timing and frequency of GH secretory episodes is regulated by GHRH and somatostatin. This study provides evidence for amplification of these GH pulses by endogenous acyl-ghrelin. Design: Blood was sampled every 10 min for 26.5 h during a fed admission with standardized meals and also during the final 24 h of a 61.5-h fast. GH secretion profiles were derived from deconvolution of 10-min sampling data, and full-length acyl-ghrelin levels were measured using a newly developed two-site sandwich assay. Setting: The study was conducted at a university hospital general clinical research center. Participants: Participants included eight men with mean (± sd) age 24.5 ± 3.7 yr (body mass index 24 ± 2.1 kg/m2). Results: Correlations were computed between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-min interval preceding each GH burst. In the fed state, the peak correlations were positive for all subjects and significantly higher than in the fasting state when acyl-ghrelin levels declined [mean (± sem): 0.7 (0.04) vs. 0.29 (0.08), P = 0.017]. In addition, long-term fasting was associated with an increase in the GH secretory pulse mass and amplitude but not frequency [fed vs. fasting pulse mass: 0.22 (0.05) vs. 0.44 (0.06) μg/liter, P = 0.002; amplitude: 5.2 (1.3) vs. 11.8 (1.9) μg/liter/min, P = 0.034; pulses per 24 h: 19.4 (0.5) vs. 22.0 (1.4), P = 0.1]. Conclusion: Our data support the hypothesis that under normal conditions in subjects given regular meals endogenous acyl-ghrelin acts to increase the amplitude of GH pulses.

Assessment of the Magnitude of Growth Hormone Hypersecretion in Active Acromegaly: Reliability of Different Sampling Models

Context: The pulsatility of GH secretion in acromegaly poses difficulty in ascertaining true daily GH milieu in patients with this disease. Intensive GH sampling [every 10–20 (Q10–20) min for 24 h] is not practical in clinical practice. Objective: Our objective was to ascertain reliability of abbreviated sampling protocols to reflect true 24-h mean GH concentrations in patients with acromegaly. Design: An analysis of previously obtained plasma GH profiles was performed. Setting: The analysis was performed at the General Clinical Research Center at the University of Michigan. Patients: A total of 115 GH profiles obtained in 94 patients with active acromegaly were examined. Intervention: Frequent blood sampling, i.e. Q10–20 min for 24 h, was performed. Main Outcome Measures: Concordance of 24-h mean GH concentrations derived from Q10- to 20-min samplings with abbreviated GH sampling schedules was performed. The study was planned after data collection. Results: All abbreviated schedules of GH sampling correlated well with the true 24-h plasma GH means (i.e. Q10- to 20-min sampling) (R = 0.93–0.98; P < 0.0001 for all). In the GH range more than 20 μg/liter, only 5 and 9-h means had R values more than 0.9. Single GH concentrations less than 1 μg/liter had a positive predictive value of only 0.29, and those with less than 2.5 μg/liter had a positive predictive value of 0.67 vs. their corresponding 24-h mean GH values of the same magnitude. Conclusions: The intensity of GH sampling in patients with acromegaly may vary depending on the nature of the required information. Investigators and clinicians should be aware of the limitations of the abbreviated GH sampling protocols in acromegaly.

Functional Significance of Polycystic-Size Ovaries in Healthy Adolescents

Context: The relevance of adult polycystic ovary criteria to adolescence is unclear. Objective: The objective was to determine the functional significance of polycystic-size ovaries (PSO) in healthy adolescents. Design/Setting/Participants/Interventions: Healthy 11- to 18-yr-old postmenarcheal volunteers (n = 22) were recruited and divided into groups with normal size ovaries (VNSO; n = 10) or a polycystic-size ovary (VPSO; n = 12). They were secondarily compared with adolescents with polycystic ovary syndrome (PCOS; n = 8) matched for gynecological age and a PSO. All underwent GnRH agonist (GnRHag), oral glucose tolerance, and ACTH1–24 testing in our General Clinical Research Center. Results: VPSO had a higher peak 17-hydroxyprogesterone (17PROG) response to GnRHag than VNSO (146 ± 14 ng/dl, mean ± sem, vs. 85 ± 11; P = 0.008), as well as larger ovaries (13.3 ± 0.7 cc vs. 8.5 ± 0.8 cc). VPSO peak 17PROG was elevated (>137 ng/dl) in 42% (5 of 12). However, VPSO and VNSO androgen levels were similar, with the exception of one VPSO subject who had hyperandrogenemia and thus met criteria for PCOS. VPSO were similar to VNSO in LH, FSH, estradiol, and adrenal androgenic function. Although the VPSO group resembled the PCOS group in their 17PROG response to the GnRHag test, they differed in having significantly smaller ovaries and lower body mass index and in lacking evidence of peripheral androgen excess and of insulin resistance. Conclusion: A PSO in asymptomatic adolescents seems typically to be a normal variant. However, about half have a subclinical PCOS type of ovarian dysfunction; it is unknown whether this indicates a genetic carrier state or a risk for anovulation.

Incidence of associated events during the performance of invasive procedures in healthy human volunteers

Metabolic investigations often utilize arteriovenous sampling and muscle biopsy. These investigations represent some risk to the subject. We examined 369 studies performed in the General Clinical Research Center between January 1994 and May 2003 for events related to femoral catheterization and muscle biopsies. Incidents were further examined by age (younger: 18–59 yr, n = 133; and older: 60–76 yr, n = 28). There were no clinically defined major complications associated with either procedure. The incidence of femoral catheter repositioning or reinsertion was higher in the older group (25.5 vs. 9.7%). There was no difference in the incidence of premature removal of catheters, ecchymosis or hematoma, or the persistence of pain after discharge. The occurrence of all incidents did not increase with multiple catheterizations. Muscle biopsy was associated with infrequent ecchymosis or hematoma in both groups (1.1 and 3.6% in younger and older groups, respectively). Both procedures entail a small likelihood of a vagallike response (3.3% overall), resulting in nausea, dizziness, and rarely a loss of consciousness. These results indicate that, in skilled hands and a defined clinical setting, the incidents associated with femoral catheterization and muscle biopsy in healthy volunteers are reasonable and largely controllable.