Carol J. Homko

Increase in Endoplasmic Reticulum Stress–Related Proteins and Genes in Adipose Tissue of Obese, Insulin-Resistant Individuals

OBJECTIVE—To examine fat biopsy samples from lean insulin-sensitive and obese insulin-resistant nondiabetic individuals for evidence of endoplasmic reticulum (ER) stress. RESEARCH DESIGN AND METHODS—Subcutaneous fat biopsies were obtained from the upper thighs of six lean and six obese nondiabetic subjects. Fat homogenates were used for proteomic (two-dimensional gel and MALDI-TOF/TOF), Western blot, and RT-PCR analysis. RESULTS—Proteomic analysis revealed 19 differentially upregulated proteins in fat of obese subjects. Three of these proteins were the ER stress–related unfolded protein response (UPR) proteins calreticulin, protein disulfide-isomerase A3, and glutathione-S-transferase P. Western blotting revealed upregulation of several other UPR stress–related proteins, including calnexin, a membrane-bound chaperone, and phospho c-jun NH2-terminal kinase (JNK)-1, a downstream effector protein of ER stress. RT-PCR analysis revealed upregulation of the spliced form of X-box binding protein-1s, a potent transcription factor and part of the proximal ER stress sensor inositol-requiring enzyme-1 pathway. CONCLUSIONS—These findings represent the first demonstration of UPR activation in subcutaneous adipose tissue of obese human subjects. As JNK can inhibit insulin action and activate proinflammatory pathways, ER stress activation of JNK may be a link between obesity, insulin resistance, and inflammation.

Leptin in human pregnancy: The relationship with gestational hormones

OBJECTIVES The aims of the study were (1) to examine the relationship between leptin and placental hormones by measuring serial changes in serum levels of leptin during and after pregnancy and (2) to study the effects of several gestational hormones on leptin release from fully differentiated 3T3-L1 adipocyte cell cultures. STUDY DESIGN Serum levels of leptin were measured throughout pregnancy and at 3 months post partum in 29 healthy women and were also measured in 18 healthy women at delivery by cesarean section and on postpartum day 3. In addition, 3T3-L1 mouse adipocytes were incubated for 24 hours in media containing various reproductive hormones and leptin production was measured. RESULTS Serum leptin levels increased significantly (8.4 +/- 0.9 vs 13.5 +/- 1.5 ng/mL; P <.001) between the first 2 trimesters of pregnancy but not between the second and third trimesters. These changes in leptin did not correlate significantly with changes in body mass index. Leptin levels dropped significantly during the immediate postpartum period, from 34.1 +/- 4.9 at cesarean delivery to 7.3 +/- 1.4 ng/mL on postpartum day 3 (P <.001). Fasting insulin level did not correlate significantly with leptin level during pregnancy but did so during the postpartum period (r = 0.60; P <.05). Leptin secretion from 3T3-L1 adipocytes was increased significantly when cells were cultured with human chorionic gonadotropin (150%, P <.01) and also when they were cultured with estrogen (120%, P <.03). CONCLUSION The data suggest that leptin production by adipose tissue is stimulated by several hormones of pregnancy, which may contribute to the increased leptin levels observed during gestation.

Dietary vitamin E prophylaxis and diabetic embryopathy: Morphologic and biochemical analysis ☆ ☆☆ ★ ★★

OBJECTIVE In this study we sought to determine whether dietary supplementation with vitamin E, a known antioxidant, would reduce the incidence of diabetic embryopathy in an in vivo rat model. STUDY DESIGN Eighty-day-old Sprague-Dawley rats were assigned to one of five groups: two control groups (groups 1 and 2) and three diabetic groups (groups 3, 4, and 5). One group of controls (group 2) and one group of diabetic rats (group 4) received dietary supplements of vitamin E (440 mg/day). The other three groups (groups 1, 3, and 5) received a normal diet only. Group 5 received insulin therapy to control glucose levels. On day 6 of gestation diabetes was induced in groups 3, 4, and 5 with streptozotocin (65 mg/kg). Animals were killed on day 12; embryos were examined for size, protein content, evidence of malformations, and superoxide dismutase activity. RESULTS In both groups (groups 3 and 4) of diabetic rats the mean blood glucose level than was significantly higher in controls. Insulin-treated animals (group 5) had glucose levels that were comparable to those of controls. The unsupplemented diabetic group had a neural tube defect rate of 21.48% +/- 9.6% (percentage of neural tube defects per rat) and a resorption rate of 21.37% +/- 20.39% (percentage of resorptions per rat) as compared with rates in the supplemented diabetic group of 6.92% +/- 4.08% and 2.17% +/- 3.74%, respectively (p < 0.01). Groups 1, 2, and 5 had similar neural tube defect rates (6.63% +/- 5.0%, 5.01% +/- 4.87%, and 3.55% +/- 5.92%, respectively. Vitamin E levels, measured by high-performance liquid chromatography, were significantly higher in maternal serum and embryos in the supplemented groups (p < 0.001) than in controls. Superoxide dismutase activity was reduced in the diabetes groups and was not affected by vitamin E therapy. CONCLUSIONS Supplementation with the antioxidant vitamin E confers a significant protective effect against diabetic embryopathy and may potentially serve as a dietary prophylaxis in the future. We postulate that this protective effect is mediated by a reduction in the oxidative load induced by hyperglycemia.

Multifactorial basis of the syndrome of diabetic embryopathy

OBJECTIVE The aim of the current paper is to explore the multifactorial basis of diabetes-induced embryopathy. METHOD A review of the literature regarding congenital malformations was undertaken to elucidate new advances in our understanding of diabetic embryopathy. Data from both clinical and experimental studies were collected and analyzed. RESULTS Numerous investigators have demonstrated that hyperglycemia and other metabolic fuels produce teratogenic effects during organogenesis. However, the exact mechanism(s) involved have not been completely elucidated. We and others have shown that aberrant metabolic fuels including hyperglycemia and hyperketonemia are teratogenic and that these effects occur via the yolk sac which appears to be the target site of injury. Other proposed etiologic factors include nutrient deficient states in membrane lipids such as arachidonic acid and myo-inositol as well as the generation of excess free oxygen radicals. This review highlights the multiple theories that have been proposed and summarizes the experimental and clinical data which support a multifactorial basis. CONCLUSIONS Evidence suggests that although the teratogenic process in the diabetic pregnancy is multifactorial, it may operate via a common pathway. Prevention of malformations in offspring of diabetic rats is achieved by glycemic control during organogenesis. Similar results may be obtained in a hyperglycemic state, provided there is restoration of essential fatty acid/phospholipid deficiency state and normalization of excess free radicals which may be achieved through dietary supplementation of polyunsaturated fatty acids, myoinositol, or antioxidants. The latter approach offers great promise as an adjunct to periconceptional glycemic control and as a dietary prophylaxis against the syndrome of diabetic embryopathy.

Leptin Is Present in Human Cord Blood

It has recently been reported that the ob gene receptor was expressed on human and murine hematopoietic stem cells and that the ob gene product leptin stimulated hemato- and lymphopoiesis at the stem cell level. These findings suggest a role for leptin in hemato- and lymphopoiesis during fetal development. There is at present no evidence, however, that leptin is synthesized and released by the fetus. To investigate this possibility, we have measured plasma leptin concentrations in the cord blood of 78 newborn infants. We found that leptin was present in all 78 infants in concentrations comparable with those found in adults (0.6–55.7 ng/ml). Overall, plasma leptin concentrations in the cord blood of infants correlated with birth weight (r = 0.74, P < 0.001). These observations show that leptin is synthesized and released by fetal fat cells. In addition, they are compatible with the concept that leptin may play a role in human fetal hematopoiesis.

Longitudinal study of carbohydrate metabolism in healthy obese pregnant women.

OBJECTIVE To longitudinally characterize changes in insulin sensitivity in obese women during and after pregnancy. RESEARCH DESIGN AND METHODS Six glucose-tolerant obese women underwent a 4-h euglycemic-hyperinsulinemic (500–600 pmol/l) clamping during the second (22.5 ± 2 weeks [mean ± SD]) and third trimester (36.8 ± 0.9) of pregnancy and again 15.6 ± 1.4 weeks after delivery. Rates of total body glucose turnover (with [6.6−2H2]glucose) and oxidation (with indirect calorimetry) were measured. RESULTS There were no significant changes with respect to the action of insulin on rates of glucose disappearance (GRd), carbohydrate oxidation, or endogenous glucose production (EGP), comparing the second trimester of pregnancy with the nonpregnant (postpartum) state. The third trimester, however, was characterized 1) by reductions in insulin-stimulated GRd (−28%, P < 0.05, compared with the second trimester and −40%, P < 0.05, compared with postpartum); 2) by even larger reductions in insulin-stimulated carbohydrate oxidation (−46%, P < 0.05, compared with the second trimester and −54%, P < 0.02, compared with postpartum); and 3) by reduction of insulin suppression of EGP (−39% compared with −79% at the second trimester and −77% postpartum, P < 0.01). CONCLUSIONS Glucose-tolerant obese women developed peripheral was well as hepatic insulin resistance during the third trimester of pregnancy. These alterationswere reversed after delivery and appeared to be adaptive mechanisms to copewith the increased demand for glucose of the growing fetus.

Cardiovascular disease knowledge and risk perception among underserved individuals at increased risk of cardiovascular disease.

Background: Cardiovascular disease (CVD) risk factor awareness and knowledge are believed to be prerequisites for adopting healthy lifestyle behaviors. The purpose of this study was to examine knowledge of CVD risk factors and risk perception among individuals with high CVD risk. Methods: The sample consisted of inner city and rural medically underserved patients at high risk of CVD. To be eligible for the trial, subjects were required to have a 10% or greater CVD risk on the Framingham risk score. Knowledge of CVD was assessed with a 29-item questionnaire created for this study. Subjects also rated their perception of risk as compared with individuals of their own sex and age. Results: Data were collected from 465 subjects (mean [SD] age, 60.5 [10.1] years; mean [SD] Framingham risk score, 17.3% [9.5%]). The mean (SD) CVD knowledge score was 63.7% (14.6%), and mean (SD) level of risk perception was 0.35 (1.4). Men and women had similar Framingham risk scores, but women perceived their risk to be significantly higher than that of their male counterparts. Women were also more knowledgeable than men about CVD. Urban participants had significantly higher actual risks than did their rural counterparts (18.2% [10.7%] vs 16.0% [8.9%], respectively; P = .01) but were significantly less knowledgeable about heart disease and also perceived their risk to be lower. Conclusions: These results indicate a low perception of risk and cardiovascular knowledge especially among men and inner city residents. Innovative educational strategies are needed to increase risk factor knowledge and awareness among at-risk individuals.

Dietary polyunsaturated fatty acid prevents malformations in offspring of diabetic rats.

OBJECTIVE The purpose of the current study was to determine whether a dietary source of arachidonic acid could serve as a pharmacologic prophylaxis to obviate the teratogenic effects of hyperglycemia. STUDY DESIGN Eighty-day-old Sprague-Dawley rats were mated, and after conception were randomly allocated to five groups: two groups were nondiabetic normal controls and three groups had diabetes experimentally induced with streptozocin. Of the two control groups, one was fed a normal diet (group 1) and the other group (group 2) received a normal diet and 1.0 ml of safflower oil, a polyunsaturated fatty acid known to increase serum arachidonic acid levels. In the three diabetic groups (groups 3, 4, and 5) glucose levels were allowed to remain > 350 mg/dl by withholding daily insulin therapy. Group 3 received a normal diet without supplementation; group 4 received a normal diet plus normal saline solution sham feedings, whereas group 5 received a normal diet supplemented with 1.0 ml of safflower oil. The oral agents (normal saline solution and polyunsaturated fatty acid) were administered with a tuberculin syringe. RESULTS Diabetic rats not receiving insulin therapy and receiving normal diets produced offspring with malformation rates of 20% compared with control rates of 4.8%. Supplemental normal saline solution or safflower oil given orally to controls did not alter the growth or malformation rates. These rates were similarly unaffected in the diabetic rats receiving oral supplementation of normal saline solution. However, with safflower oil supplementation to diabetic rats the incidence of neural tube defects was decreased from 20.0% to 7.6% (p < 0.0001). An inverse relationship was observed between the malformation rate and the serum arachidonic acid level: 17.83 (SD 5.84 micrograms/ml) in the nondiabetic controls, with a malformation rate of 4.8%, versus 14.18 (SD 2.58 micrograms/ml) in the diabetic rats, with a malformation rate of 20.0% (p < 0.05). With safflower oil supplementation serum levels of arachidonic increased from 14.18 +/- 2.58 micrograms/ml to 19.99 +/- 7.99 micrograms/ml (p < 0.05); this was associated with a concomitant decline in the malformation rate. CONCLUSION These data demonstrate that diabetic embryopathy is associated with a deficiency state in essential fatty acid, corroborating our previous in vitro findings. Furthermore, the use of a dietary polyunsaturated fatty acid that specifically increases arachidonic levels significantly reduced the incidence of diabetic embryopathy. These findings may serve as a basis for developing strategies of pharmacologic prophylaxis against diabetes-induced congenital malformations.

Methods of measuring insulin sensitivity.

Insulin resistance is a component of several health disorders, most notably impaired glucose tolerance and type 2 diabetes mellitus. Insulin-resistant individuals have an impaired biological response to the usual action of insulin; that is, they have reduced insulin sensitivity. Various methods are used to assess insulin sensitivity both in individuals and in study populations. Validity, reproducibility, cost, and degree of subject burden are important factors for both clinicians and researchers to consider when weighing the merits of a particular method. This article describes several in vivo methods used to assess insulin sensitivity and presents the advantages and disadvantages of each.

The Role of Free Radicals and Membrane Lipids in Diabetes-Induced Congenital Malformations

Objective: The incidence of major congenital malformations is approximately 6-9% in pregnancies complicated by diabetes mellitus. This incidence is 3-4-fold higher than that in the general population. Congenital malformations are now ranked as the leading cause of death in the offspring of women with diabetes. The precise mechanism(s) by which these anomalies are induced is unknown. It is also not clear what predisposes women to deliver malformed infants, which infants are at risk, and why some are spared even when exposed to presumably high risk conditions. The purpose of this report is to determine, from the literature, the primary etiologic factors associated with diabetes-induced embryopathy and its prevention. Methods: A review of the current literature regarding congenital malformations in diabetic pregnancies was conducted to elucidate dominant concepts in the pathogenic mechanism(s) of these anomalies and to discuss current and future strategies for their prevention. Results: Numerous investigators have demonstrated that hyperglycemia has a teratogenic effect during organogenesis. However, the exact mechanisms involved have not been completely elucidated. Dietary supplementation of deficient substrates (arachidonic acid or myo-inositol), either in vitro or in vivo, has been shown to reduce the incidence of diabetes-related malformations in offspring of diabetic pregnant animals. In addition, free oxygen radical-scavenging enzymes and antioxidants aimed at reducing the excess load of radicals also result in a reduced malformation rate. Clinical evidence has demonstrated that the teratogenic effects of hyperglycemia may be obviated by maintaining euglycemia throughout organogenesis. Numerous studies have demonstrated that participation in a preconception care program can reduce the incidence of malformations in women with diabetes to the background rate. Unfortunately, less than 10% of women with diabetes currently enter these programs. Conclusions: Diabetic embryopathy remains the single most common lethal problem affecting diabetic pregnancies today. Although preconception planning and glycemic control can reduce the incidence of malformations, it is often difficult to get women to attend such programsand to achieve and maintain euglycemia. The use of dietary supplements, which presumably would override the teratogenic effects of aberrant metabolic fuels, holds great promise for the future as a prophylaxis against diabetic embryopathy.