Britt G. Gabrielsson

Separation of human adipocytes by size: hypertrophic fat cells display distinct gene expression

Enlarged adipocytes are associated with insulin resistance and are an independent predictor of type 2 diabetes. To understand the molecular link between these diseases and adipocyte hypertrophy, we developed a technique to separate human adipocytes from an adipose tissue sample into populations of small cells (mean 57.6±3.54 pm) and large cells (mean 100.1±3.94 pm). Microarray analysis of the cell populations separated from adipose tissue from three subjects identified 14 genes, of which five immune‐related, with more than fourfold higher expression in large cells than small cells. Two of these genes were serum amyloid A (SAA) and transmembrane 4 L six family member 1 (TM4SF1). Real‐time RT‐PCR analysis of SAA and TM4SF1 expression in adipocytes from seven subjects revealed 19‐fold and 22‐fold higher expression in the large cells, respectively, and a correlation between adipocyte size and both SAA and TM4SF1 expression. The results were verified using immunohistochemistry. In comparison with 17 other human tissues and cell types by microarray, large adipocytes displayed by far the highest SAA and TM4SF1 expression. Thus, we have identified genes with markedly higher expression in large, compared with small, human adipocytes. These genes may link hypertrophic obesity to insulin resistance/type 2 diabetes.—Jernås, M., Palming, J., Sjöholm, K., Jennische, E., Svensson, P.‐A., Gabrielsson, B. G., Levin, M., Sjögren, A., Rudemo, M., Lystig, T. C., Carlsson, B., Carlsson, L. M. S., Lönn, M. Separation of human adipocytes by size: hypertrophic fat cells display distinct gene expression. FASEB J. 20, E832–E839 (2006)

Cardiac Insulin-like Growth Factor I and Growth Hormone Receptor Expression in Renal Hypertension

The aim of the present study was to investigate the role of insulin-like growth factor I in the development of cardiac hypertrophy in two-kidney, one clip hypertension by relating growth hormone receptor and insulin-like growth factor I receptor mRNA levels to insulin-like growth factor I gene transcription using a solution hybridization/RNase protection assay. Two-kidney, one clip hypertension was induced in male Wistar rats, and experiments were performed 2, 4, 7, and 12 days after surgery. Systolic blood pressure was elevated 2, 7, and 12 days after clipping (P < .001). Left ventricular weights were increased 2, 4, 7, and 12 days after surgery (P < .01). Associated with the rise in blood pressure, left ventricular insulin-like growth factor I mRNA was increased 2, 7, and 12 days after surgery (P < .01). Furthermore, growth hormone receptor and insulin-like growth factor I receptor gene expression increased specifically in the left ventricle of renal hypertensive rats (P < .05 and P < .001, respectively). Left ventricular growth hormone receptor mRNA peaked 7 days after induction of renal artery stenosis. These results show that insulin-like growth factor I, growth hormone receptor, and insulin-like growth factor I receptor mRNA increase in the pressure-overloaded left ventricle of two-kidney, one clip rats, suggesting a role for insulin-like growth factor I and the growth hormone/insulin-like growth factor I axis in the development of cardiac hypertrophy.

Neonatal losartan treatment suppresses renal expression of molecules involved in cell-cell and cell-matrix interactions.

Lack of neonatal angiotensin II type 1 receptor (AT(1)) stimulation produces renal abnormalities characterized by papillary atrophy and impaired urinary concentrating ability, but the mechanisms involved are still unclear. DNA microarray was used to identify genes that are differentially expressed in renal medulla in response to neonatal treatment with AT(1) receptor antagonist losartan (30 mg/kg per d), which commenced within 24 h after birth. The data showed that losartan treatment for 48 h downregulated 68 genes, approximately 30% of which encode various components of cytoskeleton and cytoskeleton-associated proteins, extracellular matrix, and enzymes involved in extracellular matrix maturation or turnover. With the use of immunohistochemistry and Western immunoblot, the microarray data were confirmed and it was demonstrated that losartan suppressed renal expression of syndecan 2, alpha-smooth muscle actin, MHC class II, and leukocyte type 12-lipoxygenase by day 4. In addition, losartan inhibited medullary expression of integrin alpha6 and caused relocalization of integrins alpha6 and alpha3. Moreover, losartan inhibited cell proliferation in medullary tubules by day 9, as detected by Ki-67 immunostaining. This study provides new data supporting the contention that a lack of AT(1) receptor stimulation results in abnormal matrix assembly, disturbed cell-cell and cell-matrix interactions, and subsequent abnormal tubular maturation. Moreover, regulation of the expression of leukocyte type 12-lipoxygenase and alpha-smooth muscle actin by the renin-angiotensin system in the immature kidney adds new knowledge toward the understanding of renal vascular development.

Effects of divalent metal ions on the uptake of glutamate and GABA from synaptosomal fractions

The effects of divalent metal ions on high affinity uptake glutamate and GABA were examined, using crude and purified synaptosomal fractions prepared from brains of DBA/2CBI. The uptake velocities of both amino acids are severely reduced in the presence of Cu2+, Fe2+ and Zn2+ but remain unaffected by Co2+.

Glucocorticoids and growth problems

Both acute stimulation and chronic inhibition of growth hormone (GH) release by glucocorticoids have been well documented in animals and humans ( 1 , 2 ) , but the exact roles and relative importance of the different sites of action for the control of GH secretion by adrenal steroids remain unclear. Evidence for the effects of exogenous glucocorticoids on GH synthesis within the somatotroph (3), as well as on somatostatin synthesis and output from the hypothalamus (4), has accumulated. Some of the differences in the results of these studies may be due to differences in dose and time of steroid exposure. Many in vitro systems show increased responses to glucocorticoids when compared with their basal activities in culture medium in the total absence of glucocorticoids; however, this may reflect a more general improvement in cellular protein synthesis rather than specific effects of such steroid treatments. On the other hand, deleterious effects of glucocorticoids are usually seen following exposure to pharmacologically high levels of steroid, and it is difficult to relate this to normal physiology.

Maternal Dietary Intake of Essential Fatty Acids Affects Adipose Tissue Growth and Leptin mRNA Expression in Suckling Rat Pups

We have previously shown that maternal intake of essential fatty acids during late gestation and lactation affects the level of serum leptin in pups. The aim of the present study was to investigate the effect of dietary essential fatty acids on leptin content in the milk of rat dams and leptin expression in white adipose tissue of pups during the suckling period. During late gestation and throughout lactation, rats were fed a control or an essential fatty acid–deficient (EFAD) diet. Milk of the EFAD dams contained more saturated and less polyunsaturated fatty acids compared with the control dams. Milk leptin levels were higher in the EFAD dams than in the control dams at 3 wk of lactation. The weight of inguinal white adipose tissue depots and the serum leptin levels of the EFAD pups were significantly lower than in the control pups during the whole suckling period. In addition, semiquantitative reverse transcriptase-PCR analysis of leptin mRNA levels in inguinal white adipose tissue showed a reduction in the EFAD pups compared with the control pups at 3 wk of age. We conclude that maternal dietary essential fatty acid intake affects serum leptin levels in pups by regulating both the amount of adipose tissue and the leptin mRNA expression.

Regulation of human aldoketoreductase 1C3 (AKR1C3) gene expression in the adipose tissue

Aldoketoreductase 1C3 (AKR1C3) is a functional prostaglandin F synthase and a negative modulator of the availability of ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ). AKR1C3 expression is known to be associated with adiposity, one of the components of the metabolic syndrome. The aim of this study was to characterize the expression of AKR1C3 in the adipose tissue and adipocytes and to investigate its potential role in the metabolic syndrome. Using microarray analysis and realtime PCR, we studied the expression of AKR1C3 in adipose tissue samples from obese subjects with or without metabolic complications, during very low calorie diet-induced weight loss, and its expression in isolated human adipocytes of different sizes. The adipose tissue AKR1C3 expression levels were marginally lower in obese subjects with the metabolic syndrome compared with the levels in healthy obese subjects when analyzed using microarray (p = 0.078) and realtime PCR (p < 0.05), suggesting a secondary or compensatory effect. The adipose tissue mRNA levels of AKR1C3 were reduced during and after dietinduced weight-loss compared to the levels before the start of the diet (p < 0.001 at all time-points). The gene expression of AKR1C3 correlated with both adipose tissue mRNA levels and serum levels of leptin before the start of the diet (p < 0.05 and p < 0.01, respectively). Furthermore, large adipocytes displayed a higher expression of AKR1C3 than small adipocytes (1.5-fold, p < 0.01). In conclusion, adipose tissue AKR1C3 expression may be affected by metabolic disease, and its levels are significantly reduced in response to dietinduced weight loss and correlate with leptin levels.

Molecular characterization of a local sulfonylurea system in human adipose tissue.

ATP-sensitive potassium (KATP) channels are present in many cell types and link cellular metabolism to the membrane potential. These channels are heterooctamers composed of two subunits. The sulfonylurea receptor (SUR) subunits are targets for drugs that are inhibitors or openers of the KATP channels, while the inwardly rectifying K+ (Kir) subunits form the ion channel. Two different SUR genes (SUR1 and SUR2) and two different Kir6.x genes (Kir6.1 and Kir6.2) have been identified. In addition, isoforms of SUR2, SUR2A and SUR2B, have been described. We have previously performed expression profiling on pooled human adipose tissue and found high expression of SUR2. Others have reported expression of SUR1 in human adipocytes. The aim of this study was to characterize the expression of the sulfonylurea receptor complex components in human adipose tissue.RT-PCR analysis, verified by restriction enzyme digestions and DNA sequencing, showed that SUR2B, Kir6.1 and α-endosulfine, but not SUR1, SUR2A or Kir6.2, are expressed in human adipose tissue. Real-time RT-PCR showed that SUR2B was expressed at higher levels in subcutaneous compared with omental adipose tissue in paired biopsies obtained from seven obese men (p < 0.05). Analysis of tissue distribution showed that SUR2B expression in adipose tissue was lower than that in muscle, similar to that in heart and liver, while the expression in pancreas was lower. The effect of caloric restriction was tested in obese men (n = 10) treated with very low calorie diet for 16 weeks, followed by a gradual reintroduction of ordinary food for two weeks. Biopsies were taken at week 0, 8 and 18. There was no consistent effect of weight reduction on SUR2B or Kir6.1 expression.We conclude that the necessary components for a local sulfonylurea system are expressed in human adipose tissue and that the sulfonylurea receptor complex in this tissue is composed of SUR2B and Kir6.1. The expression of SUR2B was higher in subcutaneous compared with omental adipose tissue and was not affected by weight loss.We conclude that the necessary components for a local sulfonylurea system are expressed in human adipose tissue and that the sulfonylurea receptor complex in this tissue is composed of SUR2B and Kir6.1. The expression of SUR2B was higher in subcutaneous compared with omental adipose tissue and was not affected by weight loss.

Identifying molecular effects of diet through systems biology: influence of herring diet on sterol metabolism and protein turnover in mice

Background Changes in lifestyle have resulted in an epidemic development of obesity-related diseases that challenge the healthcare systems worldwide. To develop strategies to tackle this problem the focus is on diet to prevent the development of obesity-associated diseases such as cardiovascular disease (CVD). This will require methods for linking nutrient intake with specific metabolic processes in different tissues. Methodology/Principal Finding Low-density lipoprotein receptor-deficient (Ldlr −/−) mice were fed a high fat/high sugar diet to mimic a westernized diet, being a major reason for development of obesity and atherosclerosis. The diets were supplemented with either beef or herring, and matched in macronutrient contents. Body composition, plasma lipids and aortic lesion areas were measured. Transcriptomes of metabolically important tissues, e.g. liver, muscle and adipose tissue were analyzed by an integrated approach with metabolic networks to directly map the metabolic effects of diet in these different tissues. Our analysis revealed a reduction in sterol metabolism and protein turnover at the transcriptional level in herring-fed mice. Conclusion This study shows that an integrated analysis of transcriptome data using metabolic networks resulted in the identification of signature pathways. This could not have been achieved using standard clustering methods. In particular, this systems biology analysis could enrich the information content of biomedical or nutritional data where subtle changes in several tissues together affects body metabolism or disease progression. This could be applied to improve diets for subjects exposed to health risks associated with obesity.

Postnatal deficiency of essential fatty acids in mice results in resistance to diet-induced obesity and low plasma insulin during adulthood

Our objective was to investigate the long-term metabolic effects of postnatal essential fatty acid deficiency (EFAD). Mouse dams were fed an EFAD diet or an isoenergetic control diet 4 days before delivery and throughout lactation. The pups were weaned to standard diet (STD) and were later subdivided into two groups: receiving high fat diet (HFD) or STD. Body composition, energy expenditure, food intake and leptin levels were analyzed in adult offspring. Blood glucose and plasma insulin concentrations were measured before and during a glucose tolerance test. EFAD offspring fed STD were leaner with lower plasma leptin and insulin concentrations compared to controls. EFAD offspring fed HFD were resistant to diet-induced obesity, had higher energy expenditure and lower levels of plasma leptin and insulin compared to controls. These results indicate that the fatty acid composition during lactation is important for body composition and glucose tolerance in the adult offspring.