Susan A. Phillips

Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis

Adipose tissue plays important roles in regulating carbohydrate and lipid homeostasis, though less is known about the regulation of amino acid metabolism in adipocytes. Here we applied isotope tracing to pre–adipocytes and differentiated adipocytes to quantify the contributions of different substrates to tricarboxylic acid metabolism and lipogenesis. In contrast to proliferating cells that use glucose and glutamine for acetyl–coenzyme A (AcCoA) generation, differentiated adipocytes increased branched chain amino acid (BCAA) catabolic flux such that leucine and isoleucine from media and/or protein catabolism accounted for as much as 30% of lipogenic AcCoA pools. Medium cobalamin deficiency caused methylmalonic acid accumulation and odd–chain fatty acid synthesis. B12 supplementation reduced these metabolites and altered the balance of substrates entering mitochondria. Finally, inhibition of BCAA catabolism compromised adipogenesis. These results quantitatively highlight the contribution of BCAAs to adipocyte metabolism and suggest that BCAA catabolism plays a functional role in adipocyte differentiation.

Mechanisms of adiponectin regulation and use as a pharmacological target.

Adiponectin is an insulin-sensitizing and anti-inflammatory fat cell hormone that has immense potential as a therapeutic target for a multitude of obesity-associated diseases, including type 2 diabetes, NASH and atherosclerosis (Chandran M, Phillips SA, Ciaraldi T, Henry RR: Adiponectin: more than just another fat cell hormone?Diabetes Care 2003, 26:2442-2450). The adiponectin gene is located in chromosome 3q27, a susceptibility locus for T2DM and metabolic disorders (Saito K, Tobe T, Minoshima S, Asakawa S, Sumiya J, Yoda M, Nakano Y, Shimizu N, Tomita M: Organization of the gene for gelatin-binding protein (GBP28). Gene 1999, 229:67-73). Increased circulating levels of adiponectin are associated with improvement in the metabolic syndrome and reductions are strongly predictive of diabetes risk (Li S, Shin HJ, Ding EL, van Dam RM: Adiponectin levels and risk of type 2 diabetes: a systematic review and meta-analysis. JAMA 2009, 302:179-188. Extensive efforts have been made to understand how adiponectin levels can be elevated. The complex post-translational processing and secretion of adiponectin provides a rich area where pharmacologic manipulation may be developed to increase adiponectin levels in humans. Circulating adiponectin levels are increased by many commonly used drugs, such as statins, angiotensin converting enzyme (ACE) inhibitors, and thiazolidinediones (TZDs) providing an important opportunity to gain insight into the mechanisms underlying their effects. This review describes the cellular processes by which adiponectin is synthesized and secreted, current therapeutics known to affect this pathway and the potential for therapeutic manipulation in human subjects.

Adipose tissue depot and cell size dependency of adiponectin synthesis and secretion in human obesity.

Adiponectin is an insulin sensitizing fat cell (FC) hormone whose levels are related to adipose tissue (AT) mass and depot distribution. We hypothesized that the nature of AT expansion (hypertrophy vs. hyperplasia) contributes to obesity-related reductions in serum adiponectin and that this effect is influenced by the regional distribution of AT to subcutaneous (S) and visceral (V) depots. Thirteen obese subjects provided paired AT biopsies. Serum total and high molecular weight (HMW) adiponectin levels were determined by ELISA. Secretion was quantified following 24-h explant culture. FC size, number, % large, and % small FC were determined by microscopic analysis. Secretion of total adiponectin was highest by SAT (P = 0.008) and correlated more strongly with serum adiponectin (total: P = 0.015, r = 0.77; HMW: P = 0.005, r = 0.83) than did secretion by VAT (P = 0.05, r = 0.66 for both). FC size was greatest in SAT and correlated negatively with both serum (total: P = 0.01, r = −0.74; HMW: P = 0.03, r = −0.69) and secreted (total: P = 0.05, r = −0.72; HMW: P = 0.02, r = −0.87) adiponectin. The % small FC in SAT correlated positively with both serum (total: P = 0.006, r = 0.87; HMW: P = 0.009, r = 0.79) and secreted (total: P = 0.03, r = 0.75; HMW: P = 0.01, r = 0.92) adiponectin. VAT FC size correlated negatively with serum HMW adiponectin (P = 0.01, r = −0.76) but not with any measure of secretion. VAT had the greatest % small FC, which related positively to serum HMW (P = 0.004, r = 0.81) and to secreted total adiponectin (P = 0.02, r = 0.78). These studies indicate that differences in fat cell size and depot distribution of AT expansion are important influences on adiponectin in obesity.

Metabolic and Hormonal Changes Induced by Pioglitazone in Polycystic Ovary Syndrome: A Randomized, Placebo-Controlled Clinical Trial

CONTEXT Polycystic ovary syndrome (PCOS) is characterized by insulin resistance, compensatory hyperinsulinemia, increased prevalence of impaired glucose tolerance, and increased ovarian androgen biosynthesis. OBJECTIVE The aim of the study was to evaluate effects of pioglitazone on whole body insulin action and ovarian androgen biosynthesis in PCOS. DESIGN We performed a randomized placebo-controlled trial. SETTING The study was conducted at the Special Diagnostic and Treatment Unit of the Veterans Affairs Medical Center, San Diego, and the University of California, San Diego, General Clinical Research Center. PATIENTS OR OTHER PARTICIPANTS A total of 23 subjects with PCOS were evaluated at baseline and end of treatment. Six age- and body mass index-matched women without PCOS were normal controls for baseline evaluation. INTERVENTION Subjects with PCOS were randomized to oral placebo or pioglitazone 45 mg daily for 6 months. MAIN OUTCOME MEASURE(S) The primary outcome measures were whole body insulin action as measured by hyperinsulinemic euglycemic clamp and ovarian androgen biosynthesis as measured by leuprolide-stimulated production of 17-hydroxyprogesterone (17-OHP). RESULTS Compared with placebo, pioglitazone treatment significantly improved multiple measures of insulin action, including glucose disposal rate (P < 0.01), 2-h glucose during 75-g oral glucose tolerance test (P < 0.01), area under the curve glucose during oral glucose tolerance test (P < 0.01), serum adiponectin (P < 0.01), and fasting hyperinsulinemia (P < 0.01). Compared to placebo, pioglitazone treatment reduced the increment of leuprolide-stimulated 17-OHP (P < 0.02). Improvements in glucose disposal rate correlated with reductions in 17-OHP stimulation (P < 0.02). CONCLUSIONS Compared to placebo, pioglitazone treatment in PCOS was associated with improvements in insulin action and glucose homeostasis and ameliorated the hyperandrogenic ovarian response.

Selective regulation of cellular and secreted multimeric adiponectin by antidiabetic therapies in humans

Adiponectin, an insulin-sensitizing factor secreted from adipose tissue, is decreased in individuals with type 2 diabetes (T2D) and increased in response to thiazolidinedione (TZD) therapy. Changes in its secretion and assembly into higher-order forms affect insulin sensitivity. To determine the relative potency of TZDs on intra-adipocyte multimerization and secretion of adiponectin, we assessed the impact of in vivo low- or high-dose rosiglitazone treatment alone or combined with metformin in subjects with T2D. T2D subjects received high-dose rosiglitazone (8 mg/day), high-dose metformin (2,000 mg/day), or low-dose combination rosiglitazone-metformin therapy (4 mg + 1,000 mg/day) for 4 mo. All subjects were then switched to high-dose rosiglitazone-metformin combination therapy (8 mg + 2,000 mg/day) for another 4 mo. Low-dose rosiglitazone increased serum adiponectin, whereas the high dose increased both adipocyte content and serum adiponectin levels. TZDs selectively increased the percentage of circulating adiponectin in the potent, high-molecular-weight (HMW) form. No TZD effects were evident on multimer distribution in the cell. Expression of the chaperone protein ERp44, which retains adiponectin within the cell, was decreased by TZD treatment. No changes occurred in Ero1-Lalpha expression. Metformin had no effect on any of these measures. Increases in adiponectin correlated with improvements in insulin sensitivity. In vivo, TZDs have apparent dose-dependent effects on cellular and secreted adiponectin. TZD-mediated improvements in whole body insulin sensitivity are associated with increases in circulating but not cellular levels of the HMW adiponectin multimer. Finally, TZDs promote the selective secretion of HMW adiponectin, potentially, in part, through decreasing the expression of the adiponectin-retaining protein ERp44.

Adiponectin secretion and response to pioglitazone is depot dependent in cultured human adipose tissue

The subcutaneous (S) and visceral (V) adipose tissue (AT) depots are increasingly recognized as distinct. To test the hypothesis that depot differences exist for adiponectin, fresh and cultured human VAT and SAT from obese type 2 diabetic (T2D) and obese nondiabetic (ND) subjects was examined to determine whether differences in adiponectin content and secretion occurred as a function of depot studied, diabetic status, and response to thiazolidinedione treatment. VAT and SAT were obtained by biopsy and AT explants cultured in defined media for 7 days. Protein expression was assessed by Western blot. Adiponectin content of conditioned medium was determined by radioimmunoassay. Diabetic status had no effect on adiponectin secretion over days 0-2 of culture. In ND SAT, secretion fell over days 2-4 but was sustained at greater levels vs. T2D SAT. In both ND and T2D VAT, adiponectin secretion was low, similar to T2D SAT. Over the 7-day culture period, cellular adiponectin increased in ND SAT and VAT; it remained unchanged in T2D SAT and VAT. Pioglitazone increased adiponectin secretion and content in all SAT. Pioglitazone failed to increase adiponectin secretion from either ND or T2D VAT and increased cellular content only in ND VAT. AT depot differences exist in the secretion of adiponectin and responsiveness to thiazolidinedione treatment. These data suggest that SAT, not VAT, appears to be the major contributor to increased circulating adiponectin levels in response to pioglitazone treatment.

The Effect of Cysteamine Bitartrate on Adiponectin Multimerization in Non-Alcoholic Fatty Liver Disease and Healthy Subjects

OBJECTIVE To determine the effects of cysteamine on adiponectin multimerization in sera of patients with nonalcoholic fatty liver disease (NAFLD). STUDY DESIGN Sera from 10 children with biopsy-proven NAFLD treated with cysteamine were assayed for adiponectin multimers at baseline, after 24 weeks of treatment, and again 16 weeks after discontinuing treatment. Pretreatment sera from subjects with NAFLD and from adult controls without NAFLD controls (n = 8) were incubated in cysteamine and multimers were measured 1 hour later. A cysteamine/adiponectin multimer dose-response curve was created. RESULTS Following 24 weeks of cysteamine therapy, the mean percentage increase for high, medium (MMW), and low (LMW) molecular weight multimers and total adiponectin from baseline was 53% (P = .02), 19% (P = .02), 29.4% (P = .03), and 49.3% (P = .05), respectively. Levels returned to baseline at 16 weeks after stopping therapy, unlike hepatic transaminase levels which remained low. Sera from 0 week, incubated in cysteamine for 1 hour, showed a significant mean percent increase in LMW adiponectin levels and a mean percent reduction in MMW levels compared with baseline in adults with and without NAFLD. CONCLUSIONS Cysteamine impacts adiponectin multimerization. Long-term cysteamine therapy increases levels of all multimers, whereas, in vitro short-term exposure causes a rapid increase in LMW and reduction in MMW multimers in NAFLD and healthy controls. Cysteamine may be a potential therapeutic agent for conditions associated with insulin-resistance, oxidative stress, and depressed adiponectin levels.

Response of adiponectin and its receptors to changes in metabolic state after gastric bypass surgery: dissociation between adipose tissue expression and circulating levels

BACKGROUND Adiponectin is an adipokine with anti-atherogenic and insulin-sensitizing properties. Specific adiponectin receptors, adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2), are present in adipose tissue, indicating adiponectin might have autocrine/paracrine effects on its production or action. In addition, endoplasmic reticulum oxidoreductase 1-Lalpha might mediate regulation of its secretion. The study aim was to determine the subcutaneous adipose tissue (SAT) adiponectin gene and protein expression and their correlation to metabolic parameters during metabolically distinct times after gastric bypass surgery. METHODS A total of 12 morbidly obese male patients underwent SAT biopsy during gastric bypass surgery, active weight loss (negative energy state), and at weight stabilization (steady state energy). The SAT mRNA and protein content of adiponectin, AdipoR1 and AdipoR2, and endoplasmic reticulum oxidoreductase 1-Lalpha protein levels and the serum levels of adiponectin were assessed. RESULTS SAT adiponectin, AdipoR1, and AdipoR2 gene expression increased significantly at the negative energy state, with no further change at steady state energy (P<.05, P<.05, and P=.04, respectively), without significant increases in protein at any stage. Changes in SAT adiponectin protein correlated with changes in AdipoR1 and AdipoR2 during steady state energy (P=.003 and P=.002, respectively). Changes in SAT adiponectin expression did not correlate with those in circulating levels. Changes in endoplasmic reticulum oxidoreductase 1-Lalpha did not correlate with either SAT or circulating levels of adiponectin. CONCLUSION Our data indicate distinct functions of adiponectin receptors, AdipoR1 and AdipoR2, mediate the autocrine/paracrine actions of adiponectin. The lack of correlation between changes in SAT adiponectin gene and protein expression and its circulating levels suggests that adipose tissue synthesis and release of adiponectin are highly regulated pathways.

Mutational Analysis in Pediatric Thyroid Cancer and Correlations with Age, Ethnicity, and Clinical Presentation

Background: Well-differentiated thyroid cancer (WDTC) incidence in pediatrics is rising, most being papillary thyroid carcinoma (PTC). The objective of the study was to assess the prevalence of different mutations in pediatric WDTC and correlate the genotype with the clinical phenotype. Methods: This is a single-center retrospective study. Thyroid tissue blocks from 42 consecutive pediatric WDTC patients who underwent thyroidectomy between 2001 and 2013 were analyzed at Quest Diagnostics for BRAFV600E, RAS mutations (N,K,H), and RET/PTC and PAX8/PPARγ rearrangements, using validated molecular methods. Thyroid carcinomas included PTC, follicular thyroid carcinoma (FTC), and follicular variant of PTC (FVPTC). Results: Thirty-nine samples (29 females) were genotyped. The mean age at diagnosis was 14.7 years (range 7.9–18.4 years), and most were Hispanic (56.4%) or Caucasian (35.9%). The mean follow-up period was 2.9 years. Mutations were noted in 21/39 (53.8%), with both BRAFV600E (n = 9), and RET/PTC (n = 6) detected only in PTC. Mutations were detected in 2/5 FTC (PAX8/PPARγ and NRAS) and 3/6 FVPTC cases (PAX8/PPARγ). Of 28 PTC patients, 57.1% had mutations: 32.1% with BRAFV600E, 21.4% with RET/PTC, and 3.6% with NRAS. Of patients with BRAFV600E, 77.8% were Hispanic and 88.9% were >15 years, while all RET/PTC-positive patients were ≤15 years (p = 0.003). Tumor size, lymph node involvement, and distant metastasis at diagnosis (or soon after 131I ablation) did not vary significantly based on the mutation. Conclusions: BRAFV600E was the most common mutation, especially in older and Hispanic adolescents. A larger, ethnically diverse pediatric cohort followed long term will enable the genotypic variability, clinical presentation, and response to therapy to be better assessed.

Subcutaneous adipose tissue fatty acid desaturation in adults with and without rare adipose disorders

BackgroundElevated stearoyl-CoA desaturase activity has been described in obese states, with an increased desaturation index (DI) suggesting enhanced lipogenesis. Differences in the DI among various phenotypes of abnormal adiposity have not been studied. Abnormal accumulation of subcutaneous adipose tissue occurs in rare adipose disorders (RADs) including Dercums disease (DD), multiple symmetric lipomatosis (MSL), and familial multiple lipomatosis (FML). Examining the DI in subcutaneous fat of people with DD, MSL and FML may provide information on adipose tissue fatty acid metabolism in these disorders. The aims of this pilot study were: 1) to determine if differences in adipose tissue DIs are present among RADs, and 2) to determine if the DIs correlate to clinical or biochemical parameters.MethodsSubcutaneous adipose tissue was obtained from human participants with DD (n = 6), MSL (n = 5), FML (n = 8) and obese Controls (n = 6). Fatty acid composition was determined by gas chromatography/mass spectrometry. The DIs (palmitoleic/palmitic, oleic/stearic, vaccenic/stearic ratios) were calculated from the gas chromatogram peak intensities. SCD1 gene expression was determined. Spearmans correlations between the DIs and available clinical or biochemical data were performed.ResultsIn DD subjects, the vaccenic/stearic index was lower (p < 0.05) in comparison to Controls. Percent of total of the saturated fatty acid myristic acid was higher in DD compared with Controls and FML. Percent of monounsaturated vaccenic acid in DD trended lower when compared with Controls, and was decreased in comparison to FML. In MSL, total percent of the polyunsaturated fatty acids was significantly lower than in the Control group (p < 0.05). In the total cohort of subjects, the palmitoleic/palmitic and oleic/stearic DIs positively correlated with age, BMI, and percent body fat.ConclusionsThe positive associations between the DIs and measures of adiposity (BMI and percent body fat) support increased desaturase activity in obesity. The lower vaccenic/stearic DI in DD SAT compared with Controls suggests presence of other factors involved in fat accumulation in addition to lifestyle. Other mechanisms driving fat accumulation in DD such as inflammation or lymphatic dysfunction should be investigated.