Dr Laura Gathercole

Objective:Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome. Steroidhormones and bile acids are potent regulators of hepatic carbohydrate and lipid metabolism. Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates steroid hormones and catalyzes afundamental step in bile acid synthesis.Methods:Human liver biopsies were obtained from 34 obese patients and AKR1D1 mRNA expression levels weremeasured using qPCR. Genetic manipulation of AKR1D1 was performed in human HepG2 and Huh7 liver celllines. Metabolic assessments were made using transcriptome analysis, western blotting, mass spectrometry, clin-ical biochemistry, and enzyme immunoassays.Results:In human liver biopsies,AKR1D1expression decreased with advancing steatosis,fibrosis and inflamma-tion.Expression was decreasedin patients with type 2 diabetes. In human liver cell lines,AKR1D1knockdown de-creased primary bile acid biosynthesis and steroid hormone clearance. RNA-sequencing identified disruption ofkey metabolic pathways, including insulin action and fatty acid metabolism.AKR1D1knockdown increased he-patocyte triglyceride accumulation, insulin sensitivity, and glycogen synthesis, through increasedde novolipo-genesis and decreasedβ-oxidation, fueling hepatocyte inflammation. Pharmacological manipulation of bileacid receptor activation prevented the induction of lipogenic and carbohydrate genes, suggesting that the ob-served metabolic phenotype is driven through bile acid rather than steroid hormone availability.Conclusions:Genetic manipulation of AKR1D1 regulates the metabolic phenotype of human hepatoma cell lines,driving steatosis and inflammation. Taken together, the observation thatAKR1D1mRNA is down-regulated withadvancing NAFLD suggests that it may have a crucial role in the pathogenesis and progression of the disease.

Objective and Context. Increasing adiposity, ageing and tissue‐specific regeneration of cortisol through the activity of 11β‐hydroxysteroid dehydrogenase type 1 have been associated with deterioration in glucose tolerance. We undertook a longitudinal, prospective clinical study to determine if alterations in local glucocorticoid metabolism track with changes in glucose tolerance. Design, Patients, and Measurements. Sixty‐five overweight/obese individuals (mean age 50.3 ± 7.3 years) underwent oral glucose tolerance testing, body composition assessment, subcutaneous adipose tissue biopsy and urinary steroid metabolite analysis annually for up to 5 years. Participants were categorized into those in whom glucose tolerance deteriorated (“deteriorators”) or improved (“improvers”). Results. Deteriorating glucose tolerance was associated with increasing total and trunk fat mass and increased subcutaneous adipose tissue expression of lipogenic genes. Subcutaneous adipose tissue 11β‐HSD1 gene expression decreased in deteriorators, and at study completion, it was highest in the improvers. There was a significant negative correlation between change in area under the curve glucose and 11β‐HSD1 expression. Global 11β‐HSD1 activity did not change and was not different between deteriorators and improvers at baseline or follow‐up. Conclusion. Longitudinal deterioration in metabolic phenotype is not associated with increased 11β‐HSD1 activity, but decreased subcutaneous adipose tissue gene expression. These changes may represent a compensatory mechanism to decrease local glucocorticoid exposure in the face of an adverse metabolic phenotype.

Background and Aims

Alström syndrome (AS) is a recessive monogenic syndrome characterized by obesity, extreme insulin resistance and multi-organ fibrosis. Despite phenotypically being high risk of non-alcoholic fatty liver disease (NAFLD), there is a lack of data on the extent of fibrosis in the liver and its close links to adipose in patients with AS. Our aim was to characterize the hepatic and adipose phenotype in patients with AS.

Methods

Observational cohort study with comprehensive assessment of metabolic liver phenotype including liver elastography (Fibroscan®), serum Enhanced Liver Fibrosis (ELF) Panel and liver histology. In addition, abdominal adipose histology and gene expression was assessed. We recruited 30 patients from the UK national AS clinic. A subset of six patients underwent adipose biopsies which was compared with control tissue from nine healthy participants.

Results

Patients were overweight/obese (BMI 29.3 (25.95–34.05) kg/m2). A total of 80% (24/30) were diabetic; 74% (20/27) had liver ultrasound scanning suggestive of NAFLD. As judged by the ELF panel, 96% (24/25) were categorized as having fibrosis and 10/21 (48%) had liver elastography consistent with advanced liver fibrosis/cirrhosis. In 7/8 selected cases, there was evidence of advanced NAFLD on liver histology. Adipose tissue histology showed marked fibrosis as well as disordered pro-inflammatory and fibrotic gene expression profiles.

Conclusions

NAFLD and adipose dysfunction are common in patients with AS. The severity of liver disease in our cohort supports the need for screening of liver fibrosis in AS.

Background & Aims

Insulin resistance and lipotoxicity are pathognomonic in non-alcoholic steatohepatitis (NASH). Glucagon-like peptide-1 (GLP-1) analogues are licensed for type 2 diabetes, but no prospective experimental data exists in NASH. This study determined the effect of a long-acting GLP-1 analogue, liraglutide, on organ-specific insulin sensitivity, hepatic lipid handling and adipose dysfunction in biopsy-proven NASH.

Methods

Fourteen patients were randomised to 1.8 mg liraglutide or placebo for 12-weeks of the mechanistic component of a double-blind, randomised, placebo-controlled trial (ClinicalTrials.gov-NCT01237119). Patients underwent paired hyperinsulinaemic euglycaemic clamps, stable isotope tracers, adipose microdialysis and serum adipocytokine/metabolic profiling. In vitro isotope experiments on lipid flux were performed on primary human hepatocytes.

Results

Liraglutide reduced BMI (−1.9 vs. +0.04 kg/m2; p 

Conclusions

Liraglutide reduces metabolic dysfunction, insulin resistance and lipotoxicity in the key metabolic organs in the pathogenesis of NASH. Liraglutide may offer the potential for a disease-modifying intervention in NASH.

Context:

It is widely believed that glucocorticoids cause insulin resistance in all tissues. We have previously demonstrated that glucocorticoids cause insulin sensitization in human adipose tissue in vitro and induce insulin resistance in skeletal muscle.
 

Objective:

Our aim was to determine whether glucocorticoids have tissue-specific effects on insulin sensitivity in vivo.
 

Design:

Fifteen healthy volunteers were recruited into a double-blind, randomized, placebo-controlled, crossover study, receiving both an overnight hydrocortisone and saline infusion. The tissue-specific actions of insulin were determined using paired 2-step hyperinsulinemic euglycemic clamps incorporating stable isotopes with concomitant adipose tissue microdialysis.
 

Setting:

The study was performed in the Wellcome Trust Clinical Research Facility, Queen Elizabeth Hospital, Birmingham, United Kingdom.
 

Main Outcome Measures:

The sensitivity of sc adipose tissue to insulin action was measured.
 

Results:

Hydrocortisone induced systemic insulin resistance but failed to cause sc adipose tissue insulin resistance as measured by suppression of adipose tissue lipolysis and enhanced insulin-stimulated pyruvate generation. In primary cultures of human hepatocytes, glucocorticoids increased insulin-stimulated p-ser473akt/protein kinase B. Similarly, glucocorticoids enhanced insulin-stimulated p-ser473akt/protein kinase B and increased Insulin receptor substrate 2 mRNA expression in sc, but not omental, intact human adipocytes, suggesting a depot-specificity of action.
 

Conclusions:

This study represents the first description of sc adipose insulin sensitization by glucocorticoids in vivo and demonstrates tissue-specific actions of glucocorticoids to modify insulin action. It defines an important advance in our understanding of the actions of both endogenous and exogenous glucocorticoids and may have implications for the development and targeting of future glucocorticoid therapies.

Background: Antiretroviral (ARV) treatment has been associated with abnormalities in lipid and mitochondrial metabolism. We compared patterns of gene expression in the subcutaneous adipose tissue (SAT) of HIV-positive subjects before and after 18–24 months of ARV therapy with HIV-negative controls.

Methods: HIV patients naive to ARV were randomized to receive zidovudine (AZT), lamivudine (3TC) with efavirenz (EFV) or tenofovir disoproxil fumarate (TDF) with emtricitabine (FTC) and EFV. Healthy controls (n=15) were matched for age, ethnicity and gender. Patients on a regimen containing abacavir (ABC), 3TC and EFV for 18–24 months were also tested. Genes involved in adipocyte glucocorticoid, lipid and mitochondrial metabolism, and adipocyte differentiation, were profiled with real-time PCR.

Results: AZT led to increased visceral adipose tissue (VAT; P=0.012) and VAT:SAT ratio (P=0.036), whereas TDF increased SAT (P=0.047) and peripheral fat/lean body mass ratio (P=0.017). HIV treatment-naive patients had lower plasma lipoprotein lipase (LPL) activity (P=0.0001) versus controls (remaining below controls after ARV; P=0.038–0.0001). The overall pattern of gene expression was similar across all treatment groups, being most marked with AZT and least with TDF. There was up-regulation of peroxisome proliferator-activated receptor-γ coactivator-1α, uncoupling protein-2 and hexose 6-phosphate dehydrogenase, and down-regulation of nuclear respiratory factor-1, cytochrome oxidase B, cytochrome c oxidase-4, uncoupling protein-3, 11β-hydroxysteroid dehydrogenase type-1, glucocorticoid receptor-α, fatty acid synthase, fatty acid binding protein-4, LPL and hormone sensitive lipase (18–24 months post-treatment versus pretreatment levels and controls; P<0.05 to <0.0001).

Conclusions: The decreased expression of genes involved in lipid and mitochondrial metabolism 18–24 months post-ARV treatment in SAT of HIV patients, in conjunction with the increase in uncoupling protein-2 and decrease in cytochrome oxidase B gene expression, provides evidence of mitochondrial dysfunction and a shift to anaerobic metabolism within SAT in EFV-containing ARV regimens.

Background: Endogenous or exogenous glucocorticoid (GC) excess (Cushing's syndrome) is characterized by increased adiposity and insulin resistance. Although GCs cause global insulin resistance in vivo, we have previously shown that GCs are able to augment insulin action in human adipose tissue, contrasting with their action in skeletal muscle. Cushing's syndrome develops following chronic GC exposure and, in addition, is a state of hyperinsulinemia.

Objectives: We have therefore compared the impact of short- (24 h) and long-term (7 days) GC administration on insulin signalling in differentiated human adipocytes in the presence of low or high concentrations of insulin.
 

Results: Both short- (24 h) and long-term (7 days) treatment of chub-s7 cells with dexamethasone (Dex) (0.5 μm) increased insulin-stimulated pTyr612IRS1 and pSer473akt/PKB, consistent with insulin sensitization. Chronic high-dose insulin treatment induced insulin resistance in chub-s7 cells. However, treatment with both high-dose insulin and Dex in combination still caused insulin sensitization.
 

Conclusions: In this human subcutaneous adipocyte cell line, prolonged GC exposure, even in the presence of high insulin concentrations, is able to cause insulin sensitization. We suggest that this is an important mechanism driving adipogenesis and contributes to the obese phenotype of patients with Cushing's syndrome.

Context: Thyroid-associated ophthalmopathy (TAO) is a sight-threatening autoimmune disease in which de novo adipogenesis has been identified as a fundamental pathogenic mechanism. 11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) increases cortisol bioavailability and is pivotal in mediating glucocorticoid responses in adipose tissue and inflammation.

Objective: In this study we characterize 11β-HSD1 as a determinant of the adipogenic and inflammatory pathways in TAO orbital fat (OF) compared with normal OF.

Patients and Methods: OF was harvested from 46 TAO and 44 control patients undergoing orbital surgery. Samples were examined by a combination of immunohistochemistry, real-time RT-PCR, primary cell culture, specific enzyme assays, colorimetric proliferation assays, and bead-based ELISA.

Results: Glucocorticoid (glucocorticoid receptor-α,11β-HSD1, hexose-6-phosphate dehydrogenase) and inflammatory cytokines (IL-1β, IL-1 receptor, IL-6, TNF-α, TNF-α inductible protein, TGF-β2) target genes together with markers of late adipocyte differentiation (fatty-acid-binding-protein-4, glycerol-6-phosphate-dehydrogenase) were highly expressed in TAO whole OF (P < 0.05) compared with controls. Primary cultures of TAO OF stromal cells demonstrated greater 11β-HSD1 oxoreductase activity (P < 0.05), which was regulated by cytokines, most notably TNF-α (P < 0.01), compared with controls. Activity increased across differentiation, and this was most marked in TAO cells (P < 0.01). Similarly, stromal cell proliferation was limited by incubation with cortisol in TAO cells only. Furthermore, cortisone decreased IL-6 (P < 0.005), IL-8 (P < 0.05), and macrophage chemoattractant protein-1 (P < 0.05) production by cultured TAO cells only, an effect that was abrogated by inhibition of 11β-HSD1.

Conclusions: Induction of 11β-HSD1 activity and expression by inflammatory cytokines (TNF-α, IL-6) may enhance orbital adipocyte differentiation (adipogenesis) and limit proliferation in TAO. 11β-HSD1 may also have a role in regulating the local orbital inflammatory response.

OBJECTIVE Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11β-HSD1 inhibitors improve insulin sensitivity.

RESEARCH DESIGN AND METHODS Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11β-HSD1 inhibition upon insulin signaling and action.

RESULTS Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer307 insulin receptor substrate (IRS)-1. 11β-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11β-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer307 IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11β-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer307 IRS1 decreased and pThr308 Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression.

CONCLUSIONS Prereceptor facilitation of glucocorticoid action via 11β-HSD1 increases pSer307 IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11β-HSD1 inhibition decreases pSer307 IRS1, increases pThr308 Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.

Background: Abnormal lipid metabolism and cell oxidative mechanisms are reported in patients on antiretroviral treatment. We compared the expression of several key adipocyte genes in HIV-infected patients randomized to antiretroviral regimens containing zidovudine (AZT) or tenofovir disoproxil fumarate (TDF).

Methods: Subcutaneous fat was sampled from 32 HIV-positive treatment-naive patients before and 6 months after randomization to AZT/lamivudine/efavirenz (n=15) or TDF/emtricitabine/efavirenz (n=17) plus 15 HIV-negative matched controls. Expression of genes involved in adipocyte differentiation, lipid metabolism, mitochondrial function and glucocorticoid generation were profiled using real-time PCR. Lipoprotein lipase and hepatic lipase activity were assessed.

Results: Before treatment, 11β-hydroxysteroid dehydrogenase expression was down-regulated compared with controls. Following 6 months treatment with AZT, there was a significant increase in visceral adipose tissue (VAT; P=0.02) and the ratio of VAT to subcutaneous adipose tissue (P=0.008), down-regulation of cytochrome B (P=0.003) and cytochrome oxidase (COX)-3 gene expression (P=0.03), up-regulation of NADH dehydrogenase (P=0.008) and nuclear-encoded COX-4 (complex IV) gene expression (P=0.012). Genes involved with adipocyte cortisol generation, fatty acid metabolism and the tricarboxylic acid cycle were up-regulated. In the TDF-treated patients, there was no significant change in regional body fat or mitochondrial genes compared with pretreatment values. Changes in the expression of genes involved with cortisol and fatty acid metabolism were less marked with TDF.

Conclusions: Interference with the mitochondrial electron transport chain appears to occur early in an AZT-containing regimen and occurs at a time when there is increased visceral fat and up-regulation of genes involved with adipocyte differentiation and fatty acid flux.

Context: Glucocorticoid (GC) excess is characterized by central obesity, insulin resistance, and in some cases, type 2 diabetes. However, the impact of GC upon insulin signaling in human adipose tissue has not been fully explored.
 

Objective: We have examined the effect of GC upon insulin signaling in both human sc primary preadipocyte cultures and a novel human immortalized sc adipocyte cell line (Chub-S7) and contrasted this with observations in primary cultures of human skeletal muscle.
 

Design and Setting: This is an in vitro study characterizing the impact of GC upon insulin signaling in human tissues.
 

Patients: Biopsy specimens were from healthy volunteers who gave their full and informed written consent.
 

Interventions: Combinations of treatments, including GC, RU38486, and wortmannin, were used.
 

Main Outcome Measures: Insulin signaling cascade gene and protein expression and insulin-stimulated glucose uptake were determined.
 

Results: In human adipocytes, pretreatment with GC induced a dose-dependent [1.0 (control); 1.2 ± 0.1 (50 nm); 2.2 ± 0.2 (250 nm), P < 0.01 vs. control; 3.4 ± 0.2 (1000 nm), P < 0.001 vs. control] and time-dependent [1.0 (1 h); 3.2 ± 2.0 (6 h); 9.1 ± 5.9 (24 h), P < 0.05 vs. 1 h; 4.5 ± 2.2 (48 h)] increase in insulin-stimulated protein kinase B/akt phosphorylation. In addition, whereas insulin receptor substrate (IRS)-1 protein expression did not change, IRS-1 tyrosine phosphorylation increased. Furthermore, GC induced IRS-2 mRNA expression (2.8-fold; P < 0.05) and increased insulin-stimulated glucose uptake [1.0 (control) 1.8 ± 0.1 (insulin) vs. 2.8 ± 0.2 (insulin + GC); P < 0.05]. In contrast, in primary cultures of human muscle, GC decreased insulin-stimulated glucose uptake [1.0 (control) 1.9 ± 0.2 (insulin) vs. GC 1.3 ± 0.1 (insulin + GC); P < 0.05].
 

Conclusions: We have demonstrated tissue-specific regulation of insulin signaling by GC. Within sc adipose tissue, GCs augment insulin signaling, yet in muscle GCs cause insulin resistance. We propose that enhanced insulin action in adipose tissue increases adipocyte differentiation, thereby contributing to GC-induced obesity.