Lipid Metabolism and Insulin Signalling in Adipocytes : enhanced autophagy in type 2 diabetes

Energy storage in the adipose tissue, to an extent leading to obesity, is associated with local as well assystemic insulin resistance. When insulin-producing beta-cells in the pancreas gradually fail tocompensate, plasma levels of glucose rise and overt type 2 diabetes is diagnosed. Adipocytes are largecells, mostly consisting of one big central lipid droplet, with the surrounding plasma membrane full ofsmall invaginations called caveolae. As caveolae contain the insulin receptor and several other insulinsignallingproteins, we have investigated several aspects of caveolae. We have also mapped mechanismsand defects in the insulin-signalling network in adipocytes from type 2 diabetic patients. In paper I, we show that a subtype of caveolae has the capability to synthesize triglycerides from fattyacids and glycerol-3-phosphate. The triglyceride-synthesizing caveolae subtype also contains perilipin,suggesting the existence of a mechanism to protect newly made triglycerides from hydrolysis. In paper II, we demonstrate that adipocytes from patients with type 2 diabetes have an attenuated insulinstimulatedphosphorylation of IRS-1 at Ser-307 (human sequence), which correlates with reduced insulinstimulatedphosphorylation of IRS-1 at tyrosine residues. Insulin-stimulated phosphorylation of IRS-1 atSer-307 is dependent on the nutrient sensor TORC1. This finding indicates that adipocytes from type 2diabetic patients have reduced TORC1 activity. In paper III, we focus on the mechanisms for RBP4-induced insulin resistance. We also continue ourmapping of insulin-resistance in adipocytes from type 2 diabetes. These cells exhibit, in addition toimpaired insulin-stimulated glucose uptake and the defects presented in paper I, impaired insulinstimulatedphosphorylation of ERK. We do, however, not see any defects in PKB signalling. Neither dowe se any enhanced insulin-stimulated phosphorylation of IRS-1 at Ser-312 (human sequence), a site thatin mice is hyper-stimulated in response to high-fat feeding. Incubation with RBP4 recapitulates all defectswe so far have seen in type 2 diabetes except reduced insulin-stimulated glucose uptake. These results aremirrored by blockade of endogenously produced RBP4 in the incubations with adipocytes from type 2diabetic patients. In other words, RBP4-blocking antibodies restore all insulin-signalling defects we havefound in adipocytes from type 2 diabetic patients, except insulin-stimulated glucose uptake. In paper IV we show by several approaches that TORC1 activation is down-regulated in adipocytes fromtype 2 diabetic patients. The main finding is that there is enhanced autophagy in those adipocytes.Interestingly, autophagy may be a mechanism to enhance the breakdown of stored triglycerides in theadipocyte. In conclusion, our data suggest that caveolae, in addition to being micro-domains for insulin-signallingare metabolic platforms. We describe defects in insulin-signalling in adipocytes from type 2 diabeticpatients where the main finding is enhanced autophagy in these obese patients. The perceived starvationin adipose tissue might via secretion of adipokines, such as RBP4, have implications for local as well assystemic insulin-resistance

Opponent

autophagosome in an adipocyte from a type 2 diabetic patient. Autophagosomes are formed in response to starvation and participate in digestion of intracellular components to release energy thus promoting cell survival during starvation in a process called autophagy. Autophagy is derived from Greek roots: auto, meaning “self”, and phagy, “to eat”. © 2009 Anita Öst Published articles have been reprinted with the permission of the respective copyright holders

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-104290 The Concentration of b-Carotene in Human Adipocytes, but Not the Whole-Body Adipocyte Stores, Is Reduced in

We have examined the concentration of b-carotene in the fat of isolated abdominal subcutaneous adipocytes obtained from lean (BMI,23 kg/m 2), non-obese with higher BMI (23#BMI,28 kg/m 2), obese (BMI$28 kg/m 2), and from a group of obese subjects with type 2 diabetes. The concentration of b-carotene was 50 % lower in the adipocytes from the obese and obese/diabetic groups compared with the lean and non-obese groups. Interestingly, the total amount of b-carotene in the adipocyte stores of each subject was constant among all groups. Triacylglycerol constituted 9261 % (by weight) of the adipocyte lipids in the lean group and this was increased to 9962 % in the obese group with diabetes (p,0.05). The concentration of cholesteryl esters was in all cases,0.1 g per 100 g of total lipids, demonstrating that mature human adipocytes have negligible stores of cholesteryl ester. Our findings demonstrate that adipocyte concentrations of b-carotene are reduced in obese subjects. The lower concentrations in adipocytes from subjects with type 2 diabetes apparently reflect subjects obesity. Our finding that whole-body stores of b-carotene in adipocytes are constant raises new questions regarding what function it serves, as well as the mechanisms for maintaining constant levels in the face of varie

The Concentration of beta-Carotene in Human Adipocytes, but Not the Whole-Body Adipocyte Stores, Is Reduced in Obesity

We have examined the concentration of beta-carotene in the fat of isolated abdominal subcutaneous adipocytes obtained from lean (BMIless than23 kg/m(2)), non-obese with higher BMI (23 less than= BMIless than28 kg/m(2)), obese (BMI greater than= 28 kg/m(2)), and from a group of obese subjects with type 2 diabetes. The concentration of b-carotene was 50% lower in the adipocytes from the obese and obese/diabetic groups compared with the lean and non-obese groups. Interestingly, the total amount of beta-carotene in the adipocyte stores of each subject was constant among all groups. Triacylglycerol constituted 92 +/- 1% (by weight) of the adipocyte lipids in the lean group and this was increased to 99 +/- 2% in the obese group with diabetes (pless than0.05). The concentration of cholesteryl esters was in all cases less than0.1 g per 100 g of total lipids, demonstrating that mature human adipocytes have negligible stores of cholesteryl ester. Our findings demonstrate that adipocyte concentrations of beta-carotene are reduced in obese subjects. The lower concentrations in adipocytes from subjects with type 2 diabetes apparently reflect subjects obesity. Our finding that whole-body stores of beta-carotene in adipocytes are constant raises new questions regarding what function it serves, as well as the mechanisms for maintaining constant levels in the face of varied adipose tissue mass among individuals over a period of time

The Concentration of beta-Carotene in Human Adipocytes, but Not the Whole-Body Adipocyte Stores, Is Reduced in Obesity

We have examined the concentration of beta-carotene in the fat of isolated abdominal subcutaneous adipocytes obtained from lean (BMIless than23 kg/m(2)), non-obese with higher BMI (23 less than= BMIless than28 kg/m(2)), obese (BMI greater than= 28 kg/m(2)), and from a group of obese subjects with type 2 diabetes. The concentration of b-carotene was 50% lower in the adipocytes from the obese and obese/diabetic groups compared with the lean and non-obese groups. Interestingly, the total amount of beta-carotene in the adipocyte stores of each subject was constant among all groups. Triacylglycerol constituted 92 +/- 1% (by weight) of the adipocyte lipids in the lean group and this was increased to 99 +/- 2% in the obese group with diabetes (pless than0.05). The concentration of cholesteryl esters was in all cases less than0.1 g per 100 g of total lipids, demonstrating that mature human adipocytes have negligible stores of cholesteryl ester. Our findings demonstrate that adipocyte concentrations of beta-carotene are reduced in obese subjects. The lower concentrations in adipocytes from subjects with type 2 diabetes apparently reflect subjects obesity. Our finding that whole-body stores of beta-carotene in adipocytes are constant raises new questions regarding what function it serves, as well as the mechanisms for maintaining constant levels in the face of varied adipose tissue mass among individuals over a period of time

Seqpac: a framework for sRNA-seq analysis in R using sequence-based counts

Motivation: Feature-based counting is commonly used in RNA-sequencing (RNA-seq) analyses. Here, sequences must align to target features (like genes or non-coding RNAs) and related sequences with different compositions are counted into the same feature. Consequently, sequence integrity is lost, making results less traceable against raw data.Small RNA (sRNA) often maps to multiple features and shows an incredible diversity in form and function. Therefore, applying feature-based strategies may increase the risk of misinterpretation. We present a strategy for sRNA-seq analysis that preserves the integrity of the raw sequence making the data lineage fully traceable. We have consolidated this strategy into Seqpac: An R package that makes a complete sRNA analysis available on multiple platforms. Using published biological data, we show that Seqpac reveals hidden bias and adds new insights to studies that were previously analyzed using feature-based counting.We have identified limitations in the concurrent analysis of RNA-seq data. We call it the traceability dilemma in alignment-based sequencing strategies. By building a flexible framework that preserves the integrity of the read sequence throughout the analysis, we demonstrate better interpretability in sRNA-seq experiments, which are particularly vulnerable to this problem. Applying similar strategies to other transcriptomic workflows may aid in resolving the replication crisis experienced by many fields that depend on transcriptome analyses.Funding Agencies|Swedish Research Council [2015-03141]; Knut and Alice Wallenberg Foundation [2015.0165]; Ragnar Soderberg; Swedish Research Council for Sustainable Development [2020-01042]</p

Dietary Sugar Shifts Mitochondrial Metabolism and Small RNA Biogenesis in Sperm

Aims: Increasing concentrations of dietary sugar results in a linear accumulation of triglycerides in male Drosophila, while inducing a U-shaped obesity response in their offspring. Here, using a combination of proteomics and small RNA (sRNA) sequencing, we aimed at understanding the molecular underpinning in sperm for such plasticity.Results: Proteomic analysis of seminal vesicles revealed that increasing concentrations of dietary sugar resulted in a bell-shaped induction of proteins involved in metabolic/redox regulation. Using stains and in vivo redox reporter flies, this pattern could be explained by changes in sperm production of reactive oxygen species (ROS), more exactly mitochondria-derived H2O2. By quenching ROS with the antioxidant N-acetyl cysteine and performing sRNA-seq on sperm, we found that sperm miRNA is increased in response to ROS. Moreover, we found sperm mitosRNA to be increased in high-sugar diet conditions (independent of ROS). Reanalyzing our previously published data revealed a similar global upregulation of human sperm mitosRNA in response to a high-sugar diet, suggesting evolutionary conserved mechanisms.Innovation: This work highlights a fast response to dietary sugar in mitochondria-produced H2O2 in Drosophila sperm and identifies redox-sensitive miRNA downstream of this event.Conclusions: Our data support a model where changes in the sperm mitochondria in response to dietary sugar are the primary event, and changes in redox homoeostasis are secondary to mitochondrial ROS production. These data provide multiple candidates for paternal intergenerational metabolic responses as well as potential biomarkers for human male fertility.Funding Agencies|Swedish Research Council [2015-03141]; Ragnar Soderbergs foundation; Knut and Alice Wallenberg foundation [2015.0165]</p

Subjects and clinical parameters (mean ± SD).

<p>Subjects and clinical parameters (mean ± SD).</p

Concentration of TAG and β-carotene in lipid extracts of adipocytes.

<p>The concentrations of TAG (A) and of β-carotene (B) were determined in lipid extracts of isolated adipocytes from subjects that were divided into groups of lean (BMI<23 kg/m²), non-obese (23≤BMI<28 kg/m²), obese (BMI≥28 kg/m²), or obese subjects with type 2 diabetes (as indicated). Lines indicate significant differences between indicated groups (p<0.05).</p

Total adipocyte stores of β-carotene in subjects.

<p>Whole body content of β-carotene stored in adipocytes was determined for each subject as the adipocyte concentration of β-carotene adjusted for total body fat. No statistically significant difference was found between the mean values in the groups using one-way analysis of variance (ANOVA), p>0.5.</p