BADERC- Harvey Lodish

Harvey Lodish, Ph.D.

Regulation of glucose and fatty acids by adiponectin and its orthologs and by adipocyte- specific microRNAs

Whitehead Institute for Biomedical ResearchDepartments of Biology and Bioengineeriing, MIT
Research in the Lodish Laboratory rlated to Diabetes and Obesity falls into two broad areas: understanding the role of adiponectin and its orthologs in regulating glucose and fatty acid metabolism, and on the roles of microRNAs (miRNAs) in the biology and pathophysiology of adipocytes.

1. Testing the hypothesis that FATPs and acyl-CoA synthases are signal transduction proteins essential for AMPK activation by adiponectin.

Adiponectin plays a crucial role in regulating whole body fat and glucose metabolism through activation of AMP-activated protein kinase (AMPK). Decreased adiponectin expression induced by obesity is associated with several metabolic changes, including hyperglycemia and dyslipidaemia, both of which lead to increased risk of type II diabetes and cardiovascular disease. How adiponectin activates AMPK is not known. AMPK is activated by one or more upstream AMPK kinases (AMPKKs) as well as by AMP. Several years ago we showed that AMPK activation by adiponectin is accompanied by an increased concentration of 5’ AMP, implying the presence of uncharacterized pathways connecting adiponectin receptors with AMPK by producing AMP. Activation of free fatty acids to the CoA derivative is one important metabolic process that generates AMP, and at least some of the acyl-CoA synthase isoforms are localized on the plasma membrane. We showed that free fatty acids, essential substrates for the production of AMP by acyl-CoA synthases, are required for AMPK activation by adiponectin in C2C12 myocytes, suggesting that adiponectin receptor activation is coupled to activation of an acyl-CoA synthase. Using a siRNA knockdown technique, we identified that FATP1 is the acyl-CoA synthase isoform transducing the adiponectin signal to AMPK. Exactly how adiponectin - receptor binding induces FATP1 activation is under further investigation.

We are also testing the role of T- cadherin in adiponectin signaling, and trying to identify other cell surface proteins that bind adiponectin and/or are involved in its signaling.

2. Identification and characterization of CTRP9, a novel secreted glycoprotein from adipose tissue that reduces serum glucose in mice and forms a heterotrimer with Adiponectin

Recently we reported the cloning and characterization of CTRP9, a secreted glycoprotein homologous to adiponectin and with multiple posttranslational modifications in its collagen domain that include hydroxylated prolines and hydroxylated/glycosylated lysines. It is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum. Furthermore, CTRP9 and adiponectin can be secreted as hetero-oligomers when co-transfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally co-immunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, using gel filtration chromatography combined with co-immunoprecipitation analysis, we showed that adiponectin and CTRP9 form heterotrimers. Because different oligomeric forms of adiponectin have distinct biological activities, identification of CTRP9 that can heterotrimerizes with adiponectin impacts the study of adiponectin function.

In differentiated cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. In animals, adenovirus-mediated overexpression of CTRP9 significantly lowered serum glucose levels in obese (ob/ob) mice compared to controls. Collectively, these results suggest that CTRP9 is a novel adipokine and further study of CTRP9 will yield novel mechanistic insights into its physiologic and metabolic function. We are trying to clone the receptor(s) for CTRP9 and identify the signal transduction pathways they activate.

3. MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity

MicroRNAs (miRNAs) are important post-transcriptional regulators and affect diverse biological processes and many diseases. We profiled miRNA expression during in vitro adipogenesis of the preadipocyte 3T3-L1 cells using miRNA microarrays and validated by RT-PCR eight miRNAs that are significantly upregulated and four that are downregulated. Similar changes in miRNA expression were observed by comparison of mature primary adipocytes and enriched primary preadipocytes. We also profiled miRNA expression in purified mature adipocytes and compared miRNA profiles in epididymal adipocytes from normal and leptin- deficient and diet-induced obese mice. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice. Conversely miRNAs that were decreased during adipogenesis were elevated in adipocytes from obese mice. These changes are likely associated with the chronic inflammatory environment in obese adipose tissue as they were mimicked by TNFa treatment of differentiated adipocytes.

Ectopic expression of two adipocyte- enriched miRNAs, miR-103 or miR-143, in preadipocytes accelerated adipogenesis, as measured both by the upregulation at an early stage of adipogenesis of many adipocyte-important genes including adiponectin and the key transcription factor PPARg and by an increase in triglyceride accumulation.

Our results provide the first experimental evidence for miR-103 function in adipose biology. The remarkable inverse regulatory pattern for many miRNAs during adipogenesis and obesity has important implications for the understanding adipose tissue dysfunction in obese mice and humans and the link between chronic inflammation and obesity with insulin resistance. An understanding of the role of miRNAs in adipose biology may lead to novel RNA-based therapies that complement current anti-obesity treatments.

References

Wong, G., J. Wang, C. Hug, T.-S. Tsao, and H. F. Lodish. A family of Acrp30/adiponectin structural and functional paralogs Proc. Natl. Acad. Sci. USA 101: 10302 - 10307 (2004)
Hug, C., J. Wang, N. Ahmad, J. Bogan, T.-S. Tsao, and H. F. Lodish. T-cadherin is a receptor for hexameric and high molecular weight forms of Acrp30/adiponectin. Proc. Natl. Acad. Sci. USA 101: 10308 - 10313 (2004)
Ruan, H. and H. F. Lodish. Role of Adipose-Tissue-Derived Hormones and Inflammatory Cytokines in Obesity-Linked Type 2 Diabetes Curr. Opin Lipidology 15:297-302 (2004)
Wong, G.W., S. A. Krawczyk, C. Kitidis-Mitrokostas, T. Revett, R. Gimeno, and H. F. Lodish,Molecular, biochemical and functional characterizations of C1q/TNF family members: adipose-tissue-selective expression patterns, regulation by PPAR-gamma agonist, cysteine-mediated oligomerizations, combinatorial associations and metabolic functions. Biochem. J. 416:161-177 (2008).
Xie, H., B. Lim, and H. F. Lodish. MicroRNAs Induced during Adipogenesis That Accelerate Fat Cell Development Are Downregulated in Obesity Diabetes 58: 1050-1057 (2009).
Wong, G.W., S. A. Krawczyk, C. Kitidis-Mitrokostas, G. Ge, E. Spooner, C. Hug, R. Gimeno, and H. F. Lodish.Identification and characterization of CTRP9, a novel secreted glycoprotein from adipose tissue that reduces serum glucose in mice and forms heterotrimers with adiponectin FASEB Journal 23: 241 - 258 (2009).






			Harvey Lodish, Ph.D. Regulation of glucose and fatty acids by adiponectin and its orthologs and by adipocyte- specific microRNAs Whitehead Institute for Biomedical ResearchDepartments of Biology and Bioengineeriing, MIT Research in the Lodish Laboratory rlated to Diabetes and Obesity falls into two broad areas: understanding the role of adiponectin and its orthologs in regulating glucose and fatty acid metabolism, and on the roles of microRNAs (miRNAs) in the biology and pathophysiology of adipocytes. 1. Testing the hypothesis that FATPs and acyl-CoA synthases are signal transduction proteins essential for AMPK activation by adiponectin. Adiponectin plays a crucial role in regulating whole body fat and glucose metabolism through activation of AMP-activated protein kinase (AMPK). Decreased adiponectin expression induced by obesity is associated with several metabolic changes, including hyperglycemia and dyslipidaemia, both of which lead to increased risk of type II diabetes and cardiovascular disease. How adiponectin activates AMPK is not known. AMPK is activated by one or more upstream AMPK kinases (AMPKKs) as well as by AMP. Several years ago we showed that AMPK activation by adiponectin is accompanied by an increased concentration of 5’ AMP, implying the presence of uncharacterized pathways connecting adiponectin receptors with AMPK by producing AMP. Activation of free fatty acids to the CoA derivative is one important metabolic process that generates AMP, and at least some of the acyl-CoA synthase isoforms are localized on the plasma membrane. We showed that free fatty acids, essential substrates for the production of AMP by acyl-CoA synthases, are required for AMPK activation by adiponectin in C2C12 myocytes, suggesting that adiponectin receptor activation is coupled to activation of an acyl-CoA synthase. Using a siRNA knockdown technique, we identified that FATP1 is the acyl-CoA synthase isoform transducing the adiponectin signal to AMPK. Exactly how adiponectin - receptor binding induces FATP1 activation is under further investigation. We are also testing the role of T- cadherin in adiponectin signaling, and trying to identify other cell surface proteins that bind adiponectin and/or are involved in its signaling. 2. Identification and characterization of CTRP9, a novel secreted glycoprotein from adipose tissue that reduces serum glucose in mice and forms a heterotrimer with Adiponectin Recently we reported the cloning and characterization of CTRP9, a secreted glycoprotein homologous to adiponectin and with multiple posttranslational modifications in its collagen domain that include hydroxylated prolines and hydroxylated/glycosylated lysines. It is secreted as multimers (predominantly trimers) from transfected cells and circulates in the mouse serum. Furthermore, CTRP9 and adiponectin can be secreted as hetero-oligomers when co-transfected into mammalian cells, and in vivo, adiponectin/CTRP9 complexes can be reciprocally co-immunoprecipitated from the serum of adiponectin and CTRP9 transgenic mice. Biochemical analysis demonstrates that adiponectin and CTRP9 associate via their globular C1q domain, and this interaction does not require their conserved N-terminal cysteines or their collagen domains. Furthermore, using gel filtration chromatography combined with co-immunoprecipitation analysis, we showed that adiponectin and CTRP9 form heterotrimers. Because different oligomeric forms of adiponectin have distinct biological activities, identification of CTRP9 that can heterotrimerizes with adiponectin impacts the study of adiponectin function. In differentiated cultured myotubes, CTRP9 specifically activates AMPK, Akt, and p44/42 MAPK signaling pathways. In animals, adenovirus-mediated overexpression of CTRP9 significantly lowered serum glucose levels in obese (ob/ob) mice compared to controls. Collectively, these results suggest that CTRP9 is a novel adipokine and further study of CTRP9 will yield novel mechanistic insights into its physiologic and metabolic function. We are trying to clone the receptor(s) for CTRP9 and identify the signal transduction pathways they activate. 3. MicroRNAs induced during adipogenesis that accelerate fat cell development are downregulated in obesity MicroRNAs (miRNAs) are important post-transcriptional regulators and affect diverse biological processes and many diseases. We profiled miRNA expression during in vitro adipogenesis of the preadipocyte 3T3-L1 cells using miRNA microarrays and validated by RT-PCR eight miRNAs that are significantly upregulated and four that are downregulated. Similar changes in miRNA expression were observed by comparison of mature primary adipocytes and enriched primary preadipocytes. We also profiled miRNA expression in purified mature adipocytes and compared miRNA profiles in epididymal adipocytes from normal and leptin- deficient and diet-induced obese mice. Importantly, miRNAs that were induced during adipogenesis were downregulated in adipocytes from both types of obese mice. Conversely miRNAs that were decreased during adipogenesis were elevated in adipocytes from obese mice. These changes are likely associated with the chronic inflammatory environment in obese adipose tissue as they were mimicked by TNFa treatment of differentiated adipocytes. Ectopic expression of two adipocyte- enriched miRNAs, miR-103 or miR-143, in preadipocytes accelerated adipogenesis, as measured both by the upregulation at an early stage of adipogenesis of many adipocyte-important genes including adiponectin and the key transcription factor PPARg and by an increase in triglyceride accumulation. Our results provide the first experimental evidence for miR-103 function in adipose biology. The remarkable inverse regulatory pattern for many miRNAs during adipogenesis and obesity has important implications for the understanding adipose tissue dysfunction in obese mice and humans and the link between chronic inflammation and obesity with insulin resistance. An understanding of the role of miRNAs in adipose biology may lead to novel RNA-based therapies that complement current anti-obesity treatments. References Wong, G., J. Wang, C. Hug, T.-S. Tsao, and H. F. Lodish. A family of Acrp30/adiponectin structural and functional paralogs Proc. Natl. Acad. Sci. USA 101: 10302 - 10307 (2004) Hug, C., J. Wang, N. Ahmad, J. Bogan, T.-S. Tsao, and H. F. Lodish. T-cadherin is a receptor for hexameric and high molecular weight forms of Acrp30/adiponectin. Proc. Natl. Acad. Sci. USA 101: 10308 - 10313 (2004) Ruan, H. and H. F. Lodish. Role of Adipose-Tissue-Derived Hormones and Inflammatory Cytokines in Obesity-Linked Type 2 Diabetes Curr. Opin Lipidology 15:297-302 (2004) Wong, G.W., S. A. Krawczyk, C. Kitidis-Mitrokostas, T. Revett, R. Gimeno, and H. F. Lodish,Molecular, biochemical and functional characterizations of C1q/TNF family members: adipose-tissue-selective expression patterns, regulation by PPAR-gamma agonist, cysteine-mediated oligomerizations, combinatorial associations and metabolic functions. Biochem. J. 416:161-177 (2008). Xie, H., B. Lim, and H. F. Lodish. MicroRNAs Induced during Adipogenesis That Accelerate Fat Cell Development Are Downregulated in Obesity Diabetes 58: 1050-1057 (2009). Wong, G.W., S. A. Krawczyk, C. Kitidis-Mitrokostas, G. Ge, E. Spooner, C. Hug, R. Gimeno, and H. F. Lodish.Identification and characterization of CTRP9, a novel secreted glycoprotein from adipose tissue that reduces serum glucose in mice and forms heterotrimers with adiponectin FASEB Journal 23: 241 - 258 (2009).