Birgit Knebel

Liver-specific expression of transcriptionally active SREBP-1c is associated with fatty liver and increased visceral fat mass.

The pathogenesis of fatty liver is not understood in detail, but lipid overflow as well as de novo lipogenesis (DNL) seem to be the key points of hepatocyte accumulation of lipids. One key transcription factor in DNL is sterol regulatory element-binding protein (SREBP)-1c. We generated mice with liver-specific over-expression of mature human SREBP-1c under control of the albumin promoter and a liver-specific enhancer (alb-SREBP-1c) to analyze systemic perturbations caused by this distinct alteration. SREBP-1c targets specific genes and causes key enzymes in DNL and lipid metabolism to be up-regulated. The alb-SREBP-1c mice developed hepatic lipid accumulation featuring a fatty liver by the age of 24 weeks under normocaloric nutrition. On a molecular level, clinical parameters and lipid-profiles varied according to the fatty liver phenotype. The desaturation index was increased compared to wild type mice. In liver, fatty acids (FA) were increased by 50% (p<0.01) and lipid composition was shifted to mono unsaturated FA, whereas lipid profile in adipose tissue or serum was not altered. Serum analyses revealed a ∼2-fold (p<0.01) increase in triglycerides and free fatty acids, and a ∼3-fold (p<0.01) increase in insulin levels, indicating insulin resistance; however, no significant cytokine profile alterations have been determined. Interestingly and unexpectedly, mice also developed adipositas with considerably increased visceral adipose tissue, although calorie intake was not different compared to control mice. In conclusion, the alb-SREBP-1c mouse model allowed the elucidation of the systemic impact of SREBP-1c as a central regulator of lipid metabolism in vivo and also demonstrated that the liver is a more active player in metabolic diseases such as visceral obesity and insulin resistance.

Insulin-activated Erk-mitogen-activated Protein Kinases Phosphorylate Sterol Regulatory Element-binding Protein-2 at Serine Residues 432 and 455 in Vivo

The transcription factor sterol regulatory element binding protein (SREBP)-2 plays a pivotal role in lipid metabolism. Previously, we have shown that the mature form of SREBP-2 is a substrate of Erk-mitogen-activated protein kinases (MAPK). The aim of the present study was to identify Erk-specific phosphorylation sites. Using a protein chemistry approach, we could identify Ser-432 and Ser-455 as major phosphorylation sites. Further characterization by electrophoretic mobility shift assay and promoter reporter gene analyses revealed that phosphorylation does not influence protein/DNA interaction, but enhances trans-activity. In intact cells, SREBP-2 is phosphorylated by insulin, which seems to be related to their bio-responses on low density lipoprotein receptor activity. These results suggest that activation of Erk-MAPK pathways by hormones such as insulin might be related to a novel regulatory principle of SREBP-2.

The peroxisome proliferator activated receptor gamma Pro12Ala polymorphism is associated with a lower hirsutism score and increased insulin sensitivity in women with polycystic ovary syndrome.

Background  Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and chronic anovulation. The genetic background of the insulin resistance frequently associated with PCOS is unclear.

Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo.

Phosphorylation of sterol regulatory element-binding protein (SREBP)-1a links growth hormone action to lipid metabolism in hepatocytes

OBJECTIVE Increased lipid accumulation in cells and tissues is a key phenomenon in the development of obesity, insulin resistance, and atherosclerosis. In the regulation of lipid content of cells and tissues the SREBPs play a dominant role as transcription factors. METHODS Since growth hormone (GH) affects lipid metabolism and function of fat as well as liver cells, we have investigated the role of SREBP-1a, SREBP-1c and SREBP-2 in the gene regulatory action of GH in the human liver cell line HepG2 and primary mouse hepatocytes. RESULTS These experiments showed that SREBP-1a couples the stimulatory effect of GH on cholesterol regulated genes, e.g. LDL receptor gene, via sterol sensitive binding cis-element (sre-1). This effect was not depending on RNA expression, but related to phosphorylation of SREBP-1a protein. The result was supported by experiments with the mutant variant SREBP-1a S117A, which is not phosphorylated by Erk-MAP kinases. To analyse a possible role of GH-induced SREBP-1a phosphorylation in cellular physiology we investigated an expression profile of genes coding for central players in lipid transport or lipid metabolism as well as for transcription factors by real time PCR in primary mouse hepatocytes and human hepatoma cell line stably overexpressing the mature form of SREBP-1a or mutated form. CONCLUSION These experiments emphasize the role SREBP-1a and its phosphorylation for gene regulatory effects of GH.

Metabolic syndrome and hypertension: pathophysiology and molecular basis of insulin resistance.

Abstract Several recent studies indicate that type 2 diabetes, arterial hypertension, lipid disorders as well as visceral obesity are coronary risk factors which might belong to a syndrome which is caused by decreased insulin sensitivity with compensatory hyperinsulinaemia. More than 50% of patients with essential hypertension have some degree of insulin resistance, but in contrast to dyslipoproteinaemia and glucose intolerance the causal relation between insulin resistance and elevated arterial blood pressure appears not to be as evident. One explanation is that the link between blood pressure and insulin sensitivity might be mainly related to concomitant obesity.Accordingly, obesity can be associated with an increased activity of the sympathetic nervous system, elevated plasma levels of the vasoconstrictor endothelin-1, and decreased insulin-induced endothelium-dependent vasodilation. Furthermore, adipocytes can secrete vasogenic peptides, such as angiotensinogen. Since insulin resistance is a polygenic disorder, the two basic genetic approaches we follow is to identify genetic defects of insulin action in cells of patients with inherited syndromes of insulin resistance and to characterize molecular mechanisms of insulin regulated gene expression. The results show that insulin can affect the expression rate of various genes, e.g. involved in cholesterol and fatty acid metabolism, by modulating the activity of transcription factors coupled to the MAP kinase cascade and that a genetic postreceptor defect in these intracellular signaling pathways might have a pleiotropic effect on cell metabolism and clinical phenotype.

Tissue-Specific Differences in the Development of Insulin Resistance in a Mouse Model for Type 1 Diabetes

Although insulin resistance is known to underlie type 2 diabetes, its role in the development of type 1 diabetes has been gaining increasing interest. In a model of type 1 diabetes, the nonobese diabetic (NOD) mouse, we found that insulin resistance driven by lipid- and glucose-independent mechanisms is already present in the liver of prediabetic mice. Hepatic insulin resistance is associated with a transient rise in mitochondrial respiration followed by increased production of lipid peroxides and c-Jun N-terminal kinase activity. At the onset of diabetes, increased adipose tissue lipolysis promotes myocellular diacylglycerol accumulation. This is paralleled by increased myocellular protein kinase C θ activity and serum fetuin A levels. Muscle mitochondrial oxidative capacity is unchanged at the onset but decreases at later stages of diabetes. In conclusion, hepatic and muscle insulin resistance manifest at different stages and involve distinct cellular mechanisms during the development of diabetes in the NOD mouse.

Increased low grade inflammatory serum markers in patients with Polycystic ovary syndrome (PCOS) and their relationship to PPARgamma gene variants.

The Polycystic ovary syndrome (PCOS) is the most frequent endocrine disorder in premenopausal women and is associated with features of the insulin resistance syndrome, altered glucose homeostasis, and central obesity. Inflammation appears to be a link between obesity and insulin resistance, because adipose tissue is one major source of proinflammatory cytokines. Since peroxisome proliferator-activated receptor (PPAR)gamma affects adipocyte differentiation as well as insulin sensitivity, we investigated whether the levels of proinflammatory factors in PCOS patients are related to sequence variations of the PPAR gamma gene. Proinflammatory cytokine levels, i.e. IL-1 beta, IL-6, IL-7, IL-8, IL-17 and TNFalpha, were evaluated in PCOS patients (n=21) in comparison to obese controls (n=120). Next to this the complete coding sequence of the PPAR gamma gene was investigated by resequencing all probands. We show that the levels of IL-8 and IL-17 were unchanged, IL-1 beta, IL-6 and TNFalpha were elevated and the level of IL-7 was decreased in PCOS patients compared to obese controls. Sequence analyses of the PPAR gamma gene indicated that neither the common polymorphisms P12A or H478 H, nor novel polymorphisms (E79Q, V32G, -39 T>C, c.480 +33 t > g,) or unique sequence variations (S22S, A23A, T41A, S226C, K272 T, I484I, c.819 +24 a>c) detected in this investigation revealed evidence for a direct association of PPAR gamma with altered IL-7, IL-1beta, IL-6 and TNFalpha levels in PCOS patients. So, alterations in inflammatory serum markers appear to be a feature of PCOS per se, and are independent of PPAR gamma variants.

Mechanisms of Insulin Resistance in Primary and Secondary Nonalcoholic Fatty Liver.

Nonalcoholic fatty liver disease is associated with hepatic insulin resistance and may result primarily from increased hepatic de novo lipogenesis (PRIM) or secondarily from adipose tissue lipolysis (SEC). We studied mice with hepatocyte- or adipocyte-specific SREBP-1c overexpression as models of PRIM and SEC. PRIM mice featured increased lipogenic gene expression in the liver and adipose tissue. Their selective, liver-specific insulin resistance was associated with increased C18:1-diacylglycerol content and protein kinase Cε translocation. SEC mice had decreased lipogenesis mediated by hepatic cholesterol responsive element–binding protein and featured portal/lobular inflammation along with total, whole-body insulin resistance. Hepatic mitochondrial respiration transiently increased and declined with aging along with higher muscle reactive oxygen species production. In conclusion, hepatic insulin resistance originates from lipotoxicity but not from lower mitochondrial capacity, which can even transiently adapt to increased peripheral lipolysis. Peripheral insulin resistance is prevented during increased hepatic lipogenesis only if adipose tissue lipid storage capacity is preserved.

Phosphorylation of sterol regulatory element-binding protein (SREBP)-1c by p38 kinases, ERK and JNK influences lipid metabolism and the secretome of human liver cell line HepG2

Abstract The transcription factor sterol regulatory element binding protein (SREBP)-1c plays a pivotal role in lipid metabolism. In this report we identified the main phosphorylation sites of MAPK-families, i.e. p38 stress-activated MAPK (p38), ERK-MAPK (ERK) or c-JUN N-terminal protein kinases (JNK) in SREBP-1c. The major phosphorylation sites of p38, i.e. serine 39 and threonine 402, are identical to those we recently identified in the splice-variant SREBP-1a. In contrast, ERK and JNK phosphorylate SREBP-1c at two major sites, i.e. threonine 81 and serine 93, instead of one site in SREBP-1a. Functional analyses of the biological outcome in the human liver cell line HepG2 reveals SREBP-1c phosphorylation dependent alteration in lipid metabolism and secretion pattern of lipid transporting proteins, e.g. ApoE or ApoA1. These results suggest that phosphorylation of SREBP-1c by different MAPKs interferes with lipid metabolism and the secretory activity of liver cells.