Cristina M. Rondinone

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

On the Control of Lipolysis in Adipocytes

ABSTRACT: The lipolytic reaction in adipocytes is one of the most important reactions in the management of bodily energy reserves, and dysregulation of this reaction may contribute to the symptoms of Type 2 diabetes mellitus. Yet, progress on resolving the molecular details of this reaction has been relatively slow. However, recent developments at the molecular level begin to paint a clearer picture of lipolysis and point to a number of unanswered questions. While HSL has long been known to be the rate‐limiting enzyme of lipolysis, the mechanism by which HSL attacks the droplet lipids is not yet firmly established. Certainly, the immunocytochemical evidence showing the movement of HSL to the lipid droplet upon stimulation leaves little doubt that this translocation is a key aspect of the lipolytic reaction, but whether or not HSL phosphorylation contributes to the translocation, and at which site(s), is as yet unresolved. It will be important to establish whether there is an activation step in addition to the translocation reaction. The participation of perilipin A is indicated by the findings that this protein can protect neutral lipids within droplets from hydrolysis, but active participation in the lipolytic reaction is yet to be proved. Again, it will be important to determine whether mutations of serine residues of PKA phosphorylation sites of perilipins prevent lipolysis, and whether such modifications abolish the physical changes in the droplet surfaces that accompany lipolysis.

Low cellular IRS 1 gene and protein expression predict insulin resistance and NIDDM.

We examined the gene and protein expression of IRS 1 (insulin receptor substrate 1) in adipocytes from two groups of healthy individuals with an increased propensity for non‐insulin‐dependent diabetes mellitus (NIDDM): those with two first‐degree relatives with diabetes and another group with massive obesity. A low expression of IRS 1(<50% of the matched control group) was seen in «30% of both groups and these individuals were characterized by insulin resistance and its hallmarks: higher levels of insulin, glucose, and triglycerides. Two individuals with previously unknown NIDDM were diagnosed and both had low IRS 1 expression. Low IRS 1 protein expression was associated with low mRNA levels but not with the common Gly972Arg polymorphism of the IRS 1 gene. Taken together, our present and previous findings show that a low expression of IRS 1 in fat cells predicts insulin resistance and NIDDM. Furthermore, they support the likelihood that an impaired transcriptional activation may play a key role in the pathogenesis of NIDDM.—Carvalho, E., Jansson, P.‐A., Axelsen, M., Eriksson, J. W., Huang, X., Groop, L., Rondinone, C., Sjostrom, L., Smith, U. Low cellular IRS 1 gene and protein expression predict insulin resistance and NIDDM. FASEB J. 13, 2173–2178 (1999)

Rapamycin partially prevents insulin resistance induced by chronic insulin treatment

Chronic insulin exposure induces serine/threonine phosphorylation and degradation of IRS-1 through a rapamycin-sensitive pathway, which results in a down-regulation of insulin action. In this study, to investigate whether rapamycin (an mTOR inhibitor) could prevent insulin resistance induced by hyperinsulinemia, 3T3-L1 adipocytes were incubated chronically in the presence of insulin with or without the addition of rapamycin. Subsequently, the cells were washed and re-stimulated acutely with insulin. Chronic insulin stimulation caused a reduction of GLUT-4 and IRS-1 proteins with a correlated decrease in acute insulin-induced PKB and MAPK phosphorylations as well as a reduction in insulin-stimulated glucose transport. Rapamycin prevented the reduction of IRS-1 protein levels and insulin-induced PKB Ser-473 phosphorylation with a partial normalization of insulin-induced glucose transport. In contrast, rapamycin had no effect on the decrease in insulin-induced MAPK phosphorylation or GLUT-4 protein levels. These results suggest that chronic insulin exposure leads to a down-regulation of PKB and MAPK pathways through different mechanisms in adipocytes.

Protein–Tyrosine Phosphatase 1B–Deficient Myocytes Show Increased Insulin Sensitivity and Protection Against Tumor Necrosis Factor-α–Induced Insulin Resistance

Protein–tyrosine phosphatase (PTP)1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. In this study, we have assessed the role of PTP1B in the insulin sensitivity of skeletal muscle under physiological and insulin-resistant conditions. Immortalized myocytes have been generated from PTP1B-deficient and wild-type neonatal mice. PTP1B−/− myocytes showed enhanced insulin-dependent activation of insulin receptor autophosphorylation and downstream signaling (tyrosine phosphorylation of insulin receptor substrate [IRS]-1 and IRS-2, activation of phosphatidylinositol 3-kinase, and serine phosphorylation of AKT), compared with wild-type cells. Accordingly, PTP1B−/− myocytes displayed higher insulin-dependent stimulation of glucose uptake and GLUT4 translocation to the plasma membrane than wild-type cells. Treatment with tumor necrosis factor-α (TNF-α) induced insulin resistance on glucose uptake, impaired insulin signaling, and increased PTP1B activity in wild-type cells. Conversely, the lack of PTP1B confers protection against insulin resistance by TNF-α in myocyte cell lines and in adult male mice. Wild-type mice treated with TNF-α developed a pronounced hyperglycemia along the glucose tolerance test, accompanied by an impaired insulin signaling and increased PTP1B activity in muscle. However, mice lacking PTP1B maintained a rapid clearance of glucose and insulin sensitivity and displayed normal muscle insulin signaling regardless the presence of TNF-α.

Impaired glucose transport and protein kinase B activation by insulin, but not okadaic acid, in adipocytes from subjects with Type II diabetes mellitus

Aims/hypothesis. To study the effects of insulin and okadaic acid, a serine/threonine phosphatase inhibitor which does not increase PI3-kinase activity, on the rate of glucose transport and protein kinase B activation in adipocytes from healthy subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus. Methods. Adipocytes were incubated with or without insulin or okadaic acid or both and glucose transport, protein kinase B activity, phosphorylation and protein expression measured. Results. Insulin and okadaic acid alone increased glucose uptake to a similar degree in adipocytes from healthy subjects and, when combined, exerted a partial additive effect. The effect of insulin was reduced by about 60 % in adipocytes from Type II diabetic patients, whereas the effect of okadaic acid was essentially unchanged and no further increase was seen when okadaic acid and insulin were combined. Okadaic acid increased protein kinase B activity to a greater extent (two to threefold) than insulin but only slightly increased the serine phosphorylation of protein kinase B. Adipocytes from Type II diabetic subjects exhibited both an impaired sensitivity as well as a reduced total serine phosphorylation and activation of protein kinase B in response to insulin but protein kinase B activity in response to okadaic acid was intact. Conclusion/interpretation. These results show that the ability of insulin to increase glucose transport and activate protein kinase B is reduced in fat cells from Type II diabetic subjects. Protein kinase B can, however, be activated by agents like okadaic acid which bypass the upstream defects in the insulin signalling pathway in Type II diabetic cells and, thus, increase glucose uptake. [Diabetologia (1999) 42: 819–825]

Insulin resistance in fat cells from obese Zucker rats--evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4.

The effect of insulin on glucose transport, glucose transporter 4 (Glut4) translocation, and intracellular signaling were measured in fat cells from lean and obese Zucker rats of different ages. Insulin-stimulated glucose transport was markedly reduced in adipocytes from old and obese animals. The protein content of Glut4 and insulin receptor substrates (IRS) 1 and 2 were also reduced while other proteins, including the p85 subunit of PI3-kinase, Shc and the MAP kinases (ERK1 and 2) were essentially unchanged. There was a marked impairment in the insulin stimulated tyrosine phosphorylation of IRS-1 and 2 as well as activation of PI3-kinase and PKB in cells from old and obese animals. Furthermore, insulin-stimulated translocation of both Glut4 and PKB to the plasma membrane was virtually abolished. The phosphotyrosine phosphatase inhibitor, vanadate, increased the insulin- stimulated upstream signaling including PI3-kinase and PKB activities as well as rate of glucose transport. Thus, the insulin resistance in cells from old and obese Zucker rats can be accounted for by an impaired translocation process, due to signaling defects leading to a reduced activation of PI3-kinase and PKB, as well as an attenuated Glut4 protein content.

Liver-specific Knockdown of JNK1 Up-regulates Proliferator-activated Receptor γ Coactivator 1β and Increases Plasma Triglyceride despite Reduced Glucose and Insulin Levels in Diet-induced Obese Mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor γ coactivator 1β, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.

Liver X receptor agonists ameliorate TNFα-induced insulin resistance in murine brown adipocytes by downregulating protein tyrosine phosphatase-1B gene expression

Aims/hypothesisThe nuclear receptors, including nuclear receptor subfamily 1, group H, member 3 (NR1HR, also known as liver X receptor [LXR]), are sensors of cholesterol metabolism and lipid biosynthesis that have recently been proposed as insulin sensitisers. TNFα has been described as a link between obesity and the development of insulin resistance, an important contributor to the pathogenesis of type 2 diabetes. Therefore, we decided to investigate the ability of NR1HR agonists to ameliorate TNFα-induced insulin resistance in brown adipocytes.MethodsPrimary brown adipocytes from rat fetuses, and from wild-type neonate mice and neonate mice deficient in the gene encoding protein tyrosine phosphatase-1B (Ptpn1, also known as Ptp1b) were cultured in the absence or presence of TNFα and different nuclear receptor agonists. Among them, the unrelated NR1HR ligands T0901317, GW3965 and (22R)-hydroxycholesterol were tested. After insulin stimulation, glucose uptake and solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) translocation were measured. Next the insulin signalling cascade was determined by submitting cells to lysis, immunoprecipitation and immunoblotting.ResultsNR1HR agonists ameliorate TNFα-induced insulin resistance restoring completely insulin-stimulated glucose uptake and SLC2A4 translocation to plasma membrane. This effect is parallel to the recovery of the insulin cascade insulin receptor/IRS-2/phosphatidylinositol 3-kinase/protein kinase B, and could be due to the fact that T0901317 prevents the increase of PTPN1 production and phosphatase activity produced by TNFα. In this regard, Ptpn1-deficient brown adipocytes showed protection against insulin resistance by TNFα. Moreover, we observed that T0901317 produced in itself a significant increase over basal glucose uptake consistent with an increase of SLC2A4 protein content in plasma membrane, attributable to the activation of protein kinase ζ and/or the increase of Slc2a4 expression.Conclusions/interpretationNuclear receptors NR1HR are interesting potential targets for drug treatment of insulin resistance.

PTP1B antisense-treated mice show regulation of genes involved in lipogenesis in liver and fat

Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.