Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes.

The liver is a major insulin-responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin-induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin-induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin-induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML-12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin-induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin-induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin-induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin-induced glucose uptake and glycogenesis in hepatocytes.

Immunohistochemical localization of CD31, NOTCH1 and JAGGED1 proteins in experimentally induced polycystic ovaries of immature rats.

We analyzed histomorphometrical changes and blood vessel immunohistochemical staining of CD31, NOTCH1 and JAGGED1 in induced polycystic ovaries of immature female Wistar rats, as well as serum hormone levels. The rats were randomly divided into control (n=18) and treated (n=18) groups. Treated animals received intramuscularly testosteronenantat weekly (0.1mg/g). Controls received the same amount of ricinus oil. Rats were weighed daily. Control and treated subgroups (6 rats per subgroup) were subsequently sacrificed after 21, 28 and 35 days of treatment. In ovaries of treated rats we found large cystic follicles, thick stromal tissue, many atretic preantral follicles, no ovulation and a thinner granulosa cell layer. CD31 stained blood vessels in the theca layer were reduced, with reduced JAGGED1 and NOTCH1 immunostaining. In controls, preantral and antral follicles were larger than in the treated group. Treated animals showed statistically significant lower progesterone and higher testosterone levels. They gained more weight than controls. Reduced immunostaining for NOTCH1 and JAGGED1 of reduced blood vessels of the theca layer was found in all stages of folliculogenesis with a distinct reduction in cystic and atretic follicles. Our results provide evidence of intrinsic abnormality during all stages of folliculogenesis in polycystic ovaries and this may result from crosstalk between circulating gonadotropins and follicular angiogenesis.

Substituting dietary polyunsaturated fat with monounsaturated fat increases insulin sensitivity in cultured rat hepatocytes.

Increased dietary fat intake generally, and saturated fat specifically, will lead to impairment of insulin action and to development of metabolic syndrome. The aim of this study was to find out changing of hepatic glucose output in dependence of high monounsaturated and high polyunsaturated fat diet and possible direct action of insulin in hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high polyunsaturated fat diet, as well as those isolated from rats on high monounsaturated fat diet was significantly higher than in standard controls. Insulin significantly decreased glucose production but only in hepatocytes obtained from rats on high monounsaturated fat diet. On the other side energy expenditure was significantly higher in rats on high monounsaturated fat diet and significantly lower in rats on high polyunsaturated fat diet comparable with animals on standard diet. Monounsaturated fat in Mediterranean diet could have beneficial effect on the development of the metabolic syndrome by increasing insulin sensitivity and energy expenditure.

Effects of tumor necrosis factor-alpha on insulin stimulated amino acid transport in cultured rat hepatocytes.

It has been suggested that tumor necrosis factor alpha (TNF-alpha) plays a pivotal role in the pathogenesis of insulin resistance. It could act directly or indirectly in liver. The aim of this study was to determine direct short time (4 h) and long time (24 h) action of TNF-alpha on amino acid transport in cultured rat hepatocytes and possible role of protein kinase C (PKC) in insulin signal pathway and insulin resistance. Hepatocytes were isolated by a modified collagenase perfusion technique and cultured for 24 h in M 199 medium. In the presence of insulin basal alpha-amino isobutyric acid (AIB) uptake was increased 55%. TNF-alpha in short time action did not change basal AIB transport, but significantly (25%) increased insulin stimulated uptake. Short time action of TNF-alpha was ameliorated by phorbol ester treatment. These results indicated that PKC activation is important in insulin signaling and TNF-alpha action. TNF-alpha acting directly did not cause insulin resistance in cultured hepatocytes.

Effects of vanadate on glucose production in cultured hepatocytes isolated from rats on high saturated fat diet.

Insulin resistance is a common phenomenon in obesity and Type 2 diabetes. Common factor important for development of diabetes and insulin resistance is intake of saturated fat. Vanadate treatment improves glucose homeostasis in vivo. The aim of this study was to find out changing of hepatic glucose output in dependence of saturated fat diet and possible direct action of vanadate in cultured hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high saturated fat diet was significantly 139% higher comparable to standard controls. Glucagon 100% increased glucose production in hepatocytes from rats on standard diet and 200% in hepatocytes on saturated high fat diet. The addition vanadate significantly decreased basic glucose production and did not influence glucagon stimulated glucose production. Presence of insulin did not influence either glucagon or vanadate effect. High saturated fat diet not only increases insulin resistance but also decreases chances of successful therapy of diabetes.

Effects of troglitazone and insulin on glucose production in cultured hepatocytes isolated from rats on high fat diet.

Increased dietary fat intake in general, and saturated fat specifically, will lead to the impairment of insulin action. The aim of this study was to find out the changes in hepatic glucose output in dependence of fat diet and a possible direct action of insulin and trogitazone in hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high fat diet (unsaturated fat) was 79% higher compared to control and even 139% higher than in rats on high-fat diet (saturated fat). Troglitazone significantly decreased the glucose production in hepatocytes obtained from rats on unsaturated fat diet. The troglitazone in presence of insulin totally normalized glucose production but also only in hepatocytes obtained from rats on unsaturated-fat diet. The troglitazone showed an insulinomimetic as well as insulin-sensitizing effect but only in rats on unsaturated-fat diet.

Insulin-like and non-insulin-like action of vanadate in hepatocytes cultured in vitro.

The aim of this study was to determine possible insulin-mimetic effects of vanadate on amino acid transport, glycogen synthesis and glucose production in hepatocytes cultured in vitro. Vanadate, like insulin, caused a significant (100%) increase of amino acid transport. However, vanadate produced a significant (31%) decrease of 14C-glucose incorporation into glycogen, in contrast to insulin which caused a 54% increase. Vanadate also caused a significant decrease (24%) of basal glucose production but had no effect on glucagon-stimulated glucose production. Vanadate and insulin given together reduced more strongly the basal glucose production (68%) and also effectively prevented the glucagon-stimulated effect on glucose production. In hepatocytes cultured in vitro vanadate had insulin-like and non-insulin-like action. Our results clearly demonstrated that non-insulin-like effects of vanadate could have a beneficial influence on the therapy of diabetes type 2 causing a decrease of glucose production from the liver.