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G-protein-coupled receptor kinases (GRKs) play a key role in agonist-induced desensitisation of G-protein-coupled receptors (GPCRs) that are involved in metabolic regulation and glucose homeostasis. Our aim was to examine whether small peptides derived from the catalytic domain of GRK2 and -3 would ameliorate Type 2 diabetes in three separate animal models of diabetes.Synthetic peptides derived from a kinase-substrate interaction site in GRK2/3 were initially screened for their effect on in vitro melanogenesis, a GRK-mediated process. The most effective peptides were administered intraperitoneally, utilising a variety of dosing regimens, to Psammomys obesus gerbils, Zucker diabetic fatty (ZDF) rats, or db/db mice. The metabolic effects of these peptides were assessed by measuring fasting and fed blood glucose levels and glucose tolerance.Two peptides, KRX-683(107) and KRX-683(124), significantly reduced fed-state blood glucose levels in the diabetic Psammomys obesus. In animals treated with KRX-683(124) at a dose of 12.5 mg/kg weekly for 7 weeks, ten of eleven treated animals responded with mean blood glucose significantly lower than controls (4.7+/-0.4 vs 16.8+/-0.8 mmol/l, p/=0.0001). Significant reductions in blood glucose compared with controls were also seen in ZDF rats administered KRX-683(124) and in db/db mice, which had significantly reduced fasting and 2-hour postprandial glucose levels after the treatment.Sequence-based peptides derived from GRK2/3 have an antidiabetic effect demonstrated in three different animal models of Type 2 diabetes. By modulating GRK2/3 activity, these peptides enhance GPCR-initiated signal transduction, resulting in improved glucose homeostasis. Sequence-based peptide modulation of GRK could prove useful in the treatment of Type 2 diabetes.

The desert gerbil Psammomys obesus (“sand rat”), a model of nutritionally induced insulin resistance and non-insulin-dependent diabetes mellitus, was treated after weaning with exogenous insulin implants in the normoglycaemic, normoinsulinaemic state. Albino rats matched for weight and age served as high energy diet adjusted reference animals. Insulin administration, elevating the serum insulin to 6000 pmol/l resulted in only a mild reduction in blood glucose levels in Psammomys, but caused a severe, often fatal hypoglycaemia in the albino rats. The hepatic response to insulin-induced hypoglycaemia in rats involved a significant loss in glycogen and suppression of phosphoenolpyruvate carboxykinase (PEPCK) activity. In Psammomys under similar hyperinsulinaemia no appreciable changes in liver glycogen and PEPCK activity were evident, indicating that blood glucose was replenished by continuing gluconeogenesis. Euglycaemic, hyperinsulinaemic clamp caused a complete shut-down of hepatic glucose production in albino rats. However, in both diabetes-prone and diabetes-resistant Psammomys lines, mean hepatic glucose production was reduced by only 62 to 53 % respectively, despite longer lasting and higher levels of hyperinsulinaemia. These results indicate that Psammomys is characterized by muscle and liver insulin resistance prior to diet-induced hyperglycaemia and hyperinsulinaemia. This is assumed to be a species feature of Psammomys, exemplifying a metabolic adjustment to survival in conditions of food scarcity of both animal and human populations. It may reflect a propensity to insulin resistance and hyperglycaemia in population groups exposed to affluent nutrition. [Diabetologia (1996) 39: 1269–1275]

The possibility that free fatty acids (FFA) and their CoA esters may directly inhibit the activity of enzymes of glycolysis and lipogenesis was studied in liver and adipose tissue of acutely diabetic rats. Despite a marked elevation in tissue FFA, the activity of glucose-6phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, α-glycerophosphate dehydrogenase, aldolase, citrate synthetase, and several other enzymes was not affected 3 or 6 h after anti-insulin serum injection. The activities of hepatic and adipose tissue acetyl-CoA carboxylase, hepatic glucokinase and adipose tissue hexokinase were decreased. The tissue FFA levels were compared to linoleate concentrations required for halfmaximal inhibition (Ki) of several enzymes in tissue fractions afterin vitro contact of 30 min at 37 °C. Linoleate irreversibly inactivated the enzymes to a varying degree but its effect was dependent on the protein content of the system. The Ki of linoleate increased linearly with cellular protein concentration; when extrapolated to the protein level in the intact cell it became unphysiologically high, markedly exceeding the liver or adipose tissue FFA concentration of acutely diabetic rats. The Ki linoleate and the actual FFA concentrations were particularly discrepant in the cytoplasmic compartment, comprising roost of the investigated enzymes, but only a small proportion of tissue FFA, as determined by measurements of intracellular FFA distribution. Similar discrepancy was found for the interaction of palmityl-CoA with glucose-6-phosphate dehydrogenase and acetyl-CoA carboxylase. Thus, the selective decrease in the activity of glucokinase and acetyl-CoA carboxylase in acute diabetes was attributed to factors other than FFA or their CoA esters. The nature of FFA-enzyme interaction, causing enzyme inactivation rather than specific inhibition is discussed, concluding that FFA do not act as direct homeostatic modifiers of enzymes regulating carbohydrate and fat metabolism.

The metabolic basis for glycogen accumulation in the placenta of rats with diabetes induced by streptozotocin on day 12 of pregnancy was studied on days 15 and 20. On day 15 glycogen content of the placenta was 1.5-fold higher in the diabetic than in the control rats and this difference increased to greater than fivefold on day 20 of gestation whether calculated per g tissue or per total placenta. Accumulation of glycogen was associated with increased specific activities of both glycogen synthase and phosphorylase. The activities of these enzymes regulating synthase and phosphorylase activities and the activity of acid alpha-glucosidase were not significantly affected by diabetes. Glucose-6-phosphate concentration of the placenta was 67 and 23 nmol/g in diabetic and control rats, respectively. Incubation of placental homogenates with glucose increased the rate of inactivation of phosphorylase and activation of glycogen synthase. These results indicate that the enhanced glucogenesis in diabetes is not due to changes in the activities of these enzymes, as measured in vitro under standard conditions. The factors promoting glycogen accumulation in vivo are related to the abundance of glucose and glucose-6-phosphate as substrates for glycogen synthesis, which may also cause an increase in the activity ratio glycogen synthase a/phosphorylase a. In addition, the high intracellular glucose-6-phosphate concentration is likely to enable glycogen synthase b to contribute to glycogen synthesis.

Rats were made diabetic by the injection of streptozotocin on day 12 of pregnancy and were investigated on days 17 and 20 of pregnancy. A significant correlation between both maternal plasma triglyceride and nonesterified fatty acid levels with placental or fetal triglyceride content was found, although fetal weight did not change significantly. In order to investigate the source of the placental and fetal fat the rats received, intragastrically 24 h earlier, 1-14C-triolein (as a preformed fatty acid tracer) and tritiated water (as a marker of de novo fatty acid synthesis). Several maternal tissues, placenta, and fetuses were extracted and analysed for fatty acid radioactivity. Compared with non-diabetic pregnant rats, maternal 14C-label storage was reduced. In contrast, the placental and fetal tissues demonstrated a significant rise in 14C-label, similar in magnitude to the rise in their triglyceride content. The pattern of 3H incorporation also indicated a pronounced decrease in maternal fatty acid synthesis, but no increase in de novo fatty acid synthesis in the fetus or placenta. The findings show that fetal lipids in the pregnant rat originate both from maternal fatty acids and those synthesized in situ. The diabetes-induced increment in fetal triglyceride content is derived, however, from preformed maternal triglycerides or non-esterified fatty acids secondary to the increase in their concentration in the maternal circulation.

Summary: A substantial portion of rat milk triglycerides was hydrolyzed in the ligated stomach of suckling rats with excised lingual gland and pancreas, due to the action of gastric lipase. Free fatty acids were the main lipolytic products. There were some diglycerides and traces of monoglycerides. Medium chain length (C8-C12) fatty acids were predominantly recovered in the free fatty acid fraction, whereas the remaining tri- and diglycerides became richer in long chain (≥C14) fatty acids suggesting a preferential lipolysis of medium chain fatty acid ester bonds. The lipase activity in extracts of stomach wall and sublingual gland tissue was more stable at acid pH and more resistant to the action of pepsin than the activity of pancreatic lipase. Trypsin strongly affected lingual lipase activity but only moderately reduced gastric and pancreatic lipase activity. Presence of sodium taurocholate made the lingual and gastric lipases less sensitive to proteolytic attack. It was also found that the activity of gastric lipase, related to the tissue protein content, decreased with the age of rats, whereas that of lingual lipase increased. The joint capacity of the stomach and lingual gland lipases amounted to about 50% of the total digestive lipolytic capacity 6 days after rat birth but decreased to about 20% at 60 days of life. This was due mainly to the considerable increase in the pancreatic gland size. Speculation: Lingual and gastric lipases are distinct enzymes with a mildly acidic pH optimum. They are stable in acid medium, resistant to peptic proteolysis and able to split all three ester bonds on the glycerol molecule. These properties enable them to efficiently digest milk triglycerides in the stomach of the newborn. There is a carry-over of digestive activity by lingual and gastric lipases into the upper intestine where they supplement the action of pancreatic lipase and complement it with respect to lipid emulsification. Gastric lipase seems important in intestinal lipolysis because since it is activated by bile salts, the presence of which protects the enzyme against tryptic proteolysis. Although the relative contribution of gastric and lingual lipases to the overall lipolytic capacity falls short of that of pancreatic lipase, it is likely that the extrapancreatic lipases are compensatory in conditions of pancreatic lipase deficiency.