Albert Renold Memorial Lecture: Molecular Background of Nutritionally Induced Insulin Resistance Leading to Type 2 Diabetes – From Animal Models to Humans

Albert Renold strived to gain insight into the abnormalities of human diabetes by defining the pathophysiology of the disease peculiar to a given animal. He investigated the Israeli desert-derived spiny mice (Acomys cahirinus), which became obese on fat-rich seed diet. After a few months hyperplasia and hypertrophy of β-cells occurred leading to a sudden rupture, insulin loss and ketosis. Spiny mice were low insulin responders, which is probably a characteristic of certain desert animals, protecting against insulin oversecretion when placed on an abundant diet. We have compared the response to overstimulation of several mutant diabetic species and nutritionally induced nonmutant animals when placed on affluent diet. Some endowed with resilient β-cells sustain long-lasting oversecretion, compensating for the insulin resistance, without lapsing into overt diabetes. Some with labile beta cells exhibit apoptosis and lose their capacity of coping with insulin resistance after a relatively short period. The wide spectrum of response to insulin resistance among different diabetes prone species seems to represent the varying response of human beta cells among the populations. In search for the molecular background of insulin resistance resulting from overnutrition we have studied the Israeli desert gerbil Psammomys obesus (sand rat), which progresses through hyperinsulinemia, followed by hyperglycemia and irreversible beta cell loss. Insulin resistance was found to be the outcome of reduced activation of muscle insulin receptor tyrosine kinase by insulin, in association with diminished GLUT4 protein and DNA content and overexpression of PKC isoenzymes, notably of PKCε. This overexpression and translocation to the membrane was discernible even prior to hyperinsulinemia and may reflect the propensity to diabetes in nondiabetic species and represent a marker for preventive action. By promoting the phosphorylation of serine/threonine residues on certain proteins of the insulin signaling pathway, PKCε exerts a negative feedback on insulin action. PKCε was also found to attenuate the activity of PKB and to promote the degradation of insulin receptor, as determined by co-incubation in HEK 293 cells. PKCε overexpression was related to the rise in muscle diacylglycerol and lipid content, which are prevalent on lascivious nutrition especially if fat-rich. Thus, Psammomys illustrates the probable antecedents of the development of worldwide diabetes epidemic in human populations emerging from food scarcity to nutritional affluence, inappriopriate to their metabolic capacity.

Albert Renold strived to gain insight into the abnormalities of human diabetes by defining the pathophysiology of the disease peculiar to a given animal. He investigated the Israeli desert-derived spiny mice (Acomys cahirinus), which became obese on fat-rich seed diet. After a few months hyperplasia and hypertrophy of/J-cells occurred leading to a sudden rupture, insulin loss and ketosis. Spiny mice were low insulin responders, which is probably a characteristic of certain desert animals, protecting against insulin oversecretion when placed on an abundant diet. We have compared the response to overstimulation of several mutant diabetic species and nutritionally induced nonmutant animals when placed on affluent diet. Some endowed with resilient //-cells sustain long-lasting oversecretion, compensating for the insulin resistance, without lapsing into overt diabetes. Some with labile beta cells exhibit apoptosis and lose their capacity of coping with insulin resistance after a relatively short period. The wide spectrum of response to insulin resistance among different diabetes prone species seems to represent the varying response of human beta cells among the populations. In search for the molecular background of insulin resistance resulting from overnutrition we have studied the Israeli desert gerbil Psammomys obesus (sand rat), which progresses through hyperinsulinemia, followed by hyperglycemia and irreversible beta cell loss. Insulin resistance was found to be the outcome of reduced activation of muscle insulin receptor tyrosine kinase by insulin, in association with diminished GLUT4 protein and DNA content and overexpression of PKC isoenzymes, notably of PKCe. This overexpression and translocation to the membrane was discernible even prior to hyperinsulinemia and may reflect the propensity to diabetes in nondiabetic species and represent a marker for preventive action. By promoting the phosphorylation of serine/threonine residues on certain proteins of the insulin signaling pathway, PKCe exerts a negative feedback on insulin action. PKCe was also found to attenuate the activity of PKB and to promote the degradation of insulin receptor, as determined by co-incubation in HEK 293 cells. PKCe overexpression was related to the rise in muscle dia-INTRODUCTION This lecture is dedicated to the memory of Albert Renold (Fig. 1). I shall not dwell on his biography, which is well known to most of us. I would like just to say that he was endowed with a generous personality, free of any scientific or personal prejudice, and unbound enthusiasm for experimental .research of diabetes. His leadership capacity, art of dialogue and negotiations, enabled him to establish the EASD in 1965 and perform outstandingly as the President of IDF 1979-1983. He attracted a cohort of renowned international scientists to his Geneva Department, and created there a European Mecca of diabetes research and teaching from which close to 500 outstanding contributions emanated. He passed away suddenly on March 21, 1988, about 13 years ago.
I had the privilege to share with him the excitement of experimental diabetes research, in animal models and to launch in 1982 the International Workshops on Lessons from Animal Diabetes in Jerusalem, (Fig. 2) which have continued to take place in several locations, and today, the 8th Workshop, in Tokyo. Albert Renold strived to gain insight into the abnormalities of human diabetes by defining the pathophysiology of the disease peculiar to a given animal. He was convinced that the elucidation of pathogenesis of human diabetes will be better understood by the integration FIGURE 1 Professor Albert E. Renold in his laboratory at the Institute de Biochimie Clinique in Geneva. of pathogenesis of several genetically transmitted or experimental diabetes syndromes. His belief was that whether induced by surgical, chemical, endocrine, immunologic or genetic treatments, models of diabetes can be extremely informative and helpful, but may be misinterpreted by equating a single model with human diabetes. Ill With the advances in research, it may be said that the existence of numerous variants of animals with characteristics close to Type 2 diabetes allows to uncover different mechanisms leading to insulin resistance,/3-cell demise, disrupted glucoregulation and species specific complications. Consolidation of this knowledge may pave the way to classification of human diabetes into better defined entities.
Among the models Renold investigated were the Israeli desert-derived spiny mice (Acomys cahirinus), (Fig. 3) which became obese on fat-rich seed diet. After a few months/3-cell hyperplasia and hypertrophy developed leading to a sudden islet rupture and resulting in ketosis and insulin deficiency. Prior to overt diabetes spiny mice exhibited only intermittent hyperglycemia and impaired glucose tolerance. They were characterized as "low insulin responders". Different secretagogues failed to elicit sufficient /3-ceil insulin response. This was attributed to several anatomic and biochemical features of their /3cells, e.g., low adenylate cyclase activity, low FIGURE 3 A spiny mouse (Acomys cahirinus) compared with an albino mouse. cAMP content, low amount of vincristine precipitable material in microtubules through which insulin is extruded and low innervation (reviewed in 2,3). These properties were initially attributed to a genetic mutation, which might have occurred during 15 years and 40 generations of maintenance in captivity on fat-rich seed diet. However, it is now most probable that these are typical characteristics of desert animals subsisting on scarce nutrition requiring only limited capacity of insulin response. I41 The low insulin response to glucose and other secretagogues may be a protection against/3-cell overstimulation by sudden availability of nutrients with which spiny mice organism is unable to cope.
A particular characteristic of the spiny mice was the proliferation of /5-cells within islets, accompanying the obesity. The islets increased several fold in number, diameter and /8-cell content (Fig. 4). The resulting very high density of B-cells in the islets was the highest in comparison with other diabetic obese species (Fig. 5). Thus, spiny mice represent a particular example of obesity associated with enormous enlargement of islets.
Among the protective mechanisms which spiny mice were able to mobilize to cope with the increased nutrient intake were increased plasma levels and hepatic production of triiodothyronine (T3) which induced some energy waste and was especially evident on high sucrose diet (Tab. I).  [96].  We have devoted particular attention to the gerbil Psammomys obesus (often nicknamed sand rat) The main native nutrient of Psammomys is a leafy halophilic plant, Atriplex halimus, (saltbush) (Fig. 6). This gerbil never exhibits diabetes in its native desert habitat but was known to develop fatal diabetes when transferred from the Nile Delta in Egypt to the USA. [9] In the 1980s Adler and colleagues have transferred Psammomys from the desert shores of the Dead Sea to the laboratory, I1,111 maintaining the animals on low energy (LE) diet, and succeeded to establish a stable, reproducible colony. The animals became diabetic on standard laboratory diet, which is high energy (HE) with respect to Psammorays (Fig. 7). The animals are not hyperphagic but when offered their diet ad libitum gradually lapse from normalcy (stage A) into stages of hyperinsulinemia (stage B), hyperinsulinemia with hyperglycemia (stage C), and insulin   Figure 8 and described in detail in several publications and reviews. [12][13][14] Similar observations of the progress to diabetes have been published regarding Psammomys from Algeria I151 and a branch, of Israeli Psammomys colony bred in Australia. [16,17] It should be emphasized that insulin resistance and hyperinsulinemia appear before weight gain in Psammomys but they may contribute to adipose tissue accretion, a condition which we term: diabesity. Triglyceride deposition in adipose and nonadipose tissues, primarily muscles is driven by hepatic lipogenesis, which continues unabated despite insulin resistance, as demonstrated in rats. [18] Beacon hypothalamic gene recently discovered by Collier and colleagues [9,2] promotes diabesity on ad libitum feeding. It is remarkable that the progress of Psammomys to diabesity may be reversed by reducing the nutrition for just a few days, in stage C, before apoptosis and ]3-cell degranulation set in. The recovery by diet restriction has been described in our colony [2] and in Psammomys bred in Australia. [221 Psammomys maintained on HE diet for a few weeks undergoes massive /3-cell degranulation, loss of insulin immunostaining, apoptosis and necrosis set in. [23][24][25][26][27] J6rns and colleagues [281 have followed in recent ultrastructural studies the time course of progression of Psammomys to diabetes. A gradual loss of /3-cell insulin, glucokinase and GLUT2 transporter immunoreactivities was visualized, occuring subsequently to hyperglycemia. After one week on HE diet the /3-cell volume became reduced by about 1/3 and immunostaining of glucokinase, and GLUT2 by >50% (Fig. 9). After 3 weeks on HE diet this reduction became 70-95% in correlation with the rising blood glucose level. Ultrastructurally Set 1: Control pancreas from Psammomys on LE diet. Semithin sections stained for insulin, glucokinase (cytoplasm) and GLUT2 transporter (membrane). Magnification 300x; Set 2: Hyperinsulinemic Psammomys after one week on HE diet. Compared with control pancreas the immunoreactivities of insulin glucokinase and GLUT2 are fainter and moderately reduced. GLUT2 exhibits large gaps. Magnification 400x; Set 3: Hyperglycemic, hyperinsulinemic Psammomys after I week on HE diet. The immunoreactivities of insulin, glucokinase and GLUT2 are markedly decreased compared with nonglycemic hyperinsulinemic animal. Only a few cells exhibited immunostainable insulin. Magnification 500x; Set 4: Hyperglycemic Psammomys after 3 weeks on HE diet. Extensive ]-cell death. The remaining/-cells show vacuolization and very faint immunostaining for insulin and glucokinase. GLUT2 is present in the cytoplasm rather than membrane. Magnification 800x. different signs of necrotic destruction of pancreatic ]3-cells such as the pyknosis of nuclei and a massive vacuolization in the cytoplasm were evident. These findings were accompanied by swollen mitochondria and dilated cisternae of the Golgi complex and of the rough endoplasmic reticulum. At one week on the HE diet most secretory granules were still intact even in the face of marked degranulation. Other endocrine cells of the islet did not show structural lesions. These changes in/3cells were particularly severe in animals after 3 weeks on the HE diet.
The/3-cells in the pancreas removed from hyperglycemic, insulin deficient animals (stage D), after several weeks on HE diet were also found to exhibit apoptosis and DNA cleavage I21,251 (Fig. 10). DNA fragments were seen in the cell nucleus and in the cytoplasm. Also Donath et al. [26] and Nesher et al. [27] have observed both apoptosis and necrosis in pancreases removed from Psammomys after several weeks on HE diet. It is relevant that/3-cell apoptosis was also evident in the diabetic, obese hyperphagic ZDF rats. [29] Figure 9 clearly shows that the HE diet induced pancreatic/3-cell dysfunction in the Psammomys and disintegration of cellular architecture as a consequence of developing hyperglycemia. Hyperinsulinemia by itself does not appear to be responsible for the observed deterioration of the pancreatic/3-cell function, except by the imposed oversecretion. Prolonged incubation of isolated /3-cells from ZDF rats I31 and from humans [31l in high glucose media markedly impaired basal and stimulated insulin secretion. Unger [321 also FIGURE 10 /3-cell apoptosis in stage D Psammomys revealed by Tdt-mediated dUTP nick end labeling (TUNEL) and staining of the biotin labeled DNA cleavage nick ends with 3-aminoethyl carbazole. Note the nuclear fragmentation and spreading of nuclear fragments in the cytoplasm, indicated by the brownish flecks. Magnification 500x. Reproduced with permission of the Editor of Pancreas, Ref. [18].
pointed out the deleterious effect of FFA and triglyceride (TG) accumulation in ]3-cells on the insulin secretion function in ZDF rats, termed by him as "lipotoxicity".
There is no direct evidence for the involvement of glucoor lipotoxicity in the necrosis of/5-cells in Psammomys unlike the findings in ZDF rats. An attempt to prevent the possible toxic action of advanced glycation end products or of nitrous oxide by including the advanced glycation inhibitor, aminoguanidine in the hyperglycemic incubation medium was not effective in protecting /5-cells in Psammomys. I271 However, Kaneto et al. [33] obtained evidence that reducing sugars may trigger apoptosis in B-cells of albino rats by provoking oxidative stress of glycation products. In their hands the antioxidants N-Acetyl-Lcysteine and aminoguanidine inhibited the apoptosis. We presume that the damage to/8-cell architecture with loss of the insulin biosynthetic and secretory capacity in Psammomys occurs promptly and is most probably the result of exhaustion chiefly due to the hypersecretion pressure prior to eventual cytotoxicity.
Psammomys in stage C shows increased proinsulin levels in the circulation, up to one half of the circulating immunoassayable total insulin. I341 The inordinate secretion pressure may cause a swift exocytosis of immature insulin granules escaping the C peptide cleavage before the release into the circulation. Similar disproportionate elevation of proinsulin in human and experimental type 2 diabetes and insulin resistance has been observed. I351 This indicates, on one hand, that the compensation of the delayed glucose removal or suppression of gluconeogenesis are not effective since proinsulin has only a minute fraction of insulin activity. On the other hand, the high level of circulating proinsulin does not mean that its secretion equals that of insulin since the halflife of proinsulin is much longer than that of insulin.[ 36

Insulin Resistance and Tyrosine Kinase
Attenuation in Psammomys obesus Attenuation of tyrosine kinase (TK) is the basic event responsible for deficient function of the insulin receptor (IR) causing insulin resistance. To investigate the development of insulin resistance, the activity of TK, the initiator of insulin signaling pathway was studied in the liver and muscle of Psammomys. Kanety and colleagues I371 found that the binding of insulin to the liver and muscle preparations was very low, even in stage A, indicating the low IR content, about one fifth of the laboratory albino rat. However, insulin binding and TK activity per receptor was completely normal, both in vitro and in vivo. The TK activity was measured in stages B and C of progression to diabetes as compared to the normoglycemic stage A. Basal phosphorylation of the isolated IR was comparable in these stages to that in the normoglycemic stage A, but the extent of TK activation by insulin was markedly lower in stages B and C in the liver and muscle (Fig. 11). [37] The reduced insulin activation was accompanied by a marked decrease in muscle GLUT4 protein and mRNA (Fig. 12). Both could be reversed by nutritional restriction to one half of their daily food intake for a few days. The recovery of TK activity was complete when the animals returned to normoinsulinemia. The recovery was partial when hyperglycemia was corrected but the insulin  levels did not return to normal (Fig. 11). This finding points out the attenuating effect of hyperinsulinemia on the function of the IR, indicating that it is an important cause of insulin resistance.

Deleterious Effect of Hyperinsulinemia on IR Function
The deleterious effect of hyperinsulinemia, even in non-nutritionally induced conditions, can be demonstrated in several animal species and humans. A few cogent examples can be quoted. Transgenic mice enriched with 8 or 32 insulin gene copies, resulting in circulating hyperinsulinemia, exhibited both IGT and hypertriglyceridemia in correlation with the amount of insulin gene copies in their B-cells (Fig. 13). [38] In other transgenic mice insulin oversecretion was related to deleterious overexpression of glutamine: fructose 6 phosphate amidotransferase associated with/3-cell malfunction. [39] Hyperinsulinemia and insulin resistance was also achieved by targeted disruption of genes Among other detrimental results of hyperinsulinemia is the uncoupling of the TK activity in 3T3 cells after initial activation. I421 In HepG2 cells the activation of IR TK by insulin was attenuated; only incompetent receptors remained on cell surface [43] and in rat adipocytes the Vma of TK was reduced.
[441 It was also found that hyperinsulinemia inhibits myocardial protein degradation in patients with cardiovascular disease, which is a potential mechanism contributing to cardiomegaly. I451 Hyperinsulinemia of endogenous or exogenous origin should be considered, also in humans, not only as a compensatory response to insulin resistance, but as an inducer of a defect in insulin action. In nondiabetic human volunteers the infusion of insulin for several days followed by euglycemic hyperinsulinemic clamp, resulted in the reduction of nonoxidative wholebody glucose metabolism by up to 40o/0. [46] Also, patients with insulinoma exhibited insulin resistance that was related to the extent of their hyperinsulinemia. I471 Furthermore, fasting hyperinsulinemia in diabetes-prone Pima Indians has been found to exert a primary role in the progress to type 2 diabetes by being the antecedent of the decline in response to i. Among the several PKC isoenzymes probed with specific antibodies PKCe was most significantly overexpressed in the skeletal muscle of Psammomys, in the hyperglycemic-hyperinsulinemic stage C compared with the nondiabetic stage A (Fig. 14). It was also translocated from the cytosol to muscle membrane to a larger extent than other PKC isoenzymes, which indicates not only PKCe overexpression but increased activity as well. [49] As shown in Table III   showed highest degree of membrane association in Stage A Psammomys but was surprisingly low in Stage C. The membranal PKCc and ]3 were also elevated. PKC" was elevated but did not change between stages A and C. PKC ,and (the atypical isoenzymes) are known to promote phosphorylations integral to the insulin signal transduction. We have compared the expression of several PKC isoenzymes in diabetes resistant (DR) and diabetes prone (DP) Psammomys lines. The DR line was isolated from the parent Psammomys colony by assortative mating of individuals, which did not exhibit hyperglycemia and hyperinsulinemia on HE diet. I531 It was found that the difference between the DR and DP animals is related to the efficiency of nutrient utilization: the cost of weight gain upon growth is in Psammomys DR 9.3 kcal/g and DP 6.0kcal/g. Interestingly, a significant overexpression of PKCe was also observed in the normoglycemic stage A of DP Psammomys compared with the DR line ( Fig. 15), which indicates that PKCe overexpression precedes the onset of overt insulin resistance. Thus, PKCe overexpression in stage A may be considered as a marker of "prediabetic" or "preinsulinemic" stage and of propensity of a given individual to progress to overt diabetes on affluent nutrition. It is, however, without untoward consequences as long as the diet is LE. PEPCK, the rate limiting enzyme of gluconeogenesis, I541 as well as the hepatic glucose output (Fig. 16). This may be a typical characteristic of a desert animal in which muscle insulin resistance saves glucose for the support of other glucose obligatory tissues.
Since PKCe overexpression resulted in impaired TK activation by insulin and reduced GLUT4 mRNA and protein, which indicates an impaired PI3K activation, it was of interest to investigate whether PKC overexpression induces a further negative downstream defect in insulin signaling. The activity of PKB/Akt was determined, an enzyme regarded as responsible for the activation of pleiotropic metabolic systems within the cell, subsequent to PI3K activakon on IRS. The transfection of HEK 293 cells with IR and/or PKCe plasmids, followed by stimulation with insulin or TPA respectively, clearly showed the activation of PKCe by TPA coupled with a significant reduction of PKB expression and inhibition of PKB activation (Fig. 17). These results indicate that PKCe inhibits PKB activation by insulin and has a far-reaching negative effect on metabolic reactions dependent on insulin signaling as illustrated in the insulin signaling scheme (Fig. 18 The activity of PKC isoenzymes in the membrane, in IR proximity, may be the reason for the inhibitory influence on the IR TK activation. Several PKC isoenzymes were reported to reduce the TK catalyzed phosphorylation of the IR ]3-subunit and IRS-1. I55- 61 We have found that PKCe overexpression was associated with reduced binding of insulin by muscle IR (Fig. 19). This was not likely to be attributed to the impaired binding capacity of IR but to the reduction in the number of IR per cell. Indeed, the downregulation of IR was demonstrated in HEK 293 cells which were transfected with human IR and PKCe plasmids and activated by TPA (Fig.  20). Evaluation  This is in accord with previous observations of downregulation of IR, by the conventional PKCo [61] and possibly other DAG sensitive PKC enzymes.
Several in vitro studies indicate that PKC isoenzymes directly interfere with insulin signaling through serine/threonine phosphorylation of either the IR itself or one of its major substrates. [62][63][64] PKC may also mediate the tumor necrosis factor (TNFce) inhibition of IR function, the major cause of diabesity-linked insulin resistance. [65,66] TNFc was reported to induce phosphorylation of IRS-1 on serine 307. [67] Interestingly, high insulin levels also induced the phosphorylation of this serine in association with insulin resistance in signal transduction. This observation suggests the possibility of PKC involvement. Several other serine sites have been indicated to be phosphorylated on IR or IRS with negative effects on signal transduction. I68-71 Muscle PKC activation was also seen in insulin resistant Goto-Kakizaki rats. I711 It may be therefore assumed that serine/threonine phosphorylation of IRS-1, inhibits the TK activity of the IR via a feedback loop and is responsible for the deficient TK activation by insulin in Psammomys as described earlier, I371 with insulin resistance accentuated at stages B and C on the HE diet.

PKCe Overexpression and Muscle Lipid Content
The enhanced PKCe activity and/or expression in Psammomys was found to be correlated with the increased muscle content of DAG (Fig. 21). [491 DAG is an intermediate of both fatty acid esterificatio to TG and TG breakdown to fatty acids and glycerol. The raised muscle concentration of DAG results from increased TG deposition and turnover in muscle, which occurs in the situation of hyperinsulinemia and hyperglycemia, characteristic of stages B and C of Psammomys. An increase in incorporation of glucose carbon into DAG was also seen in the soleus muscle incubated with glucose and insulin I721 probably in relation to increased intracellular TG synthesis. Also, the rise in plasma FFA in conditions of IGT may contribute to muscle fat deposition. Indeed, in vitro uptake of saturated FFA was recently reported by Yu et al. 731 to raise rat muscle DAG levels and lead to PKC activation (Fig. 22). Exogenous lipid infusion in rats resulted in the  deposition of a significant fraction of fatty acids in muscle, particularly in the fasted state I741 and FFA infusion to human subjects was found to elicit insulin resistance and activation of PKC0. I751 Muscle TG content is also increased in patients with type I diabetes although it did not correlate in this condition with insulin sensitivity. [76] Increased TG deposition in muscles was studied extensively by Kraegen and colleagues [77][78][79] in rats maintained on a fat-rich diet. Insulin resistance developed in these rats both in muscles and liver. The hepatic insulin resistance was associated with increased gluconeogenesis, whereas the muscle insulin resistance markedly reduced the insulin stimulated glucose uptake. The accumulation of muscle fat was inversely correlated with insulin resistance and delayed glucose disposal also in human subjects. [8] Schmitz-Pfeiffer et al. [8] found that in high fat fed rats, TG and DAG accumulated in muscle and activated PKC isoenzymes interfering with IR function.
The total expression of PKC c, e and " isoenzymes was not increased in muscles of these rats but their activity and distribution between cytosolic and membrane compartments was shifted in favor of the latter. This was particularly prominent in the case of PKCe showing a sixfold increase in the membrane/cytosol ratio in correlation with muscle TG content (Fig. 23). There was no accumulation of TG and DAG in control rats fed a starch diet. Also in Psammomys the muscle and liver TG content increased on HE diet but the increase was moderate in comparison with fat-fed rats (Fig. 24). [14] This does not necessarily mean that muscle lipid deposition is a result of outright obesity, but even a small weight gain usually occurring prior to marked hyperglycemia in Psammomys leads to TG deposition, also in nonadipose tissues.

Protein Tyrosine Phosphatases and Muscle Insulin Resistance in Psammomys
Goldstein and colleagues [81][82][83] have reviewed the mode of action of PTPases and their impact on the regulation of IR signaling by modulating the tyrosine phosphorylation state of IR and of proteins that transmit the insulin signal. PTPIB is considered to play a key role in glucose homeostasis and energy expenditure and has been pointed out as an important negative regulator of insulin action. [83][84][85] Mouse transgenic and knockout models with altered expression of LAR (Leukocyte Antigen Related), PTPIB and SHP-2 PTPases generated additional insight into the involvement of these regulatory enzymes in insulin action and glucose metabolism. LAR PTPase can have a negative impact on cellular insulin signaling, although its exact physiological role has not Triglyceride (/mol/g) 8-Triglyceride (/mol/g) FIGURE 23 A. Relation of gastrocnemius muscle triglyceride and DAG contents in rats fed fat-rich and starch diets. 13 24 Tissue triglyceride content in Psammomys. Liver and muscle triglyceride levels in Psammomys in stages A-C. Values are mean_SE for groups of 10-14 animals at each stage differences significant at P < 0.05 at least. Adapted from Ref. [14].
been established. On the other hand, SHP-2 positively influences receptor signaling on mitogenic pathways in cellular studies. The reduction of tyrosine phosphorylation effected by TK activity could be caused by enhanced dephosphorylation of the receptor/3-subunit and IRS-1, carried out by the action of the tandem domain transmembrane LAR PTPase. Abundance of LAR-PTPase was observed in skeletal muscle and liver of rodents with genetically determined insulin resistance rats and in human obese patients. [86'89] The activity and expression of LAR PTPase was investigated in Psammomys. [91 In stage A, a low PTPase activity in liver and muscle was found, in parallel with the low density of insulin receptors. Fasting caused a decrease rather than increase in LAR-PTPase in stage A Psammomys (Fig. 25). However, Psammomys tissues in stage C did not show an increase in cytosolic or membranal LAR PTPase activity, compared with stage A, suggesting that LAR-PTPase is unlikely to be responsible for IR dephosphorylation in insulin resistant Psammomys. This observation parallels the findings in human nutritionally induced diabetes. Molecular and linkage analysis of type 1  To examine whether PTP 1B is involved in the susceptibility to insulin resistance or diabetes in Psammomys, Ikeda et al. [921 have measured its expression and activity towards the isolated IR of skeletal muscle of diabetic animals from DP line and control animals from the DR line. The expression level of PTP 1B in the skeletal muscle was increased by 83% in the diabetic animals compared with the control animals. However, when the activity of PTP 1B was determined there was a surprising 60% decrease in its activity in stage C Psammomys. This was seen in the total homogenate and especially in the particulate fraction when compared with the control DR animals or with the prediabetic animals (Fig. 26). In addition, PTP 1B activity was inversely correlated to serum glucose concentrations and insulin levels. The decrease in activity was assumed to be due to qualitative change in the PTP 1B molecule, probably secondary to the effect of some factor(s) in the diabetic milieu. These findings suggest that PTP 1B is not involved in the development of insulin resistance in Psammomys. The

Conclusions and Overview
Psammomys is a model of human nutritionally induced diabetes which reaches now epidemic proportions in certain populations. The underlying cause is increased food availability and consumption following a welcome improvement in lifestyle. However, the latent propensity to diabetes among these populations is related to their inborn metabolic capacity which is probably not adjustable to the dietary surplus. This may result in insulin resistance, diabesity and strain on compensatory insulin secretion with ultimate loss of ]B-cell function. Some authors refer to such socioeconomic perspective as leading to nutritional genocide of global proportions. The above described elements of insulin resistance in Psammomys represent the antecedents of the development of worldwide diabetes epidemic in human populations emerging from food scarcity to food abundance.
Insulin resistance and ]3-cell dysfunction are considered two interrelated factors in the pathogenesis of IGT and type 2 diabetes. Although a debate is still continuing on the primacy of each abnormality, the evidence from Psammomys studies clearly demonstrates that insulin resistance with hyperinsulinemia precede the ]B-cell lesion. ]3-cell dysfunction occurs only on HE diet, there is no ]B-cell lesion in animals consuming their native salt bush or laboratory LE diets. The onset of insulin resistance and hyperinsulinemia in Psammomys precede any appreciable weight gain, precluding any contribution of obesity to IGT. On the contrar)4 if hyperinsulinemia with el-cells oversecrete long enough the expansion of adipose tissue may occur and secondarily lead to overweight. A return to normalcy is possible even after a period on HE diet, either by a short term fasting, or by restricting the dietary intake. It is most probable that similar triggering of IGT and diabetes applies to the affected human populations.
The aberrant activity of PKC isoenzymes, especially of PKCe, is the potential causative mechanism in the generation of insulin resistance by phosphorylation of serine/threonine residues on IR and proteins of the signaling pathway. This may lead to TK, PI3K and PKB attenuation with negative feedback as well as to IR degradation. Thus, the compensatory hyperinsulinemia precludes the adequate function of insulin signaling.
One of the primary outcomes of the overexpression of PKC isoenzymes may include PKCI3, involved in the initiation of vascular complications of diabetes in insulin independent tissues as retina and kidneys. [94,95] The common aspect of this overexpression with Psammomys and fatfed rats is tissue accumulation of DAG. DAG is directly related to tissue TG content and this may be an especially important inducer of insulin resistance in nonadipose tissues. Insulin resistance and its corollaries may then result from enhanced muscle lipid deposition, not necessarily from hyperlipidemia. The initial fat deposition may be also promoted by hyperinsulinemia with hyperglycemia and the following diabesity.
The preventive attempts should be therefore directed to avoiding muscle TG deposition, promotion of DAG breakdown by use of modalities activating DAG kinase, which converts DAG into phosphatidic acid. Other possibility to counteract the insulin resistance is specific inhibition of PKC isoenzymes dependent on DAG.
Among those is H7a piperazine derivative, polymyxin B, bisindoxylmaleimide and staurosporine which are inhibitory to PKC isoenzymes in vitro. [96] Herbimycin was also shown to have PKC inhibitory properties. I971 Inhibition of PKCI3 with high degree of specificity has been achieved by LY 33353 I981 and tried successfully in vascular tissues, mainly ocular and renal, which are predisposed to complications in hyperinsulinemic-hyperglycemic conditions. It is also remarkable that the recently reported PKC0 knockout in mice improved insulin action and signaling defects induced by lipid infusion. [99] Potentiating insulin sensitivity at its prevalent concentrations would also lead to lowering of insulin resistance as shown by the application of IR activators. I11 Increased insulin sensitivity was also achieved by treatment with vanadyl sulfate and other vanadium compounds [11][12][13][14] including Psammomys maintained on HE diet (Fig. 27). Vanadyl sulfate restoration of normoglycemia and normoinsulinemia and increase in muscle metabolic activity appears to be distal to IR/TK signaling. I11 The overexpression of PKC isoenzymes may be the result of genetic susceptibility exemplified by Psammomys or by "thrifty gene" characteristics of desert animals or individuals in the affected populations, activated by the changing environmental influences. This course of events is illustrated by Figure 28. The inherent muscle insulin resistance aimed to spare the scarce glucose for obligatory tissues (such as the brain) fails when confronted with excess of nutrients. It turns the insulin resistance to effect a misuse of the surplus energy by creating diabesity, hyperlipidemia and elicit other complications. Such situation is most probably an integral component of the insulin resistance syndrome in animals and humans alike and may be therefore considered as "PKC overexpression syndrome".
Since the times of ancient discoveries of the causes of diabetes, emphasis was always placed on sweetness of urine, blood and other body fluids, leading to the addition of the adjective "mellitus" to diabetes. We should reconsider if this adjective is fully justified. The traditional concepts related to "sweetness" and emphasis on glucose-insulin axis do not explain the basic pathophysiological mechanisms leading to severe complications and mortality both in type 1 and type 2 diabetes as well as the reasons for the development of insulin resistance. The major causes of IGT and diabetes morbidity are strongly related to aberrant fat rather than carbohydrate metabolism. These include lack of restraint of the mobilization of FFA from adipose tissue leading to lipolysis and subsequent excessive fatty acid glucose uptake by peripheral tissues as well as enhanced hepatic gluconeogenesis and delivery of fat to muscle. It may be remarked that the restraint of lipolysis in adipose tissue is the most sensitive action of insulin which becomes compromised by hyperinsulinemia. In blood vessels the oxidative trend prevailing in diabetes directs the cholesterol esterified with unsaturated fatty acids to surrogate receptors due to nonrecognition of the oxidized molecules, which facilitates foam cell and smooth muscle proliferation, arterial coronary cholesterol plaques, atherosclerosis and thrombosis. In the pancreas the excess of lipids taken up or synthesized in ]3-cells leads to /B-cell malfunction due to lipotoxicity with apoptosis and necrosis. As indicated here and elsewhere the accumulation of muscle TG and the consequent rise in the content of the triglyceride intermediate DAG is instrumental in overexpression of PKCe and other PKC isoenzymes. In addition to the derangement in fat metabolism discussed in detail by McGarry I151 the newly discovered detrimental role of DAG accumulation may have uncovered a new culprit implicating fat metabolism as a causative metabolic deviation in diabetes. Perhaps this is the time to start using the term "diabetes lipidicus" rather than "mellitus" in order to appropriately define the diabetes pathophysiology.