https://imcjms.com/public Ibrahim Medical College Journal of Medical Science https://imcjms.com/public/registration/journal_full_text/127 Mon, 31 Oct 2016 12:14:36 +0000 The recent epidemic nature of obesity and association of obesity with the development of type 2 diabetes demands dissection of the pathophysiology of this morbid disorder which is essential for better understanding of the process of evolution of insulin resistance. Different animal models have been used to explore the mechanism linking obesity to insulin resistance and type 2 diabetes. The discovery of ob gene and its product, leptin, has revealed the signaling system regulating energy balance in rodents. The mice lacking this ob gene, ob/ob mice, display obesity, hyperglycemia and hyperinsulinemia and has been extensively used for the study of type 2 diabetes and for potential drug development. In this review,  the features and development of obese hyperglycemic syndrome, the role of leptin in the pathogenesis of the syndrome and finally the applicability of the findings in rodents to body weight regulation and pathogenesis of insulin resistance in humans have been summarized. Ibrahim Med. Coll. J. 2008; 2(2): 72-84 Introduction   Marked obesity, hypoactivity, hyperphagia, transient hyperglycemia (subsiding around 14-16 weeks), severe hyperinsulinemia and insulin resistance are the cardinal features of obese hyperglycemic syndrome when ob gene is expressed in the C57BL/6J strain background.5,12-14 In contrast to the C57BL/6J strain, BL/Ks ob/ob mice are characterized by obesity, severe hyperglycemia and glucose intolerance, transient hyperinsulinemia, islet atrophy and early death.14,15 Early signs of the obese hyperglycemic syndrome are loweroxygen consumption,16 decreasedthermogenesis17 and increased weight gain.12 Metabolism The obese mice are hypercholesterolemic.12,22 However, the increase is primarily in high-density lipoprotein cholesterol, so that atherosclerotic lesions are unusual in this mouse model.23 Plasma triglyceride levels are also elevated in the ob/ob mice. Rate of lipogenesis in the liver and adipose tissue is more than doubled and both intraperitoneal and subcutaneous deposit of fat is increased.4,18 The increased amount of lipids stored by ob/ob mice is accommodated through both hyperplasia and hypertophy of adipocytes; whereas in other genetic obesities in mice, increase in fat depots is entirely due to cell hypertrophy.24 Body weight   Fig-1. Lean C57BL/6J (top) and an obese mice (B6.V-Lepob, bottom). Life span     Islet morphology and biochemistry Immunocytochemical studies demonstrate that glucagon, gastric inhibitory polypeptide and somatostatin containing cells were intermingled with the b-cells in obese mouse islets in addition to their peripheral localization.32,36 In contrast, its lean littermates show only a peripheral localization of these cell types. There are signs of b-cell degranulations in islets from obese mice with increased nuclear and nucleolar size.1,37-39 The islets of the obese mice are remarkably hyperemic;30,39,40 pseudocysts, dilated ducts and dilated capillaries are also common findings in ob-mouse islets.32   The ob/ob mice exhibit an increased sensitivity to low levels of glucose and a left shift in the glucose-response curve.35 The threshold for glucose-induced insulin secretion from perifused islets in fed ob/ob mice is about 2 mmol/L and in 24 hrs fasted obese mice is about 3 mmol/L.47 In contrast, islets from lean mice exhibit considerably higher thresholds - about 5 and 7 mmol/L glucose in fed and 24 hrs starved lean mice, respectively. When insulin secretion was measured from equally-sized islets in ob/ob (Uppsala colony) and lean mice, no significant differences were noted between them both in basal and higher glucose level.48 However, in a similar experimental protocol, islets of ob/ob mice from Michigan colony hypersecreted insulin in response to high concentrations of glucose than that of lean mice.47  Neurotransmitters and hormones that normally potentiate insulin secretion only above 6 mM glucose in lean mice are found to stimulate insulin secretion in ob/ob mice at basal glucose levels.35,47 The ob/ob islets of Norwich colony are permanently more depolarized even in the absence of a primary stimulus to secretion than the lean ones.52 While b-cells of lean mice show continuous spike activity above 16 mM glucose, ob/ob b-cells often exhibit a burst pattern of electrical activity at glucose concentrations as high as 33 mM.52,53 They also exhibit an increased responsiveness to quinine (a KATP channel blocker) and apamin (a Ca2+-dependent K+ channel blocker) suggesting an altered sensitivity of KATP and Ca2+-activated K+ channels.54 Electrophysiological studies and 86Rb+ efflux data also suggest a modified K+ permeability in pancreatic b-cells from Norwich ob/ob mice.52,53 However, with patch-clamp measurements, KATP channels in ob/ob mice islets display a normal behavior in respect of conductance, ionic selectivity, kinetic behavior, voltage dependency, and sensitivity to glucose and ATP/ADP.55 Monoamines (dopamine and 5HT) are stored in the insulin secretory granules of b-cells and may affect the granule maturation and (or) exocytotic procedure.67 Glucose itself induces a significant suppression of islet monoamine oxidase (MAO) activity within 2 min after an intravenous injection.68 Generally, MAO levels rise as glucose levels fall, but this correlation is not observed in islets of ob/ob mice.69 Adrenals   Diffuse nodular lipohyaline deposits are present in the kidney in aging obese mice and they are primarily localized to the mesangial cells.71,72 Obese mice are sterile.2 Infertility is an absolute characteristic of the ob/ob females; however about 20% of the ob/ob male can reproduce.73 The level of LH, FSH and testosterone in serum is lower in obese mice compared to their lean littermates.18 In female obese mice the ovaries and uterus remain atrophic;74 whereas the male counterpart is characterized by smaller testes, hypoplastic seminal vesicles, atrophic interstitial Leydig cells and a slight decrease in the number of spermatozoa.73 Thus, it has been suggested that there is a persistent immaturity of the hypothalamic-pituitary axis in obese mice.75 Endocrine abnormalities Many symptoms in adult ob/ob mice, eg, their low metabolic rate, hypercholesterolemia, decreased body temperature, susceptibility to cold and hypoactivity suggest functional hypothyroidism.18,84 But conflicting results in serum concentrations of thyroid hormones (serum TSH, T3 and T4 and also hypothalamic content of TRH) in ob/ob mice have been reported – either lower,85 higher86,87 or the same88-90 as in lean mice. However, van der Kroon et al84 have tried to explain the discrepancy in results about thyroid activity in ob/ob mice and provided support for the hypothesis that the obese hyperglycemic syndrome in mice is characterized by congenital hypothyroidism. Mobley and Dubuc91 found that obese mice have significantly reduced hormone concentrations between 10 and 21 days of age. Thereafter, the values remained equal to, or above those of their lean littermates. This result is consistent with human data provided by Ozata et al.92 They have demonstrated that thyroid function is abnormal only in the obese child of the leptin gene mutant family but is normal in the adult patients. Thus, it is most likely that in adult ob/ob mice pituitary-thyroid hormone levels remain normal and their apparent hypothyroidism is largely independent of hormone availability to target tissues rather depends on defective target tissue responses.86,93 This interpretation is supported by a number of observations, including decreased 5´-deiodinase activity in brown adipose tissue,94 liver90 and kidney;89 and decreased transport of T3 across the hepatic plasma membrane and a reduced nuclear T3 receptor occupancy.95,96 These findings suggest that T3 availability to target tissues may be impaired in obese mice, which may contribute to diminished thyroid hormone expression and heat production in these animals.89 Furthermore, the thyroid-stimulated component of Na+-K+-ATPase is deficient in ob/ob mice.18 The activity of Na+-K+-ATPase is low in liver, kidney and skeletal muscle of ob/ob mice.97 And a defective regulation of this enzyme may lead to decreased thyroid action.18 Others   Time course of the developing obese hyperglycemic syndrome indicates that the obesity, hyperinsulinemia, and skeletal growth deficits that characterize mature ob/ob mice develop before weaning and are clearly defined as early as 17 days of age.13 Other studies regarding developmental sequence of the abnormalities demonstrate that obesity precedes hyperinsulinemia, which precedes the onset of insulin resistance and hyperglycemia.13,104,105 When lean and obese mice are compared as groups there is a significant difference in weight at day 6 in Jackson colony16 or at day 12 in Swedish colony.106 And at day 18, clinical diagnosis of the ob/ob syndrome could be made with 100% certainty. Already at day 17, Dubuc13 and Garthwaite et al26 found higher insulin levels in obese mice, others reported about day 20.12,106 However, there is no significant rise in blood sugar until day 22, but afterward, obese animals have higher blood glucose values than their lean littermates.106 These results suggest, since obesity and hyperinsulinemia are manifested well before the presence of hyperglycemia, neither hyperglycemia nor insulin resistance seems to be responsible for the development of the obesity nor of the hyperinsulinemia that occurs in mature ob/ob mice.13 However, based on the manifestation of features, the development of obese hyperglycemic syndrome in ob/ob mice can be differentiated into three distinct phases. The first phase is the dynamic phase. After an initial asymptomatic period the dynamic phase is characterized by rapid weight gain, increasing insulin secretion and decreasing glucose tolerance with fasting hyperglycemia.12,25,107 The next phase is the transitional phase, which is characterized by shifting of glucose pattern, ie, extremely poor glucose tolerance and extremely high serum insulin level is followed by improving glucose tolerance and decreasing insulin levels. Body weight gradually reaches to its maximum value. In the final static phase, blood glucose and serum insulin levels return to near normal values and body weight slowly decreases. Ingalls et al2 first described the obese hyperglycemic syndrome in 1950 as a single gene mutation in the C57BL/6J mouse strain. Previous studies with ob/ob mice have demonstrated that several experimental techniques reduce the severity of the behavioral and metabolic abnormalities displayed by these mice. For example, treatment with b-cytotoxic agents, or the implantation of pancreatic islets from lean littermates reduces hyperinsulinemia, hyperglycemia and increased body weight.108-110 Adrenalectomies, hypophysectomy, alterations of diet and food restriction are also effective in normalizing some aspects of the obese-hyperglycemic syndrome.111-114 However, they are not enough to conclude which metabolic disturbance is directly related to the genetic lesion. In an elegant experiment, Coleman5 showed that parabiosis of obese mouse (ob/ob) with a normal one suppressed weight gain in the obese mouse, whereas parabiosis to diabetes mouse (db/db) caused profound weight lost and death of the obese one. Taken together, these results suggest that the ob gene was necessary for the production of a humoral satiety factor that regulates energy balance and due to the mutation, obese mouse does not produce sufficient satiety factor to turn off its eating drive.5,115 This long search for the precise nature of the defect come to an end in 1994, when Zhang et al,10 cloned the ob gene and confirmed Coleman’s hypothesis.   Fig-2. Physiological role of leptin in glucose homesotasis. Leptin stimulates the rate of hepatic glucose output (HGO), inhibits insulin secretion, increases glucose uptake and glycogenesis in muscle. It stimulates lipolysis and decreases lipogenesis in adipoytes.   Fig-3. Development of the features in obese hyperglycemic syndrome due to lack of circulating leptin. NPY, AGRP, POMC and CART neurons are directly responsive to leptin. NPY and AGRP stimulate feeding (orexigenic), whereas α-melanocyte stimulating hormone and CART inhibit feeding (anorexigenic). These neurons also project to the lateral hypothalamus and regulate the expression of melanin-concentrating hormone (MCH) and possibly orexins (hypocretin). Leptin inhibits the expression and secretion of NPY via Y1 receptor. NPY = Neuropeptide Y; AGRP = agouti-related peptide; MCH = melanin-concentrating hormone; POMC = proopio-melanocortin; αMSH = α-melanocyte stimulating hormone (a product of POMC); CART = cocaine- and amphetamine-regulated transcript; CRH = corticotropin-releasing hormone; TRH = thyrotropin-releasing hormone; SS = somatostatin; GHRH = growth hormone-releasing hormone; GnRH = gonadotropin-releasing hormone; ACTH = Adrenocorticotrpin hormone; TSH = Thyroid-stimulating hormone; GH = Growth hormone; LH = Luteinizing hormone; FSH = Follicle-stimulating hormone; T3 = Tri iodothyronine; T4 = Thyroxine; TH = Helper T cells. Sequences in development of ob/ob syndrome   Fig-4. Sequences in the development of ob/ob syndrome due to lack of leptin. Use of ob/ob mice   In humans, circulating leptin concentrations have been reported to correlate closely with the body mass index (BMI),124,125 total amount of body fat126 as well as the size of adipose tissue mass.127 The normal range for plasma leptin in healthy humans is 3-5 ng/ml and in obese subjects are in the range of 8-90 ng/ml.126 The increase in serum leptin concentration in obesity involves both the increase in number of adipocytes and induction of ob mRNA. The large adipocytes, which are commonly present in obese subjects due to hypertrophy and hyperplasia of adipose tissue, express more ob mRNA than small adipocytes.128 The ob mRNA expression is also upregulated by glucocorticoids.129,130 By contrast, stimulation of the sympathetic nervous system or the increase in circulating epinephrine results in inhibition of ob mRNA expression.130 In human beings, there is a highly organized pattern of leptin secretion over a 24-h period. The circadian pattern is characterized by basal levels between 08:00 and 12:00 hours, rising progressively to peak between 24:00 and 04:00 hours and receding steadily to nadir by 12:00 hours.133 However, these changes in leptin plasma concentrations are not acutely altered by food intake or changes in insulin and glucose concentrations in humans. The underlying mechanism is not fully understood, but leptin rhythm seems to be more associated with meal timing than to the circadian clock.134,135 It has been speculated that the nocturnal rise in leptin could have an effect in suppressing appetite during the night while sleeping136 and since leptin and cortisol show an inverse circadian rhythm,137 it has been suggested that a regulatory feedback is present. Glucocorticoids act directly on the adipose tissue and increase leptin synthesis and secretion in humans.138 Leptin levels are markedly increased in Cushing’s syndrome patients and in other pseudo-Cushing’s syndrome states.138 However glucocorticoids appears to play a modulatory, but not essential roles in generating leptin diurnal rhythm. Increased leptin secretion glucocorticoids in turn augments coordinated activation of anorexigenic pathways and inhibition of orexigenic pathways mediated by leptin-responsive neurons in the hypothalamus.138,139 Furthermore the modulatory role of glucocorticoids could be altered in obesity, but the precise mode of action is still unknown. Human obesity is often associated with severe insulin resistance with high circulating levels of both insulin and leptin.142 In this regard, the failure of the elevated leptin levels to restore normal energy and metabolic homeostasis is commonly viewed as evidence for leptin resistance. Resistance is likely caused by a combination of resistance at the receptor and post-receptor levels (level of signaling) as well as a decreased ability of the blood-brain barrier (BBB) to transport circulating leptin into the brain.126 Several studies have documented various types of evidence for impaired transport of leptin across the BBB. For example, obese humans have a decreased CSF-to-serum ratio for leptin despite high circulating levels of leptin, and some obese rats that no longer respond to peripherally administered leptin can still respond to leptin given directly into the CNS.126 Rising leptin levels associated with progressing obesity are generally regarded as simply a consequence rather than a causative factor in the leptin resistance and obesity. Though serum leptin levels are high in obesity, obese as well as lean subjects with type 2 diabetes display reduced leptin levels.143 This lower leptin levels in diabetics have been speculated to contribute in accumulation of cellular lipids that is associated with diabetes.143,144 The absolute requirement for leptin in controlling body fat mass and regulating reproduction is firmly shared between mouse and man.154,155 Similar to ob/ob mice, leptin helps in regulation of ovarian development and steroidogenesis and serves as either a primary signal initiating puberty or a permissive regulator of sexual maturation.156,157 Leptin regulates the growth and development of conceptus, fetal/placental angiogenesis, embryonic hemopoiesis and hormonal biosynthesis within the maternal–fetoplacental unit and could be taken as a marker of fetal mass in humans.157 Elevated leptin concentrations in cord blood are associated with macrosomia158 and recently, it has been found that leptin levels of amniotic fluid and maternal serum were higher in pregnant women who had fetuses with neural tube defect than in women with healthy fetuses.159 Human leptin has been purified and identified in milk and colostrum from nursing mothers and it has been suggested that in the neonatal period it may play a role in the regulation of neonatal food intake and in the intestinal maturation.160,161 Clinical implications The marked insulin resistance and hyperlipidemia in leptin-deficientrodent models of lipoatrophy is largely reversed by leptin administration.167-169 Low-dose leptin treatment has dramatic effects to ameliorateinsulin resistance and hyperlipidemia in patients with low leptinlevels resulting from congenital or acquired lipodystrophy.170 The beneficial metabolic effects were associated with reducedtriglyceride deposition in liver and intramyocellular lipidcontent in skeletal muscle.171,172 Leptin also improved pituitary,reproductive, and thyroid axis function in lipoatrophic patients.173 Plasma leptin concentrations are also decreased in some patients with lipodystrophy associatedwith human immunodeficiency virus infection and antiretroviraltreatment.174,175 It is possible that leptin replacement therapywould be beneficial in managing some of the metabolic abnormalities(hepatic steatosis, hyperlipidemia, and insulin resistance)in the patients with low leptin levels. 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