Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.     

Abdominal obesity in BTBR male mice is associated with peripheral but not hepatic insulin resistance

Insulin resistance is a common feature of obesity. BTBR mice have more fat mass than most other inbred mouse strains. On a chow diet, BTBR mice have elevated insulin levels relative to the C57BL/6J (B6) strain. Male F1 progeny of a B6 x BTBR cross are insulin resistant. Previously, we reported insulin resistance in isolated muscle and in isolated adipocytes in this strain. Whereas the muscle insulin resistance was observed only in male F1 mice, adipocyte insulin resistance was also present in male BTBR mice. We examined in vivo mechanisms of insulin resistance with the hyperinsulinemic euglycemic clamp technique. At 10 wk of age, BTBR and F1 mice had a >30% reduction in whole body glucose disposal primarily due to insulin resistance in heart, soleus muscle, and adipose tissue. The increased adipose tissue mass and decreased muscle mass in BTBR and F1 mice were negatively and positively correlated with whole body glucose disposal, respectively. Genes involved in focal adhesion, actin cytoskeleton, and inflammation were more highly expressed in BTBR and F1 than in B6 adipose tissue. The BTBR and F1 mice have higher levels of testosterone, which may be related to the pathological changes in adipose tissue that lead to systemic insulin resistance. Despite profound peripheral insulin resistance, BTBR and F1 mice retained hepatic insulin sensitivity. These studies reveal a genetic difference in body composition that correlates with large differences in peripheral insulin sensitivity.     

Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides

To investigate the mechanisms underlying long-term resistance of the A/J mouse strain to diet-induced obesity, we studied, over a period of 4 wk, the expression of uncoupling proteins in brown adipose tissue and the expression of hypothalamic neuropeptides known to regulate energy homeostasis and then used microarray analysis to identify other potentially important hypothalamic peptides. Despite increased caloric intake after 2 days of high-fat feeding, body weights of A/J mice remained stable. On and after 1 wk of high-fat feeding, A/J mice adjusted their food intake to consume the same amount of calories as mice fed a low-fat diet; thus their body weight and insulin, corticosterone, free fatty acid, and glucose levels remained unchanged for 4 wk. We found no changes in hypothalamic expression of several orexigenic and/or anorexigenic neuropeptides known to play an important role in energy homeostasis for the duration of the study. Uncoupling protein-2 mRNA expression in brown adipose tissue, however, was significantly upregulated after 2 days of high-fat feeding and tended to remain elevated for the duration of the 4-wk study. Gene array analysis revealed that several genes are up- or downregulated in response to 2 days and 1 wk of high-fat feeding. Real-time PCR analysis confirmed that expression of the hypothalamic IL-1 pathway (IL-1beta, IL-1 type 1 and 2 receptors, and PPM1b/PP2C-beta, a molecule that has been implicated in the inhibition of transforming growth factor-beta-activated kinase-1-mediated IL-1 action) is altered after 2 days, but not 1 wk, of high-fat feeding. The role of additional molecules discovered by microarray analysis needs to be further explored in the future.     

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.     

Fetuin-null mice are protected against obesity and insulin resistance associated with aging

alpha2-HS glycoprotein (AHSG), also known as fetuin-A, inhibits insulin receptor autophosphorylation and tyrosine kinase activity in vitro and in vivo. Earlier we have shown that fetuin-null (KO) mice demonstrate improved insulin sensitivity and resistance to diet-induced obesity. Since aging is associated with insulin resistance and impaired glucose handling, we tested the hypothesis that fetuin-null (KO) mice are resilient to changes in insulin sensitivity associated with aging. Aged (80-week-old) fetuin-null mice were leaner and demonstrated significantly lower body weights compared to age- and sex-matched wild-type (WT) littermates. Leanness in aged fetuin KO mice was accompanied by a significant increase in dark-onset energy expenditure, without marked alteration of respiratory quotient. In comparison to WT mice, fetuin KO mice demonstrated a lower fasting insulin resistance index, and significantly lower blood glucose and insulin levels, following a 4h fast. Interestingly, despite significantly decreased insulin levels during a glucose tolerance test, aged fetuin-null mice demonstrated a similar glucose excursion as WT mice, indicative of improved insulin sensitivity. Analysis of aldehyde-fuchsin stained pancreas from aged fetuin KO mice indicated no difference in islet beta-cell size or number. An insulin tolerance test confirmed the increased insulin sensitivity of aged fetuin KO mice. Further, compared to WT mice, aged fetuin-null mice demonstrated increased skeletal muscle and liver IR autophosphorylation and TK activity. Taken together, this study suggests that the absence of fetuin may contribute to the improvement of insulin sensitivity associated with aging.     

Plasma aldosterone, plasma lipoproteins, obesity and insulin resistance in humans.

Aldosterone production in vitro can be affected by many hormones, autacoids, ions, and lipids, but regulation in humans is incompletely understood. We measured plasma aldosterone in adult subjects with a wide range of obesity and insulin resistance. Aldosterone levels correlated with measures of visceral obesity in one predominantly male cohort and in the women of a second cohort. In the same subjects, aldosterone correlated with insulin resistance. Aldosterone also correlated with plasma cortisol in men and women, and with DHEA-S in women. The data suggested that visceral fat stimulates adrenal steroidogenesis. We found that certain fatty acids stimulated aldosterone production in vitro by rat adrenal cells incubated with rat hepatocytes, but not adrenal cells alone. The results suggested that fatty acids from visceral adipocytes induce hepatic formation of an adrenal secretagogue. This may explain the correlation of plasma steroids with visceral obesity. Aldosterone may contribute to vascular diseases that complicate obesity.     

Adropin deficiency is associated with increased adiposity and insulin resistance

Adropin is a secreted peptide that improves hepatic steatosis and glucose homeostasis when administered to diet-induced obese mice. It is not clear if adropin is a peptide hormone regulated by signals of metabolic state. Moreover, the significance of a decline in adropin expression with obesity with respect to metabolic disease is also not clear. We investigated the regulation of serum adropin by metabolic status and diet. Serum adropin levels were high in chow-fed conditions and were suppressed by fasting and diet-induced obesity (DIO). High adropin levels were observed in mice fed a high-fat low carbohydrate diet, whereas lower levels were observed in mice fed a low-fat high carbohydrate diet. To investigate the role of adropin deficiency in metabolic homeostasis, we generated adropin knockout mice (AdrKO) on the C57BL/6J background. AdrKO displayed a 50%-increase in increase in adiposity, although food intake and energy expenditure were normal. AdrKO also exhibited dyslipidemia and impaired suppression of endogenous glucose production (EndoR(a)) in hyperinsulinemic-euglycemic clamp conditions, suggesting insulin resistance. While homo- and heterozygous carriers of the null adropin allele exhibited normal DIO relative to controls, impaired glucose tolerance associated with weight gain was more severe in both groups. In summary, adropin is a peptide hormone regulated by fasting and feeding. In fed conditions, adropin levels are regulated dietary macronutrients, and increase with dietary fat content. Adropin is not required for regulating food intake, however, its functions impact on adiposity and are involved in preventing insulin resistance, dyslipidemia, and impaired glucose tolerance.     

Aging is associated with myocardial insulin resistance and mitochondrial dysfunction

Aging is associated with insulin resistance, often attributable to obesity and inactivity. Recent evidence suggests that skeletal muscle insulin resistance in aging is associated with mitochondrial alterations. Whether this is true of the senescent myocardium is unknown. Twelve young (Y, 4 years old) and 12 old (O, 11 years old) dogs, matched for body mass, were instrumented with left-ventricular pressure gauges, aortic and coronary sinus catheters, and flow probes on left circumflex artery. Before surgery, all dogs participated in a 6-wk exercise program. Dogs underwent measurements of hemodynamics and plasma substrates before and during a 2-h hyperinsulinemic-euglycemic clamp to measure whole body and myocardial glucose and nonesterified fatty acid uptake. Following the protocol, myocardial and skeletal samples were obtained to measure components of the insulin-signaling cascade and mitochondrial structure. There was no difference in plasma glucose (Y, 90 +/- 4 mg/dl; O, 87 +/- 4 mg/dl), but old dogs had higher (P < 0.02) nonesterified fatty acids (Y, 384 +/- 48 micromol/l; O, 952 +/- 97 micromol/l) and plasma insulin (Y, 39 +/- 11 pmol/l; O, 108 +/- 18 pmol/l). Old dogs had impaired total body glucose disposition (Y, 11.5 +/- 1 mg x kg(-1) x min(-1); O, 8.0 +/- 0.5 mg x kg(-1) x min(-1); P < 0.05) and insulin-stimulated myocardial glucose uptake (Y, 3.5 +/- 0.3 mg x min(-1) x g(-1); O, 1.8 +/- 0.3 mg x min(-1) x g(-1); P < 0.05). The impaired insulin action was associated with altered insulin signaling and glucose transporter (GLUT4) translocation. There were myocardial mitochondrial structural changes observed in association with decreased expression of uncoupling protein-3. Aging is associated with both whole body and myocardial insulin resistance, independent of obesity and inactivity, but involving altered mitochondrial structure and impaired cellular insulin action.     

The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity

Adiponectin is an adipocyte-derived hormone. Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, where the gene encoding adiponectin is located. Here we show that decreased expression of adiponectin correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin decreases insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. This effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. Moreover, insulin resistance in lipoatrophic mice was completely reversed by the combination of physiological doses of adiponectin and leptin, but only partially by either adiponectin or leptin alone. We conclude that decreased adiponectin is implicated in the development of insulin resistance in mouse models of both obesity and lipoatrophy. These data also indicate that the replenishment of adiponectin might provide a novel treatment modality for insulin resistance and type 2 diabetes.     

Childhood obesity and insulin resistance

Insulin resistance (IR) in childhood has importance to the understanding and prevention of the growing epidemic of insulin resistance syndrome (IRS) in adults with attendant obesity, type 2 diabetes (T2DM), atherosclerotic diseases, hypertension, gout, non-alcoholic, steato-hepatitis (NASH), gall bladder disease, nephropathy, polycystic ovarian disease (PCOS), infertility and premature senility. The severity of IR and its’ complications in children unfortunately and usually progresses in their pubertal transition to adulthood; affected young children are more likely than adults to have underlying causal monogenic disorders; the sequence of natural history and events give insights into disease pathogeneses, and optimal life style choices that last are best made during the early formative years. Some features of IR in children discussed herein are: a strong tendency to low birth weight for gestational age, adverse effects of adrenarche and therapeutic steroid therapy, predisposition to premature pubarche, acanthosis nigricans, tall stature despite pituitary GH suppression, allergic diathesis, hyperandrogenism and PCOS, dyslipidemia and fatty liver disease, and diagnosis by clinical and biochemical markers of IR including insulin regulated hepatic hormonal binding proteins such as IGFBP-1. The national preoccupation with the “metabolic syndrome” T2DM and obesity, should be appropriately directed to an improved understanding of IR in children and their management, if the looming health crisis in affected adults is to be seriously addressed. The nation is facing its’ first generation of children who will be less healthy and die younger than the previous generation (Marks (2005) Presentation to the American Association of Diabetes Educators 32nd Annual Meeting and Exhibition, August 10–13, Washington, DC).     

Acquired obesity is associated with increased liver fat, intra-abdominal fat, and insulin resistance in young adult monozygotic twins

We determined whether acquired obesity is associated with increases in liver or intra-abdominal fat or impaired insulin sensitivity by studying monozygotic (MZ) twin pairs discordant and concordant for obesity. We studied nineteen 24- to 27-yr-old MZ twin pairs, with intrapair differences in body weight ranging from 0.1 to 24.7 kg [body mass index (BMI) range 20.0-33.9 kg/m2], identified from a population-based FinnTwin16 sample. Fat distribution was determined by magnetic resonance imaging, percent body fat by dual-energy X-ray absorptiometry, liver fat by proton spectroscopy, insulin sensitivity by measuring the fasting insulin concentration, and whole body insulin sensitivity by the euglycemic insulin clamp technique. Intrapair differences in BMI were significantly correlated with those in intra-abdominal fat (r = 0.82, P < 0.001) and liver fat (r = 0.57, P = 0.010). Intrapair differences in fasting insulin correlated with those in subcutaneous abdominal (r = 0.60, P = 0.008), intra-abdominal (r = 0.75, P = 0.0001) and liver (r = 0.49, P = 0.048) fat. Intrapair differences in whole body insulin sensitivity correlated with those in subcutaneous abdominal (r = -0.72, P = 0.001) and intra-abdominal (r = -0.55, P = 0.015) but not liver (r = -0.20, P = 0.20) fat. We conclude that acquired obesity is associated with increases in intra-abdominal and liver fat and insulin resistance, independent of genetic factors.     

Bypassing the duodenum does not improve insulin resistance associated with diet-induced obesity in rodents

Roux-en-y gastric bypass (RYGB) surgery rapidly improves glucose tolerance and reverses insulin resistance in obese patients. It has been hypothesized that this effect is mediated by the diversion of nutrients from the proximal small intestine. We utilized duodenal-jejunal bypass (DJB) as a modification of gastric bypass to determine the effect of nutrient diversion from the foregut without gastric restriction on insulin resistance in obese rats. The effects of DJB or Sham surgery on glucose homeostasis were determined in both high-fat-fed Long-Evans and Wistar rats. Body weight and food intake were measured weekly postoperatively, and body composition was monitored before and after surgery. Glucose tolerance was tested before and as early as 1 month postoperation; additionally, in Wistar rats, insulin sensitivity was determined by a hyperinsulinemic-euglycemic clamp (HIEC). DJB did not affect body weight, body composition, glucose tolerance, or insulin concentrations over the period of the study. The average glucose infusion rate (GIR) during the HIEC was 6.2 ± 1.16 mg/kg/min for Sham rats compared to 7.2 ± 1.71 mg/kg/min for DJB rats (P = 0.62), and neither endogenous glucose production (EGP; P = 0.81) nor glucose utilization (glucose disappearance (R(d)), P = 0.59) differed between DJB and Sham rats. DJB does not affect insulin resistance induced by a high-fat diet in Long-Evans and Wistar rats. These data suggest that duodenal bypass alone is an insufficient mechanism to alter insulin sensitivity independent of weight loss in obese, nondiabetic rodents.     

Decreased AMP-activated protein kinase activity is associated with increased inflammation in visceral adipose tissue and with whole-body insulin resistance in morbidly obese humans

Inflammation and infiltration of immune cells in white adipose tissue have been implicated in the development of obesity-associated insulin resistance. Likewise, dysregulation of the fuel-sensing enzyme AMP-activated protein kinase (AMPK) has been proposed as a pathogenetic factor for these abnormalities based on both its links to insulin action and its anti-inflammatory effects. In this study, we examined the relationships between AMPK activity, the expression of multiple inflammatory markers in visceral (mesenteric and omental) and abdominal subcutaneous adipose tissue, and whole-body insulin sensitivity in morbidly obese patients (BMI 48 ± 1.9 kg/m²) undergoing gastric bypass surgery. AMPK activity was assessed by Western-blots (P-AMPK/T-AMPK) and mRNA levels of various markers of inflammation by qRT-PCR. Patients were stratified as insulin sensitive obese or insulin-resistant obese according to their HOMA-IR values. The results indicate that AMPK activity is lower in visceral than in subcutaneous abdominal adipose tissue of these patients and that this is associated with an increased expression of multiple inflammatory genes. They also revealed that AMPK activity is lower in adipose tissue of obese patients who are insulin resistant (HOMA-IR > 2.3) than in BMI-matched insulin sensitive subjects. Furthermore, this difference was evident in all three fat depots. In conclusion, the data suggest that there are close links between reduced AMPK activity and inflammation in white adipose tissue, and whole-body insulin resistance in obese humans. Whether adipose tissue AMPK dysregulation is a causal factor for the development of the inflammation and insulin resistance remains to be determined.     

Activation of Toll-like receptor 4 is associated with insulin resistance in adipocytes

Chronic inflammation is closely associated with metabolic disorders such as obesity and type 2 diabetes, however, the underlying mechanism is unclear. Toll-like receptors (TLRs) play a key role in innate immune response as well as inflammatory signals. Here, we observed that mRNA level of TLR4 was induced during adipocyte differentiation and remarkably enhanced in fat tissues of obese db/db mice. In addition, activation of TLR4 with either LPS or free fatty acids stimulated NFkappaB signaling and expression of inflammatory cytokine genes, such as TNFalpha and IL-6 in 3T3-L1 adipocytes. Furthermore, we discovered that TLR4 activation in 3T3-L1 adipocytes provoked insulin resistance. Taken together, these results suggest that activation of TLR4 in adipocyte might be implicated in the onset of insulin resistance in obesity and type 2 diabetes.