C. N. Hales

Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis

In this contribution we put forward a novel hypothesis concerning the aetiology of Type 2 (non-insulin dependent) diabetes mellitus. The concept underlying our hypothesis is that poor foetal and early post-natal nutrition imposes mechanisms of nutritional thrift upon the growing individual. We propose that one of the major long-term consequences of inadequate early nutrition is impaired development of the endocrine pancreas and a greatly increased susceptibility to the development of Type 2 diabetes. In the first section we outline our research which has led to this hypothesis. We will then review the relevant literature. Finally we show that the hypothesis suggests a reinterpretation of some findings and an explanation of others which are at present not easy to understand.

Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation.

Maternal protein restriction is a model of fetal programming of adult glucose intolerance. Perfused livers of 48-h- starved adult offspring of rat dams fed 8% protein diets during pregnancy and lactation produced more glucose from 6 mM lactate than did control livers from rats whose dams were fed 20% protein. In control livers, a mean of 24% of the glucose formed from lactate in the periportal region of the lobule was taken up by the most distal perivenous cells; this distal perivenous uptake was greatly diminished in maternal low protein (MLP) livers, accounting for a major fraction of the increased glucose output of MLP livers. In control livers, the distal perivenous cells contained 40% of the total glucokinase of the liver; this perivenous concentration of glucokinase was greatly reduced in MLP livers. Intralobular distribution of phosphenolpyruvate carboxykinase was unaltered, though overall increased activity could have contributed to the elevated glucose output. Hepatic lobular volume in MLP livers was twice that in control livers, indicating that MLP livers had half the normal number of lobules. Fetal programming of adult glucose metabolism may operate partly through structural alterations and changes in glucokinase expression in the immediate perivenous region.

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide‐sensitive channels in an insulin‐secreting cell line

1 The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch‐clamp methods in an insulinsecreting cell line derived from a rat islet cell tumour. 2 Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2–5 fold. The concentrations of tolbutamide and glibenclamide giving half‐maximum effects on insulin secretion were approximately 40 μM and 0.2 μM, respectively. 3 Diazoxide (0.6‐1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide‐induced release was also inhibited by diazoxide. 4 Tolbutamide, glibenclamide, HB699 and HB985 reduced the open‐state probability of the ATP‐K+ channel in a dose‐dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5 In whole cell recording, in which the total ATP‐sensitive K+ conductance of the cell could be measured, dose‐inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 μM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6 Diazoxide (0.6 mM) activated the ATP‐K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3–30 μM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7 Neither tolbutamide (1.0 mM) nor glibenclamide (10 μM) altered the open‐state probability of the Ca2+‐activated K+ channel or the Ca2+‐activated non‐selective cation channel which are present in this cell line. 8 It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP‐K+ channel or a protein closely associated with this channel.

Dual effects of diazoxide on ATP-K+ currents recorded from an insulin-secreting cell line.

1 The effects of diazoxide on ATP‐K+ channel currents, recorded from the insulin‐secreting cell line, CRI‐G1, were studied using patch‐clamp techniques. 2 Under current‐clamp recording conditions diazoxide (0.6 mm), inhibited action potential activity and hyperpolarized CRI‐G1 cells with a concomitant increase in membrane conductance. Recordings from voltage‐clamped whole‐cells and isolated patches indicate that activation of ATP‐K+ channel currents underlie these effects. 3 Diazoxide elicited an activation of ATP‐K+ channels which had been partially inhibited by ATP, on application to either surface of the plasma membrane, although it was more effective when applied directly to the cytoplasmic side. Activation of the ATP‐K+ currents involves an increase in the single channel open‐state probability and an apparent increase in the number of functional channels. 4 Activation was observed only when Mg‐ATP was present in the cytoplasmic bathing solution. There was no activation of currents by diazoxide when ATP, in the absence of Mg2+ ions, or Mg‐AMP‐PNP was present to inhibit the ATP‐K+ channels. 5 In the absence of ATP and Mg2+ ions in the cytoplasmic bathing solution, diazoxide (0.6 mm) produced an inhibition of ATP‐K+ currents. 6 Cromakalim (BRL 34915) at 10 μm and 100 μm had no significant effects on ATP‐K+ currents. 7 It is concluded that diazoxide‐induced activation of ATP‐K+ channel currents probably involves phosphorylation of the channel or some closely associated membrane protein.

Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells.

SummarySulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma membrane resulting in inhibition of ATP-sensitive K+ channels, membrane depolarization and thus influx of Ca2+ through voltage-dependent Ca2+ channels. Sulphonylureas can also induce hormone release at fixed membrane potentials without Ca2+ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [3H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (Kd = 1 nmol/l). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority (>90%) of binding proteins were localized to intracellular membranes with only minor levels (<10%) on plasma membranes. The 170 kDa sulphonylurea binding protein was present in both plasma and granule membrane fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcellular distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell.

The effect of insulin on 32P1 incorporation into rat fat cell phospholipids

Abstract 1. 1. The effect of insulin on the incorporation of 32Pi into the phospholipids of rat fat cells incubated in glucose-free medium has been investigated. 2. 2. The incorporation of 32Pi into fat cell phospholipid increased with time, and incubation of fat cells with insulin resulted in increased incorporation of 32Pi into phospholipid compared with controls. 3. 3. Fat cells incubated with insulin for I h showed increased specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with controls. 4. 4. ATP concentrations in fat cells were determined by the luciferase assay. Insulin incubated for 1 h with fat cells produced no significant change in ATP concentrations compared with controls. Glucose incubated for I h with fat cells either with insulin or without insulin did not result in any change in ATP concentration compared with incubated controls. 5. 5. The effect of insulin on the incorporation of 32Pi into ATP was determined and the specific radioactivity of ATP calculated. Incubation of fat cells with insulin resulted in an increase in the specific radioactivity of ATP compared with ATP in control cells, and the increase was time-dependent.

Nucleotide Regulation of a calcium-activated cation channel in the rat insulinoma cell line, CRI-G1.

The nucleotide regulation of a calcium-activated nonselective cation (Ca-NS+) channel has been investigated in the rat insulinoma cell line CRI-G1. The activity of the channel is reduced by both AMP and ADP (1–100 μm) in a concentration-dependent manner, with AMP being more potent than ADP. At lower concentrations (0.1–5 μm), both ADP and AMP activate the channel in some patches. Examination of the nucleotide specificity of channel inhibition indicates a high selectivity for AMP over the other nucleotides tested with a rank order of potency of AMP > UMP > CMP ≥GMP. Cyclic nucleotides also modulate channel activity in a complex, concentration-dependent way. Cyclic AMP exhibits a dual effect, predominantly increasing channel activity at low concentrations (0.1–10 μm) and reducing it at higher concentrations (100 μm and 1 mm). Specificity studies indicate that the cyclic nucleotide site mediating inhibition of channel activity exhibits a strong preference for cyclic AMP over cyclic GMP, with cyclic UMP being almost equipotent with cyclic AMP. Cyclic IMP and cyclic CMP are not active at this site. The cyclic nucleotide site mediating activation of the channel shows much less nucleotide specificity than the inhibitory site, with cyclic AMP, cyclic GMP and cyclic IMP being almost equally active.

Regulation of calcium-activated nonselective cation channel activity by cyclic nucleotides in the rat insulinoma cell line, CRI-G1.

The regulation of a calcium-activated nonselective cation (Ca-NS+) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 μm and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 μm, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N6-monobutyryl cyclic AMP and N6, 2′-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS+ channel at all concentrations tested and 2′-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS+, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1mm. Ca-NS+ channel activity was also predominantly activated by low concentrations of Sp-cAMPS. The activating effects of both Sp-cAMPS and cyclic AMP are antagonized by Rp-cAMPS, which by itself only produced a weak inhibition of Ca-NS+ channel activity even at concentrations of 10 μm and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS+ channel by binding to two separate sites.

The effects of pyridine nucleotides on the activity of a calcium-activated nonselective cation channel in the rat insulinoma cell line, CRI-G1

The activity of a calcium-activated nonselective (Ca-NS+) channel in a rat insulinoma cell line (CRI-G1) is inhibited by pyridine nucleotides in excised patches. The effects of all four pyridine nucleotides tested, β-NAD+, β-NADH, β-NADP+ and β-NADPH were very similar when tested at 0.1 mm, and at 1 mm the phosphorylated forms, β-NADP+ and β-NADPH, appeared to be slightly more potent than β-NAD+ and β-NADH. All the pyridine nucleotides tested reduced both the open state probability of the channel and the number of functional channels observed in a single patch.The application of β-NAD+, but not of the other nucleotides tested, to the cytoplasmic surface of isolated inside-out patches from CRI-G1 cells opened a novel nonselective cation channel (the β-NAD+-NS+ channel). The activity of this new channel is calcium sensitive and may also be inhibited by AMP.

BIOASSAY OF GLUCAGON IN HUMAN SERUM

Abstract A sensitive bioassay for glucagon is described using the lipolytic response of isolated fat cells prepared from chicken adipose tissue. The glucagon concentration in human serum was measured and found to be in the range 0·4-1·4 ng. glucagon per ml.